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Yeast As A Tool in Cancer Research 2007
Yeast As A Tool in Cancer Research 2007
Edited by
and
JOSEPH HEITMAN
Duke University Medical Center,
Durham, NC, U.S.A.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
Published by Springer,
P.O. Box 17, 3300 AA Dordrecht, The Netherlands.
www.springer.com
Foreword…………………………………………………………………….ix
List of Contributors………………………………………………………….xi
Introduction………………………………………………………………....xv
Chapter 1
FROM DNA REPLICATION TO GENOME INSTABILITY
IN SCHIZOSACCHAROMYCES POMBE: PATHWAYS
TO CANCER………………………………………………………………...1
Julie M. Bailis and Susan L. Forsburg
Chapter 2
DISSECTING LAYERS OF MITOTIC REGULATION
ESSENTIAL FOR MAINTAINING GENOMIC STABILITY…………...37
Jennifer S. Searle and Yolanda Sanchez
Chapter 3
YEAST AS A TOOL IN CANCER RESEARCH:
NUCLEAR TRAFFICKING……………………………………………….75
Anita H. Corbett and Adam C. Berger
Chapter 4
STUDIES OF PROTEIN FARNESYLATION IN YEAST …………...….101
Nitika Thapar and Fuyuhiko Tamanoi
Chapter 5
FROM BREAD TO BEDSIDE: WHAT BUDDING YEAST
HAS TAUGHT US ABOUT THE IMMORTALIZATION
OF CANCER CELLS…………………………………………………..…123
Soma S. R. Banik and Christopher M. Counter
v
vi Table of Contents
Chapter 6
HSP90 CO-CHAPERONES IN SACCHAROMYCES
CEREVISIAE……………………………………………………………...141
Marija Tesic and Richard F. Gaber
Chapter 7
YEAST AS A MODEL SYSTEM FOR STUDYING
CELL CYCLE CHECKPOINTS………………………………………….179
Carmela Palermo and Nancy C. Walworth
Chapter 8
METABOLISM AND FUNCTION OF SPHINGOLIPIDS
IN SACCHAROMYCES CEREVISIAE: RELEVANCE
TO CANCER RESEARCH…………………………………………….…191
L. Ashely Cowart, Yusuf A. Hannun and Lina M. Obeid
Chapter 9
EXPLORING AND RESTORING THE p53 PATHWAY
USING THE p53 DISSOCIATOR ASSAY IN YEAST………………….211
Rainer K. Brachmann
Chapter 10
FUNCTIONAL ANALYSIS OF THE HUMAN p53 TUMOR
SUPPRESSOR AND ITS MUTANTS USING YEAST………………....233
Alberto Inga, Francesca Storici and Michael A. Resnick
Chapter 11
ABC TRANSPORTERS IN YEAST – DRUG RESISTANCE
AND STRESS RESPONSE IN A NUTSHELL………………………….289
Karl Kuchler and Christoph Schüller
Chapter 12
THE FHCRC/NCI YEAST ANTICANCER DRUG SCREEN………..…315
Susan L. Holbeck and Julian Simon
Chapter 13
YEAST AS A MODEL TO STUDY THE IMMUNOSUPPRESSIVE
AND CHEMOTHERAPEUTIC DRUG RAPAMYCIN………………....347
John R. Rohde, Sara A. Zurita-Martinez and Maria E. Cardenas
Chapter 14
USE OF YEAST AS A MODEL SYSTEM FOR IDENTIFYING
AND STUDYING ANTICANCER DRUGS…………………………..…375
Jun O. Liu and Julian A. Simon
Table of Contents vii
Chapter 15
GENETIC ANALYSIS OF CISPLATIN RESISTANCE
IN YEAST AND MAMMALS…………………………………………...393
Seiko Ishida and Ira Herskowitz
Chapter 16
USING YEAST TOOLS TO DISSECT THE ACTION
OF ANTICANCER DRUGS: MECHANISMS OF ENZYME
INHIBITION AND CELL KILLING BY AGENTS
TARGETING DNA TOPOISOMERASES………………………………409
Anna T. Rogojina, Zhengsheng Li, Karin C. Nitiss and John L. Nitiss
Index…......….…………………………………………………………….429
FOREWORD
Leland H. Hartwell
Director, Fred Hutchinson Cancer Research Center, Nobel Laureate for Medicine, 2001
ix
x Foreword
systems. Questions like these have rarely been answered in the field of drug
discovery.
A more challenging question is: Can yeast be made more applicable for
the discovery of drugs against human targets? While many human drugs are
active in yeast many are not. The lack of effect in yeast can be due to the fact
that yeast does not have the targets at all – e.g., cell surface hormone
receptors, or because the orthologous protein in yeast is sufficiently different,
or that yeast cells have redundant proteins and inhibition of one is masked by
the second. However, even if drugs against human targets are active in yeast,
the yeast orthologue is likely to be different enough to preclude optimization
of drug identification in yeast. One way to solve this problem is by
substitution of human orthologues for yeast ones. One could even substitute
human transport proteins and drug-metabolizing proteins to further optimize
the yeast system.
Moreover, our ultimate interest in drugs is to alter physiology, which is
the product not of single proteins but of pathways and networks of proteins
acting in concert. The most effective use of yeast would probably result
from substituting entire pathways of yeast proteins with their human counter-
parts. With the aid of reporters that quantitatively reveal the activity of the
pathway at different points one could enter a new era of drug discovery that
interrogates modulation of the pathway at different sites. Finally, we should
think about using the same approach for pathways that do not normally exist
in yeast – for example pathways that synthesize hormones. By constructing
the pathway in yeast de novo with appropriate reporters one could screen for
drugs that modulate hormone synthesis and easily localize the target in the
pathway. This sounds like fun. I suspect the dough has only begun to rise on
what yeast has to offer in the arena of drug discovery.
LIST OF CONTRIBUTORS
xi
xii List of Contributors
John L. Nitiss
Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis,
TN 38105
xv
xvi Introduction
1 INTRODUCTION
1
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 1–35.
© 2007 Springer.
2 J. M. Bailis and S. L. Forsburg
The fission yeast genome, like that of other eukaryotes, is divided among
multiple linear chromosomes each requiring multiple origins of replication.
DNA synthesis must therefore be coordinated both between the different
chromosomes and within each individual chromosome. The identification of
specific fragments of the genome that could replicate autonomously on
plasmids provided the first step toward identifying S. pombe origins of
replication. The autonomously replicating sequence (ARS) elements in
S. pombe that have been described thus far are at least 500 bp in size and
correspond to intergenic regions of the genome [24, 53, 114, 203].
Interestingly, centromeric regions of S. pombe chromosomes appear to be
enriched for DNA fragments with ars activity [170]. In some chromosomal
regions, two or more origins are clustered close together [40, 147].
Replication initiates from discrete, defined sites within each origin region
[24, 39, 40, 53, 147, 203].
S. pombe replication origins preferentially lie in adenine–thymine (A–T)
rich regions of the genome where the local sequence of As and Ts is
asymmetric [210]. Although short consensus sequences similar to that of the
S. cerevisiae ARS element have been identified within some of the S. pombe
origins, these are not essential for origin activity [114]. Fine structure
examination of individual origins has revealed multiple short stretches of
DNA where replication initiates as well as adjacent accessory sequences that
promote origin function [29, 39, 85]. Thus, the organization of S. pombe
replication origins appears to be modular, with redundant, dispersed, and
degenerate elements contributing to efficient activity.
Different replication origins display distinct firing efficiencies [147]. It is
not known whether the choice of origins is regulated or stochastic. Within
those regions of the chromosome where origins are clustered, there may be a
hierarchy of preferential origin usage determined at least in part by local
enhancer sequences [84]. In the S. pombe genome, the number of potential
Chapter 1: Replication in Fission Yeast 3
involved in this process are conserved in eukaryotes, many of the details and
order of their functions are still incomplete. Figure 1 provides a model of our
current view of the assembly and activation of proteins at replication origins.
Figure 1. Model of assembly and activation of the preRC based on information from multiple
systems. Cdc18 and Cdt1 bind to ORC at the origin and facilitate the loading of the MCM
protein complex. The preRC is activated by the actions of the CDK and Hsk1/Dfp1 kinases
(“P” indicates phosphorylation). This promotes the association of other replication factors
with the complex localized to the origin, and leads to initiation of DNA synthesis. Many of
the relevant substrates of Hsk1 and CDK have yet to be identified.
The S. pombe Cdc18 (Cdc6 in other eukaryotes) and Cdt1 proteins bind
to origin sites marked by ORC [55, 95, 141]. ORC and Cdc18 physically
interact [28], as do Cdc18 and Cdt1 [141]. ORC, Cdc18, and Cdt1 together
recruit the minichromosome maintenance (MCM) proteins to the complex
assembling at origins [79, 146]. Genetic interactions between the MCM pro-
teins and Cdc18, and the MCM proteins and ORC, have been demonstrated
in S. pombe [49, 55, 101, 102]. Although each of the preRC components is
essential for DNA replication, the different proteins carry out distinct
functions in assembly and activation of replication origins.
The Cdc18/Cdc6 and Cdt1 proteins are thought to act as a “licensing
factor” that is a critical determinant of the onset of DNA replication [81,
109, 142]. Expression of S. pombe Cdc18 and Cdt1, which is controlled by
the Cdc10 transcription factor [65, 81], is restricted to the G1/S window of
the cell cycle [81, 141]. In addition, the Cdc18 protein is regulated by
phosphorylation, which targets Cdc18 for ubiquitin-mediated proteolysis
[68, 69]. It is not known whether S. pombe Cdt1 protein levels are also
regulated. In Xenopus, Cdt1 activity is inhibited by association with another
protein, geminin [16]; however, a geminin homolog has not been described
in yeast. The cell cycle-regulated activity of Cdc18 and Cdt1 both promote
Chapter 1: Replication in Fission Yeast 5
preRC assembly and prevents its reassembly until the next cell cycle [17,
80].
The MCM complex is not only a preRC component, but also a
compelling candidate for a replicative helicase [41, 80, 98, 148]. The
complex is composed of six homologous subunits (MCM2–7) with similarity
to a large family of ATPases [91]. Each subunit is conserved in other
eukaryotes, and each is essential for replication and cell viability [148, 193].
Mutation of individual S. pombe MCM genes results in defects in replication,
irreversible S-phase arrest and delocalization of the entire MCM complex
from the nucleus [34, 49, 101, 149]. The mcm mutant arrest occurs with the
bulk of replication completed and requires the DNA damage checkpoint,
suggesting that the chromosomes are damaged in mcm mutant cells [101,
102, 108, 123, 178].
Interestingly, although the MCM proteins are estimated to be at least
tenfold more abundant than the predicted number of origins, reducing the
amount of a single MCM protein results in chromosome instability and
defects in the completion of S phase [49, 102]. Replication can still initiate
with low levels of MCM protein, suggesting that the amount of MCM
protein required to initiate DNA replication is much less than that needed to
complete S phase [102]. Genetic interactions between S. pombe MCM genes
and factors involved in the elongation step of DNA replication suggest that
MCM complex function is required throughout S phase [49, 102]. This is
consistent with more direct experiments in S. cerevisiae, where specific
degradation of one of the six MCM subunits during S phase blocks further
DNA replication [94, 103]. The current model suggests that a heterohexa-
meric MCM complex with all six MCM subunits is present at replication
origins, and then travels with the replication fork [78, 148, 193].
Although all six S. pombe MCM subunits interact [1], distinct MCM
subcomplexes have been identified in vitro and in vivo, suggesting that
individual MCM proteins have different relative affinities for each other [96,
162, 163]. The Mcm4, 6, and 7 proteins are thought to form a “core” complex
that is tightly associated [96, 163]. Mcm2 associates with this core through
interactions with Mcm4 [163]. The Mcm3 and 5 proteins form a dimer that
is also loosely associated with the MCM core proteins [162], probably
through interaction with Mcm7 [101]. Similar subcomplexes of MCM
proteins have also been described in other eukaryotes [67, 160]. The core
complex of Mcm4, 6, and 7, but not the heterohexameric complex of Mcm2,
3, 4, 5, 6, and 7, demonstrates weak helicase activity in vitro in S. pombe [96]
and in human cells [67]. Curiously, point mutations of conserved residues in
each Mcm protein that are predicted to inhibit ATPase or helicase activity
display different effects in vivo in both S. pombe [49, 52] and S. cerevisiae
[160] depending on the subunit mutated. This suggests division of labor
6 J. M. Bailis and S. L. Forsburg
amongst the MCM subunits, and may explain why six related proteins are
required for MCM activity.
Although MCM nuclear localization is regulated in budding yeast, MCM
proteins localize to the nucleus constitutively throughout the cell cycle in
fission yeast and in metazoans [149, 193]. In these organisms, the
association of the MCM proteins with chromatin varies: MCM proteins
localize to chromatin, including replication origins, in late M phase and
dissociate during S phase [79, 146]. Thus, regulation of MCM chromatin
binding is one mechanism of control of MCM complex function. Part of this
regulation is provided by the Cdc18 and Cdt1 loading factors, which limit
MCM binding to M/G1 of the cell cycle [80]. In some organisms, Mcm4
is also regulated by CDK-dependent phosphorylation. In Xenopus, this
promotes Mcm4 dissociation from chromatin [62, 152]; in S. cerevisiae,
Mcm4 phosphorylation leads to its exclusion from the nucleus [140].
However, CDK consensus site mutants of S. pombe Mcm4 do not display
obvious phenotypes in vivo [52].
Figure 2. Control mechanisms that prevent rereplication. CDK activity is regulated so that the
preRC can only assembly once each cell cycle. CDK phosphoyrlates Drc1, which promotes
association with Rad4/Cut5 and replication initiation. CDK phosphorylation of Cdc18 and
Orc2 prevents origins from refiring.
Remarkably, the CDK kinase complex plays both positive and negative
roles in S phase progression (Figure 2). Replication initiation is positively
activated by CDK-mediated phosphorylation of Drc1, which results in the
association of Drc1 with Rad4/Cut5 [143]. Rad4/Cut5 is essential for the
initiation of DNA synthesis and also acts in the DNA damage checkpoint
[44, 156, 157]. Although the molecular role of Rad4/Cut5 is unclear, the
interaction between Rad4 and Drc1 may promote the association of the DNA
polymerases α and ε with origins [143]. The S. pombe homologs of
Rad4/Cut5 in S. cerevisiae (Dpb11) and in human cells (TopBP1) interact
with DNA polymerase as part of their replication function [112, 182]. Each
of these proteins contains BRCT motifs, which are also found in the human
DNA repair gene XRCC1 and the BRCA1 tumor suppressor [19].
CDK activity negatively regulates DNA replication by preventing origins
from refiring in a single cell cycle [17, 80]. CDK-mediated phosphorylation
of Cdc18 and its subsequent degradation prevent reassembly of the preRC
until the next cell cycle, because recruitment of preRC factors such as the
MCM proteins depends on Cdc18 [68]. Overproduction of Cdc18 or
expression of a nonphosphorylated version of Cdc18 induces re-replication,
presumably by resetting origins to the G1 state [80]. Mutation of the CDK
phosphorylation sites in Orp2 also allows re-replication [197], although the
mechanism by which CDK-mediated phosphorylation of Orp2 prevents
re-replication is not clear [95, 107, 197]. Unlike Cdc18, Orp2 appears to
remain associated with the ORC complex at the origin [107]; it is not known
whether phosphorylation of Orp2 changes its affinity for other origin-associated
proteins.
Manipulation of the Cdc2 protein kinase itself can result in re-
replication. Cells that lack mitotically active Cdc2 [21] or the mitotic cyclin
Cdc13 [61] re-replicate DNA, as do cells overproducing the CDK inhibitor
8 J. M. Bailis and S. L. Forsburg
Once the preRC is formed and activated by the CDK and Cdc7 protein
kinases, additional components of the replication machinery become
associated with the complex, which becomes the active replication fork.
Many of these proteins are essential for viability and conserved in all
eukaryotes, including Cdc23/Mcm10, Sna41/Cdc45, the single-strand DNA
binding protein replication protein A (RPA), DNA polymerase α DNA
polymerase δ DNA polymerase ε DNA, replication factor C (RFC), and
proliferating cell nuclear antigen (PCNA) [198].
S. pombe cdc23 mutants, like the corresponding mutants in other
eukaryotes, display defects in replication and demonstrate genetic
Chapter 1: Replication in Fission Yeast 9
interactions with other replication mutants [7, 77, 100]. A mutant of the
S. cerevisiae homolog of cdc23+, MCM10, can be complemented by
S. pombe cdc23+, suggesting conservation of gene function [7]. S. cerevisiae
MCM10 appears necessary to recruit the MCM complex to chromatin [66],
and also functions in replication elongation [116]. In both S. cerevisiae and
S. pombe, Cdc23 associates with chromatin throughout the cell cycle [66,
101]. In Xenopus, MCM10 is dispensable for MCM complex binding to
chromatin but is required to localize Cdc45/Sna41 [204].
Sna41/Cdc45 appears to be required both for replication initiation and
elongation. The association of Sna41 with chromatin requires the Sld3
protein, which has been described in both the budding and fission yeasts [74,
136]. Sna41/Cdc45 is required to load other replication factors such as RPA,
DNA polymerase α, DNA polymerase ε, and PCNA [182]; in S. pombe,
Sna41 [124, 194] has been shown to interact with DNA polymerase α [195]
and recruit it to the MCM protein complex at replication origins [195]. A
role for Sna41/Cdc45 in origin DNA unwinding has been suggested from
experiments in Xenopus [199] and in S. cerevisiae [74]. In S. cerevisiae,
Cdc45 function has been shown to be required throughout S phase [189].
Many of the genes involved in DNA replication in S. pombe are
homologous to those described in other eukaryotes, and the enzymology of
the gene products is conserved [198]. RPA is needed to recruit the DNA
polymerases α, δ, and ε to replication origins [198]. Interestingly, DNA
polymerase α may have an additional function beyond replication initiation
and DNA synthesis (see section VIII). The eukaryotic DNA polymerase
processivity factor PCNA becomes associated with the replication fork
through the actions of the clamp loader, RFC [198]. S. pombe PCNA also
interacts with DNA polymerase δ [155] and promotes its processivity [6].
Components of S. pombe RFC and primase are involved in the replication
checkpoint as well as in DNA synthesis [56, 164].
Several proteins involved in lagging strand metabolism, such as DNA
ligase [72], the Dna2 helicase [54, 75], and the homolog of the FEN1
endonuclease, Rad2 [132] have also been characterized in S. pombe. The
dna2 mutant can be suppressed by overproduction of DNA polymerase δ,
DNA ligase or Rad2, suggesting that Dna2 has a central function in Okazaki
fragment maturation [75]. Mutants of a novel S. pombe replication factor,
cdc24+, are suppressed by overproduction of Dna2, as well as by
overproduction of PCNA or RFC [54, 184]. S. pombe cdc24+ has no known
sequence homolog in other systems [54]. Mutants of cdc24+ are defective in
the completion of S phase and display chromosome breakage, which is
not typical of replication mutants [54]. Cdc24 thus may play a role in
10 J. M. Bailis and S. L. Forsburg
and subsequent progress through the cell cycle. This suggests that replication
fork structure is preserved during S phase arrest caused by HU treatment.
The checkpoint kinase Cds1 may play a role in stabilization of stalled
replication forks. S. pombe cds1 mutants arrest in HU, but are defective in
recovery from the arrest [104]. The HU-induced arrest of cds1 cells results
from the activation of the damage checkpoint kinase Chk1, suggesting that
Cds1 is needed to prevent chromosomal damage in the presence of HU
[104]. Consistent with this hypothesis, aberrant replication structures are
observed in S. pombe cds1 and rad3 mutants treated with HU [86]. Loss of
replication fork integrity has also been observed in rad53 mutants of
S. cerevisiae, suggesting a conserved mechanism of damage tolerance [106,
188].
S. cerevisiae Rad53 is phosphorylated in response to S phase damage
[150, 188] and phosphorylates components of the replication machinery to
restrain its progression [150]. Similarly, S. pombe Cds1 becomes phos-
phorrylated in response to HU treatment [104]. This in turn leads to Cds1-
dependent phosphorylation of both Hsk1 and its activator Dfp1, suggesting
that Hsk1 and Dfp1 are targets of the cellular response to replication blocks
[22, 171]. These phosphorylation events may serve to regulate the inter-
action of Hsk1/Dfp1 with proteins localized to replication origins and to
prevent the activation of origins under conditions unfavorable to the cell
[70, 110].
Although S. pombe Hsk1 and Dfp1 have an essential role in activation of
replication origins, Cds1 is dispensable during a normal S phase [104, 129].
Recently, an S-phase specific upstream activator of Cds1, Mrc1, was
identified [3, 185]; both Mrc1 and Cds1 are nonessential for cell viability [3,
129]. Future work should determine how the cell senses blocks to replication
and how this information is directed to Mrc1 and Cds1.
Recovery from HU-induced S phase arrest also requires Rqh1, the fission
yeast homolog of the Escherichia coli RecQ helicase [175]. The wild-type
function of Rqh1 during S phase is not known but may involve preventing
inappropriate recombination events when replication forks are stalled [175].
Mitotic recombination is elevated in rqh1 mutants that are treated with HU
[131, 175]. Mutants of rqh1+ also display synthetic genetic interactions with
components of the replication machinery [131, 171]. In S. cerevisiae, the
Rqh1 homolog SGS1 localizes with the Rad53 kinase and promotes its
phosphorylation in response to HU [51]. However, SGS1 mutants are
hyperrecombinant even in the absence of HU, suggesting there may be some
differences in the function between SGS1 and Rqh1 [51].
12 J. M. Bailis and S. L. Forsburg
in S. pombe and other eukaryotes, which are likely to be significant for the
maintenance of overall genome stability.
Figure 3. Model for replication restart (based on 33,119). When cells are treated with HU,
replication forks stall. If the structure of the fork can be maintained through the arrest, then
the fork may resume synthesis once HU is removed from the media. If the fork structure
cannot be maintained, the fork may collapse, generating DNA double-strand breaks.
Recombination is one mechanism that may repair DNA breaks and reestablish stalled
replication forks.
The events of S phase are closely linked with the later events of the cell
cycle. As described above, maintenance of the order of the cell cycle and
alternation of S phase with mitosis preserves the genome integrity. In
addition, chromosomal processes such as sister-chromatid cohesion and
silencing are regulated coordinately with the replication of the DNA.
Intriguingly, the replication fork is the one structure that contacts all of the
DNA, once, in a single cell cycle. This positions the replication machinery
in a unique state to monitor and modify any region of the genome during
S phase.
Cohesion between newly replicated sister chromatids holds them together
from S phase until their separation during mitosis. This arrangement is
essential for the proper attachment of kinetochores to the microtubule
spindles and correct segregation of the chromatids during mitosis [139]. The
14 J. M. Bailis and S. L. Forsburg
ACKNOWLEDGMENTS
Work in our laboratory was supported by grants from the NIH, NSF, and
the American Cancer Society. S.L.F. is a Stohlman Scholar of the Leukemia
& Lymphoma Society. J.M.B. is a fellow of the Damon Runyon Cancer
Research Fund (DRG-1634). We thank Michael Catlett and Han-Kuei
Huang for comments on the manuscript.
Chapter 1: Replication in Fission Yeast 21
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Chapter 1: Replication in Fission Yeast 33
ADDENDUM
Since this chapter was written, several important discoveries have advanced
our understanding of how DNA replication contributes to genome stability.
First, progress has been made toward uncovering the functions of proteins
associated with replication origins. Unexpectedly, an additional, conserved
protein complex essential for DNA replication has been identified. The
GINS complex consists of four subunits (Sld5/Cdc105, Psf1/Cdc101,
Psf2/Cdc102, and Psf3) that associate with replication origins and are
required for replication initiation and elongation [10, 12, 21]. In S. pombe,
the Psf2 subunit was identified in a screen for mutants that prevent
rereplication; similar to its homologs in other organisms, S. pombe Psf2 is
required for normal S-phase progression [4]. Other recent reports have
uncovered details about the requirements for replication protein association
with origins and activity on their targets. The S. pombe Cdc23 protein has
been shown to be required for Hsk1-dependent phosphorylation of the MCM
complex [13], as well as for the recruitment of Cdc45 to chromatin [6].
Cdc45 appears to be one of the last proteins to associate with the prerepli-
cative complex, as its localization requires formation of the prereplicative
complex, as well as Hsk1 and Rad4 [3, 6]. Cdc45 has been suggested to
travel with the replication fork in S. cerevisiae [1, 22] and is required for the
in vitro helicase activity of the heterohexameric MCM complex [14].
A second area of research progress has been in understanding how cells
respond to S-phase damage and replication blocks. S. pombe Cds1, like its
homolog Rad53 in S. cerevisiae, is required to prevent replication fork
collapse in the presence of HU [15]. Importantly, two proteins that mediate
Cds1 function, Mrc1 and Swi1/Tof1, have been shown to associate with the
replication fork even in the absence of DNA damage, suggesting a
mechanism to couple detection of damage by the moving fork [11, 15, 17].
In mammalian cells, HU-induced checkpoint activation leads to the
association of MCM7 with the Rad17 and ATRIP/ATR checkpoint proteins
[23] and phosphorylation of MCM2 and MCM3 [2, 9, 24]. Interestingly, the
mammalian checkpoint kinases appear to affect not only fork stabilization
during damage, but also the timing of origin firing during normal S-phase
progression [19]. Together, these findings strongly suggest a central,
conserved link between S-phase checkpoints, MCM proteins, and replication
fork stability. Interestingly, fission yeast checkpoint mutants predicted to
cause replication fork collapse do not cause cell inviability in meiosis [18].
Meiotic cells may tolerate a higher level of DNA damage because pro-
grammed DNA damage is generated during meiotic recombination.
Lastly, our understanding of the links between replication and human
disease has been strengthened. Seckel syndrome, a human disease that
predisposes to chromosome instability and shares phenotypes with DNA
34 J. M. Bailis and S. L. Forsburg
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Chapter 1: Replication in Fission Yeast 35
Cell division is the process by which a cell copies its DNA and cellular
constituents and gives rise to two cells. One complete cell division is termed
a cell cycle. (Although you can have a cell cycle without cell division.) This
process is divided into four distinct phases for most eukaryotic cells: G1, S,
G2, and M (Figure 1A). S phase (for synthesis) is the stage in which the
DNA is replicated and M phase, (for mitosis), is the stage in which the
replicated DNA condenses and the chromosomes are divided equally into
two cells. G1 and G2 are “gap phases” that separate DNA replication and
mitosis. This chapter is focused on how yeast have served as a useful model
system that was instrumental in the identification of proteins that promote
cell cycle progression in all eukaryotes. We will discuss the pioneering
work of individuals that used budding yeast to reveal the layers of regulation
that coordinate progression through the cell cycle. In addition, we will
discuss how this model organism that is amenable for genetics has been used
to identify proteins that regulate cell cycle, and in particular mitosis, in order to
safeguard the integrity of the genome. We will end by linking this work to the
specific problem of cancer, and mention the utility of this system in research
designed to establish and validate targets for anticancer drug discovery.
37
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 37–73.
© 2007 Springer.
38 J. S. Searle and Y. Sanchez
cannot live without them), some of the mutants that Hartwell identified had
defects in cell cycle progression (cdc = cell division cycle mutants). Hartwell
identified dozens of mutants that arrested at different stages of the cell cycle
when the cells were raised to the restrictive temperature. The arrest point
was easy to identify as the cells arrested with no bud (G1) or with a uniform
bud size (Figure 1B). One such mutant, cdc28, was defective in the
initiation of DNA replication [43]. The cdc28 mutation caused cells to arrest
without a bud and with unreplicated DNA at the restrictive temperature.
Paul Nurse and Pierre Thuriaux carried out a screen similar to Hartwell’s
using the fission yeast Schizosaccharomyces pombe. They identified a
mutant, cdc2, which arrested prior to mitosis under restrictive conditions
[92]. When the CDC2 gene was cloned, it was found to encode a protein
with homology to kinases. Protein kinases are enzymes that modify other
proteins by adding phosphate groups, a common mechanism used by the cell
to transmit information or regulate protein activity. David Beach, while
working with Nurse, also found that the cdc2 mutation was in a gene
homologous to the budding yeast CDC28 gene identified by Hartwell [5] and
cloned earlier by Nasmyth and Reed [87].
In the late 1980s, investigators in the fields of biochemistry and genetics
came together and initiated an explosion of cell cycle research. At that
point, Manfred Lohka and James Maller succeeded in the purification of
MPF by monitoring an activity that could promote chromosome conden-
sation. Jean Gautier and Maller’s group joined with Nurse, and William
Dunphy and John Newport joined with David Beach and both groups
discovered that MPF is composed of two subunits; a kinase that is the
product of the CDC28 and cdc2+ genes in the yeasts and the mitotic cyclin
[27, 33]. Thus, it was found that the cell cycle engine is universal or
conserved, and that in its bare bones is a complex, composed of a cyclin-
dependent kinase (Cdk) and an activating partner, the cyclin [27, 33, 65, 72].
Interaction of the Cdk and cyclin activates the complex to phosphorylate
substrates, allowing the cell to move into the next cell cycle stage. The
cyclin partner not only activates the Cdk but also provides specificity for
substrates that will drive a particular cell cycle transition. For example, a
complex containing a Cdk and a member of the G1 cyclin family promotes
entry into a new cell division cycle; whereas a Cdk/Mitotic cyclin complex
promotes entry into mitosis. Thus, the availability of the cyclins serves to
regulate the cell cycle stage-specific activity of the Cdks.
The 2001 Nobel Prize for medicine was awarded to three individuals who
used biochemistry and genetics to discover the Cdk complex. Tim Hunt
discovered the mitotic cyclin and, importantly, he found that degradation of
the cyclin coincided with the end of mitosis and, identified a layer of
regulation of the cell cycle engine. Leland Hartwell carried out the famous
40 J. S. Searle and Y. Sanchez
genetic screen that led to the identification of the Cdc28/Cdk. Paul Nurse
identified cdc2+ in a similar genetic screen and found that mammals had a
gene that could function in the place of cdc2+. In doing so, he showed that
the proteins that comprise the cell cycle engine are conserved between yeast
and mammals [68]. These individuals, combined forces with other
biochemists studying the activity of the cell cycle engine, which sparked the
intensity of the field that studies how cell division, chromosome duplication,
and segregation are coordinated. [86, 98].
Many years after the discovery of the Cdk complex, Pavletich and co-
workers analyzed the crystal structure of the cyclin/Cdk complex, which
revealed the mechanism by which the cyclin activates the Cdk. It turns out
that the helix containing a conserved amino acid involved in ATP binding is
positioned away from the active site of the Cdk. The cyclin, by virtue of its
binding, realigns the residues involved in ATP binding and keeps domains
from blocking the catalytic cleft of the enzyme. Thus, binding of the cyclin
removes a stearic block that allows the cdk to phosphorylate its substrates
[55]. This is the reason why MPF activity coincides with a peak of cyclin
levels. Phosphorylation of the activation loop is required to stabilize the
active form of the Cdk complexed with the cyclin, and thus adds another
level of regulation to this complex [95].
Figure 1A. The eukaryotic cell cycle and the universal cell cycle engine. The eukaryotic cell
cycle (with the exception of germ cells and early embryonic cells) is divided into four distinct
phases G1, S, G2, and M. S phase (for synthesis) is the stage in which the DNA is replicated
and M phase, mitosis, is the stage in which the replicated DNA condenses and the
chromosomes are divided equally into two daughter cells. G1 and G2 are “gap phases” that
separate DNA replication and mitosis. The cyclin-dependent kinase/cyclin (Cdk/cyclin)
complex which drives cell cycle transitions is regulated by phosphorylation of the Cdk.
Chapter 2: Checkpoints and Genomic Stability 41
Figure 1B. Progression of Saccharomyces cerevisiae cells through the cell cycle can be
monitored by bud and nuclear morphology. Bud emergence coincides with the initiation of
DNA synthesis (DNA replication), G2 cells have large buds and an undivided nucleus at the
neck. Cells in anaphase have an elongated bipolar nucleus (dumbbell shape) and elongated
spindles. Telophase cells have two distinctly divided nuclei in the mother and daugher cells.
Cytokinesis gives rise to two unbudded cells that are in G1. Indicated is the morphology with
which the cdc mutants arrest at each point in the cell cycle.
anaphase where the sisters separate due to the force exerted by the spindle. The separated
chromatids move to opposite poles of the cell. The mitotic Cdk/B-type cyclin complexes are
inactivated via many mechanisms, which allows the pre-replication complexes to assemble on
the chromatin so that the cell is ready to begin DNA replication at the next S phase.
proteolysis of the B-type cyclin [66]. Then Kirschner’s and Hieter’s groups
joined forces to determine that Cdc27 and Cdc16 are indeed components of a
large complex that catalyzes the conjugation of ubiquitin to the B-type
cyclins and are thus, components of the evolutionarily conserved APC/C
[60].
The biochemical experiments with the nondegradable B-type cyclin
mentioned above always led to a block in mitotic exit but not to chromosome
segregation at anaphase indicating that proteolysis of the B-type cyclins was
not required for anaphase to occur. The observation that APC mutants that
arrested in pre-anaphase did so with high levels of the B-type cyclin Clb2,
and that B-type cyclin degradation is not required for anaphase to take place
(biochemical experiments with nondegradable B-type cyclin and [118]),
suggested that the APC/C had different substrates in order to promote
anaphase and mitotic exit. The anaphase substrate of the APC/C, Pds1, was
identified in a genetic screen carried out by Doug Koshland’s group that
identified genes encoding proteins involved in chromosome segregation
[135, 136]. In this screen, the investigators were looking for mutations that
caused the cell to separate sister chromatids prematurely (Pds = precocious
dissociation of the sisters). Orna Cohen-Fix, while in Koshland’s laboratory,
showed that Pds1 was destroyed prior to anaphase and that it was a substrate
of the APC/C [20]. The APC/C components and its substrates are conserved,
thus Pds1 orthologues are now called securin, due to their function in
keeping sister chromatids together until anaphase. The mechanism by which
Pds1 keeps sister chromatids together is by acting as an inhibitor of a
caspase-like protease Esp1 (also known as separase). Esp1 cleaves Scc1, a
protein that is part of the cohesin complex (Scc1, Smc1, Smc3, and Scc3)
that forms during DNA replication to keep the sister chromatids together.
Cleavage of Scc1 by Esp1 leads to loss of cohesion along the arms of the
chromosomes, and the tension provided by the spindle apparatus is then able
to pull the sister chromatids apart at anaphase (see Figure 2). In summary,
the APC promotes progression through mitosis by the ordered ubiquitin-
mediated proteolysis of the anaphase inhibitor, Pds1 (securin), and the
B-type (mitotic) cyclins, Clb2 and Clb5 [66, 97, 123, 126, 127].
The APC relies on proteins that act as specificity factors in order to
ubiquitinate the correct substrates in an ordered and timely manner. These
proteins are the WD repeat (tryptophan and aspartic acid repeats) containing
proteins Cdc20 and Hct1/Cdh1. Thus, cdc20, a mutant identified in the
Hartwell screen that arrests prior to anaphase, is defective in the ubiquitination
and destruction of both Pds1 (securin) and Clb2 (B-type cyclin). Since
destruction of the B-type cyclins is not required for anaphase [118], this
finding suggested that there existed a regulatory step that ensured that the
46 J. S. Searle and Y. Sanchez
in place that regulated the order and timing of cell cycle transitions. These
mechanisms are referred to as checkpoint controls [98].
The controls identified by Rao and Johnson become essential when the
relative timing of cell cycle transitions is challenged and cannot be merely
regulated by an oscillator, or the “cell cycle clock”; such is the case when
DNA replication is prolonged due to DNA damage or other stress conditions
(Figures 4 and 5). Elegant experiments carried out by Smythe and Newport
with cell free extracts from Xenopus eggs provided biochemical evidence of
this response [111] by showing that incompletely replicated DNA blocks the
activation of the mitotic Cdk/cyclin complex by maintaining the Cdk subunit
in a phosphorylation form. The mechanisms that are activated when the
relative timing of cell cycle transitions is altered are also important when the
chromosomes are not correctly attached or oriented on the spindle or when a
cell has not reached a critical size prior to division. These checkpoint
controls are mediated by signal transduction pathways that ensure the
interdependence of cell cycle events. For example, during DNA replication
a signal is produced (probably at the replication fork, or when forks pause or
stall] that will cause the cell to delay mitosis until all forks have completed
replication. Because of their function, these biochemical pathways are called
checkpoints, a term coined by Hartwell, and they represent the next layer of
regulation of the cell cycle that is covered in this chapter.
the anaphase inhibitor, Pds1 (securin), and the B-type (mitotic) cyclins, Clb2, and Clb5. The
APC/Cdc20 targets Pds1 for ubiquitination, this leads to activation of the separase and loss of
cohesion (see Figure 2). The APC/Cdc20 then targets the Clbs for ubiquitination leading to
the activation of the mitotic exit network (MEN) and release of the phosphatase Cdc14 from
the nucleolus. Dephosphorylation of Hct1 and Sic1 serve to further inactivate the mitotic
Cdk/Cyclin complexes and allows the assembly of pre-replication complexes and exit from
mitosis.
The Rad screen. By the late 1960s, many groups had carried out screens
to identify yeast mutants that were sensitive to ionizing radiation, UV
radiation, or both; these mutants were named rad mutants. Brian Cox, John
Game, and Robert Mortimer among others, identified a collection of
radiation sensitive mutants [22]. In 1970 scientists came to an agreement that
mutations that conferred sensitivity to ionizing radiation would continue to
be named rad mutants, numbered from RAD50 upward and they would be
loci separate from those identified in screens for UV-sensitive mutants.
50 J. S. Searle and Y. Sanchez
At around the same time, Kato and Ogawa characterized a mutant named
esr1, which showed sensitivity to the alkylating agent methyl–methane–
sulfonate (MMS) and to UV radiation [57]. In addition, the esr1 mutants
displayed meiotic defects. David Stern’s group took a biochemical approach
and isolated Spk1 in a screen that used a bacterial system to identify dual
specificity kinases (kinases that can phosphorylate proteins on serines/
threonine and tyrosine residues) from yeast [141].
When all of the genes mutated in these strains were cloned it turned out
that many alleles of the same genes had been identified in the different
screens and this pointed not only to their important role in various responses
but also provided hints as to their biochemical functions. From the genes
that were cataloged by Game and Mortimer as x-ray sensitive [30], RAD51,
RAD52, and RAD54–RAD57 encoded proteins involved in recombination-
mediated DNA repair. However, RAD53 encoded an essential kinase that is
required for the cell to arrest not only in response to DNA damage but also
when DNA replication is slowed down or blocked [2, 133, 141]. RAD53
alleles were identified not only in the rad screens but also in the sad (sad1),
and mec (mec2) screens and Rad53 was the kinase identified in David
Stern’s screen for dual specificity kinases (Spk1) (Table II). Another
essential kinase that regulates this response is Mec1, and it was identified in
the mec screen, sad screen (sad3) and in Ogawa’s screen as the gene mutated
in esr1.
Dun1 turned out to be another kinase [143] that shared phosphor–peptide
recognition domains (FHA forked-head associated domains [47]) with
Rad53 outside of the kinase domain. Dun2 is the catalytic subunit of DNA
polymerase epsilon [89]. The fact that a component of DNA polymerase
showed defects in a checkpoint response sparked enthusiasm and speculation
that the DNA polymerase complexes, by the nature of their function, made
attractive candidates for sensors and scanning machines [88]. Three kinases
had been identified that are required in order to signal DNA damage and
replication blocks. Many groups began to organize the rad, mec, and sad
mutants (and other mutants that displayed sensitivity to agents that damage
DNA or cause replication blocks) into the DNA repair category or as
components of signal transduction pathways that signal the presence of these
lesions.
There were several criteria and several readouts that were used by many
laboratories in order to piece together the checkpoint signal transduction
pathways. The readouts included the effect of mutations on: (1) the cell’s
ability to delay cell cycle progression when encountering damage at different
stages of the cell cycle; (2) the activation of the kinases by phosphorylation;
and (3) the upregulation of RNR3 mRNA and other damage-inducible gene
transcripts. Using these readouts, proteins required for the checkpoint
52 J. S. Searle and Y. Sanchez
differentiate between the scenarios where the RNA primer is the signal or
where the synthesis of the primer is a step required for the sensor complexes
to assemble on DNA [138] (Figure 5). Nevertheless, with some of the known
players in hand, the answer to these questions may be around the corner.
The transducers. In S. cerevisiae, replication blocks and DNA damage
which occur in S-phase activate a checkpoint response regulated by the kinase
Mec1 (Atr orthologue), which acts upstream of the kinase Rad53 to stabilize
replication forks, prevent firing of late replicating origins and prevent
anaphase entry [2, 106, 120, 133]. A role for Rad53 in the regulation of repli-
cation origin stability and firing would suggest that Rad53 complexes may
also be localized in close proximity to proteins involved in DNA replication.
Mec1 has also been shown to mediate the DNA-damage checkpoint that
regulates mitotic progression by signaling to both Chk1 and Rad53. While in
the Elledge laboratory we showed that Chk1 and Rad53 via Dun1 form
parallel branches in the DNA-damage checkpoint that function downstream
of Mec1 to regulate anaphase entry and mitotic exit, respectively (Figure 4,
[103]). This is accomplished by blocking the destruction of two substrates
of the anaphase-promoting complex or cyclosome.
The effectors. The effectors of the S-phase checkpoint that mediate a
block to anaphase in the budding yeast remain elusive, although in the
fission yeast and vertebrates Chk1 and Cds1/Chk2 (Rad53 orthologue) target
the Cdc25 phosphatase to elicit a G2/M arrest. However the effectors of the
DNA damage checkpoint in the budding yeast have been identified.
The DNA damage-induced checkpoint arrest that prevents progression
through mitosis in S. cerevisiae occurs after the formation of a spindle,
a stage that requires high activity of the mitotic cyclin/Cdk kinase
(Clb2/Cdc28). At the checkpoint-induced arrest, which occurs at the
metaphase to anaphase transition (M–A), the sister chromosomes are paired,
held together by cohesins (such as Scc1, Mcd1) and can presumably interact
with the bipolar pre-anaphase (short) spindle that has assembled at this stage.
Progression through mitosis in all eukaryotic cells is regulated by the activity
of the APC or cyclosome, which triggers chromosome segregation and
mitotic exit through the ubiquitin-mediated degradation of anaphase
inhibitors, such as Pds1 and mitotic cyclins, respectively. Orna Cohen-Fix,
when in Koshland’s laboratory, showed that Pds1 was phosphorylated in
response to DNA damage in a Mec1-dependent manner [18]. Phosphorylation
of Pds1 correlates with accumulation of Pds1 following DNA damage [104].
While in the Elledge laboratory, we also showed that Chk1 inhibits the M–A
transition by phosphorylation and stabilization of Pds1 and Rad53 blocks
Clb2 degradation and possibly exit from mitosis by blocking the activation
of the MEN. The Elledge laboratory continued to look for the targets of the
56 J. S. Searle and Y. Sanchez
Rad53 pathway and found that Bfa1, a component of the MEN pathway is
regulated by Rad53 and Dun1. Phosphorylation of Bfa1 in a Rad53-
dependent manner inhibits the activity of the MEN pathway to help maintain
the levels of Clb2 and thus allow cells to maintain their arrest in mitosis by
maintaining active Cdk/B-type complexes [50]. However, the first wave of
Clb2 destruction occurs at or right after anaphase, and the Cross laboratory
showed that the first wave of Clb2 destruction could be sufficient for mitotic
exit [131] indicating that Rad53 must have other targets in order to maintain
high levels of Clb2/Cdk activity.
Other roles of the checkpoint pathways involve the transcriptional
induction of genes encoding products involved in DNA metabolism and
DNA repair. The ribonucleotide reductase (RNR) genes are transcriptional
targets of the S phase and DNA damage checkpoints that have been studied
extensively in yeast and are conserved in mammals. The Rnr proteins
catalyze the rate-limiting step of DNA synthesis, which is the generation of
the deoxyribonucleotide pools. The Sphase and DNA damage checkpoint
mediated by Mec1, Rad53, and Dun1 controls the transcriptional upregu-
lation of the RNR genes in yeast [51, 139, 142]. Two of the effectors for the
checkpoint-induced upregulation of Rnr activity are the transcriptional
repressor Crt1 and the Rnr regulatory subunit Sml1 [139, 143]. In response
to DNA damage or stalled replication forks, Crt1 is hyperphosphorylated in
a Dun1-dependent manner and released from the DNA allowing
transcription of the RNR genes [51]. In addition, phosphorylation of Sml1 in
a Dun1-dependent fashion allows the activation of ribonucleotide reductase,
presumably by mediating the degradation of Sml1 [141].
The Chk1 and Rad53 (Cds1/Chk2) proteins from both S. pombe and
S. cerevisiae function downstream of the Atm homologues Rad3 and Mec1
and regulate mitotic progression following DNA damage, albeit through
different mechanisms. Although S. cerevisiae displays a unique cell cycle
organization, orthologues of both Pds1 and Esp1 have been identified in
S. pombe (cut2 and cut1, respectively) and mammals (separin and securin,
see Table II).
Chapter 2: Checkpoints and Genomic Stability 57
Figure 4. The DNA damage checkpoint regulates mitotic progression by blocking the
ubiquitin-mediated destruction of Pds1 and the inactivation of the mitotic Cdk/Cyclin
complexes. Checkpoint activation is achieved by the interaction of two complexes that are
independently recruited to sites of DNA damage Mec1-Ddc2/Lcd1/Pie1 and Mec3-Ddc1-
Rad17 (9-1-1 complex in mammals). The Rad24-RFC “clamp loading” complex recruits the
Ddc1-Mec3-Rad17 “clamp like” complex following recognition or processing of the lesion.
Mec1 and Ddc2/Lcd1/Pie1 requires both the Rad24 and Mec3 complexes for activation of the
DNA damage response. The Ddc1 and Ddc2 proteins are phosphorylated by Mec1. The
Rad24 and Mec3 complexes provide substrate specificity to the central checkpoint kinase
Mec1 to direct phosphorylation of substrates such as Rad9, and the effector kinases Rad53
and Chk1, which then associate with Rad9. Chk1 and Rad53 form parallel branches in the
DNA damage checkpoint to regulate anaphase entry and mitotic exit. Chk1 phosphorylates
and blocks the ubiqutin-mediated destruction of the securin Pds1, and Rad53 activates Dun1
to block the inactivation of Bfa1 and activation of MEN. Dun1 also phosphorylates the Crt1
58 J. S. Searle and Y. Sanchez
transcriptional repressor and the RNR inhibitor Sml1 (not shown) in order to elicit a
transcriptional response.
Andrew Murray, who has studied the regulation of mitosis using both
biochemistry (MPF) and yeast genetics, identified mitotic arrest deficient
(mad) mutants with increased sensitivity to anti-microtubule drugs. These
mutants were defective in the mitotic checkpoint arrest in response to a drug
that interferes with mitotic spindle assembly by depolymerizing micro-
tubules [70]. Murray identified mad1, mad2, and mad3 in this screen.
Andrew Hoyt also identified mutants that were sensitive to microtubule
destabilizing agents and failed to restrain subsequent cell cycles as
evidenced by budding and DNA replication when in the presence of an anti-
microtubule drug; these mutants were named bub (budding uninhibited by
benzimidazole) [49]. Bub1 was later shown by Hoyt to be a kinase, again
pointing to a signal transduction pathway in checkpoint control that is
activated in response to microtubule perturbation. Mark Winey identified
mutants defective in the duplication of the spindle poles. Duplication of
spindle pole bodies is a requisite step in order to form a bipolar spindle that
will separate the chromosomes at mitosis. He called his mutants mps for
(monopolar spindle) [134]. One of the proteins identified by Whiney, Mps1,
is a kinase [67], and the function of Mps1 is required for both spindle pole
body duplication and the spindle checkpoint.
The spindle checkpoint pathways, like the DNA damage checkpoint,
bifurcate to control anaphase and mitotic exit [1, 8, 69, 119]. Epistasis
analyses determined that the Mad2 and Bub2 acted in two separate
pathways: Mad/Bub pathway functions to block M–A and the Bub2 pathway
functions to block mitotic exit. The kinase Mps1 is thought to act upstream
of the identified spindle checkpoint proteins as its overexpression activates
both the Mad/Bub pathway and the Bub2 pathway, and causes cells to arrest
both in metaphase and before mitotic exit [41].
Chapter 2: Checkpoints and Genomic Stability 59
Figure 5. What is the signal that activates the S/M checkpoint during DNA replication and in
response to replication problems? Some of the same proteins involved in the DNA damage
response, such as the clamp loader proteins and the clamp-like complex, are involved in
activation of the S/M phase checkpoint during DNA replication. During DNA replication the
primase and DNA polymerase α complex synthesizes an RNA primer followed by short
stretch of DNA synthesis. This acts to recruit the PCNA clamp via the RFC complex to the
primer/template junction. PCNA and other proteins have a role in recruiting the processive
DNA polymerase enzymes δ and ε. Genetic and biochemical experiments implicate proteins
that are located at the replication fork (Rpa, DNA polymerase α/Primase complex) in the
recruitment of both the clamp loader, clamp and Atr (Mec1) kinases in order to signal ongoing
replication and stalled forks and to activate a checkpoint response that will block mitotic
progression until DNA replication has been completed.
3.3.2 The biochemical pathways that are emerging from the studies
of the genes identified in the genetic screens
As was the case for the DNA damage and S-phase-checkpoint proteins,
the biochemical properties of the proteins that regulate the spindle
checkpoint comprise a signal transduction pathway. Because the spindle and
DNA damage checkpoints both regulate mitotic progression, they share
some of the same targets, however, the transducers and sensors are different
due to the nature and location of the signal. In the spindle checkpoint the
60 J. S. Searle and Y. Sanchez
Figure 6. The spindle checkpoint blocks mitotic progression when the chromosomes are not
attached or bi-oriented to the spindle. Unattached kinetochores as well as mono-oriented
kinetochores (not bi-oriented) activate the spindle checkpoint. The spindle checkpoint is a
signal transduction pathway that bifurcates to control anaphase and mitotic exit. Mps1 is
thought to act upstream and it signals to both the Mad/Bub pathway and the Bub2 pathway, to
bock anaphase and mitotic exit. Mps1 phosphorylates Mad1, and this phosphorylation is
dependent on Bub1, Mad2, and Mad3. Phosphorylated Mad1 interacts with Mad2 and Bub3,
Mad3 and Mad2, can bind to Cdc20. Interaction of these proteins with Cdc20 prevents the
activation of the APC/Cdc20 blocking the ubiquitination of Pds1 and the Clbs. Mps1
activation of Bub2 blocks activation of the MEN. Ipl1, an Aurora kinase that localizes to
kinetochres, activates the spindle checkpoint when the spindles bound to the kinetochores are
not connected to opposite spindle pole bodies. The signal for this event is the lack of tension
on the spindles bound to the sister chromatids. There are two possible mechanisms for Ipl1
mediated activation of the spindle checkpoint, (a) Ipl1 causes the dissociation of the spindle
from a kinetochore which is detected by Mad2 and activates the Mps1-dependent checkpoint,
62 J. S. Searle and Y. Sanchez
and (b) Ipl1 could activate the Mad2 checkpoint directly. The detailed mechanisms of this
surveillance pathway remain to be determined.
From the studies carried out in S. cerevisiae, the following roles have
been elucidated for the checkpoint that monitors DNA replication status and
DNA integrity during S phase:
The sensors are part of or associated with the replication machinery, or
their structure mimics the complexes involved in DNA replication. The
kinases Mec1 and Rad53 regulate effectors that (1) stabilize replication forks
and allow reinitiation of DNA replication once the problem has been
overcome [26, 73, 120]; (2) prevent firing of late replication origins [106];
(3) block anaphase until DNA replication is complete [2, 133]; and (4)
through regulation of Dun1, activate transcription of genes encoding proteins
involved in DNA metabolism and DNA repair [51, 139, 142, 143].
The DNA damage checkpoint pathway that operates at G1/S functions to
block entry into S phase until the damage is repaired. The targets of this
response have been better characterized in metazoans than in yeast. Due to
differences in the organization of the cell cycle, the effectors for the DNA
damage-induced arrest at G2/M differ between the budding yeast and
mammals. In the budding yeast, the arrest occurs at the metaphase to
anaphase transition (Figure 4); whereas in metazoans and fission yeast, the
Chk1 and Cds1/Chk2 (sp and hRad53)-mediated arrest occurs in G2 by
inactivation of the Cdc25 phosphatase and activation of the Wee1 kinases
[29, 38, 96, 100, 105]. Budding yeast cells do activate a G2/M checkpoint in
response to perturbations in bud construction and/or bud size [45, 79];
therefore, it remains to be determined whether mammalian cells also regulate
the metaphase to anaphase transition in response to DNA damage.
The mitotic or spindle checkpoint monitors the proper attachment and
orientation of chromosomes on the spindle. This ensures the accurate
transmission of genetic material to daughter cells.
The signals that activate the S phase and spindle checkpoints originate at
the replication fork or the kinetochore, and serve to indicate lack of
completion of that particular cell cycle stage, much like the checkpoint
mechanisms identified by Rao Johnson. Similarly the checkpoints that
monitor replication forks and DNA integrity use similar mechanisms, and
sometimes some of the same proteins, to delay cell cycle transitions, and in
addition play an important role in the stability of replication forks and in
DNA repair.
Chapter 2: Checkpoints and Genomic Stability 63
cancer. When the checkpoint pathways go awry, they become targets for
cancer drug discovery. Thus, studies that shed light on the mechanisms of
the DNA damage response in mammalian cells will allow us to further
understand both the therapeutic effects and the individual risk of exposure to
genotoxic agents, chemotherapeutic agents, and to radiation.
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Chapter 3
YEAST AS A TOOL IN CANCER RESEARCH:
NUCLEAR TRAFFICKING
75
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 75–100.
© 2007 Springer.
76 A. H. Corbett and A. C. Berger
All macromolecular traffic between the cytoplasm and the nucleus flows
through a large channel called the nuclear pore complex (NPC). The NPC is
a large macromolecular machine that spans the double membrane of the
nuclear envelope (see [28, 98, 100] for recent reviews). Studies over the
years have revealed that nuclear pores have eightfold rotational symmetry
within the membrane but have distinct nuclear and cytoplasmic faces. The
cytoplasmic face has filaments that emanate from the pore and the nuclear
face has a basket-like structure. These distinct faces are thought to contribute
to the mechanism of targeting and transport through the pores.
Nuclear pores are composed of proteins termed nucleoporins or Nups. A
large number of these nucleoporins contain characteristic phenylalanine–glycine
(FG) repeat motifs that are likely to play a critical role in the translocation of
substrates through the pore. Several recent studies have identified all the
proteins that make up both the yeast and the vertebrate NPC. This work was
led by a pioneering study in S. cerevisiae where Rout and colleagues used a
proteomic approach to identify the proteins that comprise the yeast NPC
[86]. This work paved the way for a similar study in higher eukaryotes [19],
which revealed that although the vertebrate pore complexes are somewhat
larger than those in yeast, the number of distinct proteins is approximately
the same.
Spatial analysis of the yeast nuclear pore revealed that many of the
nuclear pore proteins exist in multiple copies that are symmetrically
distributed between the nuclear and cytoplasmic faces of the pore [86].
Chapter 3: Nuclear Trafficking in Yeast 77
2 PROTEIN TRAFFICKING
NLS sequence is typified by the NLS found in the SV40 large T antigen
(PKKKRKV), whereas the bipartite NLS is typified by the nucleoplasmin
NLS (KRPAATKKAGQAKKKK). These sequences mediate binding to a
classical nuclear import receptor, importin/karyopherin α (discussed in more
detail in section 2.2 below). Although numerous NLS sequences have been
identified, the best consensus sequence that has emerged thus far is:
K(K/R)x(K/R) where x is any amino acid [44].
The classical NES is comprised of a series of hydrophobic amino acids,
generally leucine, isoleucine, or valine [32]. The founding member of the
NES family is the protein kinase A inhibitor protein, which contains the NES
sequence LALKLAGLDI, where the underlined residues are critical for NES
function [108]. Numerous variations on this theme have been identified and
thus far the best consensus sequence for an NES is LxxxLxxLxL, where the
spacing between the leucines can vary and, in fact, the leucines can be
substituted with virtually any hydrophobic amino acid.
Obviously, it is possible to scan the sequence of any given protein and
identify putative classical NLS or NES sequences, however, substantial
experimental evidence is required to prove that an amino acid sequence is
both necessary and sufficient to serve as a functional targeting signal [21].
This is particularly critical given the rather weak consensus sequences for
the known nuclear targeting signals. Current efforts are being made to better
define the important characteristics of both NLS and NES sequences. Recent
studies have examined the interaction between NLS sequences and the
classical NLS receptor, importin α, by determining the binding energy
contributed by each residue within NLS sequences [44]. This study has been
extended by using budding yeast to determine how the binding affinity of the
NLS for the NLS receptor relates to the actual accumulation of the cargo
within the nucleus [43]. A complementary study used an in vivo rando-
mization-selection assay to determine which residues within an NES are
absolutely required for export function [11].
Mutations or modifications that alter the nuclear-targeting signals of
growth regulatory proteins are associated with some instances of cellular
transformation. There is evidence that phosphorylation that occurs proximal
to an NLS sequence can alter the nuclear targeting of the cargo protein [40,
51]. One notable example where an alteration in nuclear targeting is asso-
ciated with cellular transformation is the v-jun oncogene. Normal cellular jun
(c-jun) contains a classical NLS with a flanking cysteine [13]. In the v-jun
oncogene the sequence is altered such that the cysteine is replaced by a
serine [13, 102]. It seems that phosphorylation of this serine regulates the
nuclear import of v-jun and thus imparts an inappropriate function on the
oncoprotein [13, 102]. There are a number of other mechanisms that can
be used to regulate the function of a nuclear targeting signal. For example,
there are cases where an NLS is masked by a binding partner as in the case
Chapter 3: Nuclear Trafficking in Yeast 79
of IκB binding to the transcription factor NFκB. IκB binds to NFκB and
masks the NLS and only when IκB is degraded in a signal-dependent
manner does NFκB enter the nucleus, trigger gene expression, and cause
changes in cell growth [71, 80]. As we learn more about the signals that
target cargo proteins to and from the nucleus, it is likely that we will uncover
other regulatory mechanisms that are critical for proper control of cell
growth. In fact, recent work with the Bcr–Abl oncoprotein indicates that
trapping this protein within the nucleus in its active state can induce
transformed cells to undergo apoptosis [105]. Thus, there is potential
therapeutic value in uncovering ways to alter the nuclear transport properties
of certain proteins. Experiments carried out in the yeast model system are
making important contributions to our understanding of nuclear targeting
signals.
Nuclear transport
receptors*
With the exception of the NLS adaptor protein, Kap60p, Kaps share a
common domain structure [34, 35]. Each has a conserved N-terminal domain
that mediates binding to the small GTPase Ran, which regulates cargo
binding to the Kap proteins as described in more detail in the following
section (2.3) on the Ran GTPase cycle. The central domain of the Kaps is an
FG binding domain that mediates interactions with the FG-repeat-containing
nucleoporins located in the nuclear pore. Finally, the least well understood
domain is the C-terminal domain, which mediates binding to the cargoes.
Some of the receptor/cargo interactions are starting to be defined at the
molecular level by structural studies of both yeast and vertebrate proteins.
The most structural information has been gathered on the heterodimeric
import receptor for classical NLS-containing cargoes. Structures of both
mouse and yeast importin α/Kap60p have provided important information
about how an NLS is recognized and how binding to the NLS cargo is
regulated [17, 61]. These studies also revealed that despite relatively low
sequence conservation between the yeast and vertebrate NLS receptor
proteins (~30% identity), the overall structure of the proteins is identical.
The structural basis for the interaction between vertebrate importin α and
importin β has also been defined [16]. In addition, crystallization of a
complex of vertebrate importin β with an FG-repeat peptide has revealed the
structural basis for the interaction of the transport receptor with the FG-
containing nucleoporins [5]. Although many of these structural studies have
examined vertebrate proteins, subsequent studies in yeast, which exploited
this structural information to generate rationally designed site-directed
mutants, strongly argue for functional conservation of these proteins
between yeast and higher eukaryotes [4, 82]. This lends confidence to the
assumption that studies of these nuclear transport proteins in the yeast model
system can provide important insight into their function in higher eukaryotes.
Alteration in several nuclear transport receptors have been implicated in
human cancer. The export receptor required for the recycling of the NLS
receptor, importin α, is a protein named CAS, which stands for cellular
apoptosis susceptibility [7]. The human CAS gene is amplified in several
transformed cell lines including breast, colon, and bladder cancer [12]. In
addition, its expression pattern and intracellular localization is altered in
82 A. H. Corbett and A. C. Berger
One of the major requirements for efficient transport of cargoes into and
out of the nucleus is that the system must have inherent directionality. For
example, in the case of nuclear protein import, the cargo protein must be
bound by the receptor in the cytoplasm, translocated into the nucleus, and
then dissociated from the receptor. There must be some mechanism to switch
the receptor from cargo pick up to cargo delivery. For most nuclear transport
processes, this switch is the Ran GTPase [22, 97, 107].
Work from a number of laboratories over the years has provided an
insight into how Ran imparts directionality on nuclear transport (For review
see [35]). The current model (Figure 1) relies on the compartmentalization of
RanGDP and RanGTP, where RanGDP is primarily cytoplasmic and RanGTP
is within the nucleus associated with chromatin [78]. The underlying
mechanism for this compartmentalization is the differential localization of
the proteins that regulate the GTPase activity of Ran. Like many other small
GTPases [70], isolated Ran hydrolyzes GTP very slowly. The rate of
hydrolysis is increased ~10,000-fold in the presence of the Ran GTPase
Activating Protein (RanGAP) [8], which is called Rna1p in S. cerevisiae [9].
The Ran Guanine nucleotide Exchange Factor (RanGEF) enhances the rate
of conversion of RanGDP to RanGTP [10]. The S. cerevisiae RanGEF is
Prp20p [30]. RanGAP is localized to the cytoplasm [46], which increases the
cytoplasmic pool of RanGDP. In contrast, RanGEF is enriched within the
nucleus where it is bound to chromatin at least in part through interactions
with histones [78, 79]. This enriches the nuclear pool of RanGTP.
Many of the studies that helped to elucidate the Ran cycle were carried
out in yeast. All proteins of the Ran cycle are highly conserved. For
example, the amino acid sequence of human Ran is 85% identical to that of
the S. cerevisiae Ran protein, Gsp1p. Human RanGEF, RanGAP, and NTF2
can all functionally replace their yeast counterparts [18, 30, 72]. This
Chapter 3: Nuclear Trafficking in Yeast 83
[88, 106, 122]. In this case, the RanGTP likely releases nuclear pore
proteins, which are required for the association and assembly of early NPCs
with the chromatin. In all three cases, nuclear transport, mitotic spindle
assembly, and nuclear envelope assembly, RanGTP serves to dissociate
proteins from the transport receptor in the vicinity of chromatin. This
concentrates proteins at the chromatin or within the nucleus where they
function. Thus, although RanGTP was originally identified as a critical
regulator of nuclear transport, this role is probably only one aspect of Ran
function.
Ran
GEF
Ran
GAP
Ran Ran
GTP GTP
Ran
GTP
Ran
GTP
Import
Cargo
Cargo
Ran β β Ran
GDP Ran GTP
Ran GTP Ran
GDP GTP
Ran
GDP Cargo Ran
GTP
Cargo
Export β β
Ran
Ran GTP
GDP
Figure 1. The Ran GTPase cycle regulates the directionality of nuclear protein transport. The
RanGAP protein is localized to the cytoplasm, which leads to a high level of cytoplasmic
RanGDP. The RanGEF is localized to the nucleus, which leads to a high level of nuclear
RanGTP. This asymmetric distribution of RanGTP and RanGDP regulates the assembly of
receptor cargo complexes. For Import, the transport receptor (β) binds to the import cargo in
the cytoplasm. This complex is translocated through the nuclear pore into the nucleus. In the
nucleus RanGTP binds to the transport receptor, which causes a conformational change that
releases the cargo. For Export, the export receptor (β) binds to the export cargo in the nucleus
in an obligate trimeric complex with RanGTP. The trimeric complex is translocated through
the nuclear pore into the cytoplasm. In the cytoplasm, the complex encounters the RanGAP,
which hydrolyzes the RanGTP to RanGDP and dissociates the complex to release the cargo
into the cytoplasm.
cytoplasm nucleus
(1)
Cargo (2) α
β NLS
β β
α (3) Ran
β GTP
α α
Ran α
α GAP α α Cse1
Cse1 Cse1 Cse1
Cse1 Ran Ran Ran
GTP (5) GTP GTP
Ran
GDP
Figure 2. The nuclear import pathway for cargoes that contain a classical NLS is the best
characterized nuclear transport pathway. This pathway can be dissected into at least five
distinct steps: (1) recognition and binding of the NLS cargo to the Kap60p(α)/Kap95p(β)
heterodimeric import receptor in the cytoplasm; (2) targeting to the nuclear pore through
interactions between Kap95p and the nuclear pore; (3) translocation through the pore
through transient interactions between Kap95p and the FG-repeat containing nucleoporins;
(4) delivery into the nucleus when RanGTP binds to Kap95p to cause a conformational
change that releases the Kap60p/NLS cargo; and (5) recycling of Kap60p to the cytoplasm in
a heterotrimeric complex with the export receptor, Cse1p, and RanGTP.
Kap95 presumably in complex with RanGTP. Note that the only energy
expenditure in this process occurs when the karyopherin proteins are
recycled to the cytoplasm and the accompanying RanGTP is hydrolyzed.
Export of cargoes from the nucleus is very similar to the import process
except that the export complex forms in the presence of RanGTP (in the
nucleus) and is dissociated upon GTP hydrolysis (in the cytoplasm). Thus far
all export mechanisms seem to involve the direct recognition of the cargo
protein by the export Kap protein, but the molecular details of the
recognition have not been elucidated for the NES export receptor/cargo
complex. The export of cargoes that contain a leucine rich NES, which is
recognized by the export Kap, Crm1p/Xpo1p (CRM1 in vertebrates), serves
as a paradigm for transport receptor mediated nuclear export. The steps
involved in NES export are: (1) recognition and binding of the NES cargo in
a trimeric complex with Crm1p/Xpo1p; (2) targeting to the nuclear pore
through interactions between Crm1p/Xpo1p and the FG nucleoporins; (3)
translocation through the pore; (4) delivery of the cargo upon RanGTP
hydrolysis in the cytoplasm; and (5) recycling of the export receptor and
RanGDP to the nucleus. This cycle of export is virtually identical to the
recycling of Kap60p(α) by Cse1p that is illustrated in Figure 2. This
demonstrates the conservation of mechanism in the nuclear transport
process.
3 RNA EXPORT
importin-β family carrier exportin-t/Los1p [1, 42, 67, 90] and is exported as
a complex with RanGTP, which is disassembled in the cytoplasm when
RanGTP is hydrolyzed to RanGDP. Preferential export of mature tRNAs
seems to be achieved at least in part by the specificity of exportin-t for the
mature processed, modified, and appropriately aminoacylated tRNAs [94]. U
snRNAs are synthesized in the nucleus, transported to the cytoplasm where
they associate with protein components of mature snRNPs, and are then
reimported to the nucleus where they function in mRNA splicing [50]. The
monomethylated G cap of the initial export substrate is recognized by the
cap-binding complex (CBC) [50] and then exported from the nucleus in a
CRM1-dependent manner [31]. As there is no evidence that the CBC binds
directly to CRM1, it seems likely that an adaptor protein mediates this
interaction. rRNAs are exported in the context of large assembled RNP
complexes. Although export depends on Ran [48, 74], it is controversial
whether Ran plays a direct role in export or whether instead its activity may
be essential for the import of components required for RNP assembly [87].
Export of poly (A)+ RNA remains the least well understood of the RNA
export mechanisms. mRNAs are not exported to the cytoplasm as naked
nucleic acids, but rather as ribonucleoprotein complexes and it is generally
agreed that the export machinery recognizes signals within the proteins of
these complexes rather than the RNA itself [49, 77]. For example, export of
unspliced HIV transcripts from the nucleus is mediated by Rev through its
export by the NES receptor [20]. While this mechanism is exploited by HIV,
it seems that none of the karyopherin receptors, or even Ran itself, play a
central role in mRNA export.
Although the mechanistic details of mRNA export have not yet been fully
elucidated, it appears that there are two classes of proteins that are required
to achieve export of mature messages. First, there is a family of evolutionarily
conserved heterogeneous nuclear ribonucleoproteins (hnRNPs) that interact
with poly (A)+ RNA in vivo [25]. A number of these hnRNP proteins shuttle
between the cytoplasm and the nucleus and escort the poly (A)+ RNAs as
they are exported through the NPC [76, 93]. Current models suggest that at
least some of the hnRNP proteins may be involved in RNA processing steps
that occur co-transcriptionally. The hnRNPs that remain bound to the
maturing messages may serve as markers that the different processing steps
have been successfully accomplished. The second class of proteins consists
of those proteins that have been implicated more directly in the export
process, including the helicase, Sub2p (UAP56 in humans), and the
heterodimeric export receptor, Mex67p/Mtr2p (TAP/p15 in humans)
[20, 58, 92]. TAP was originally identified as a factor necessary for
88 A. H. Corbett and A. C. Berger
the export of simian type D viral RNAs that contain a CTE (constitutive
transport element). Subsequent experiments showed that TAP/Mex67p is
also required for the export of endogenous mRNAs [57, 58] and that
mutations in the yeast MEX67 gene cause a rapid onset defect in the export
of poly (A)+ RNA [92]. TAP/Mex67p shuttles between the cytoplasm and
the nucleus [58], and in complex with p15/Mtr2p, binds both to mRNA and
to nucleoporins [2]. Thus, it could potentially target bound RNAs directly to
the NPC for export. Recent work has led to the identification of the TREX
(TRanscription/EXport) complex, which links mRNA transcription and
export from the nucleus [83]. This evolutionarily conserved complex
contains both factors required for transcription of mRNA and factors that
will be required for export of the mature message. A great deal of effort is
currently focused on understanding how the hnRNP proteins, the export
factors, and other accessory proteins cooperate to export mature mRNA from
the nucleus.
There are a number of studies that demonstrate increased expression of
several different hnRNP proteins in tumor cells as compared to normal
control cells. For example, Snead et al. [96] found that hnRNP B1 was
overexpressed in 84% of malignant tumors in lung disease as compared to
only 18% of benign tumors. This and other related studies have led to the
suggestion that hnRNP expression could be a useful marker for the early
detection of specific tumors [112].
P P
P
NLS Cdc25 Ran
P P
NLS GTP
Cdc25
α
P
Cdc25
α
P
β
14-3-3
β Cdc2 Ran
GTP NLS
P P
NLS
P P
P
P
P P Cdc2
P
Cdc2 Cyclin B
α
Cyclin B Dephos- NLS β Cdc2
α P
NLS phorylation Ran Cyclin B
GTP NLS
β
P
Dephos-
CRM1 phorylation
Cdc2 Cdc2
Cyclin B CR Cyclin B CR
M
NES
S
S
M
NES
1 Cdc25 Cdc25
NE
NE
CRM1 CRM1 1
Pho4 P P Pho4
Pse1
transcription
low Pho4
Pho4 Pse1 Ran
P
Pho4
P
GTP
P phosphate Pse1 Ran
GTP
high Pho85
Ran Ran
GTP GTP phosphate Pho80
Msn5 Msn5
Msn5 P P P P P P
P
Pho4
P
P
P
GDP P P
P
Figure 3. Cyclin B. Mitotic progression through the cell cycle depends on translocation of the
Cdc2–Cyclin B complex into the nucleus. The dimeric complex is dephosphorylated on
Cdc2p by Cdc25p, allowing access to the import–receptor complex consisting of importin
α/β. The quaternary complex translocates through the nuclear pore and dissociates upon
RanGTP binding to β in the nucleus. The Cdc2–Cyclin B complex is retained in the nucleus
by inhibitory phosphorylation of Cyclin B. At the end of mitosis, the complex is
dephosphorylated, allowing CRM1 to bind to the NES of Cyclin B. Upon binding, the
complex is exported from the nucleus and targeted for destruction by the 26S proteasome.
Cdc25. The 14-3-3 protein binds to and holds Cdc25p in an inactive state in the cytoplasm
during interphase by blocking access to its intrinsic NLS. Upon activating phosphorylation of
Cdc25p at prophase, importin α/β binds to the Cdc25p NLS and the trimeric complex is
translocated into the nucleus. The complex is dissociated by binding of RanGTP to β. Cdc25p
loses activity upon dephosphorylation, exposing its intrinsic NES, which is then bound by
CRM1. The dimeric complex is then translocated through the nuclear pore into the cytoplasm
where the complex is dissociated. Pho4. Under low phosphate conditions, the activity of the
Pho80p/Pho85p kinase complex is downregulated, leading to an underphosphorylated form of
Pho4p. Dephosphorylation reveals the NLS of Pho4p and allows binding by its import
receptor Pse1p. The dimeric complex is translocated into the nucleus where it is dissociated
by RanGTP binding to Pse1p. After Pho4p activates transcription of the phosphate responsive
genes, Pho4p becomes hyperphosphorylated, creating a binding site for its export receptor
Msn5p to form a trimeric complex with RanGTP. Following binding by Msn5p, Pho4p is
rapidly recycled to the cytoplasm where the trimeric complex is dissociated by hydrolysis of
RanGTP to RanGDP.
Chapter 3: Nuclear Trafficking in Yeast 91
4.2 Cdc25
Evidence from S. pombe has provided much of the foundation for the
regulation of mitotic entry in eukaryotic cells (Figure 3). This entry is
triggered by the cyclin-dependent kinase, Cdc2/Cdc28p. As discussed above,
the activity of Cdc2/Cdc28p is dependent upon interaction with its regula-
tory subunit, cyclin B/Clb2p. Prior to mitosis, Cdc2–cyclin B complexes are
held in an inactive state by phosphorylation of Cdc2/Cdc28p. The cell
division cycle 25C protein phosphatase, Cdc25C/Cdc25p, acts on the Cdc2–
cyclin B complex to dephosphorylate Cdc2/Cdc28p, thus activating the
Cdc2–cyclin B complex to promote mitosis. In order to prevent early
activation, the cell must regulate the activity of Cdc25C/Cdc25p. This is
accomplished by regulating the nuclear transport of Cdc25C/Cdc25p.
Studies in both mammalian cells and fission yeast have shown that
Cdc25C/Cdc25p shuttles between the nucleus and cytoplasm throughout
the cell cycle, but has a steady state cytoplasmic localization during inter-
phase and a nuclear localization during prophase [41, 69]. Cdc25C/Cdc25p
contains both a classical NLS and an NES and studies using Xenopus and
S. pombe have shown that Cdc25C/Cdc25p is imported through interactions
with importin-α/Kap60p [65, 115] and importin-β/Kap95p [15]. Posttransla-
tional modifications near these targeting sequences control the steady state
localization of the protein by interfering with the recognition of these signals
by the receptors.
During interphase in both Xenopus [66] and S. pombe, [121] phos-
phorylation of Cdc25C/Cdc25p near its intrinsic NLS creates a binding
site for the phosphoserine-binding protein, 14-3-3. The 14-3-3 protein binds
to Cdc25C/Cdc25p and inhibits nuclear import probably by sterically
hindering the access of importin α to the NLS. This causes a redistribution
of Cdc25C/Cdc25p from the nucleus to the cytoplasm. This model is
supported by experiments from fission yeast, which show that in the absence
of the 14-3-3 protein, Rad24p, Cdc25C/Cdc25p is predominantly nuclear
92 A. H. Corbett and A. C. Berger
4.3 Pho4p
with the unphosphorylated form of Pho4p [54]. Pho4p then activates the
transcription of the phosphate-responsive genes. When phosphate levels rise,
the Pho80p–Pho85p complex regains activity and hyper-phosphorylates
Pho4p. This phosphorylation causes Pho4p to be rapidly recycled to the
cytoplasm through its export receptor, Msn5p [53], an importin β family
member that specifically recognizes the phosphorylated form of Pho4p [53],
thus repressing the transcription of the phosphate-responsive genes.
The Pho80p–Pho85p complex phosphorylates Pho4p on multiple serine
resides [64]. Phosphorylation of Pho4p on two sites is necessary and
sufficient to cause nuclear export by Msn5p [64]. Additional phospho-
rylation of Pho4p on a third residue within the nuclear localization signal
inhibits its interaction with the import receptor, Pse1p [64]. Both of these
phosphorylation events serve to ensure that Pho4p remains cytoplasmic
under conditions of high phosphate levels. Thus, phosphorylation of these
residues serves to regulate the nucleocytoplasmic transport of Pho4p. By
examining nuclear transport of Pho4p, we can better understand how the
activity of transcription factors is regulated in response to environmental
signals.
5 CONCLUSIONS/IMPLICATIONS
FOR THE FUTURE
ACKNOWLEDGMENTS
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Chapter 4
1 PROTEIN FARNESYLATION
101
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 101–122.
© 2007 Springer.
102 N. Thapar and F. Tamanoi
CaaX
CaaX
aaX
CaaX Protease
Carboxyl Methyltransferase
C Me
Rac proteins [24, 39, 86]. The modification involves a 20-carbon geranyl-
geranyl group added to a cysteine in the CaaL motif (similar to the CaaX
motif except that the C-terminal amino acid is leucine or phenylalanine) [74,
108]. GGTase I shares a common α-subunit with FTase, while its β-subunit
shares about 30% homology with the β-subunit of FTase. GGTase II
catalyzes the addition of a geranylgeranyl group to both cysteines within the
CC or the CXC motif that are found in a number of Rab protein [88]. In
addition to α- and β-subunits that share homology with their counterparts in
FTase, GGTase II contains an additional component-Rep (Rab escort
protein) [96].
Recently, anticancer drugs based on the inhibition of protein farnesylation
have been developed. These small molecular weight compounds called
farnesyltransferase inhibitors (FTIs) selectively inhibit farnesyltransferase
[7, 38, 84, 94]. A variety of compounds including peptidomimetic inhibitors,
farnesyl pyrophosphate analogues, bisubstrate inhibitors, and natural
compounds have been identified. FTIs have been shown to reverse ras-
mediated phenotypes in ras-transformed cells. FTIs inhibit the growth of
tumors or even regress tumors in animal model systems [62]. Currently,
FTIs are being evaluated in a variety of clinical trials [57].
In this review, we discuss how yeast studies contributed to the overall
study of protein farnesylation. We will first describe yeast studies concerning
FTase as well as yeast-based assays that led to the identification of one of
the first generation FTI compounds. We will then focus on identification and
characterization of farnesylated proteins. Yeast has provided a compre-
hensive analysis of farnesylated proteins that was instrumental in the iden-
tification of a number of novel farnesylated proteins. Of particular interest
is Rheb, a novel family of the Ras-superfamily G-proteins. This protein is
highly conserved and plays a role in the regulation of cell cycle at the G1/S
phase. We will summarize the current understanding of Rheb in S. cerevisiae
and S. pombe.
104 N. Thapar and F. Tamanoi
One of the unique properties of FTase is its ability to recognize the CaaX
motif. This observation was critical in the development of peptidomimetic
FTIs, as they were derived from a tetrapeptide such as CVFM that inhibited
the activity of FTase. Thus, it was of interest to understand which amino
Chapter 4: Studies of Protein Farnesylation in Yeast 105
Table 1a. S. cerevisiae CaaX motif-containing proteins which have been shown to be
farnesylated or are candidates for farnesylation based on sequence similarity
Protein CAAX motif Function/cellular role
Ras1 -CIIC G-protein, cAMP pathway
Ras2 -CIIS G-protein, cAMP pathway
Mfa1 -CVIA Mating pheromone a-factor
Mfa2 -CVIA Mating pheromone a-factor
Stel8 -CTLM Gγ involved in mating
Ydj1 -CASQ DnaJ homolog, protein transport
Xdj1j -CCIQ DnaJ homolog
Rho3 -CTIM Cell polarity
Rho4 -CIIM Cell polarity/Actin cytoskeleton organization
Rheb -CSIM GTP binding/Amino acid metabolism
Skt5 -CVIM Cell wall maintenance
Gis4 -CAIM Signal transduction/Cell stress
Rcy1 -CCIM Vesicular transport
Pex19 -CKQQ Peroxisome biogenesis
Atr1 -CTVA Small molecule transport
N1142 -CSIM Hemoprotein
Ykt6 -CIIM v-SNARE, vesicular transport
YCP4 -CTVM Unknown
YDL009C -CAVS Unknown
YPL191C -CVVM Unknown
YGL082W -CVIM Unknown
YMR265C -CSNA Unknown
YML133C -CCPS Unknown
YPR203W -CCPS Unknown
YFL065C -CCPS Unknown
YHL049C -CCPS Unknown
YDL151C -CYPA Unknown
YJL118W -CCCS Unknown
YIR007W -CVIS Unknown
The fission yeast S. pombe has a few proteins functionally related to the
S. cerevisiae proteins which have been shown to be farnesylated. As
summarized in Table 1b, these include Mfm1, 2 and 3, the functionally
redundant precursor polypeptides for the mating pheromone M factor
Chapter 4: Studies of Protein Farnesylation in Yeast 109
Table 1b. S. pombe CaaX motif-containing proteins which have been shown to be farnesylated
or are candidates for farnesylation based on sequence similarity
Protein CAAX motif Function/cellular role
SpRas -CVIC GTP binding/mating response
Mfm1 -CVIA Signal transduction/mating response
Mfm2 -CVIA Signal transduction/mating response
Mfm3 -CVIA Signal transduction/mating response
Rhb1 -CVIA Cell cycle regulator
Spj1 -CAQQ Protein folding/cell stress
Rho2 -CIIS Cell polarity/cell wall maintenance
Rho3 -CIIA Cell polarity/cell wall maintenance
Git11 -CTIS Signal transduction/mating response
SPBC405.06 -CQAQ Unknown (has DnaJ domain)
SPBC13G1.11 -CIIA Unknown (predicted SNARE)
SPCC417.05C -CIIS Unknown (predicted chitin biosynthesis)
SPAC24B11.10C -CVVM Unknown (predicted chitin biosynthesis)
SPAC607.09C -CALT Unknown (predicted cellular pH homeostasis)
SPAC17C9.14 -CPTQ Unknown (predicted peroxisome biogenesis)
There are features unique to the Rheb subfamily. Recent studies indicate the
need of Rheb to be farnesylated for its activity. In the following sections, we
describe characterization of this novel member of the Ras family.
G1
ScRheb MEYATMSSSNSTHNFQRKIALIGARNVGKTTLTVRFVESR 40
SpRheb M----------APIKSRRIAVLGSRSVGKSSLTVQYVENH 30
HsRheb M----------PQSKSRKIAILGYRSVGKSSLTIQFVEGQ 30
DmRheb M----------P-TKERHIAMMGYRSVGKSSLCIQFVEGQ 29
* . *.**..* *.***..* ...**..
Effector Domain G3
ScRheb FVESYYPTIENEFTRIIPYKSHDCTLEILDTAGQDEVSLL 80
SpRheb FVESYYPTIENTFSKNIKYKGQEFATEIIDTAGQDEYSIL 70
HsRheb FVDSYDPTIENTFTKLITVNGQEYHLQLVDTAGQDEYSIF 70
DmRheb FVDSYDPTIENTFTKIERVKSQDYIVKLIDTAGQDEYSIF 69
**.** *****.*.. .... ...******* *..
G4
ScRheb NLPVILVGTKADLGRSTKGVKRCVTKAEGEKLASTIGSQD 160
SpRheb WVPIVVVGNKSDLHM-----QRAVTAEEGKALANE----- 140
HsRheb QIPIMLVWNKKDLHM-----ERVISYEEGKALAES----- 140
DmRheb YVPVVLVGNKIDLHQ-----ERTVSTEEGKKLAES----- 139
.*...* .* ** .* .. .**. **..
G5
ScRheb KRNQAAFIECSAELDYNVEETFMLLLKQMERVEGTLGLDA 200
SpRheb --WKCAWTEASARHNENVARAFELIISEIEKQAN--PSPP 176
HsRheb --WNAAFLESSAKENQTAVDVFRRIILEAEKM-D--GAAS 175
DmRheb --WRAAFLETSAKQNESVGDIFHQLLILIENE-N--GNP- 173
. *. * ** . .. * .. *. .
CAAX
ScRheb ENNNKCSIM 209
SpRheb GDGKGCVIA 185
HsRheb QGKSSCSVM 184
DmRheb QEKSGCLVS 182
. . * .
Figure 2. Amino acid sequence alignment of Rheb protein from various species (Sc –
S. cerevisiae; Sp – S. pombe; Hs – Human; Dm – D. melanogaster). The G-boxes (G1–G5)
are indicated by overlining. The invariant arginine in the G1 box, the effector domain and the
CaaX motif are shown in bold. Asterisks represent identical residues and dots represent
similar residues.
112 N. Thapar and F. Tamanoi
1 G1 G2 G3 G4 G5 209
1 G1 G2 G3 G4 G5 185
Figure 3. Structure of S. cerevisiae and S. pombe Rheb proteins. The G-boxes are represented
as dark segments. The invariant arginine in the G1 box as well as the C-terminal CaaX motif
are indicated.
and cardiac muscle [43]. Rheb mRNA has been found to be induced in
cerebral-ischemia elicited events through NMDA receptor action in rat brain
[58] as well as in human fibroblasts exposed to UV radiation implying a
possible role in UV sensitization of cells [59].
Human Rheb was reported to be upregulated in several transformed cells
[43]. The growth regulatory activities of Rheb were explored by Clark et al.
[23] who found that neither the wild type nor the presumed constitutively
active (Q64L) Rheb protein could induce transformation in normal NIH3T3
cells. At the same time, a mutant Rheb, S20N that carries an amino acid
change analogous to the dominant negative form of Ras (S17N) did not
inhibit growth of the cells. Rheb was found to behave more like Rap1A [23],
which is a negative regulator of Ras function, as it was also found to
antagonize the oncogenic potential of Ras. It was also speculated that since
Rheb and Ras show strong identity in the effector domain region which is
crucial for Raf binding, Rheb may be binding to Raf in a nonproductive
manner and hence titrating it away from Ras and preventing the downstream
cascade [23]. On the other hand, Yee and Worley [107] found that Rheb
stimulates transformation of NIH3T3 fibroblasts when expressed in
conjunction with Raf-1. Thus, a synergistic interaction of Rheb with Raf-1
Chapter 4: Studies of Protein Farnesylation in Yeast 113
was suggested, as the transforming potential was much lower when Rheb or
Raf-1 alone was expressed singly. A report showing that Rheb inhibits
B-Raf has been published [53].
The Rheb protein became a prime focus of attention recently after
Drosophila and mammalian studies showed that it is a component of the
insulin/TOR/S6K pathway. Rheb activates S6K-mediated by TOR. Rheb
was found to be the direct target of the Tsc1/Tsc2 complex which serves as a
GAP for Rheb [6, 78]. Mutations in the Tsc1/Tsc2 genes have been shown to
be responsible for the development of tuberous sclerosis (TSC), a genetic
disease characterized by the presence of benign tumors known as hamar-
tomas in various organs and occurrence of seizures and mental retardation.
Studies involving Drosophilia and human Rheb indicate a potentially critical
role of Rheb in the pathogenesis of this disease. Thus, a further investigation
of the function of Rheb may provide insights into possible therapies for
TSC.
5 SUMMARY
S. cerevisiae S. pombe
Figure 4. Schematic drawing showing the function of Rheb in S. cerevisiae and S. pombe.
Rheb is farnesylated at the C-terminal cysteine residue and localized to the plasma membrane.
In S. cerevisiae, Rheb regulates arginine uptake; in S. pombe, Rheb regulates cell cycle
progression as well as arginine uptake.
ACKNOWLEDGMENTS
We thank Angel Tabancay and Dr. Jun Urano for discussion. This work
is supported by NIH grant CA41996.
Chapter 4: Studies of Protein Farnesylation in Yeast 117
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120 N. Thapar and F. Tamanoi
1 INTRODUCTION
2 CELLULAR IMMORTALIZATION
It has long been appreciated that normal human somatic cells adapted to
grow outside the body, or in culture, divide a limited number of times [29,
123
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 123–139.
© 2007 Springer.
124 S. S. R. Banik and C. M. Counter
3 TELOMERES
6 TELOMERASE
upon the cloning of hTERT, the pace of research in cancer and cellular
immortalization hastened dramatically. First, it was shown that the hTERT
mRNA was indeed upregulated during the immortalization process in
cultured cells, and present in cancer cells but generally not in normal
somatic cells [43, 48, 57, 61]. More importantly, ectopic expression of the
hTERT cDNA in telomerase-negative mortal cells was shown to restore
telomerase activity, arrest telomere shortening and immortalize cells,
resembling all of the characteristics of telomerase function that had already
been delineated in yeast [10, 19, 38, 62, 76, 81].
The ability to immortalize normal human cells has now given rise to a
plethora of opportunities with respect to both research and therapeutics. For
example, isogenic normal cells from patients can now be immortalized to
provide isogenic controls for cancer cell lines isolated from tumours from
the same patient, thus representing an invaluable resource for assessing a
normal baseline in studies of drug action and gene activation [28]. In
addition, the ability to immortalize normal cells has widespread applications
beyond the realm of cancer research. In the case of often rare genetic
disorders, fibroblasts can be isolated and stably immortalized to provide an
unlimited supply of cells that can be used for disease characterization and to
develop possible therapeutic interventions [65]. With respect to tissue
engineering, activation of telomerase in normal cells may prove to be
important for increasing the proliferative lifespan necessary for making
genetic manipulations in vitro [70].
While the role of telomerase had been documented in the process of cell
immortalization, its function in tumourigenesis was conclusively established
in the first study that converted normal cells into cancer cells through the
introduction of defined genetic elements. Human kidney or mammary
epithelial, fibroblast, or astrocyte cells were made to be tumourigenic upon
infection with the SV40 early region to derail cell cycle checkpoints,
oncogenic ras to provide proliferative signals, and hTERT to allow for
cellular immortalization [23, 35, 66]. These cells clearly showed hallmarks
of cancer, namely, having the capability to form colonies in a semi-solid
medium and giving rise to tumours upon injection into nude mice; however,
these phenotypes were markedly absent in cells not infected with hTERT.
Thus, the link between the processes of hTERT expression, cellular immor-
talization, and tumourigenesis was firmly established.
Chapter 5: From Bread to Bedside 131
11 BACK TO BASICS
Having identified the core components of human telomerase and the basic
mechanism through which telomerase functions, we are now poised to
dissect telomerase function and the mechanisms governing cancer cell
immortalization in more precise detail. In particular, to further understand
telomerase function, it will be necessary to elucidate the mechanism by
which telomerase-dependent elongation of telomeres occurs in immortal
human cells. Once again, studies done in yeast are leading the way.
Much work has been accomplished in yeast towards understanding one
mode of telomerase-dependent elongation of telomeres; namely to regulation
of telomerase access to telomeres. Specifically, several of the proteins now
known to be involved in telomere-targetting were in fact discovered in initial
genetic screens that identified the proteins Est1, Est2, Est3, and Cdc13
which function in the telomerase pathway [50, 55]. While Est1p has been
shown to associate with telomerase, it does not appear to directly target
telomerase to the telomere end. Cdc13p on the other hand, binds the extreme
end of the telomere, having high affinity for single-stranded telomeric DNA
[63]. This implies that telomerase may be recruited to telomere ends through
these two intermediate proteins. This model was further substantiated in
experiments where the fusion of Est1 and Cdc13 led to greatly elongated
telomeres [24], and fusion of the telomerase enzyme to Cdc13 was able to
bypass the requirement for a functional Est1 protein within the cell [24].
As the mechanism of telomerase recruitment may also be conserved
between yeast and humans, considerable effort is now being aimed at
understanding this process in human cells. Support for the existence of this
mode of telomerase regulation in humans has come from telomerase fusion
experiments done in human cells. Specifically, we identified a mutant of
hTERT that retained telomerase activity yet was unable to elongate
telomeres [4] – a phenotype reminiscent of yeast mutants defective in
telomerase recruitment [24, 42, 52]. To test if this mutant of hTERT was
indeed defective in telomere targetting, it was fused to the telomere-binding
protein TRF2. While the mutant hTERT protein alone was unable to
elongate telomeres or immortalize cells, the function of the mutant protein
was rescued upon artificially targetting it to telomeres via the TRF2 protein
[5], indicating that human telomerase is regulated by its access to telomeres.
Presumably such regulation is mediated by protein–protein interactions.
Chapter 5: From Bread to Bedside 133
In this regard we note that the human protein hPot1 [8] may be a functional
homologue of the yeast protein Cdc13, which is involved in the recruitment
of telomerase to telomeres. While a possible telomerase recruitment function
for Pot1 analogous to that of Cdc13 remains speculative at this time, future
studies guided by the yeast literature will certainly help in defining this
function.
12 CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
1. Allsopp, R. C., E. Chang, M. Kashefi-Aazam, E. I. Rogaev, M. A. Piatyszek, J.
W. Shay, and C. B. Harley. 1995. Telomere shortening is associated with cell
division in vitro and in vivo. Exp. Cell Res. 220:194–200.
2. Allsopp, R. C., and C. B. Harley. 1995. Evidence for a critical telomere length in
senescent human fibroblasts. Exp. Cell Res. 219:130–136.
134 S. S. R. Banik and C. M. Counter
141
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 141–177.
© 2007 Springer.
142 M. Tesic and R. F. Gaber
cerevisiae (see Table 1). On the one hand, this facilitates (the) analysis of
Hsp90 co-chaperones in vivo with a facile and genetically tractable system.
On the other hand, the absence of certain mammalian homologs of the
Hsp90 complex in yeast affords an opportunity to use yeast as a “bag of
enzymes” to study in vivo precisely those components that are missing.
In budding yeast, two genes, HSP82 and HSC82, encode Hsp90 proteins
that are 97% identical. HSP82 was initially identified using differential
plaque filter hybridization performed to find genes expressed specifically
in heat shocked cells [54]. HSC82 was subsequently identified by hybri-
dization of HSP82 to yeast genomic library [12]. Cells harboring deletions
of either HSP82 or HSC82 are viable, but the double deletion is lethal
[12]. Under normal conditions, Hsc82 is present at approximately 20-fold
greater levels than Hsp82, and is induced only about twofold upon heat
shock. In contrast, when cells are heat shocked, HSP82 is induced some 20-
fold, to a level approximately the same as Hsc82 levels [12]. Deletions of
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 145
Most Hsp90 substrates identified to date are signaling molecules and fall
largely into two groups: transcription factors and protein kinases (for review,
see [130]), although there is a growing list of substrates not involved in
signal transduction. Common to several Hsp90 substrates is their propensity
to adopt multiple conformations. The requirement for the Hsp90 complex
arises from the need to chaperone each client into its proper conformation, in
which the molecule is poised to receive a signal and perform its cellular role.
Table 2 outlines heterologous Hsp90 substrates that have been expressed in
budding yeast cells and known endogenous substrates. In this section,
selected substrates are treated in detail according to the following criteria:
historical importance and widespread use in the studies of Hsp90 (steroid
receptors), exemplification of regulation by a co-chaperone (aryl hydro-
carbon receptor), and involvement in cellular proliferation and tumorigenesis
(p53, Ste11, and telomerase).
Table 2. Hsp90 substrates that have been expressed or identified in S. cerevisiae. The right
column lists known S. cerevisiae co-chaperones that have been experimentally demonstrated
to interact with the listed substrates either physically, genetically, or in functional assays. The
applicable references are given in the text.
Hsp90 substrates in S. cerevisiae S. cerevisiae co-chaperones
interacting with substrates
Ydj1, Sti1, Sba1, Cpr7, Cns1,
Steroid receptors
AhR is a nuclear receptor that, like steroid receptors, binds its ligands
(mostly planar aromatic molecules such as dioxin) inside the cell. Upon
ligand binding, AhR heterodimerizes with ARNT (AhR nuclear
translocator). Both AhR and ARNT contain bHLH domain for DNA
binding. AhR was initially expressed in yeast cells as a LexA fusion in order
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 147
to eliminate the need for ARNT coexpression, demonstrating for the first
time the need for Hsp90 in the signaling of this nuclear receptor [18].
Another fusion, with the DNA-binding domain of human GR to eliminate
the need for the dioxin-responsive elements, has also been expressed in yeast
[175]. In order to more faithfully replicate the mammalian system, AhR and
ARNT were coexpressed in yeast cells [106] where they exhibited depen-
dence on components of the Hsp90 complex [105].
An AhR co-chaperone, ARA9/XAP2/AIP, was identified in a yeast two-
hybrid screen for proteins that interact with AhR [17]. Its expression in
S. cerevisiae, which does not harbor a homolog, enhanced the ligand respon-
siveness of AhR [17]. It is evident from the work of several laboratories that
ARA9/XAP2/AIP determines the specificity of the Hsp90 complex for AhR
[6, 19, 82, 83, 90, 102, 103, 125].
Several important lessons can be learned from the studies of ARA9/
XAP2/AIP that might also apply to other Hsp90 co-chaperones. First,
ARA9/XAP2/AIP interacts directly both with Hsp90 and AhR [102], and the
interaction with AhR is mediated by the TPR domain of ARA9/XAP2/AIP, a
domain that is primarily implicated in binding to Hsp90. This suggests a
possibility that the TPR domains of other TPR-containing Hsp90 co-
chaperones direct the interaction between the co-chaperones and various
substrates, in addition to mediating the interaction between co-chaperones
and Hsp90. Second, ARA9/XAP2/AIP regulates AhR at least in part by
increasing the proportion of the receptor in the cytoplasm [82, 90, 125].
Other co-chaperones may also play roles in the proper localization of Hsp90
substrates, possibly even bringing together the substrates and the Hsp90
complex. Third, it has been shown that ARA9/XAP2/AIP protects AhR
against proteasome-dependent degradation [82]. ARA9/XAP2/AIP may have
a chaperone activity on its own, possibly mediated by the FKBP-like
immunophilin domain situated in the amino terminus of the protein. Several
co-chaperones (p23, CyP40, FKBPs) have in vitro chaperoning activities
[13, 58], which may be important in the maintenance of specific Hsp90
substrates in proper folded states.
It has been known for some time that the oncogenic protein kinase v-Src
can be co-immunoprecipitated with Hsp90 [14]. Expression of v-Src in
S. cerevisiae cells is toxic, presumably due to nonspecific phosphorylation of
yeast proteins [15, 88]. Hsp90 maintains normal levels and specificity of the
kinase [177]. It was initially thought that only v-Src, but not c-Src depends
on the presence of the functional Hsp90 complex. However, use of a
particularly stringent Hsp90 mutant in S. cerevisiae revealed that c-Src also
148 M. Tesic and R. F. Gaber
requires Hsp90 for full activity [178]. The regulation of v-Src by Hsp90
complex reveals aspects of both positive and negative control: the first step,
in which Hsp90 helps the kinase adopt the proper conformation, is followed
by a second step in which Hsp90 prevents the kinase from being activated.
The intrinsic structural differences between the c-Src and v-Src presumably
contribute to the varying extent of dependence on Hsp90 [178]. The
comparison between the extent of dependence of the two kinases on Hsp90
could provide useful insights into the nature of the Hsp90 substrate
recognition.
2.1.4 p53
2.2.1 Ste11
the presence of a mating pheromone [93]. In Hsp90 mutant cells, both the
accumulation of Ste11 and its activity are decreased. A screen for multicopy
suppressors of this phenotype led to the isolation of Ste5, a component of the
MAPK-signaling module that functions as a scaffold to maintain association
of the other components of the cascade (Ste11, Ste7, Fus3/Kss1) [93].
It is possible that the specificity of Hsp90 interaction with Ste11 is
determined by the Hsp90 co-chaperone Cdc37. Cdc37 has been dubbed a
“kinase-targeting subunit” of the Hsp90 complex [165], and cdc37 mutant
cells have reduced Ste11 function [2]. Cdc37, Ste11, and Hsp90 form a
heterocomplex in yeast cells [2]. Since such complexes have also been
identified in mammalian cells [60, 160], there appears to be strong
conservation in the way components of the MAPK cascade are regulated by
the Hsp90 complex, thus allowing researchers to extend studies of yeast cell
cycle to mammalian systems.
2.2.2 Telomerase
experiments. The picture of Hsp90 that arises from these studies is that of a
highly dynamic molecule that joins and departs from complexes containing
its substrates and co-chaperones (Figure 1). During the course of activation
by the Hsp90 complex, the substrate is transformed from an inactive and
possibly partially unfolded state, to a conformation in which it is poised to
respond to appropriate cellular signals.
Figure 1. Sequence of events leading to the activation of a model substrate by Hsp90 and
Hsp70 and their co-chaperones. Although steps in this sequence have been elucidated using
mammalian cells, protein names indicated here are those of S. cerevisiae (co-chaperones).
Details of dynamic interactions between the proteins are described in the text.
Steroid receptors have been used as model substrates that are chaperoned
by Hsp90 from a conformation incapable of binding the hormone ligand to a
hormone-binding form [31, 131]. A highly conserved and essential cha-
perone, Hsp70, is thought to be the first to bind the substrate, thus defining
the early phase of the steroid receptor maturation cycle [74, 132, 155]. A
recent report suggests, however, that Hsp40/Hdj1, a co-chaperone of Hsp70,
may, in fact, be the first to bind, at least to PR [66]. Another Hsp70
regulator, Hip, is also associated with progesterone receptors [129], and
might aid the formation of this early complex by stabilizing the interaction
between Hsp70 and the substrate [59]. Regardless of their relative order, it is
by now well established that Hsp70 and its co-chaperones Hip and
Hsp40/Hdj1 participate in the early stages of substrate activation. Hsp90 is
the next component to enter the complex. Its initial binding may be mediated
by the co-chaperone, Hop, which is capable of binding both Hsp90 and
Hsp70 concurrently [27, 28, 43, 76, 156]. The presence of Hop defines the
intermediate complex. Hop is replaced by another set of co-chaperones,
which bind to the same acceptor site on Hsp90 as Hop. These are most often
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 151
3.1.1 Hop/Sti1
domain of Hop with a very similar dissociation constant (Kd = 11 µM) to the
entire C-terminal domain of Hsp90 (Kd = 6 µM). This indicates that the main
(and possibly sole) structural determinants for binding to TPR domains lie
within this small region of Hsp90.
Reconstitution of steroid receptor complexes in reticulocyte lysates and
formation of complexes from purified components defined the minimal
chaperone machinery required to transform steroid receptors into an active
state: Hsp90, Hsp70, Hop, Hsp40, and p23 [130, 131]. Because of the
enhancement in the rate of steroid receptor activation in the presence of Hop
[109], it can be concluded that this co-chaperone at least enhances the
assembly of the complex and its activity on substrates, although there may
not be an absolute requirement for Hop in Hsp90 complexes.
Combined genetic and biochemical data from a number of laboratories
suggest that Hop/Sti1 helps Hsp90 in the formation of productive complexes
i.e., those capable of chaperoning substrates. In sti1∆ cells, signaling through
eIF2α kinase Gcn2 is decreased [46]. Deletion of STI1 in S. cerevisiae leads
to at least a threefold decrease in the activation of a GRE-LacZ reporter
gene, and significant loss of v-Src activity [21]. While sti1∆ affects only the
activity and not the level of v-Src protein, mutations in hsp90 decrease both.
It is therefore possible that Sti1 is involved specifically with one aspect of v-
Src chaperoning by Hsp90, aimed at maintaining its activity. However, a
closer look at the Hsp90 and Sti1 complexes in S. cerevisiae reveals a more
puzzling picture. When Hsp90 and GR complexes are precipitated from wild
type and sti1∆ cells, there is no observable difference in the association
between Hsp90 and Hsp70 [21]. Therefore, either there is a considerable
difference between the yeast and mammalian complexes with respect to the
requirement for Hop/Sti1 to link Hsp90 and Hsp70, or Hop/Sti1 is not
required to bring together the two chaperones in vivo. Indeed, because Hsp70
and Hsp90 can interact independently with substrate molecules, Hop/Sti1
may be required only under some circumstances.
How Hop/Sti1 exerts the observed stimulatory influence on the
maturation of Hsp90 substrates remains an important question. In addition to
its bridging role, which may or may not be important in vivo, Hop/Sti1
protein is apparently involved in the regulation of Hsp90 ATPase activity. In
the presence of purified Sti1, the ATPase activity of purified yeast Hsp90 is
reduced [135]. Sti1 also elicits a significant conformational change in Hsp90
upon binding to the chaperone in vitro [135]. Since the ATPase and
chaperoning activities of Hsp90 are tightly coupled, Sti1 may regulate the
chaperoning of the Hsp90 substrates through its effect on the ATPase
activity.
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 153
3.1.3 TTC4/Cns1
1:1 molar ratio, although Cpr6 is a monomer in solution [99, 135]. These
findings suggest that two TPR proteins can bind to the Hsp90 dimer
simultaneously and that TPRs may not necessarily compete for binding to
Hsp90; instead, their binding may be independent of each other, or even
cooperative. Consistent with this, others and we have shown that Cns1 and
Cpr7 can be found in the same Hsp90 complexes in vivo [44, 98]. This
model of binding corresponds well with our current understanding of the
interactions between TPR proteins and Hsp90, mediated by the MEEVD
motif. Since each of the two Hsp90 monomers contains this motif, it is easy
to envision how two TPR domains can be accommodated by the Hsp90
dimer. Interestingly, the observation of at least one mixed TPR hetero-
complex, containing Cns1 and Cpr7, suggests that other combinations may
exist and that they may play a role in Hsp90 substrate specificity. By
combining even a small set of different TPR co-chaperones, a large number
of distinct Hsp90 complexes can be achieved, perhaps providing the
specificity of the Hsp90 complexes for their substrates.
Cns1 belongs to a protein family with homologs in S. pombe, Caenor-
habditis elegans, Drosophila melanogaster, and humans. The Drosophila
Dpit47 interacts with the DNA polymerase alpha subunit [36]. The human
Cns1 homolog, TTC4, is of particular interest as a potential tumor
suppressor. The TTC4 gene maps to the region of chromosome 1p31 which
undergoes loss of heterozygosity (LOH) in up to 50% of breast cancers [69,
167]. In addition, in samples from patients with malignant melanoma, six
different point mutations in TTC4 were found [128]. We have found that
TTC4 can interact with yeast Hsp82 in vivo (Tesic, M. and R. Gaber,
unpublished data), providing a possible connection between the Hsp90
chaperone machinery and human tumorigenesis.
3.1.4 PP5/Ppt1
been investigated and may provide interesting insights into the intricacies of
the mechanism of action of this phosphatase in Hsp90 complexes.
The analysis of PP5-containing Hsp90 complexes demonstrates that this
phosphatase competes with other TPR domain co-chaperones for binding to
the Hsp90 [151]. Interestingly, PP5 is also able to compete with the binding
of Hop, which could not be out-competed by the immunophilins [151]. This
lends credence to the hypothesis that there is a hierarchy of binding to the
TPR-accepting pocket of Hsp90, which is determined by the relative-binding
constants, concentration, and availability of particular chaperones.
The crystal structure of the amino terminus of PP5 was the first structure
of a Hsp90-binding TPR domain to be solved [38], providing insight into the
structure-function relationships of this highly versatile protein–protein
interaction motif [89]. Each individual TPR consists of two antiparallel α
helices at a 24° angle, which are tandemly repeated. Eight conserved
residues, mostly small and large hydrophobic amino acids, determine the
highly degenerate signature of any TPR motif, and are buried within the
structure. Certain exposed charged residues likely to be involved in protein–
protein interactions were mutated in PP5 and shown to be important for the
binding of PP5 to Hsp90 [141]. These lysines and arginines are highly
conserved among all TPR-domain containing Hsp90-binding co-chaperones.
A structure of the TPR domain of Hop co-crystallized with the MEEVD
peptide from Hsp90 corroborated these findings by showing that the very
same charged residues made crucial interactions with Hsp90 [148].
Interestingly, mutations in the MEEVD sequence of Hsp90 significantly
reduce, but do not abrogate, the binding of PP5 TPRs to Hsp90 [141],
suggesting that there may be additional binding determinants between Hsp90
and its TPR co-chaperones.
It is still unclear whether the phosphatase domain of PP5 is involved in
the regulation of Hsp90-dependent events. The simplest model would
suggest a role for PP5 in targeting the Hsp90 multichaperone complex to a
substrate to be dephosphorylated. However, it is possible, as was shown for
Cpr7 in S. cerevisiae [50], that the phosphatase domain of PP5 is dispensable
for its Hsp90-related role. In support of the idea that TPR domains regulate
Hsp90 function, the TPR domain of PP5 is emerging as a highly versatile
functional unit. It can negatively regulate the phosphatase activity of PP5 by
steric hindrance, which is released by the addition of polyunsaturated fatty
acids [25, 154]. A study of a series of mutations in PP5 made in order to
investigate how the TPR domain accommodates the interactions both with
Hsp90 and the phosphatase domain of PP5 itself reveals that the residues
involved in binding to Hsp90 are distinct from those involved in the
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 157
autoregulatory role [81]. In general, multiple functions may exist for TPR
domains of other Hsp90 binding proteins.
Although ppt1∆ cells grows normally, treatment with antisense PP5
oligonucleotides leads to G1 cell cycle arrest in S. cerevisiae cells [186].
Investigation of possible downstream effects of PP5 loss of function reveals
a strong increase in the level of p53 phosphorylation, and increased affinity
for its genomic response elements; PP5 is also able to dephosphorylate p53
in vitro. Given these results, and the observed negative regulation of the GR,
it is possible that PP5 is involved in cancers through effects on both GR and
p53 regulation [187]. Since both GR and p53 interact with Hsp90, PP5 may
be one of the co-chaperones dedicated to these Hsp90 substrates.
3.2.1 p23/Sba1
Although it has been known for some time that steroid receptor
complexes contain a protein of 23 kDa in size termed p23 [158], the
S. cerevisiae homolog, Sba1, was identified only recently [10, 52]. SBA1 is
constitutively expressed, and sba1∆ cells are viable at 30°C and 37°C, but
grow slowly at 16°C. In S. pombe, a p23 homolog Wos2 interacts with the
Hsp90 substrate Wee1, which is involved in cell cycle control [111]. In
L cell cytosol and rabbit reticulocyte lysate reconstitution assays, p23
stabilizes Hsp90-steroid receptor complexes [42].
There are at least two requirements for the high-affinity binding of p23 to
the amino terminus of Hsp90 [29]: dimerization of Hsp90 [62] and ATP
binding (but not ATP hydrolysis) [20, 133]. p23 seems to play a role in the
“coupling” of the ATPase activity and the substrate release by Hsp90 [180].
p23 may accomplish this, at least in part, by inhibiting the ATPase activity
of Hsp90, thus extending the duration of the interaction between the client
protein and the chaperone, allowing for the productive folding to occur
[100]. This observation is in contrast with a previous report in which no
effect on the ATPase activity of purified yeast Hsp90 was observed in the
presence of purified Sba1 [117].
It has been somewhat difficult to establish whether Sba1 in S. cerevisiae
is truly a functional homolog of p23. sba1∆ cells are sensitive to
benzoquinoid ansamycins, a class of molecules such as GA, a potent
inhibitor of Hsp90 ATPase activity. In general, sensitivity to GA of yeast
cells carrying mutations in certain genes points to involvement of those
genes in Hsp90-related pathways. Sba1 was found in Hsp90 complexes,
along with Cpr6 and a small amount of Sti1; this interaction was inhibited by
158 M. Tesic and R. F. Gaber
ydj1∆ cells, mutations in the J-domain can reduce both mRNA and protein
levels of v-Src [40]. Interestingly, mutations in Ydj1 have differential effects
on various Hsp90 substrates. In ydj1∆ cells, androgen receptor-mediated
signaling is compromised [56]. In contrast, in most ydj1 mutant cells,
signaling mediated by GR and ER was enhanced, yet the levels and the
activity of v-Src were mostly decreased [78]. Together with Hsp70, Ydj1
represses the activation of an endogenous yeast substrate, Hap1, in the
absence of heme [71]. The negative effects of Ydj1 on the activation of
Hsp90 substrates appear to be mediated by its J domain [71, 78].
Collectively, these findings suggest that Ydj1 is involved in multiple steps of
client protein regulation by the Hsp90 machinery, including direct
interactions with both the chaperones as well as the substrates themselves.
3.2.3 Sch9
3.2.4 Hsp110/Sse1
3.2.5 Cdc37
It has been known for a long time that Cdc37 is involved in the regulation
of cell cycle at start [139]. Both Cdc37 and Hsp90 are required for the
signaling by the sevenless receptor tyrosine kinase in D. melanogaster [37].
Like Hsp90, Cdc37 in S. cerevisiae is an essential protein and a dimer, but
unlike Hsp90, its cellular concentration is low (about 0.01% total protein)
and is not increased by heat shock [85]. Cdc37 was first seen in steroid
receptor complexes as a protein of about 50 kDa size [124], and virtually all
cytosolic Cdc37 is associated with Hsp90 [174].
Cdc37 has been dubbed the “protein kinase targeting subunit” of Hsp90
[164, 165]. It interacts with a number of kinases that also bind to Hsp90,
including cell cycle regulator Cdk4 [165], Raf-1 [160], and v-Src [41].
However, it is now clear that its cellular role in general, and its role as an
Hsp90 co-chaperone in particular, is more versatile than just as a
determinator of specificity for Hsp90.
Cdc37 is also a molecular chaperone in its own right. Like Hsp90, it is
capable of maintaining unfolded β-galactosidase in a folding-competent state
[85]. Genetic interactions between HSP90 and CDC37 in both budding yeast
and fruit flies indicated that their roles overlap, but are not identical.
Consistent with the idea that Cdc37 interacts solely with Hsp90 kinase
substrates, overexpression of Cdc37 in the Hsp82G170D mutant restored
normal v-Src, but not GR, activity [85]. However, signaling by a different
steroid receptor, AR, was strongly affected by deletion of CDC37 [55].
Extending these findings, AR, but not GR, interacted with recombinant
Cdc37 in a GA-sensitive manner [137]. Therefore, Cdc37 is found
predominantly, but not exclusively, in Hsp90-protein kinase complexes.
Apparently, even if the substrates of Hsp90 belong to the same functional
class, like AR and GR, and share some structural similarities, they can have
distinct requirements from the Hsp90 complex. It is possible that the
structural differences between these substrates are significant enough to
require the use of a different set of chaperones and co-chaperones by the
cell. Binding to the Hsp90 complex may be determined by the folded state of
the substrate, rather than simply by the structure of folded domains. It is
conceivable that, in a partially folded state, AR resembles protein kinases
more closely than does GR, and is therefore recruited by Cdc37–Hsp90
complexes.
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 161
Cdc37 binds to Hsp90 at a site adjacent to the PP5 binding site. Full
length PP5, but not the TPR domain alone, competes with Cdc37 for the
binding to Hsp90, suggesting steric obstruction between Cdc37 and the
phosphatase domain of PP5 [153].
A direct role for Cdc37 in neoplastic processes is beginning to emerge.
Mouse mammary tumor virus promoter driven MMTV-Cdc37 transgenic
mice develop tumors at a similar rate as MMTV-cyclin D1 mice [163]. In
addition, human prostate cancer cells show increased expression levels of
CDC37 [166]. How CDC37 realizes its oncogenic property is not clear. A
possible mechanism involves its known interaction with oncogenes such as
Cdk4 and Raf1. In addition, both Cdc37 and Hsp90 have been found to
associate with oncogenic protein-tyrosine kinases, v-Yes, v-Fps, and v-Fgr
[73]. Cdc37 may regulate on its own, and help Hsp90 in regulating, a
number of proteins involved in cellular proliferation. Understanding the
mechanism and selectivity of this co-chaperone will remain particularly
important in the attempts to prevent cellular transformation by targeting
Hsp90 and its substrates.
remains unanswered. In vitro, two chaperones alone, Hsp70 and Hsp90, have
been reported to mediate maturation of steroid receptors [136], arguing
against an unconditional requirement for the co-chaperones. Furthermore,
although the Hsp90 MEEVD sequence is considered to be sufficient for the
binding of TPR domain co-chaperones, its deletion has no effect on the
viability of S. cerevisiae cells [94]. This suggests that interactions of Hsp90
with all proteins that bind to it through their TPR motifs are dispensable. If
this is the case, then TPR domain co-chaperones may simply be modulators
of Hsp90 functions. Alternatively, it may not be the physical association
with Hsp90 per se that is necessary for substrate regulation, but the presence
of co-chaperones in the Hsp90 complex, which can also be achieved through
binding to substrates directly. While some co-chaperones, like p23, are most
likely generally required by Hsp90, others may be specific for certain classes
of substrates, or only in some cell types, and under certain cellular
conditions.
Although it chaperones a large number of substrates, Hsp90 does not
interact indiscriminately with partially unfolded or inactive proteins,
suggesting that triage decisions are made in the cell that determine Hsp90
substrate specificity. One of the models of co-chaperone action, which posits
that co-chaperones determine specificity of the Hsp90 complex for indivi-
dual substrates, has been difficult to prove. Some Hsp90 co- chaperones
show clear preference for one class of substrates. ARA9/XAP2/AIP is the
only co-chaperone identified so far that apparently interacts with only one
Hsp90 substrate, AhR [6, 19, 82, 83, 90, 102, 103, 125]. All other co-
chaperones show a less stringent predilection for Hsp90 substrates. Cdc37,
for example, is present almost exclusively in protein kinase–Hsp90
complexes. However, Cdc37 also interacts with at least one steroid
receptor, AR. Proteins containing TPR domains are the most obvious
candidates for co-chaperones that determine specificity of the Hsp90
complex because they bind Hsp90 directly through the TPR domain, while
the other domain in each co-chaperone (isomerase in the case of
immunophilins, or phosphatase in the case of PP5/Ppt1) can mediate binding
to the appropriate substrate. Especially intriguing is the idea that Hsp90 can
accommodate more than one TPR domain co-chaperone (Cns1 and Cpr7, for
example), thus increasing the number of possible co-chaperone combinations
and, consequently, the number of substrates Hsp90 acts upon. However, it is
still equally likely that co-chaperones determine specificity of Hsp90
function not by bridging the chaperone to the substrate, but by keeping
Hsp90 itself in a conformation that favors some client proteins over others.
Consistent with this idea, defining the substrate recognition site for Hsp90
has proven to be difficult, suggesting that this is a highly versatile chaperone
capable of discriminating between similar proteins. Future work should be
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 165
Because a large number of proteins depend on Hsp90 for normal levels and
activity, it is a concern that indiscriminate inhibition of Hsp90 function, as is
achieved by inhibiting its function through GA, may have widely pleotropic
effects. If co-chaperones indeed determine the specificity for Hsp90, instead
of inhibiting Hsp90 function directly, it might be more advantageous to
inhibit the function of a specific co-chaperone involved in a complex with a
tumorigenic substrate. Additionally, instead of completely inhibiting the
function of a certain co-chaperone, specific inhibition of the physical
interaction between Hsp90 and the co-chaperone might provide even greater
specificity for the desired antitumor agent. In order to accomplish this, it will
be necessary to understand the details of the physical interaction between the
co-chaperones and Hsp90, as well as the various aspects of regulation by the
co-chaperones of both Hsp90 and the substrates, in the context of an in vivo
cell; for this approach, the yeast system is particularly well suited.
ACKNOWLEDGMENTS
The authors would like to thank Ellen Nollen, Joshua Schnell, and
Sricharan Bandhakavi for their critical reading of the manuscript and helpful
suggestions.
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Chapter 7
YEAST AS A MODEL SYSTEM FOR STUDYING
CELL CYCLE CHECKPOINTS
179
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 179–189.
© 2007 Springer.
180 C. Palermo and N. C. Walworth
required at both the onset of S-phase and M-phase [4, 24]. When activated,
Cdc2 controls mitotic entry via phosphorylation of a number of proteins.
The activation of Cdc2 is itself tightly regulated requiring association with a
cyclin regulatory subunit (cyclin B), appropriate subcellular localization, as
well as critical phosphorylation and dephosphorylation events on an activa-
ting threonine residue and inhibitory tyrosine (Tyr 15) residue [20].
Figure 1. A schematic model of the DNA damage checkpoint pathway. DNA damage
triggers several proteins to localize to presumptive DNA damage sites. Chk1 becomes
phosphorylated by Rad3/Rad26, concomitant with binding to the 14-3-3 protein, Rad24 or
Rad25. Chk1 phosphorylates Cdc25 and Wee1 to control their activity and/or subcellular
localization resulting in a delay to activation of Cdc2 and therefore, a delay to mitotic entry.
cells harboring mutant checkpoint components bypass the arrest and proceed
through subsequent phases of the cycle with unrepaired DNA damage,
ultimately resulting in cell death.
might structurally disrupt the complex and hence lead to complete failure of
the signal transduction system. This finding began to illustrate the complex
interactions and multiple feedback controls that exist, yet converge on a
single signal transduction pathway, whose ultimate task is to delay mitotic
progression. Interestingly, the predictions made based on genetic obser-
vations are now supported by biochemical evidence that the Rad1, Rad9, and
Hus1 gene products indeed form a heterotrimeric complex that is related in
structure to the PCNA processivity factor for DNA polymerase δ [7].
As sophisticated genetic screens began to establish the checkpoint
pathway in S. pombe, serendipitous findings also contributed candidates for
the central cast of players that couple the checkpoint pathway to the mitotic
apparatus. As one example, a genetic screen aimed to identify elements that
directly interact with Cdc2 utilized a cold-sensitive cdc2-r4 mutant that
grows as wild type at 36°C, yet undergoes cell cycle arrest at the restrictive
temperature of 25°C [17]. The cdc2-r4 mutation itself suppressed the
lethality resulting from simultaneous loss-of-function of the inhibitory
kinases wee1 and mik1, allowing cells to survive in the absence of tyrosine
phosphorylation of Cdc2 and indicating that other mechanisms of Cdc2
regulation are in operation [17]. Introduction of a multicopy gene library into
the cdc2-r4 strain led to isolation of plasmids that suppressed the effects of
the cold-sensitive mutation at 18°C [33]. Consequently, this screen revealed
one of the core components of the checkpoint pathway known as chk1 [33].
Deletion of the chk1 gene does not affect cell viability; however, a
characteristic checkpoint defect is evident when cells are exposed to UV
irradiation. Notably the UV sensitivity of a chk1 disrupted strain can be
largely rescued by imposing an “artificial” G2 delay, discriminating this
checkpoint component from gene products that are involved in DNA repair
processes [33].
Chk1 is a member of the serine/threonine family of protein kinases and
itself was found to undergo phosphorylation in response to DNA damage
[34]. The phosphorylated form, observable by SDS-PAGE as a decreased
mobility form of the protein, has proven to be a useful tool to dissect how
previously isolated and novel checkpoint components interact with Chk1 to
arrest cell cycle progression in response to DNA damage. In fact, epistatic
analysis with the rad checkpoint genes indicated that damage induced
phosphorylation of Chk1 depends on the function of rad1, rad3, rad9,
rad17, and rad26, [34], rad24 [8], and crb2 [28].
To further define the role of Chk1 in the checkpoint pathway, the full-length
gene was utilized as bait in a yeast two-hybrid screen of an S. pombe cDNA
library for proteins that interact with Chk1. Two fission yeast 14-3-3
proteins, Rad 24 and Rad25, emerged from this screen [8]. Structural data
Chapter 7: Yeast as a Model 185
ACKNOWLEDGMENT
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188 C. Palermo and N. C. Walworth
Over the last two decades, sphingolipids have emerged as important cell
regulators. Included in this lipid class are the lipid mediator sphingosine, its
phosphorylated derivative sphingosine-1-phosphate, and ceramide. In
humans, the major known functions of sphingolipid-mediated cell regulation
is the modulation of signaling pathways which control fundamental cellular
processes including cell division, senescence, apoptosis, angiogenesis, and
differentiation [40, 41, 74], all of direct relevance to cancer pathogenesis and
progression.
Because of these myriad activities, the enzymes that generate sphingo-
lipid mediators are potential targets for cancer treatment. Thus, characteri-
zation of these enzymes with respect to activity, regulation, localization, and
cellular function is fundamental to developing strategies for modulating
sphingolipid levels in vivo. The yeast system has emerged as an invaluable
tool for the identification, cloning, and characterization of enzymes of
sphingolipid metabolism. Furthermore, insights gained from yeast studies
of sphingolipid metabolism and function continue to push the forefront of
sphingolipid research, especially since the roles of sphingolipids in stress
and other cell responses appear to be conserved from yeast to human.
191
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 191–210.
© 2007 Springer.
192 L. A. Cowart, Y. A. Hannun and L. M. Obeid
Figure 1. Pathways of sphingolipid metabolism. Steps represented in bold text are shared by
yeast and mammalian cells. Yeast-specific enzymes and compounds in italics and
mammalian-specific enzymes and compounds in plain font.
Yeast enzymes required for sphingoid base N-acylation are LAG1 and LAC1
[91]. It is not yet established whether the enzymes encoded by these genes
produce ceramide directly or act as necessary activators of an as yet
unidentified ceramide synthases; however, the dependence of ceramide
formation on these proteins as well as their ability to control intracellular
ceramide levels have been well established biochemically [91]. (Longevity
assurance gene LAG1) was initially identified as a gene whose deletion
conferred increased lifespan in yeast [91]. This is particularly interesting as
studies in mammalian cells indicate that increased ceramide is correlated
with cell senescence (see below). The functions of lag1p and lac1p are
partially redundant, thus, deletion strains of LAG1 or LAC1 are viable;
however, deletion of both genes causes a severe slow growth phenotype,
indicating the importance of ceramide biosynthetic pathways in yeast [91].
Very interestingly, LAG1 homologues have been identified throughout
eukaryotic organisms, including tomato [9], Caenorhabditis elegans [51],
Drosophila [97], and human [51] based on sequence similarity to the yeast
gene. In particular, a mouse homologue (UOG) was recently identified as
necessary for N-stearoyl-sphinganine biosynthesis, and of particular interest
is that this gene had a suspected role in the regulation of cell differentiation
prior to discovery of its role in ceramide synthesis, suggesting not only
biochemical conservation but possibly some degree of functional conser-
vation as well [98].
Ceramide synthase was found to be a target of the group of fungal toxins
known as fumonisins. Fumonisin inhibition of ceramide synthase was first
reported in rat liver microsomes, but provided insight into mechanisms of
the observed correlation between fumonisin consumption and development
of cancer [99]. It appears that not all LAG1 homologues are fumonisin
targets; however, the bulk of ceramide synthase activity in mammalian cells
is inhibited by fumonisins.
Similarly to sphingoid bases, ceramide can be phosphorylated at C1. A
mammalian ceramide kinase was cloned based on sequence similarity to
mouse and human isoforms of sphingosine kinase [93]. Interestingly, no
yeast homologue for ceramide kinase has been identified. Presently,
however, at least 50% of the yeast genome (i.e., protein-encoding open
reading frames) are of unknown function. Thus, further characterization of
ceramide kinase may well lead to identification of a yeast isoform, and, if so,
yeast systems may be useful in functional characterization of ceramide-
1-phosphate.
Chapter 8: Metabolism and Function of Sphingolipids 197
Hopefully further knowledge of the yeast orthologue will assist in this goal
as well.
Though mannose is the only sugar in S. cerevisiae sphingolipids,
mammalian complex sphingolipids incorporate a variety of carbohydrate
groups including lactose, glucose, galactose, and sialic acid. These activities
in mammalian cells give rise to glycosphingolipids, gangliosides, and
cerebrosides. It should be noted that in some other fungal species including
Candida albicans, glucosylceramide synthase has been detected [60],
though the significance of this activity is not known [59].
2 SPHINGOLIPID FUNCTIONS
Figure 2. Heat stress induces many activities in yeast, some of which are mediated by stress-
induced de novo sphingolipid synthesis. Direct targets for sphingolipids in yeast include
ceramide-activated protein phosphatase, comprised of sit4p, cdc55p, and tpd3p. Additionally,
heat stress induces sphingolipid synthesis in some mammalian cell types. Interestingly,
Chapter 8: Metabolism and Function of Sphingolipids 201
several chemotherapeutic agents induce de novo sphingolipid production and thus, their
effects are likely mediated in part by sphingolipid biosynthesis. (See text for further
discussion.)
Another key feature of the yeast heat stress response is the internalization
and ubiquitin-mediated proteolysis of nutrient permeases. Previous studies
in yeast have demonstrated that de novo sphingolipid biosynthesis is
required for these activities, and that exogenously added phytosphingosine
initiates this degradation in the absence of heat stress [15, 16]. This
sphingolipid activity is at least somewhat conserved in some mammalian
cells, as ceramide has been demonstrated to modulate the ubiquitin-mediated
proteolysis of c-myc [82]. More investigation is needed in both yeast and
mammalian systems to determine the specific mechanisms of sphingolipid
modulation of this pathway. Interestingly, a recent study implicates lipid
rafts in the transport and degradation of the nutrient permease Fur4p [44],
and thus, the propensity of sphingolipids to form lipid microdomains [10, 34,
36, 47] may facilitate these activities.
These findings in yeast led to the hypothesis that mammalian heat stress
responses may depend at least partially on de novo generated sphingolipids.
Indeed, subsequent studies in MOLT-4 human leukemia cells [48] confirmed
this hypothesis. Heat stress in these cells induced a flux through sphingolipid
biosynthetic pathways similar to those observed in yeast. Heat-induced
de novo sphingolipid biosynthesis in MOLT-4 cells was shown to influence
mRNA splicing by the modulation of SR protein phosphorylation state [48].
One difference between heat-induced de novo sphingolipid biosynthesis in
yeast and in the MOLT-4 cells is that yeast accumulate sphingoid bases prior
to ceramide formation, whereas the MOLT-4 cells showed no accumulation
in sphingoid bases after heat stress, but accumulated ceramide, which is
thought to be the active intermediate in modulation of many mammalian
signaling pathways. Whether this situation is typical of mammalian systems
and whether the sphingoid bases may play specific roles remains to be
determined.
Downstream regulatory roles of sphingolipids generated during stress
responses is of interest to cancer research, as several chemotherapeutic
agents including etoposide, daunorubicin, and gemciytabine have been
demonstrated to cause increased de novo sphingolipid synthesis, and
furthermore, many effects of these drugs are blocked by inhibitors of
ceramide synthesis ([8], reviewed in [81, 89]). Thus, these agents act at least
partially through the de novo pathway of sphingolipid generation.
Therefore, understanding the mechanisms of formation and action of
sphingoid bases and ceramides may lead to direct insight into the
mechanisms of action of several chemotherapeutic agents and, especially,
the mechanisms by which they activate stress response pathways.
202 L. A. Cowart, Y. A. Hannun and L. M. Obeid
3 CONCLUSIONS
ACKNOWLEDGMENTS
This work was supported by a VA merit award (LMO), and NIH grants
AG16583 (LMO), GM62887 (LMO), GM63465 (YAH), and GM4825
(YAH).
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Chapter 9
EXPLORING AND RESTORING THE p53
PATHWAY USING THE p53 DISSOCIATOR
ASSAY IN YEAST
Rainer K. Brachmann
Department of Medicine, University of California at Irvine, Irvine, California 92697, USA
For more than a decade, the tumor suppressor protein p53 has been
recognized as a, and maybe the, central protein that protects humans from
cancer. Its role as “guardian of the genome” [51] and its frequent
inactivation in human cancers has drawn numerous researchers into the p53
field, guaranteeing a continuous flow of new research data that is regularly
summarized in excellent reviews (see for example, 20, 49, 56, 79, 97, 98; see
also the chapter by Inga and colleagues for an in-depth review of p53
biology). Therefore, this introduction will be short, but nevertheless
sufficient to appreciate the p53-related questions that are being pursued in
the p53 dissociator assay.
The p53 protein was initially isolated in 1979 as a 53 kD protein that was
associated with SV40 large T antigen [53, 57]. It was a decade before p53
was recognized as an important tumor suppressor because of its frequent
mutation in human cancers [38, 52, 73]. This key finding raised two central
questions: Why was p53 so important and what was its mode of action?
It was quickly established that wild-type p53 exerts its effects, at least in
part, as a transcription factor that recognizes its direct target genes through
binding of its core domain (amino acids 96–292) to specific DNA
sequences. In unstressed cells, p53 is relatively inactive and has a short half-
life of 15 min because of negative feedback loops, such as with MDM2.
211
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 217–232.
© 2007 Springer.
212 R. K. Brachmann
p53 drives expression of MDM2, and the Mdm2 protein then binds to the
p53 amino-terminus and induces ubiquitin-mediated degradation [55].
Activation of the p53 protein occurs through upstream signals, such as DNA
damage, hypoxia, overexpression of oncogenes and others. These signals
typically lead to posttranslational modifications of p53, in particular amino-
terminal phosphorylation and carboxy-terminal acetylation. These modify-
cations stabilize and activate p53. p53 is then able to induce its numerous
target genes and causes cell cycle arrest, DNA repair, and/or apoptosis [20,
49, 56, 79, 97, 98].
The central role of p53 in protecting humans from cancer is reflected in
the enormous number of human cancers with p53 mutations. It is estimated
that 50% of all human cancers have p53 mutations, and such mutations have
been catalogued in large international databases [5, 18, 32, 36, 37, 62, 77].
The latest update of the IARC TP53 Mutation Database contains 19,809
somatic p53 mutations. 14,123 or 71% of these are missense mutations
affecting the p53 core domain, and close to 1,000 different amino acid
changes have been described for this p53 region (R9, http://www.
iarc.fr/p53/).
It has been, and continues to be, a significant challenge to understand the
complexity of wild-type p53, p53 cancer mutants and downstream target
genes. The chapter by Inga and colleagues summarizes how powerful
genetic approaches in yeast assays have made important contributions to
characterizing the p53 network. This chapter will focus on two aspects of
p53 research that are addressed in the p53 dissociator assay: restoring
function to p53 cancer mutants and identifying proteins that play important
roles in p53 biology, be it as a p53 inhibitor, a p53 coactivator, or a protein
that is regulated by p53.
the dissociator assay has a significant difference to the other systems in that
it also allows for selection against a particular interaction. This is possible
because the URA3 gene product is involved in converting 5-fluoro-orotic
acid (Foa) into a toxic substance, thereby arresting cell growth [7]. This twist
has been exploited as the “reverse one- or two-hybrid system”, a powerful
tool that allows for the selection of novel factors, “dissociators”, able to
interfere with a macromolecular interaction [10, 95, 103].
The p53 dissociator assay consists of a tightly repressed reporter
construct, 1cUAS53::URA3, typically integrated into the yeast genome, in
which a p53 consensus DNA-binding site in the context of the artificially
introduced SPO13 promoter is placed upstream of the URA3 gene [11, 95].
p53 binds to its DNA-binding site and activates transcription of URA3. The
activity of wild-type p53 can be phenotypically scored in two ways: yeast
growth on plates without Uracil (Ura+) and lack of growth on Foa-containing
plates, i.e., Foa-sensitive (FoaS).
Figure 1. Principles of the p53 dissociator assay in yeast. The p53-dependent reporter gene
URA3 allows for selection against active p53, because URA3 expression results in conversion
of 5-fluoro-orotic acid (Foa) into a toxic substance, thus preventing yeast growth. The concept
of counterselection can be exploited for the identification of human proteins that reduce p53
activity in the yeast assay. Not only p53 inhibitors score as “dissociators”, but also p53
coactivators and proteins that are regulated by p53. This is likely the case because the human
proteins are overexpressed, often amino-terminally truncated (due to the process of the library
construction) and evaluated in an artificial yeast assay. Since the p53 dissociator assay relies
on native p53 and a consensus p53 DNA-binding site (contrary to, for example, two-hybrid
assays for p53), a variety of p53-inhibitory mechanisms can be envisioned: for example, a
“dissociator” (“?” in the figure) may conceal the p53 transactivation domain (A), but could
also achieve its effect by binding to the p53 DNA-binding site (B).
214 R. K. Brachmann
The FoaS phenotype of the p53 dissociator assay provides the essential
tool to perform cDNA expression library screens (“dissociator screens”).
Proteins that interfere with wild-type p53 activity will reduce URA3
expression and allow growth on Foa plates, i.e., Foa-resistant (FoaR). p53
dissociators might utilize a variety of mechanisms, such as binding to and
masking the transactivation domain of p53 (Figure 1A) or the actual p53
DNA-binding site (Figure 1B), p53 degradation, or posttranslational
modifications of p53. The p53 dissociator assay should clearly identify
proteins that negatively regulate p53. But proteins with quite different
functions in human cells might be isolated as well, since the yeast assay is
different from human cells in many ways. The library proteins are expressed
at higher levels than p53 and outside their usual context, thus potentially
lacking important regulators of their activity. For example, a p53 enhancer
may require a protein complex for its activity in human cells. Missing these
important cofactors in yeast, the p53 enhancer may now have the opposite
effect by binding to and sequestering p53. Additionally, due to the mode of
library construction, most of the cDNA expression plasmids encode for
amino-terminally truncated proteins that may behave in a dominant-negative
fashion because they lack, for example, a regulatory domain.
Figure 2. Recapitulating the biology of p53, E6-associated protein and high-risk human
papilloma virus (HPV) E6 in the p53 dissociator assay. Infection with high-risk HPV is
associated with more than 80% of human cervical cancers. (A) The E6 protein induces
ubiquitin-mediated degradation of p53 through recruitment of the host factor E6-associated
protein (E6-AP), an E3 in the ubiquitin cascade whose cysteine 833 forms a direct thioester
with ubiquitin (“~S-” in the figure). (B) The complex interplay between two human and one
viral protein(s) can be recreated in the p53 dissociator assay, since the combination of E6-AP
and high-risk HPV E6 results in an Foa resistant (FoaR) phenotype on plates containing
Foa (“+Foa0.1%”). The interference with p53 is highly specific, since low-risk E6 from
an HPV unable to cause cervical cancer does not confer Foa resistance. (C) The FoaR
phenotype corresponds to reduced p53 protein levels that are seen not only in galactose
Chapter 9: p53 Dissociator Assay 217
media (high expression of E6-AP), but also in glucose media (minimal expression of E6-AP).
(D) E6-AP mutants in which cysteine 833 is changed to alanine or serine suggest that
thioester formation between E6-AP and ubiquitin is essential for inhibition of p53. In all
experiments (B–D), E6-AP proteins were expressed under the control of the GAL1 promoter
that is induced on galactose, “Gal”, but not induced on glucose-containing media. E6 was
expressed under the control of the constitutive ADH1 promoter. The two plasmids containing
the marker genes LEU2 or TRP1 were selected on media lacking leucine and tryptophane
(Leu–Trp). The ADH1-p53 expression cassette was either integrated into the genome at the
LYS2 locus (B and D) or maintained on a plasmid with the HIS3 marker gene (C).
Figure 3. Summary of three p53 dissociator screens. The use of a diploid yeast reporter strain
with two copies each of the assay components (p53 expression cassette and p53-dependent
URA3 reporter gene) results in very stringent screens that allow only 1 in 50,000 plasmids to
score with an FoaR phenotype in the p53 dissociator assay.
hADA3 was one of the first proteins to be isolated in the HeLa p53
dissociator screen. At the time, this human homologue of the yeast protein
Ada3p had just been described as a part of histone acetyltransferase (HAT)
complexes containing the human HATs hGCN5 or PCAF [76]. The yeast
gene ADA3 had previously been identified because its inactivation protected
Saccharomyces cerevisiae from the toxic effects of an overexpressed
artificial transcription factor, the GAL4–VP16 fusion protein [78]. Sub-
sequent work established that yeast Ada3p physically interacts with Ada2p
and the yeast HAT Gcn5p, forming HAT complexes termed Ada or SAGA
(if additional proteins are present) [29]. These HAT complexes acetylate
histones thereby relaxing tightly packed chromatin and giving transcription
factors access to promoters. They also appear to regulate other proteins by
acetylating them. Both Ada2p and Ada3p interact with the transactivation
domains of transcription factors, and the whole Ada complex is required for
transcriptional activity of many transcription factors [29, 60, 89, 90].
218 R. K. Brachmann
Considering the central role of p53 and its frequent inactivation through
p53 gene mutations, it is very likely that the remaining human cancers with
wild-type p53 must significantly reduce the activity of the p53 pathway. But
compared to the detailed knowledge regarding p53 mutations, much less is
Chapter 9: p53 Dissociator Assay 219
known about the p53 status of the remaining 50% of human cancers with no
p53 mutations.
One obvious way of eliminating the p53 pathway would be to abrogate
p53 gene expression. This is indeed the case for a subset of breast cancers.
Raman and colleagues determined that the transcription factor HOXA5 is
important for expression of the p53 gene [81]. In breast cancers with loss of
p53 expression, the promoter of the HOXA5 gene was frequently methylated
resulting in lack of HoxA5 expression and HOXA5 protein. It is currently
unknown whether this mechanism is found in other cancers as well.
A second efficient mode to eliminate the p53 pathway is direct
inactivation of the p53 protein. There are two well-documented examples: a
large fraction of sarcomas, as well as a small percentage of other human
cancers show overexpression of MDM2, thus turning the physiological
negative feedback loop for p53 into a tool to rid the cell of p53 activity [20,
28, 56, 59, 68, 96]. Also, high-risk HPV cause more than 80% of cervical
cancers, and the HPV E6 protein is responsible for inducing the degradation
of p53 [20, 28, 70, 84, 96]. Firm numbers are not available, but it is unlikely
that the majority of human cancers with wild-type p53 utilize inactivation of
p53 by high-risk HPV E6 or MDM2 overexpression.
There are two additional ways of inactivating wild-type p53 protein
whose underlying molecular mechanisms have not yet been fully elucidated.
A variety of human cancers show complete or partial mislocalization of p53
to the cytoplasm. This has been described for primary tissues and/or cell
lines of breast cancers, colon cancers, neuroblastomas, malignant, melanomas
and retinoblastomas [8, 9, 65, 67, 85, 91, 102], and impairs p53’s ability to
induce G1 arrest and cause growth inhibition [48, 66]. Several underlying
genetic alterations can be envisioned, including loss of factors required for
active import of p53 into the nucleus (Figure 4A, I) and overexpression or
constitutive activation of factors that actively hold p53 in the cytoplasm
(Figure 4A, II). The latter model is supported by indirect evidence from
several studies that suggest that an unidentified short-lived anchor protein
might tether p53 to the vimentin scaffold [26, 47, 48] and the identification
of a cytoplasmic anchor protein, Parc, for p53 [45, 74]. Stommel and col-
leagues provided strong support for a third possibility, inappropriate nuclear
export of p53 [88]. They showed that disruption of p53 homo-tetramers
results in exposure of a nuclear export signal in the carboxy-terminus.
Inappropriate activity of the nuclear factor(s) involved in this mechanism
could thus result in excessive nuclear export of p53 (Figure 4A, III).
220 R. K. Brachmann
Figure 4. p53 dissociator screens to elucidate specific modes of wild-type p53 inactivation in
human cancers. (A) A significant percentage of human cancers carry wild-type p53 that is
mislocalized to the cytoplasm, thus rendering it inactive. Several mechanisms may underlie
this finding. I. p53 may be imported into the nucleus inefficiently. II. p53 may be actively
held in the cytoplasm. III. p53 may be excessively exported from the nucleus because of
inappropriate exposure of a normally hidden nuclear export signal (NES) in the region of the
tetramerization domain. (B) Testicular cancers carry wild-type p53 that is stabilized and
transcriptionally inactive, suggesting the presence of a p53-inhibitory protein in the nuclei of
these cancers. Targeted p53 dissociator screens with cDNA expression libraries representative
of these p53 inactivations should be able to decipher the underlying molecular mechanisms.
Because of its central role in tumor suppression, p53 and its pathways
have been recognized as a prime target for developing new cancer therapies
[13, 24, 27, 35, 40, 46, 58, 64], and various strategies have been pursued.
Wild-type p53 may be delivered through gene therapy approaches regardless
of the p53 status of the cancer [3, 19, 71, 83, 92, 105]. Cancer cells lacking
p53 function can be specifically targeted with mutant adenovirus [6, 63] or
adeno-associated virus [80]. Inactive wild-type p53 and some p53 cancer
mutants may be reactivated through targeting of the very carboxy-terminal
putative autoregulatory domain of p53 with antibodies [1, 33, 41, 72] or
peptides spanning part of this region [2, 42, 86]. If MDM2 overexpression is
responsible for p53 inactivation, the inappropriate interaction between
MDM2 and p53 may be disrupted by peptides and compounds [21, 40, 87].
The pursuit of this strategy has resulted in very potent small-molecule
inhibitors of MDM2 [54, 94]. Yet, all these strategies have their limitations,
such as lack of efficient local and/or systemic delivery, undesirable res-
ponses of the host immune system to the therapeutic agent, lack of
specificity for the cancer cell and/or targeting of a small subset of human
cancers.
Besides these strategies, one approach appears particularly appealing:
small chemical compounds that endow p53 cancer mutants with function by
restoring the integrity of the p53 core domain. Such compounds are highly
desirable, because of the high frequency of p53 mutations and the large
number of patients who could potentially benefit. It has been estimated that
every year approximately 360,000 patients in the USA and 2.6 million
patients worldwide are diagnosed with cancers that contain p53 mutations
[34, 35]. This subset of human cancers (including lung, prostate, colorectal,
breast, head and neck, pancreatic, and gastric cancers) is often resistant to
conventional therapies and difficult to treat at advanced stages [5, 18, 24, 31,
35, 38, 58].
222 R. K. Brachmann
The strategy is, at least in theory, possible because of the unique pattern
of p53 missense mutations in human cancers. Typically, p53 cancer mutants
are full-length proteins with an intact transactivation domain and carboxy-
terminal tetramerization domain. The problem is the altered core domain
(amino acids 96–292) whose structural integrity needs to be restored in order
to have functional p53. Compounds able to achieve this goal are predicted to
be specific for the cancer cells, since the structurally intact wild-type p53
core domain of surrounding normal cells should not be affected. Further-
more, p53 cancer mutants are typically present at very high levels (thus
providing a large drug target), since their lack of transcriptional activity
abrogates negative feedback loops, such as with MDM2 [55]. The approach
also has the advantage of combining systemic delivery with lack of a host
immune response.
The attractive features of this strategy are easily rivaled by the enormous
challenges it poses: such compounds may simply not exist, or, if they do,
they may rescue only a very small fraction of the close to 1,000 different
known p53 cancer mutants that have one amino acid change in the core
domain. On a more optimistic note, looking at missense mutations of the p53
core domain (71% of the database entries; R9, http://www.iarc.fr/p53/), the
50 most frequent amino acid changes account for 55% of the total number,
and of these 50, the 8 most common amino acid changes comprise the
majority of them (30% of the total). Thus, chemical compounds that rescue a
subset of these p53 mutants are likely to correspond to a large number of
cancer patients.
Also, as a proof of principle, two classes of compounds have already
been identified in large drug screens that appear to have the desired
characteristics. The first screen utilized an antibody-based screening strategy
that relied on partially unfolded wild-type p53 due to elevated temperatures.
The isolated compounds appeared to partially restore function to a few p53
cancer mutants [25]. However, further characterization of the lead
compound CP-31398 has thus far not established the exact mechanism of
rescue. In addition, CP-31398 appears to have strong p53-independent
activities [13, 16, 82, 93].
The second class of compound was isolated using a mammalian growth
suppression assay [16]. Very impressively, this compound rescued the most
common p53 cancer mutant R175H whose core domain is very likely
entirely denatured [13–16, 106]. It still remains to be determined how many
others of the most common p53 cancer mutants will be rescued by this
compound and what the exact mechanism of rescue is.
Chapter 9: p53 Dissociator Assay 223
The p53 yeast dissociator assay has allowed a broader look at the
challenge of rescuing p53 cancer mutants; it has been used for identifying
intragenic suppressor mutations that overcome the deleterious effects of
cancer mutations. Such suppressor mutations are highly informative, as they
identify key regions within the p53 core domain that, upon alteration,
provide increased stability to the core domain. Such suppressor mutations
have also been pursued by computer modeling, but this strategy generated
only one successful suppressor mutation and several false predictions [104].
Additionally, “suppressor mutation design” was not as wide in scope as
genetic screens in yeast.
Figure 5. Search for intragenic suppressor mutations that endow function to common p53
cancer mutants. (A) Starting with a common p53 cancer mutant (with mutation “C”) that is
transcriptionally inactive (Ura– phenotype), (B) the up- or downstream regions of the cDNA
are mutagenized using PCR mutagenesis. (C) In a separate reaction, the cDNA expression
plasmid is gapped in the same region. (D) Both products are transformed into yeast, and the
overlap between PCR product and gapped plasmid allows the yeast to repair the plasmid by
homologous recombination. After confirmatory experiments, the plasmid is eventually
sequenced to identify the suppressor mutation(s) (“S”).
The initial findings with the p53 dissociator assay were very promising,
yet several technical limitations needed to be overcome to allow for a
systematic analysis of the most common p53 cancer mutants. To this end, an
engineered open reading frame for p53 was constructed [39] that contained
numerous silent restriction sites [4].
Analysis for the eight most common p53 cancer mutants (30% of core
domain missense mutants) identified suppressor mutations for four of them
(G245S, R249S, R273C, and R273H; 12% of core domain missense
mutants). Several combinations of suppressor mutations were identified,
and, intriguingly, N239Y or S240R were involved in the rescue of all four
p53 mutants [4]. This suggested that amino acids 239 and 240 may be part of
a global suppressor motif, a hypothesis that was further pursued by
oligonucleotide-mediated mutagenesis of a small region of the core domain,
codons 225–241. The mutagenesis included directed changes of codons 239
and 240 to all other 19 possible amino acids plus a background mutagenesis
resulting in approximately 1 in 100 nucleotide misincorporations. This stra-
tegy resulted in the rescue of 16 out of 30 of the most common p53 mutants
tested, representing 18% of all reported p53 core domain missense mutants.
As expected, the suppressor mutations included changes of codons 239
and/or 240, but, in addition, changes of amino acid 235 to lysine were also
frequently found. The resulting p53 mutants were not only transcriptionally
active in yeast, but had also activity in mammalian reporter gene and
apoptosis assays [4].
These findings strongly argue that manipulation of a small region of the
p53 core domain is sufficient to endow p53 cancer mutants with function.
This region therefore comprises a global suppressor motif. Structural studies
Chapter 9: p53 Dissociator Assay 225
of this motif will determine the exact rescue mechanism and provide the
basis to exploit this motif further by designing small compounds able to
recreate the suppressor mechanism (Figure 6). By restoring function to p53
cancer mutants, these small molecules would, in effect, reactivate a major
apoptotic pathway and “relicense” the cancer cell to kill itself.
Figure 6. Strategy for the development of a new anticancer therapy that aims to endow p53
cancer mutants with wild-type function. (A) A pilot study established that the concept of and
search for intragenic suppressor mutations was a viable strategy to achieve a better
understanding of how mutant p53 core domains can be stabilized. (B) The subsequent
comprehensive screen using a new engineered p53 open reading frame identified amino acids
235, 239, and 240 a part of an important global suppressor motif that rescued 16 of the 30
most common p53 cancer mutants. (C) Crystal structures for p53 cancer mutants and p53
suppressor/cancer mutants will establish the underlying mechanism that allows the 235–239–
240 suppressor motif to rescue p53 cancer mutants. (D) This information will lay the
foundation to pursue the most challenging aspect of the project: design, identify, and
characterize small compounds able to recreate the rescue mechanism of the suppressor motif.
This step requires a multidisciplinary approach that combines the expertise of computer
scientists, chemists, structural biologists, and molecular biologists.
4 CONCLUSIONS
yeast, as it exploits the concept of selection against p53 activity, a twist that
allows for powerful screens for proteins important to p53 biology.
As the biology of p53 is bound to become more and more complex, p53
yeast assays will continue to contribute to our understanding of this crucial
tumor suppressor protein. The focus of this review was p53, but many of the
discussed concepts can be readily applied to the studies of other fundamental
cellular mechanisms. Undoubtedly, many human proteins, particularly
transcription factors, are just waiting to be plugged into a yeast assay of their
own.
ACKNOWLEDGMENTS
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Chapter 10
FUNCTIONAL ANALYSIS OF THE HUMAN p53
TUMOR SUPPRESSOR AND ITS MUTANTS
USING YEAST
The tumor suppressor gene p53 plays a major role in cellular response to
various environmental stresses [107, 114]. The p53 protein is a sequence-
specific transcription factor that can lead to transactivation of over 50 genes,
many of which are involved in apoptotic or cell cycle arrest responses
(Figure 1). Its role in biological responses may also relate to the ability to
interact with a vast number of proteins [164]. Functional alterations in the
p53 pathway likely occur in nearly all human cancers. In almost half the
human malignancies, there is a mutation in the p53 gene itself [71, 79].
Interestingly, ~80% of p53 mutations are missense changes that lead to
single amino acid substitutions, a feature that distinguishes p53 from other
tumor suppressor genes (e.g., APC, NF1, BRCA1) [22, 67, 155]. The
incidence of p53 mutations and the types of mutations can vary among
tumors in different tissues or populations [71]. Yeast provides unique
advantages for finding and characterizing human p53 mutations.
Figure 1. The tumor suppressor p53 is a sequence-specific transcription factor. Many types of
stress induce signal transduction pathways that lead to p53 stabilization and activation, mainly
by posttranslational modifications. The p53 protein is shown as a tetrameric (dimer of dimers)
sequence-specific transcription factor that recognizes promoter response elements (RE). The
solid arrows indicate the organization of the RE sequence as a closely spaced pair of
Chapter 10: Analysis of p53 and its Mutants Using Yeast 235
dimer-binding sites each consisting of two monomer-binding sites in inverted orientation (see
text for details). A list of the principal p53 target genes and their functions is shown.
In this chapter we first describe the role of p53 in mammalian cells and
the approaches that are commonly used to identify and evaluate the
consequences of p53 mutations. This is followed by a broad discussion of
the many yeast-based assays that have been developed for investigating p53.
The human p53 protein consists of 393 amino acids. Starting from
the amino terminus (Nter), the following functional domains have been
identified: 2 transactivation domains, a proline-rich sequence likely involved
in protein–protein interactions, a DNA-binding domain, a tetramerization
domain and a basic carboxy-terminal (Cter) region with regulatory functions
[189] (see Figure 2). The protein contains three nuclear localization signals
toward the Cter and one or possibly two nuclear export signals [107, 219].
A database of p53 mutations reported in tumor samples and cell lines has
been developed and is updated yearly at the International Agency for Cancer
Research (IARC), Lyon, France [75, 79]. Most (~80%) of the p53 mutations
associated with cancer occur as missense changes in the sequence-specific
DNA-binding domain that comprises about half of the entire protein
sequence (amino acid 100–300). This suggests that most tumor-associated
changes are likely to affect the ability to transcribe p53 responsive genes.
The unusually high frequency of missense changes in the p53 mutation
spectrum has provided opportunities in the field of molecular epidemiology
to investigate the role of carcinogens as etiologic agents of cancer [83]. The
similarity between p53 mutational fingerprints in tumors among exposed
populations and mutational fingerprints obtained following in vitro treatment
with an agent has been used to indicate its role in the etiology of some
cancers [15, 78] (discussed in section 2).
Germline p53 mutations lead to the dominant tumor-susceptibility Li–
Fraumeni syndrome (LFS) characterized by a high incidence of various
cancers with clear tissue-specific effects that in some cases relate to the
nature of the p53 mutations in terms of impact on protein function [74]. The
germline p53 mutation spectrum is similar to the somatic tumor spectrum.
An LFS-like phenotype has also been identified in families that do not
harbor germline p53 defects. Mutations in the checkpoint kinase Chk2 gene,
an upstream regulator of p53 activity, have been observed in these families
suggesting that the p53 pathway can be altered in multiple steps [112].
Alterations in the upstream regulators or downstream effectors of p53
may lead to a hypomorphic functional state of the protein that could impact
Chapter 10: Analysis of p53 and its Mutants Using Yeast 237
on the selective pressure for additional p53 gene mutations during cancer
development. An issue that has received relatively little attention is that
partial inactivation or changes in the spectrum of p53 functions may be
sufficient for, or even favor, tumor progression depending on the cell type
and physiological state (see sections 1.4, 2.1.3, and 2.5). This is relevant to
the prognostic use of tumor p53 status to estimate tumor aggressiveness and
therapeutic responses. Although reports differ possibly also because of lack
of standardization, p53 status can be a prognostic factor based on
multivariate analyses, with loss-of-function p53 mutations correlating with
poor prognosis [34, 51, 120, 124, 153, 181, 188].
The analysis of p53 mutation spectra from tumors suggests that much of
the p53 gene is a target for missense mutations that can inactivate its
function. In fact, more than 1,300 different amino acid changes have been
identified in the protein domain that provides for sequence-specific DNA
binding [126]. Although there are several strong hotspots, the majority of
tumor-associated p53 alleles do not occur at hot spot codons [79] (Figure 2).
Information on the specific functional defects associated with p53 mutations,
particularly at non-hot spot codons, is limited. These functional defects
could range from total loss of transactivation potential to partial loss that
affects only a subset of p53 target genes, to subtle differences in activity
compared with the wild-type (WT) protein, and also to enhanced and altered
DNA binding specificity leading to gain of functions (see sections 2.1.3, 2.5,
and 2.8).
The crystal structure of the human p53 DNA-binding domain and the
comparison of p53 sequences among many species show that the most
frequent mutations affect amino acids directly involved in establishing DNA
contacts and that hot spots tend to occur at evolutionary conserved residues
[36, 209]. The p53 DNA-binding domain has an immunoglobulin-like fold.
Three loops are involved in DNA contacts, and hydrophobic interactions of
two beta-sheets plus the coordination of a zinc atom provide for correct
positioning of the loops. This conformation may explain why many single
amino acid changes would affect the activity of the protein, even if distant
from the DNA contact sites. However, structural information for the
complete protein is not available, which limits structure/function analysis.
Several tumor-associated mutations affect amino acids that belong to the
DNA-binding domain but do not face the DNA [72]. Since the active form
of p53 is a tetramer (a dimer of dimers) (Figure 1), these mutations might
affect protein–protein interactions that stabilize the tetramer and indirectly
lead to inefficient DNA contacts [130].
The observation that p53 is active as a tetramer has led to the hypothesis
that the selection for missense changes in the DNA-binding domain can be
explained by the capacity of the mutant proteins to form hetero-tetramers
238 A. Inga, F. Storici and M. A. Resnick
of p53 activity also involves one major, and possibly more, negative
feedback loop(s). The MDM2 gene is directly activated by p53 [163]. The
importance of this negative regulation is exemplified by the oncogenic
potential of Mdm2 gene amplification and by the observation that Mdm2
knockout (KO) mice are embryonic lethal and exhibit rapid and massive
apoptosis, while the double knockout with p53 is viable [135].
from the consensus sequence. Also, there are variations in the number or
repeats, position relative to the ATG start site, and the arrangement of REs.
In vitro DNA-binding experiments with naked DNA indicate that the
ability of p53 to act as a transcription factor depends on posttranslational
modifications that activate the protein, enabling it to bind DNA [82]. In
particular, Cter modifications have been proposed to relieve its inhibitory
effect by triggering a conformational switch. Models whereby covalent
modifications activate p53 DNA-binding and transactivation function are
consistent with the observation that p53 is induced by stress responses.
Moreover, different posttranslational modifications might have differential
effects on p53 activity, thus providing a direct link between a particular
activating signal and a specific response [48].
Conclusions from gel shift assays, however, should be taken with caution
since it is questionable how reliably this in vitro assay reproduces the
nuclear environment. Recently the role of chromosome structure in p53
transactivation potential has been examined in vitro using chromatinized
DNA preparations and in vivo using ChIP assays [12, 52, 101, 196]. Inter-
estingly, acetylation of p53 does not appear to affect its DNA-binding affinity,
yet the histone acetylase activity of p300/CBP is important in p53-mediated
transactivation [12]. It is possible that by virtue of its specific DNA-binding
capacity and protein–protein interaction with p300/CBP, p53 can stimulate
chromatin modifications at promoter sites that stimulate transcription. The
acetylation of p53 might be a by-product of these molecular interactions
[165]. Thus, p53 intrinsic DNA-binding affinity might be an important
component in the regulation of p53-dependent transcriptional modulation.
Since p53 is active as a tetramer, its intranuclear concentration may
strongly affect binding kinetics and transactivation. The importance of p53
levels is often overlooked (discussed below), especially for mutants with
altered function, since assays in mammalian cells usually involve high
expression. In fact, both the chromatin structure of the reporter plasmid and
the high levels of p53 protein expressed from constitutive viral promoters
result in assessments of transactivation activity under conditions that are not
physiological.
There are now many examples where the yeast Saccharomyces cerevisiae
has proven an invaluable in vivo test tube for examining diverse human gene
functions and disease including, for example, cell signaling, DNA
metabolism, and mitochondrial function [102, 169].
No p53 homologue has been reported in yeast. In fact, the evolution of
the p53 gene seems to coincide with the development of multicellular
organisms, possibly because of the need for tissue remodeling via
programmed cell death and also because the elimination of individual cells
in an organism in response to DNA damage can be beneficial. However,
damage recognition and cell cycle responses induced by signaling pathways
244 A. Inga, F. Storici and M. A. Resnick
in yeast are very sensitive and elaborate and they involve protein kinases and
complexes that are well conserved during evolution [208]. The high
evolutionary conservation between yeast and mammals is also evident in
basic cellular processes such as DNA replication and repair, chromosome
segregation, transcription, and translation [14]. Because of these features and
its genetic tractability, we have referred to yeast as honorary mammal [169].
As described in the following sections, yeast-based assays have provided
the means for structure/function characterization of p53, functional
classification of p53 mutant alleles, identification and evaluation of p53
response elements, and screening of chemicals capable of reactivating p53
mutants. These assays currently provide the only practical means to rapidly
identify p53 mutations from individual patients and to assess functional
status. In a broader sense, the development of a p53 mutant functionality
database along with linkage to the vast IARC human p53 tumor mutation
database will be valuable in understanding the correlation between p53
functional status, tumor aggressiveness, and responsiveness to therapy.
2.1.1 p53 functional assays for screening cancer cell lines and tumor
samples
also been used. Its expression is repressed on glucose media but is induced
to very high levels on media containing galactose as carbon source.
Figure 3A. General features of the p53 functional assay in yeast. Expression of p53 in yeast
and transactivation by p53. The complete cDNA of a given p53 allele can be expressed under
either a constitutive or an inducible promoter from a selectable plasmid. The centromeric
plasmids are stably transmitted at low copy number. WT p53 can act as a transcription factor
in yeast stimulating the activity of a promoter whose upstream activating sequences have been
replaced with a p53 response element. Mutant p53 would change the level of transcription of
the reporter gene thus leading to phenotypic changes. If the p53 is not active, no
transactivation is observed. If the p53 allele is less active, higher levels of GAL1 induction are
needed; if more active, less induction is required for a phenotypic change.
Reporter gene:
- -
Assay: growth on his growth on ura growth on plates with low adenine
or 5-FOA
Response:
Wild type p53: growth growth on ura- and 5-FOA sensitive growth, white color
-
p53 mutant: no growth no growth on ura and growth on 5-FOA slow growth, red color
Figure 3B. Summary of reporter genes and the assays used for phenotypic analysis of p53
mutant alleles under conditions of high expression.
Figure 3C. Example of phenotypic assays using the ADE2 red/white reporter gene. Under
conditions of high constitutive expression of WT p53, transactivation of ADE2 results in
white colonies. Loss-of-function p53 mutants will result in red colonies since they cannot turn
on ADE2. When the GAL1-based inducible expression system is used, transactivation is
proportional to the levels of WT (or mutant) p53 protein expressed. Presented are yeast
transformants with WT p53 grown on plates containing different amounts of galactose
inducer (plus raffinose carbon source). Also shown are western blots of protein extracts from
cells grown under the same conditions. The appearance of colonies extends from red to pink
to white as the amount of p53 is increased.
Tumors containing p53 mutations may have cells that are heterozygous
or homozygous for WT p53 or have more than one p53 mutation. As
indicated above, the yeast-based approaches provide for the isolation and
functional analysis of separated alleles (FASAY) [28, 41, 185]. The
procedure utilizes the exquisite recombination abilities of yeast as described
in Figure 3D. First, total cDNAs are prepared by reverse transcription of
total RNA extracted from fresh or frozen tumor biopsies. The p53 cDNA is
then amplified using a pair of specific primers, typically for amino acids 43
through 374 [54]. This region corresponds to part of exon 4 through the
beginning of exon 11 and includes the entire sequence-specific DNA-binding
domain where most tumor-associated mutations are found (see Figure 2).
248 A. Inga, F. Storici and M. A. Resnick
Unpurified PCR products are directly transformed into yeast together with
an expression plasmid that contains a selectable marker, an origin of
replication, a centromere for stable propagation at low copy number, and
consecutive sequences corresponding to the Nter and Cter regions of p53,
coding for amino acids 1–64 and 351–393, respectively. The vector is
linearized between these regions. Thus, the PCR product and plasmid have
63 and 69 nucleotides of common sequence at each end.
Figure 3D. Diagram of the gap repair assay for isolation of individual p53 alleles directly
from tumor samples. Total RNA is extracted from fresh or frozen tumor biopsies and total
cDNA is prepared by RT-PCR. The p53 cDNA is PCR amplified using specific primers.
Unpurified PCR products are transformed into yeast together with a linearized p53 expression
vector whose ends are identical to the ends of the PCR fragment. The highly efficient
homologous recombination system of yeast generates a complete p53 expression vector. The
functionalily of the p53 allele is then assessed using a transactivation assay.
from the red colonies or using a colony PCR approach followed by DNA
sequencing.
There are two limitations with the yeast functional assay. It requires
extraction from fresh or frozen samples and errors may result during
transcription and amplification. These limitations hold true for many
analyses of clinical samples by PCR. Typically there are 2–5% red colonies,
possibly due to RT-PCR-generated errors. Amplification fidelity can be
assessed by parallel RT-PCR of each sample [34]. The critical requirement
for RNA can be avoided by modifying the functional assay into an exon-by-
exon DNA analysis that uses formalin-fixed, paraffin-embedded archival
samples [41].
When compared to other systems for p53 mutant identification, the yeast
functional assay has greater sensitivity than electrophoresis-based methods
(SSCP, DGGE) [113, 132, 197, 198] and is comparable to the GeneChip
approach [213]. In addition, the yeast assay is rapid and relatively
inexpensive and, furthermore, it enables functional defects in p53 to be
assessed. Functional analysis based on transactivation capacity in yeast is
robust and appears more sensitive than transient transfection gene reporter
assays in mammalian cells. Over 100 different mutant p53 alleles isolated
from clinical samples were found to lack transactivation function in the
red/white ADE2-based assays [31, 55, 172]. The tissues have included fresh
and frozen specimens from brain (glioblastoma and astrocytoma), upper
aero-digestive tract, breast, colon, bladder, prostate, lymphocytes, and skin
[29, 30, 34, 46, 65, 93, 103, 123, 125, 134, 145, 151, 159, 160, 168, 172,
186, 204, 211, 215]. Included in these analyses are dysplasias, primary
carcinomas, and advanced tumors.
The physical and functional separation of p53 alleles also provides an
assay to monitor intra-tumor WT p53 delivery by viral vectors for cancer
gene therapy approaches to treat brain, head and neck, ovarian, and lung
cancer [38, 106, 195, 199, 214]. Specific primers can be used to amplify the
mRNA obtained from endogenous p53 alleles or the virally encoded WT
cDNA to estimate the contribution of viral WT p53 mRNA in tumor
samples.
activity of p53 mutants with different p53 REs. Most screenings of tumor
samples have utilized high p53 expression and the RGC RE to determine if
p53 is capable of transactivation. The RGC sequence was originally found
within the human ribosome gene cluster and was shown to be a bona fide
p53 RE based on both in vitro binding and transactivation
WAF1
assays [11]. The
REs of the key p53-regulated genes such as p21 and bax have also been
used [55]. Highly expressed WT p53 is capable of transactivation from these
REs and results in white colonies in the ADE2 based assay. Flaman et al.
[54] examined 51 different tumor-associated mutations observed in human
tumors. Nine of them scored as WT with P21, while none were able to
transactive the BAX or the RGC RE. Another 77 mutants were examined by
Campomenosi et al. [30] using REs of RGC, BAX, and P21. Consistent with
the previous report, about 15% of the mutants were WT toward the P21,
but none of them was able to transactivate BAX. This finding suggests
that inactivation of the apoptotic function of p53 is critical for tumor
development.
DiComo and Prives [42] further expanded the investigation of p53
transactivation ability using nine different REs upstream of the HIS3 reporter.
Nineteen tumor-derived mutants were analyzed at different temperatures.
While p53 hot spot mutants appeared transcriptionally inactive, four mutants
in the DNA-binding domain scored as WT with P21 and a strong consensus
RE at 37°C, and one of them [R283H) appeared fully active with several
REs. An additional group of four mutants was active with the same REs at
lower temperatures. These mutations affected amino acids belonging to the
beta-sheet scaffold of the p53 DNA-binding domain. All the mutations were
inactive with RGC. Consistent with this, WT p53 was less active with the
RGC and BAX REs compared to P21 and the strong consensus sequence.
A sensitive screen was recently developed to specifically isolate p53
mutants retaining partial function (in preparation). The assay was based on
the observation that in the ADE2 reporter system white and red colonies
represent the extreme phenotypes in a range of p53 transactivation
capabilities from WT to complete loss-of-function. With the view that it
might be possible to identify mutants with reduced function, p53 mutations
were generated by random mutagenesis and directly cloned into yeast using
gap repair (see Figure 3). Examination of colony color and morphology
allowed the identification of intermediate phenotypic classes (pink, sectored,
and speckled colonies) that were due to the expression of p53 mutants with
reduced transactivation activity. Approximately 60 different single amino
acid changes were found that could result in proteins with reduced activity.
The changes in activity were quantified with a luciferase reporter assay that
was developed in yeast. A group of mutants that were tested for
transactivation and growth suppression in human cells validated the yeast
Chapter 10: Analysis of p53 and its Mutants Using Yeast 251
The restoration of cellular pathways in tumor cells that lead to cell death
by apoptosis is an appealing cancer therapy approach. In this respect p53
represents a potentially powerful target. The identification of a large group
of p53 mutants that appear to retain some transactivation function and the
observation that the majority of loss-of-function alleles are recessive when
heterozygous with WT p53 [136] suggest that many tumor-associated
mutants retain native conformation or do not dramatically perturb protein
conformation.
Different types of yeast assays have been used to address the possibility
that small molecules could partially reactivate p53 function either by
stabilizing the structure of the mutants and/or by enhancing DNA-binding
capability. Brachmann et al. [25] used an elegant screen to identify p53
mutations that act as intragenic suppressors of common hot spot p53 tumor
mutations. The assay utilizes random PCR mutagenesis or degenerate
oligonucleotides [13] to induce mutations in defined regions of the p53
cDNA. Gap repair was used to clone the mutagenized fragments within a
p53 cDNA that contains a specific mutation corresponding to a p53 hot spot.
Compensating mutations were identified that restore p53 function based on
transcription of the p53 URA3 reporter. Single amino acid changes were
identified that were capable either of restoring function to specific p53
Chapter 10: Analysis of p53 and its Mutants Using Yeast 253
leads for p53 reactivating drugs. However, the results were based on the
color assay (red/pink/white) that might also be influenced by the effects of
the drug on the growth of yeast. A quantitative assessment of the effect of
amifostine both on WT and mutant p53 activity is needed.
The Nter and Cter domains play important roles in p53 structural
organization, interaction with other proteins and transactivation. The p53
functional assays can also be used to address changes in these regions.
Ishioka et al. [94] reported that the p53 Cter is generally refractory to
mutations that inactivate transactivation functions and p53 deleted for the
last 40 amino acids (Q354Stop) retains WT activity. However, single amino
acid changes in the tetramerization domain (L344P, K351E) can nearly
inactivate p53 transactivation. A germline p53 mutation in the tetrameri-
zation domain (R337C) that is found in cancer prone families [119] results in
yeast colonies that are pink in the ADE2 assay, suggesting partial retention
of transactivation activity. It is possible that this amino acid change affects
the stability of the p53 tetramer; however, the mutant protein is unaffected
by temperature changes (23°–34°C), unlike many pink colony mutants that
we have examined (unpublished). A second germline mutation has been
identified at codon 337 (R337H) in Brazilian families that were charac-
terized by increased incidence of childhood adrenal carcinoma (ACC) [170].
Surprisingly, there was no effect on the incidence of other types of cancer. A
recent study showed that R337H retained almost normal function in in vitro
DNA-binding assays, but exhibited temperature and pH-sensitivity [43]. The
reason why this specific functional defect is highly tissue-specific remains to
be determined, although it has been proposed that the adrenocortical cellular
environment would lead to functional inactivation of this p53 mutant thus
increasing the risk of cancer development. It will be interesting to compare
the R337C and R337H phenotypes in the yeast functional assay.
Candau et al. [32] used a yeast-based assay to analyze Nter functions of
p53. Instead of expressing the complete p53 cDNA, chimeras were created
between p53 and the DNA-binding domain of the GAL4 transcription factor.
The use of p53 missense and deletion mutants revealed that the Nter region
of p53 contains a second transactivation domain mapping between amino
acids 40–83 with amino acids W53 and F54 playing a critical role. The
activity of this transactivation domain has been confirmed in mammalian
cell experiments [221]. It has also been proposed that this element might
display target gene specificity, thereby contributing to p53-induced
differential transactivation. However, the underlying mechanisms for this
effect remains to be established.
Coincident changes at amino acids 22 and 23 (L22E and W23S) in the
first p53 trnsactivation domain [42] can inhibit binding to MDM2,
p300/CBP, and TAF proteins [53, 118, 152, 163]. Consistent with this, the
256 A. Inga, F. Storici and M. A. Resnick
evidence that functional alteration of the p53 pathway may occur through
upstream regulation of p53 protein activity.
Differential transactivation is important in the biological responses to
stress, particularly apoptosis vs cell cycle arrest. The finding that ASPP2 acts
as cofactor for p53 but possibly only for the activation of specific target
genes provides support to a proposed mechanism explaining how differential
transactivation by p53 and the specificity of the p53-induced cellular
responses are achieved [110, 208]. Namely, cell- type-dependent availability of
cofactors may decide which pathway, cell cycle arrest or apoptosis, is
induced by p53 in response to a stress signal. Consistent with this, a recent
2-hybrid screen identified the homeodomain-interacting protein kinase-2
(HIPK2) as a p53 cofactor. HIPK2 phosphorylates p53 at serine 46,
stimulates p53 transactivation and mediates the induction of apoptosis [40].
In addition to p53-binding factors and modifying proteins, the intrinsic
DNA-binding affinity of p53 toward the many related REs in target genes
can play an important role in differential transactivation. As discussed below
(see section 2.6), yeast-based functional assays have provided tools to assess
this possibility.
We have developed a set of isogenic yeast strains that differ only for a
single p53 response element cloned upstream of a minimal CYC1promoter-
ADE2 open reading frame at the ADE2 locus [91]. Thus, all the possible
factors affecting p53 transactivation are kept equal and the role of binding
affinity and p53 protein amount can be precisely evaluated. To construct
these strains we took advantage of a recently developed in vivo mutagenesis
approach (discussed below) that allows for rapid modifications of yeast
sequences using oligonucleotides that are transformed directly into yeast
[192]. Thirty strains, each with a different p53 RE derived from mammalian
p53-responsive genes, were constructed [91]. Genes involved in cell cycle
control, apoptosis, DNA repair/replication, and p53 regulation were included
in this broad panel of REs to examine various features of WT and mutant
p53s.
To understand the importance of p53 levels in differential transactivation,
we defined conditions for the rheostatable induction of p53 under the
GAL1 promoter. Nearly linear increase of p53 over a 100-fold range could
be accomplished using a range of galactose concentrations from 0.001% to
0.12% added to medium containing 2% raffinose [91]. Transactivation of the
ADE2 reporter gene could be assessed using the phenotypic (red/pink/white)
assay and also by quantification of ADE2 mRNA with real-time PCR
measurements. The p53 REs were ranked according to their relative activity.
Surprisingly, there was a 1000-fold range in transcriptional activation bet-
ween the various RE sequences. Statistical models that estimate binding
energy based on nucleotide usage and the degree of heterology of a RE from
the consensus [16] predicted only small variations and these did not correlate
with the functional results. We found that the DNA sequence CATG at the
junction of p53 monomer binding sites greatly affects p53 transactivation
capacity, possibly due to structural properties such as bending [143]. The
absence of spacing between dimmer-binding sites is also crucial for high
transactivation capacity by WT p53. This result contrasts with in vitro DNA-
binding assays [50] but confirms previous observation with yeast-based
transactivation [202].
Our results suggest that intrinsic DNA-binding affinity as well as p53
protein levels are important contributors to p53-induced differential trans-
activation. WT p53 had weak activity toward half the apoptotic REs,
particularly those active on the mitochondrial pathway of programmed cell
death. Activation of these targets requires high levels of p53. Possibly
specific coactivators that are missing in yeast or the combinatorial action of
multiple transcription factors play an important role in transactivation of
these REs.
Qian et al. [166] recently reported the construction of an even larger
panel of REs and surrounding promoter regions into a yeast expression
262 A. Inga, F. Storici and M. A. Resnick
Given the key role of p53 protein in the stress-induced cellular responses
it was surprising that lack of this gene does not result in severe defects
during embryonic development in mice. However, inappropriate expression
of p53 can lead to embryonic lethality or increased risk of malformations
[37, 44, 76]. Two p53 homologues, p63 and p73, have been identified [99,
115]. The p63 and p73 genes might compensate for the p53 defects in
germline and somatic cells. p73 was initially proposed to be a tumor
suppressor gene in neuroblastoma due to its chromosomal location at 1p36.2
[191]. This region is frequently associated with loss of heterozygosity (LOH)
in other types of cancer.
Although there is limited evidence of an alternative spliced form of p53
[56], multiple spliced variants at the Cter and an alternative promoter at the
Nter have been described for p63 and p73. Six variants of p73 with differing
Cter structures (alpha, beta, gamma, delta, epsilon, and xi) and three of p63
have been reported [191]. The relative abundance of these protein variants
varies according to the cell type and in tumor cells. For example, the Νter
variants of both p63 and p73 are highly expressed (as much as 10 times more
than p53) in epithelial and brain tumors where they appear to have
oncogenic properties [39, 45]. The structural organization of the p63 and p73
proteins resembles p53 and comprises a transactivation domain (~20–30%
homology to p53), a DNA-binding domain (~60–70% homology), a
tetramerization domain (~30–40% homology), and a long carboxy terminus
of uncertain function [6]. KO mice have been obtained both for p63 and p73.
Unlike the p53 KO, growth defects were observed. The p73 KO showed
chronic inflammation, infections, and hippocampal dysgenesis [218]. It has
been proposed that the Nter variants act as neuroprotective factors possibly
by inhibiting p53-dependent apoptosis. Interestingly, no increase in tumor
incidence was reported in these mice. The p63 KO had a lack of mammary
glands and defects in limb formation [217].
Mutations in the DNA-binding mutations of the p63 gene have been
observed in families affected by the autosomal dominant disorder EEC
Chapter 10: Analysis of p53 and its Mutants Using Yeast 263
the p53 cDNA, enables easy production of all possible single missense
mutants between amino acids 50 and 350. The mutant construction is
achieved by simple replacement of the CORE cassette (selected on 5-FOA
and confirmed by kanamycin sensitivity) with oligonucleotides. The
oligonucleotide can be specific for a single desired mutation or degenerate in
order to randomly mutate the region of interest. Limited DNA sequencing of
the replaced fragment can confirm and/or identify the mutants [192]. The
host strain with a given p53 allele is then mated (Figure 4B) to a set of 20
isogenic strains representing a p53 functional array. Each of these strains
can test p53 transactivation using a reporter gene construct that only differs
for the sequence of a p53 RE. Cell cycle checkpoint, DNA repair/replication,
266 A. Inga, F. Storici and M. A. Resnick
Strategy for the determination of p53 allele functional fingerprints using the p53 Functional Array
x
Mate
or
MATα
MATa
p53 Functional Array:
Yeast “p53-host” strain contains an > 20 isogenic strains that differ only
integrated p53 allele or a p53 in the sequence of a p53 response
expression vector element (e.g., P21, BAX, MDM2,
PIG3, AIP1, NOXA, GADD45, etc.)
cloned upstream of the ADE2 reporter
Select diploids gene
Figure 4B. Transactivation assays using the functional array of p53 response elements. Over
20 isogenic yeast strains that differ only in the sequence of a p53 response element upstream
of the p53 reported gene ADE2 (the luciferase reporter gene can also be used to quantify
transactivation) are mated with the p53-host strain expressing a given p53 mutant. Diploids
are selected on suitable media and then tested with the phenotypic transactivation assay to
determine the functional fingerprint of the p53 allele being tested. The analysis of the
dominance potential of p53 mutants can be performed when the p53-host strain is transformed
with a second GAL1:WT p53 (or p63, or p73) expression cassette.
While many p53 mutations associated with cancer may retain partial
transactivation function and although several classification methods for p53
mutants have been attempted, it is not presently possible to predict a priori
the behavior of a mutant protein. The yeast functional assays provide an
important contribution to the functional classification of p53 alleles. It
appears that the combination of low and variable expression of p53 alleles
and the assessment of the transactivation capacity using many p53 response
elements provides a greater sensitivity for the functional classification of p53
mutants than the standard yeast assays using high-constitutive p53 expres-
sion. When combined with the efficient method for constructing p53 alleles
using in vivo mutagenesis and with the high-throughput selection and
screening system using diploids formed between “p53-host” and “the p53
functional array” strains (see Figure 4) these tools allow for rapid develop-
ment of p53 functional fingerprints. Hence, the yeast functional assay offers
a practical means to develop a p53 mutant functionality database that
enables the functional fingerprints for all the p53 mutants relevant to cancer
to be linked with the IARC p53 mutation database. This information will
become valuable in understanding the correlation between p53 functional
status and tumor aggressiveness and responsiveness to therapy, thus
providing directions for effective patient management in the clinical setting.
268 A. Inga, F. Storici and M. A. Resnick
ACKNOWLEDGMENTS
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Chapter 10: Analysis of p53 and its Mutants Using Yeast 283
ADDENDUM
RECENT DEVELOPMENTS
Figure 5. Relative transactivation capacity of p53 alleles with mutations in the DNA binding
domain [222]. Twenty-four human p53 mutations were examined in 15 isogenic yeast strains
each containing a different p53 RE that regulates expression of the ADE2 reporter gene. The
REs are ordered based on decreasing transactivation capacity with wild type p53 [91]. The
p53 mutants are ordered according to their position in the primary sequence. The trans-
activation capacity of each allele toward each RE was determined using variable expression
of p53 under the GAL1 promoter and compared to the activity of wild type p53. The relative
transactivation capacities of mutants with respect to wt is presented in a form similar to that
for expression arrays, with red greatly increased, green greatly decreased, blue loss-of-
function, and black equal to wild type. The quantification is based on the amounts of p53
protein required for transactivation with wt or with a mutant allele. This was derived from the
minimal amount of galactose required for transactivation to occur – see section 2.6.
286 A. Inga, F. Storici and M. A. Resnick
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Chapter 11
ABC TRANSPORTERS IN YEAST – DRUG
RESISTANCE AND STRESS RESPONSE
IN A NUTSHELL
Key words: Yeast, ABC protein, antifungal resistance, drug screening, detoxification,
stress response
1 SUMMARY
289
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 289–314.
© 2007 Springer.
290 K. Kuchler and C. Schüller
shall reiterate the advantages and pitfalls associated with using baker’s yeast
as a model system for the molecular analysis of mammalian ABC trans-
porters, in particular its suitability and amenability for drug screening or even
drug discovery related to the function of ABC transporters with medical
importance.
2 INTRODUCTION
Although intrinsic drug resistance has been known for quite some time
[15], MDR may develop under selective conditions such as those caused by
drug exposure or stepwise selection of cells for resistance to xenobiotics or
toxic small molecules [84]. MDR is often characterized by cross-resistance
to many unrelated compounds [14], and while this is a matter of intense
discussion, MDR of tumor cells or tissues may impair efficacy or even
prohibit anticancer therapy in vivo. At least in great many in vitro studies
using cultured cells, MDR phenotypes clearly cause cells to become
refractory to chemotherapy [14, 84].
Complex MDR phenotypes may result from several mechanisms operating
simultaneously or consecutively in a developmental process [84]. Mechanisms
of MDR include decreased drug uptake, plasma membrane permeability
changes, intracellular drug inactivation, transcriptional activation/deactivation,
gene amplification, drug target gene mutations, or even compartment-
specific sequestration of certain drugs (Figure 1). Notably, similar mecha-
nisms may counteract inhibitory effects of endogenous metabolites in case
they are toxic or detrimental for growth. Importantly, ATP-dependent drug
efflux from cells mediated by dedicated membrane transporters is considered
a major cause of MDR [14]. Such drug efflux can be accomplished by so-
called ABC transporters, which comprise one of the largest protein families
with more than 3,000 known representatives operating in all living cells
from bacteria to man [24]. Most ABC proteins are membrane-associated and
display transport activities for great many different cytotoxic drugs or
antibiotics. Substrate transport is powered by ATP hydrolysis, but ATP may
also have important roles in regulating ABC protein function or substrate
specificity. Although the domain organization of most if not all ABC
proteins is conserved throughout evolution, the molecular basis for their
broad drug substrate specificity has remained an unsolved mystery [24].
Chapter 11: ABC Transporters in Yeast 291
Figure 1. Principle mechanisms of pleiotropic drug resistance in living cells. Complex PDR
phenotypes can arise from several molecular mechanisms. PDR is typically characterized by
cross-resistance to many structurally and functionally unrelated drugs. Each mechanism may
operate on its own or in combination with another one at the same time or in a developmental
process. Yellow balls indicate environmental xenobiotics or drugs; green balls, endogenous
metabolites; N, nucleus; V, vacuole.
Table 1. Membrane-associated yeast ABC transporters and some relevant transport substrates
ABC protein Relevant substrates Length Topology Location References
Pdr5p/Sts1p Antifungal drugs, 1,511 (ABC-TMS6)2 PM [13, 9]
steroids, myco-toxins,
peptides
Snq2p Drugs, steroids, 1,501 (ABC-TMS6)2 PM [129]
mutagens, 4-NQO
Pdr15p Detergents, chlo- 1,529 (ABC-TMS6)2 PM [150]
ramphenicol, phenolic
herbicides
Pdr10p Membrane 1,511 (ABC-TMS6)2 PM [151]
detergents?
Yor1p Oligomycin, 1,477 TMS11-ABC-TMS6- PM [62]
reveromycin A, drugs ABC
Pdr12p C3-C7 weak acid 1,511 (ABC-TMS6)2 PM [111]
anions, benzoate,
sorbate
Ycf1p GSH-conjugates, 1,515 TMS11-ABC-TMS6- VAC [137]
cadmium, DNB-GS ABC
Bpt1p Cadmium, DNB-GS, 1,515 TMS11-ABC-TMS6- VAC [66, 132]
glucuronides ABC
Ybt1p/Bat1p Taurocholate, UCB, 1,515 TMS11-ABC-TMS6- VAC [105]
bilirubin ABC
Vmr1p Unknown 1,592 TMS11-ABC-TMS6- Unknown SGD
ABC
Nft1p Unknown 1,524 TMS11-ABC-TMS6- Unknown SGD [96]
ABC
Chapter 11: ABC Transporters in Yeast 293
ABC, ATP-binding cassette; TMS, transmembrane segment; PM, plasma membrane; VAC, Vacuole; GS,
glutathione S; UCB, unconjugated bilirubin; PM, plasma membrane; 4-NQO, 4-nitroguinoline-A-oxide;
DNB-GS, (2,4-dinitrobenzene)-glutathione; UCB, unconjugated bilirubin; SGD, Saccharomyces Genome
Database (http://www.yeastgenome.org/).
weak acid stress adaptation [111]. Finally, the PDR transporters Aus1p and
Pdr11p may be involved in sterol uptake under conditions of impaired
ergosterol biogenesis [150], but a distinct drug efflux activity has not been
established for them.
Transcription must play a key role in modulating PDR, since cells ought
to be able to discriminate between cellular defense against metabolites or if
there is a need to combat environmental challenges. Indeed, most yeast PDR
and MRP/CFTR genes (Table 1) form a complex genetic network known
Chapter 11: ABC Transporters in Yeast 297
The first yeast gene capable of conferring PDR was in fact PDR1 that
encoded a predicted Zn(II)2Cys6 transcription factor [8] rather than a
membrane efflux pump. Further work showed that additional Zn(II)2Cys6
298 K. Kuchler and C. Schüller
regulators (Table 2), including Pdr3p [29], Yrr1p [22, 157], Pdr8p [56],
Ucm22p and Ecm22p [145, 150], and War1p [71] play crucial roles in the
regulation of yeast ABC pumps during normal cellular growth but also in
response to adverse conditions. While several factors contribute to the
regulation of ABC transporters in the network (Figure 3), Pdr1p and
Pdr3p are still considered the master regulators of PDR [11, 68]. Pdr1p and
Pdr3p constitutively localize to the nucleus, and control PDR genes as
homo- and heterodimers [90] through so-called PDR elements (PDREs,
5′-YCCYCCGR-3′) in target promoters [30, 60]. A single cis-acting PDRE
is necessary and sufficient to attract control by Pdr1p/Pdr3p [60, 61].
Deletion of both genes causes pronounced drug hypersensitivity phenotypes,
since they regulate basal expression of several ABC transporters, including
PDR5 [60], PDR10, PDR15 [151], SNQ2 [89], YOR1 [52], as well as other
non-ABC genes implicated in the PDR phenomenon [30, 68]. In addition,
Yrr1p as well as Pdr8p are involved in the regulation of Snq2p, Pdr15p, and
Yor1p [22, 56, 157]. The presence of two PDREs in the promoters of PDR3
and YRR1 suggest autoregulatory loops in their control [21, 27, 157]. The
scenario is even more complex, since Yrm1p appears to compete with Yrr1p
for promoter occupancy on certain target genes, but a physiological
relevance for such a competition is unclear [86]. Strikingly, Pdr1p and Pdr3p
can exert opposing regulatory effects on target ABC genes that even encode
highly homologous transporters. For example, a lack of PDR1 reduces PDR5
expression, but increases PDR15 mRNA levels. In turn, only Pdr3p, but not
Pdr1p, is required for basal PDR15 expression [151].
Figure 3. ABC gene regulation within the yeast PDR network. The gene names in the
ellipses in the center represent ABC target genes of transcriptional regulators depicted about
and below. Transcription factors exerting regulatory effects are connected with target genes
by an arrow. An arrow can indicate positive or negative control. Note that the cartoon only
Chapter 11: ABC Transporters in Yeast 299
includes ABC genes for clarity and simplicity. Several non-ABC genes such as membrane
transporters of the major facilitator family are also regulated by some of the transcription
factors depicted in the cartoon. For details see text and references.
Indeed, this strategy identified the first Candida albicans ABC transporters,
Cdr1p [112, 123] and Cdr2p [122], both of which mediate clinical antifungal
resistance. The functional expression of C. albicans ABC transporters in
baker’s yeast also uncovered a hitherto unknown caspofungin transport capa-
city of Cdr2p [126]. Importantly, this defined system also established that
antifungal drug resistance can be efficiently reversed through the use of
ABC efflux pump inhibitors [125] or it can be used for the molecular analysis
of fungal drug pump function [103]. Similarly, MDR reversal has also been
attempted in cancer therapy in a clinical setting as a means to restore anti-
cancer drug response, but the success has been limited up to now [84].
To study functional conservation and to clone and identify novel drug
transporters, several mammalian ABC genes have been functionally expressed
in yeast. Cross-complementation studies yielded important information
about the function mammalian, parasite, plant, and other fungal ABC genes.
The first functional expression demonstrated that human Mdr1 P-gp is
functional when expressed in yeast, since it confers resistance to valinomycin
and actinomycin D [74, 142]. Interestingly, mouse mdr3, when expressed in
yeast cells lacking the Ste6p a-factor transporter [72], rescues the mating
defect [115], whereas human Mdr1 is unable to extrude the mating
pheromone [74]. Likewise, pfmdr1 from Plasmodium falciparum appears to
complement a loss of Ste6p, at least in certain strain backgrounds [146].
Mouse mdr3 in yeast also confers resistance to immunosuppressive drugs
such as FK520 [116] or the anticancer drug dactinomycin [55]. Yeast
expression of human Mdr2 demonstrated its physiological function as a
phospholipid translocase [120]. Further, expression of MRPs, homologues of
Ycf1p, revealed that human MRP1 as well as plant AtMRPs can
complement the heavy metal sensitivity of cells lacking Ycf1p [42, 85, 140].
Nevertheless, the signals for correct trafficking of MRPs appear to work
inefficiently in yeast, since MRPs and Mdr1 are also found in other cellular
membranes than their normal cellular location in mammalian cells [74, 140].
Yeast has also been used for the analysis and expression of human ABC
genes implicated in genetic diseases, including the CFTR [118] cystic
fibrosis transmembrane conductance regulator [64, 107, 139], and the TAP
transporters of antigen presentation, which are also functional as peptide
transporters in yeast [144]. Furthermore, the human ABC7 gene [4], which is
mutated in sideroblastic anemia with cerebellar ataxia (XLSA/A), was
produced in yeast and complemented the defect of the yeast homologue
ATM1 [12].
Baker’s yeast and other yeasts have also been exploited as single cell
“bags” for the high-level expression and purification of endogenous [38] and
Chapter 11: ABC Transporters in Yeast 303
heterologous ABC pumps [16, 59, 80]. For example, the PMA1 promoter of
the plasma membrane proton pump, significantly boosted MRP1 expression,
allowing for the production of milligram amounts of purified MRP1 [76, 77].
The related species P. pastoris is also suited for production of mammalian
ABC proteins or individual nucleotide binding domains. Purified proteins
can be reconstituted into in vitro systems, enabling studies on the catalytic
cycle of ABC transporters, structural analyses [16, 80] and perhaps even
crystallization trials [38].
Although expression of certain mammalian ABC proteins has been
accomplished in yeast, many attempts to express mammalian or human
transporters in yeast failed or gave inconsistent results. This may be because
of nonfunctional or different trafficking signals, improper protein folding,
aberrant cellular localization, lack of appropriate in vitro or in vivo assays,
unknown drug substrates or just inappropriate strain backgrounds.
Furthermore, the different lipid environment of yeast membranes may
change or even inhibit ABC function. In fact, this is the case for the retinal
ABC transporter, whose activity is even modulated by lipids in the native
membrane environment [1]. Moreover, other ABC pumps even mediate
membrane transport of lipids or sterols [75, 114, 120]. Changes in the
normal lipid composition, like those caused by defects in ergosterol
biosynthesis, dramatically modulate cell permeability [41, 113, 130] and
may therefore also affect transport activity, substrate specificity, or even the
proper folding of ABC pumps [135]. However, once expression is achieved
and verified by appropriate functional assays, yeast does represent a useful
host for the molecular genetic analysis of heterologous ABC transporters.
model for systems biology approaches that are currently receiving consi-
derable attention in the global science community [18].
After entering the post-genomic era, a series of elegant genome-wide
approaches further sparked the general interest in yeast as a tool for drug dis-
covery. For instance, genome-wide drug profiling [108], haplo-insufficiency
screening [47, 87], and global metabolomics [3] indicated that yeast can be
very useful for drug target identification. Haplo-insufficiency screening may
even provide clues about targets of orphane drugs and disclose possible
mechanisms of drug action [87]. The global description of transcription
factor promoter-binding sites undoubtedly indicates incredible complexity,
but at the same time suggest meaningful genetic and biochemical approaches
to better understand transcription in general [78]. We are just beginning to
understand the genetic interactions by exploring genome-wide synthetic
lethality relationships of otherwise nonessential genes, which might be bene-
ficial for future strategies to dissect complex genetic diseases in humans
[141]. The knowledge about global protein interaction maps [7, 44] and
perhaps their dynamic changes during cell growth is closing the circle.
Nevertheless, as elegant as genome-wide approaches seem, one must not
forget that they do not alleviate the need for mechanistic approaches to
deepen the understanding of complex biological phenomena such as PDR
development or ABC protein function both at the molecular level, and in a
whole-cell context.
To come to full circle, yeast is unlikely to loose its attraction as a
valuable test tube for the functional characterization of other eukaryotic or
mammalian ABC proteins. Once physiological substrates of ABC pumps
have been identified, one will be able to produce tailor-made yeast strains
with desired drug susceptibility, facilitating drug discovery through HTS
approaches [49, 102]. The disadvantage of a generally reduced drug
susceptibility of yeast cells compared to mammalian cells can be overcome
by using supersensitive strains lacking certain ABC pumps [49, 58]. Another
argument stems from the financial aspect, since whole cell-based assays in
yeast are more than order of magnitude cheaper than comparable assays in
mammalian cell culture. This way, yeast will perhaps contribute to a better
understanding of other medically relevant ABC proteins, since the closed
nutshell opens a door to a defined genetic system for analysis at the
molecular level. Therefore, yeast must not be neglected as a model system
for functional approaches on eukaryotic ABC transporters of medical
importance, even in cases where no cellular function is known.
Chapter 11: ABC Transporters in Yeast 305
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314 K. Kuchler and C. Schüller
1 INTRODUCTION
315
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 315–346.
© 2007 Springer.
316 S. L. Holbeck and J. Simon
3.1.1 MGT1
3.1.2 MLH1
3.1.3 RAD14
3.1.4 RAD18
3.1.5 RAD50
of Rad50, Mre11, and NBS1. Patients with defects in NBS1 have the cancer-
prone Nijmegen breakage syndrome and are hypersensitive to ionizing
radiation. The breast cancer-associated gene BRCA1 associates with human
Rad50 [57]. Rad50p also has a role in telomere maintenance [8, 12].
Structural data on Rad50p suggest that Zn++ coordinated hooks on 2
Rad50p molecules may function to keep the DNA strands involved in repair
of a DSB connected with one another [28].
3.1.6 RAD52
3.1.7 SGS1
mms4, top1, and pol32 [49]. Sgs1p interacts with the mismatch repair protein
Mlh1p – this interaction is also seen with the human BLM and MLH1
proteins [37]. Sgs1p can unwind G-quadraplexes, a structure that can be
formed by the G-rich single strand found at telomeres. This unwinding is
inhibited by compounds that bind to G-quadraplexes [26].
3.2.1 BUB3
3.2.2 CLN2
4 COMPOUNDS
Compounds for the screen came from the repository of the NCI–DTP.
This collection contains compounds submitted for screening as possible
anticancer or antiviral compounds by a large number of suppliers. The
suppliers include academic groups, government scientists, small bio-
technology companies, and large pharmaceutical companies from all over
the globe. The acquistion of compounds began in 1956 and continues to this
day, with over 500,000 compounds currently registered. Interested parties
can find details about submitting compounds at http://dtp.nci.nih.gov/. About
half of the compounds are covered by a confidentiality agreement, which
precludes the NCI from disseminating data derived from these compounds.
Data derived from “open” compounds not covered by a confidentiality
agreement is publicly available through the DTP web site, and includes
compound structures, data from the NCI 60 human tumor cell line screen,
the NCI AIDS screen, and the NCI yeast anticancer drug screen. In addition,
if sufficient inventory is available, the NCI may be able to supply qualified
researchers with small quantities of “open” compounds from the DTP
repository for nonhuman use.
The NCI does not routinely verify the structures of submitted
compounds. In addition, the stability of different chemotypes varies widely.
Thus, researchers are encouraged to verify the structure of any compounds
before investing significant resources in lead development. Indeed, one of
the compounds identified as a specific inhibitor of the DSB repair mutants
rad50 and rad52 [13] had a structure different than that indicated by the
supplier.
Compounds submitted to the NCI–DTP may or may not have an
identified mechanism of action. Many compounds were synthesized as
chemically interesting structures, or were submitted as purified natural
products, with cellular targets yet to be determined. Other submitted
compounds have been rationally designed with specific targets in mind.
Finally, many compounds are structural analogs of anticancer agents with
known mechanisms of action. The end result is that the compounds in the
Chapter 12: The FHCRC/NCI Yeast Anticancer Drug Screen 323
5 RESULTS
Figure 1. A total of 97,261 compounds were tested in Stage 0. The figure indicates the
percentage of compounds where at least one of the strains exhibited growth inhibition equal to
or greater than the indicated cutoff. 70% growth inhibition in one of more strains was chosen
as the criterion for further testing.
Chapter 12: The FHCRC/NCI Yeast Anticancer Drug Screen 325
Nearly 541,823 (93%) differed by less than 20% growth inhibition, 15,447
(2.6%) had values that differed by more than 50% growth inhibition, and
5,574 (1%) had values that differed by more than 80% growth inhibition.
Compounds that showed selective activity in Stage 1, with the strain with
the maximum growth inhibition at least five times greater than the strain
with the least growth inhibition, were selected for testing in Stage 2. A total
of 3,498 compounds were tested in duplicate at 5 doses (1.2, 3.7, 11, 33, and
100 µM). Compounds were screened against a panel of 13 strains, 2 of
which serve as wild-type controls. The remainder contained a single gene
alteration of interest. All but one of the strains was also deleted for the pdr1,
pdr3, and erg6 genes. The rad50 EPP+ strain, wild-type for ERG6, PDR1
and PDR3 genes, was included to determine whether further studies with
these compounds would be affected by these drug sensitivity mutations.
Data are publicly available for the 3,137 compounds that are not covered by
a confidentiality agreement. Stage 2 data, including bar graphs, dose-response
328 S. L. Holbeck and J. Simon
Figure 2. Structures are shown of representative compounds selective for the spindle
checkpoint mutant bub3 in Stage 2 testing. NSC 1331 was selective for bub3 at all 5 doses.
Two compounds with related structures were also bub3-selective. NSC 4000206 and two
related compounds were bub3-specific at multiple doses.
Figure 3. Growth inhibition as a function of dose is plotted for cells treated with NSC 1331.
This compound specifically inhibited the growth of the bub3 spindle checkpoint mutant at all
doses tested in Stage 2, without significant inhibition of any of the other strains.
Figure 4. These two compounds were selectively toxic to the G1 cyclin CLN2-over-
expressing strain at two of the doses tested in Stage 2. NSC 180198 is related to
ribonucleotide reductase inhibitors.
There were no compounds that inhibited only the mgt1 strain at more
than one dose. A total of 139 compounds inhibited mgt1 +/– 1 or more
strains at least 1 dose. The mgt1 mutant strain is known to be sensitive to
alkylative DNA damage in general and methylation at guanine residues at
the O6 position in particular. Even methylating agents, however, are more
toxic to the rad6 and rad18 post-replication repair mutant strains than to the
mgt1 strain. It is therefore expected that the mgt1 mutant strain would be
most sensitive among the panel of strains to few, if any, compounds.
There were no compounds that inhibited the growth of only the mlh1
mutant at 2 or more doses. A total of 164 inhibited the mlh1 strain +/– 1 or
more other strains at least 1 dose. Mlh1p is a component of mismatch repair
in yeast. There are no known agents that are selectively toxic to the mlh1
deletion strain.
and cisplatin are among the most active agents against the rad14 deletion
mutant [46]. As with the mgt1 mutant however, even with these agents, the
rad14 strain is not the most sensitive strain in the panel: the most sensitive
strains to mechlorethamine and cisplatin are rad6 and rad18 post-replication
repair mutants. Structures of two rad14-selective agents are shown in Figure 5.
Figure 5. Structures are shown for two compounds that selectively inhibited the growth of the
rad14 excision repair mutant.
Figure 6. Structures are given for representative compounds selective for the post-replication
repair mutant rad18 in Stage 2 testing. NSC 86324 is a nitrogen mustard, as were a number
of other rad18-seletive compounds. NSC 142226, an ---halocarbonyl, and NSC 3750, a
nitroaromatic, are representative of compound classes found multiple times among the rad18-
selective agents.
as well as ionizing radiation are more toxic to rad18 mutant strain than to
wild type yeast. It is not surprising then that the vast majority of compounds
showing rad18 selectivity are electrophiles capable of damaging DNA. The
most highly represented classes of electrophiles include: nitrogen mustards
(e.g., NSC 86324) (i.e., bis-[2-chloroethyl]-amines) related to the FDA-
approved agents mechlorethamine, melphalan, chlorambucil, and cyclophos-
phamide; α-halocarbonyl compounds (e.g., NSC 142226); nitroaromatics
(e.g., NSC 3750) and aza-aromatic N-oxides (e.g., NSC 5094). Interestingly,
several compounds that appear to have low potential for causing DNA
damage (e.g., NSC 136325, a bromo-fluorene derivative) also score as rad18
selectives.
5.3.7 Compounds selective for the rad50 and rad52 strains in Stage 2
Six compounds were highly specific for the rad53 mutant strain,
inhibiting only this strain in 2 of the doses. Of the 5 not covered by a
confidentiality agreement, 2 are structurally related to the antifolate
methotrexate, which is expected to trigger the S-phase DNA synthesis
checkpoint. The remaining 3 contain heavy metals (Au, Ru, Os). A total of
195 compounds inhibit the rad53 strain, either alone or in combination with
other strains, in at least 1 of the tested doses. 18 compounds (9%) (including
the 2 already mentioned) are analogs of nucleosides, antifolates, or other
compounds expected to interfere with DNA synthesis. 21 compounds (11%)
are related to topoisomerase poisons, alkylating agents, or other compounds
that introduce DNA damage.
There were no compounds highly selective for the sgs1 mutant. A total of
436 compounds inhibited sgs1, alone or in combination with a few more
strains in at least one dose. Of these 12 (3%) are analogs of compounds that
interfere with DNA synthesis, 14 (3%) are DNA damaging agents
(alkylating, topoisomerase poisons, etc.).
mutants in the yeast strain panel and the abundance of DNA damaging
agents in the NCI compound collection.
Figure 8. Growth inhibition as a function of dose is plotted for cells treated with NSC 16258.
This compound was selectively toxic to the DSB repair mutants rad50 and rad52. It is
structurally unrelated to known topoisomerase poisons, which make up the majority of the
rad50/rad52 selective agents, and thus represents a novel structure selective for the DSB
repair mutants.
Figure 9. The structure of NSC 368252, a compound with modest selectivity for the rad53
checkpoint mutant is shown. In contrast to the more highly selective rad53 compounds, this
agent is not structurally related to known antimetabolites of DNA damaging agents.
In general, the rad18 mutant is the single most sensitive strain to DNA
damage and any compound that elicits a multistrain sensitivity profile that
includes rad18 has to be viewed as a potential DNA alkylator, cross-linker,
or cutter. Table 1 lists the NSC numbers and strain selectivity for compounds
that target multiple strains.
Chapter 12: The FHCRC/NCI Yeast Anticancer Drug Screen 335
Table 1. Compounds listed in this table exhibited selectivity for multiple strains at two or
more doses. The strains inhibited, and the number of doses with this profile are indicated. For
this table, the rad50EP+ strain was not included.
Thus for the 155 compounds for which the Stage 1 and Stage 2 results
can be readily compared, the vast majority (88%) were selective for the same
strain in both stages. A further 11% were somewhat less selective in Stage 1
assays, inhibiting other strains in addition to that seen in Stage 2. Only one
of the these had discordant Stage 1 and Stage 2 results.
The erg6, pdr1, pdr3 (epp) mutations were included in all of the strains
used in Stage 0 and Stage 1 and in all but one strain in Stage 2 to increase
drug uptake (erg6) and decrease drug efflux (pdr1 and pdr3). By including
both rad50, erg6, pdr1, pdr3 (rad50epp-) and rad50, ERG6, PDR1, PDR3
(rad50EPP+) strains in the Stage 2 panel, we were able to quantify the effect
of the “drug-sensitizing” mutations. We stratified rad50-selective com-
pounds based on the following criterion. For each compound, if there were at
least two doses at which the rad50epp- strain but not rad50EPP+ had a 70%
or greater growth inhibition, then the compound was counted as having
increased activity in the mutant “drug uptake and efflux” background. Of the
116 rad50-selective compounds, 66 compounds, or 57% had increased
toxicity due to the epp mutations. For all Stage 2-tested compounds, about
one-third were aided by the drug-sensitizing mutations, with the rad50epp-
strain half a log more sensitive than the rad50EPP+ strain.
8 SUMMARY
ACKNOWLEDGMENTS
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Chapter 13
YEAST AS A MODEL TO STUDY
THE IMMUNOSUPPRESSIVE AND
CHEMOTHERAPEUTIC DRUG RAPAMYCIN
1 INTRODUCTION
347
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 347–374.
© 2007 Springer.
348 J. R. Rohde, S. A. Zurita-Martinez and M. E. Cardenas
Figure 1. TOR responds to diverse signals to regulate cell growth. TOR is activated by
compounds associated with a favorable energy status including ATP, amino acids, and
phosphatidic acid. TOR is inactivated in response to nutrient depletion, mitochondrial
dysfunction as well as rapamycin–FKBP12. Active TOR promotes cell growth while
inactivation of TOR results in growth arrest and autophagy in yeast and growth arrest and
apoptosis in higher eukaryotes.
Chapter 13: Rapamycin in yeast 349
yeast S. cerevisiae [52, 132]. Subsequently the crystal structures were solved
for both the yeast and the human protein and found to be essentially
superimposable [104, 123]. The gene encoding the yeast FKBP12 protein
was cloned and disrupted. Whereas wild-type yeast cells are exquisitely
sensitive to growth inhibition by rapamycin, with a minimum inhibitory
concentration (MIC) of 25 ng/ml, yeast cells lacking FKBP12 were
completely viable and only slightly reduced in growth rate [52, 67, 132].
Thus inhibition of an essential function of FKBP12 could not explain the
potent toxic action of FKBP12. Taken together, these findings support a
model in which both the FKBP12 protein and its ligand rapamycin are both
required to exert a toxic effect in yeast. These findings also established
unequivocally that FKBP12 plays a central role in the action of rapamycin,
and the next challenge then was to identify the molecular target of the
FKBP12–rapamycin complex.
The targets of the FKBP12–rapamycin complex, the products of the
TOR1 and TOR2 genes (target of rapamycin), were discovered in a genetic
screen in yeast searching for rapamycin resistant mutants [51]. Mutations in
three different genes were identified. First, mutations in the FPR1 gene
encoding FKBP12 were found to be recessive, and resulted from amino acid
substitutions that based on structural studies of the FKBP12–rapamycin
complex were predicted to be critical for rapamycin binding. Importantly,
mutations in two other genes identified were genetically distinct from FPR1,
mapped to two different genomic locations and conferred dominant, or
semidominant, drug resistance in genetic crosses. Based on an unusual
genetic behavior between alleles of these three genes, known as nonallelic
noncomplementation, it was proposed that the three might form a physical
complex.
The TOR1 and TOR2 genes were subsequently cloned by the Hall and
Livi laboratories, revealing that they encode extremely large proteins, ~280
kDa, that Tor1 and Tor2 are homologs of each other, and that both share a
C-terminal domain with homology to lipid and protein kinases [18, 54, 71].
Further studies demonstrated that FKBP12–rapamycin forms a physical
complex with the yeast Tor1 and Tor2 proteins [22, 76, 118]. Later, work
from five different groups converged to identify the mammalian Tor
homolog (mTor) via its ability to bind the FKBP12–rapamycin complex [15,
27, 105, 106]. In addition, it was demonstrated that yeast Tor–mTor hybrid
genes are capable of providing Tor function in yeast cells [2]. Tor homologs
were later identified in other organisms; similar to S. cerevisiae two Tor
proteins have been characterized in S. pombe, and a single Tor homolog has
been identified in C. albicans, C. neoformans, D. melanogaster, A. thaliana,
and H. sapiens [15, 32, 51, 71, 82, 92, 105, 131, 136].
Chapter 13: Rapamycin in yeast 351
[115]. This region includes Ser-2448, which was shown to be a target for
phosphorylation by protein kinase B, raising the possibility that in
mammalian cells Tor is subject to signaling by the PI3K/AKT pathway.
Figure 2. TOR controls translation in eukaryotes. In mammals and drosophila, mitogens and
nutrients inactivate Tsc1/Tsc2 to favor the GTP-bound form of Rheb. Rheb activates TOR
which in turn favors translation initiation by activating p70 S6 kinase as well as inactivating
the translational repressor PHAS-I. In yeast cells, TOR responds to nutrients and acts on
Eap1, a homolog of PHAS-I. Inactivation of TOR by nutrient limitation or rapamycin
treatment activates the Gcn2 kinase, resulting in eIF2α phosphorylation and inhibition of
overall translation. At this point it is not known if the yeast homolog of Rheb functions in the
TOR pathway or if the TOR–Gcn2 cross talk exists in mammals and drosophila.
Chapter 13: Rapamycin in yeast 353
and p70-S6 kinase [16, 19, 73, 98]. The PHAS-I protein forms a physical
complex with eIF4E, thereby blocking the ability of eIF4E to mediate
recognition of the 5′ CAP on mRNA and initiate recruitment of ribosomes to
the mRNA. Thus, PHAS-I is an inhibitor of translation initiation, and the
Tor pathway antagonizes the function of PHAS-I during culture conditions
that favor cell growth and division (reviewed in [44]). When nutrients are
limited, or rapamycin is present, the Tor signaling pathway is inactive or less
active, and PHAS-I forms a stable complex with eIF4E to prevent CAP
recognition and translation of the message. A central question in the field
has been whether or not the Tor kinase directly phosphorylates PHAS-I or
p70-S6 kinase in vivo [17]. This issue has been in part resolved by the
discovery of Tor interacting proteins that facilitate interaction with and
modification of these key substrate molecules (see below).
In yeast cells, rapamycin also leads to an inhibition of translation
initiation. Early studies implicated a different molecular target in yeast cells
as the translation initiation factor eIF4G, which physically interacts with the
5′ CAP-binding factor eIF4E and assists in the recruitment of additional
factors to the initiation complex. Rapamycin leads to degradation of eIF4G
in yeast cells and a profound block to translational initiation [10]. Two
possible distant homologs of the mammalian PHAS-I regulatory protein
have been identified in yeast and are called p20 and Eap1. Both proteins
physically interact with yeast eIF4E and thereby inhibit CAP dependent
translation initiation in a fashion analogous to the PHAS-I protein in
mammalian cells. While neither has been implicated as a direct substrate or
regulatory target of Tor in yeast cells, deletion of the EAP1 gene does confer
a modest level of resistance to rapamycin, indicating that liberating eIF4E
from this negative regulatory factor can enhance cell survival in the presence
of rapamycin [31]. It is thus quite remarkable that the role of the Tor
signaling pathway in translational control has been in large part conserved
from yeast to humans, even given that the direct molecular targets may be
distinct in these divergent organisms.
Recent studies have revealed a new and intriguing role for the Tor
pathway in regulating translation during amino acid limitation in yeast cells.
Amino acid limitation is normally sensed by the general control response, in
which the Gcn2 protein senses uncharged tRNAs and controls translation of
the mRNA encoding the global transcription factor Gcn4 [55]. The Gcn2
protein has two functional domains; a protein kinase and a domain that
shares identity with tRNA synthetases. Amino acid limitation leads to an
excess of uncharged tRNA molecules in the cell, which is sensed by the
Gcn2 kinase, leading to its dephosphorylation and activation. Gcn2 in turn
phosphorylates eIF2α, altering its activity in the cell to limit translation of
the vast majority of mRNA molecules but leads to enhanced translation of
354 J. R. Rohde, S. A. Zurita-Martinez and M. E. Cardenas
the Gcn4 mRNA. The mRNA encoding Gcn4 is unique in that it contains
four upstream open reading frames, and during normal nutrient conditions,
ribosomes initiate translation at these upstream ORFs and rarely reach the
start codon for Gcn4 itself. In cells limited for amino acids, the action of
Gcn2 on eIF2α slows overall translation initiation allowing an increased
scanning rate and initiation to produce Gcn4. In turn, Gcn4 then activates the
expression of genes involved in amino acid biosynthesis, and a plethora of
other genes.
The first hints that the Tor pathway might impinge on the general control
response were studies demonstrating that rapamycin can stimulate
expression of a GCN4-lacZ reporter gene [122]. However, given previous
studies that implicate the Tor pathway in the control of amino acid
transporter stability in yeast, an alternative explanation was that rapamycin
was inducing the general control response via an indirect effect on amino
acid uptake. More recently, two studies have converged to implicate the Tor
pathway in a more direct action on the general control response [26, 70]. The
Gcn2 kinase is regulated by both uncharged tRNAs, which stimulate its
activity, and by a kinase that phosphorylates and inactivates Gcn2 in rich
medium. Inhibition of Tor by rapamycin leads to Ser 577 dephosphorylation
in Gcn2, activation of Gcn2, and increased eIF2α phosphorylation. The net
effect of rapamycin is then an increased level of Gcn4 expression and
activation of the general control response. The power of rapamycin to
activate Gcn2 also requires the ability of Gcn2 to bind uncharged tRNAs,
and thus provides an additional regulatory input to Gcn2 that allows the Tor
signaling cascade and limiting amino acids to be coordinately detected.
The physiological significance of this expanded regulatory network is at
present not clear, but it may allow enhanced induction of the general control
response when cells are more severely starved than simple amino acid
limitation affords. Alternatively, under conditions of amino acid starvation
Tor also regulates the general control response [87], especially since Tor
responds to amino acids in mammalian cells. These mechanisms allow
coordination between two global regulatory networks, the Tor and Gcn
pathways, which together function as both translational and transcriptional
regulatory cascades.
Whether or not the cross talk between these signaling pathways occurs in
mammalian cells is not yet known. It is intriguing however that the
mammalian Gcn2 protein has been identified as a key sensor to many
signaling inputs and controls the integrated stress response. Ron and
coworkers have proposed that mammalian cells utilize this ancient pathway
to integrate diverse stress signals to regulate gene expression of the targets of
the transcription factor ATF4, which is regulated in a manner analogous to
the yeast Gcn4 factor [48].
Chapter 13: Rapamycin in yeast 355
The recent development of genome wide arrays and the high specificity
of rapamycin to inhibit Tor function have aided the unraveling of a highly
complex transcriptional program regulated by the TOR pathway in yeast
cells (Figure 3). Earlier work had established a role for TOR signaling in the
synthesis of rRNA and tRNA [80, 135]. Although the targets of this
regulation are currently unknown, mutations that affect protein phosphatase
2A (PP2A) function result in alteration of both rRNA and tRNA gene
expression [119, 124]. Thus, an intriguing possibility is that regulation of
these genes by the TOR pathway is accomplished via PP2A.
Studies employing genome wide arrays revealed that ribosomal protein
(RP) genes expressed by PolII are repressed by the addition of rapamycin in
a manner that closely resembles nutritional limitation [21, 50, 68, 97]. The
Figure 3. TOR controls transcription of genes involved in growth and adaptation to starvation
and other stresses. In response to nutrients TOR activates the transcription of genes required
for ribosome biogenesis. At the same time TOR negatively regulates genes required for
adaptation to stress and nutrient scavenging. A common strategy for the repression of this
latter class of genes is through nuclear exclusion of the transcription factors. TOR inactivates
protein phosphatase 2A, which is required for nuclear localization of Msn2, Msn4, and Gln3
as well as translation of the message for the transactivator Gcn4. TOR also excludes the bZIP
transcription factors Rtg1 and Rtg3.
356 J. R. Rohde, S. A. Zurita-Martinez and M. E. Cardenas
Figure 4. TOR signaling links nutrient sensing to histone acetylation to regulate gene
expression.
Tor1, and that Ure2 is recruited to the complex indirectly by its interaction
with Gln3 [11]. In the same study evidence was presented that Tor1 is
capable of directly phosphorylating Gln3 in vitro [11]. However, these
studies await independent confirmation.
In the phosphorylated state, Ure2 and Gln3 form a complex and
dephosphorylation of this complex by Tor inhibition promotes complex
dissociation and nuclear translocation of Gln3 [7, 11]. Indirect evidence
indicates that the rapid dephosphorylation of Ure2 and Gln3 is regulated by
Tor via protein phosphatase type 2A. Thus, rapamycin treatment or entry
into stationary growth phase induces the dissociation of the PP2A catalytic
subunit Sit4 from its regulatory subunit Tap42 [36]. Although earlier studies
suggested that Tap42 ejected an inhibitory role on Sit4 for the expression of
the NCR genes, two independent studies have concluded that both of these
factors are required for expression of these genes [7, 22, 41]. Finally it was
shown that nuclear import of Gln3 requires the importin Srp1 while
Crm1/Xpo is necessary for the nuclear export [23].
358 J. R. Rohde, S. A. Zurita-Martinez and M. E. Cardenas
STRE genes by rapamycin. Taken together these studies reveal that the Tor
pathway controls both nuclear import and nuclear export of key regulatory
transcription factors.
The Tor pathway has also been shown to control gene silencing at
subtelomeric regions. These studies capitalized on the Saccharomyces
toolbox-ordered sets of gene deletions. Zheng and coworkers screened
~4000 viable gene deletions for altered sensitivity to rapamycin [24]. One of
these mutants, sir3, displayed an increased resistance to rapamycin. Studies
of this phenomenon revealed that Tor negatively regulates MPK1, the MAP
kinase of the cell integrity pathway. MPK1 in turn phosphorylates Sir3 to
result in increased expression of subtelomeric genes involved in the repair of
cell wall damage [1]. Once again the powerful tools available in yeast were
able to quickly identify an important mode of cross talk between two
different signal transduction pathways.
Finally, observations in mammalian cells suggest that mTOR and PP2A
control the activity of the STAT3 protein in a fashion similar to the control
of Gln3 activity in yeast. STAT3 is retained in the cytoplasm until activation
by the JAK kinases that act by phosphorylating tyrosine residues in response
to cytokines. Phosphorylation of serine and threonine residues (most notably
Ser727) also occurs but it is not yet clear if phosphorylation positively or
negatively regulates the ability of STAT3 to activate transcription. Inhibition
of PP2A activity increases STAT3 phosphorylation on serine and threonine
residues and results in a concominant exclusion of STAT3 from the nucleus
[133]. Reportedly STAT3 directed transcription is blocked by rapamycin
treatment and in vitro phosphorylation studies indicate that mTOR may
phosphorylate this protein directly [134]. While these studies convincingly
show the involvement of mTOR and PP2A in regulating the Ser727
phosphorylation status of STAT3, the physiological consequences of this
phosphorylation remain unknown. Importantly, constitutive activation of
STAT3 results in malignant transformation and cancer.
characterized and were found to cause a G2/M cell cycle arrest consisting of
cells with unpolarized actin [53]. Importantly, Rho1 is known to activate the
PKC signaling pathway in yeast and overexpression of Pkc1 rescues alleles
of tor2 defective in the Tor2-unique function. In mammalian cells, mTOR
regulates the activity of PKC but it is as yet unclear how similar this
connection will be to that of yeast. A link between the actin cytoskeleton
and mTOR has not yet been established in mammalian cells and PKC
activation by mTOR is rapamycin-sensitive in mammals, whereas the Tor2-
unique function is rapamycin-insensitive in yeast [138]. Recently, careful
studies have revealed a rapamycin-sensitive and cell cycle dependent
polarization of the yeast actin cytoskeleton [127]. These studies suggest
that Tor1 may have a greater role in actin polarization than previously
appreciated.
A second function for the Tor kinases at the G2/M phase of the cell cycle
is beginning to emerge. Recent reports suggest that the Tor kinases play a
role in the assembly or function of microtubules [12, 29]. This appears to be
a rapamycin-sensitive function that is shared between yeast Tor1 and Tor2
and that is conserved from yeast to mammals [12]. Genetic and biochemical
evidence in yeast suggests that this process involves the kinesin-related
proteins (KRPs), which are important for spindle–pole separation at
anaphase [29]. Consistent with this, rapamycin treatment causes defects in
chromosome and nuclear segregation in both yeast and mammalian cells
[12, 29].
[63]; Hara, 2002 #3227]. In particular, the mammalian raptor protein can
bind to the Tor substrates p70 S6 kinase and PHAS-I via a previously
defined domain known as the TOR signaling motif (TOS) [91, 108, 109].
The TOS domain allows PHAS-I to dock onto the mTor/Raptor protein
complex, and PHAS-I is then phosphorylated to promote release and
activation of eIF4E and cell growth. In yeast cells, a second complex, called
the Torc2 complex, can be detected containing Tor2, Lst8, and the Avo1,
Avo2, and Avo3 proteins [75, 130]. FKBP12–rapamycin can physically
associate with the Torc1 complex but not with the Torc2 complex,
suggesting that this second complex mediates the rapamycin-insensitive role
of Tor2 in controlling polarization of the actin cytoskeleton. If and how any
of these newly discovered Tor associated proteins communicate nutrient
sensing via Tor to PP 2A or any of its associated subunits is as yet unknown.
lacking the Rheb homolog Rhb1 were also viable but exhibited defects in
nutrient sensing and arrested in G1 more rapidly than wild-type cells in
response to nitrogen limitation. Furthermore, in the rhb1 mutant cells several
nitrogen starvation induced genes were found to be expressed under rich
nitrogen conditions [78]. Homologs of the mammalian Tsc1 and Tsc2
proteins in fission yeast are known to regulate the proper targeting and
function of amino acid permeases, a role consistent with related studies on
the Rheb homolog [81]. The Aspergillus fumigatus Rheb homolog, rhbA, is
induced in response to nitrogen starvation. The rhbA mutant cells are viable
but their growth was compromised in poor nitrogen sources, nitrogen rich
conditions failed to repress their asexual development, and they were
hypersensitive to rapamycin [93, 94]. Taken together, these findings are
consistent with a role for the small G protein Rheb in nutrient sensing in
budding yeast, fission yeast, and pathogenic fungi and suggest Rheb might
play a globally conserved role in activating the Tor kinases in response to
certain nutritional cues. Rheb is not essential for growth in any of these
fungi, in contrast to Tor, and thus Rheb likely represents one of several
redundant regulatory inputs.
The key questions that remain are how the Tor kinases sense nutrients
and how they communicate this information to their downstream effectors.
Two recent studies suggest that the mTOR kinase is activated in response to
two small molecules: ATP and PA [35, 41]. Phosphatidic acid is produced
by phospholipase D and has been reported to bind mTOR and thereby
stimulate its kinase activity, in vitro and in vivo [41]. Phospholipase D is
conserved in yeasts, where it plays a known role in promoting morphological
changes to pheromone and in meiosis [46, 103]. As yet, no role has been
adduced for phospholipase D in controlling Tor signaling in yeasts. Recent
studies reveal that Pld1 is required for filamentous growth in Candida
albicans [56], and our studies have shown that the Tor pathway is necessary
for filamentous growth in S. cerevisiae, C. albicans, and C. neoformans [34].
Finally, Thomas and colleagues have suggested that the low intrinsic Km of
mTOR for ATP poises the enzyme to serve as a general ATP sensor for the
cell [35]. Thus PA and ATP may contribute to the activation of mTOR via or
in addition to nutrient sensing mechanisms.
including smooth muscle cells and several neoplastic cell lines. The recent
finding that cardiac stents impregnated with rapamycin can significantly
reduce restenosis, and that rapamycin can inhibit growth of a variety of
different tumors, will lead to new clinical indications for this novel natural
product. In light of these significant clinical advances, a more detailed
understanding of the targets of rapamycin and their function in growth
promoting pathways should provide considerable insight into the molecular
basis of therapeutic action and support the development of additional and
novel targets for therapeutic intervention.
Of particular interest and importance is the clinical utility of rapamycin
as a novel chemotherapy agent. A flurry of recent reports have identified
unique tumor cell lines that are exquisitely sensitive to growth inhibition by
the mTOR inhibitor rapamycin and its analogs. Particularly, in cells and
mice lacking the tumour suppressor PTEN, a lipid phosphatase that controls
signaling via the PI3 kinase pathway, rapamycin inhibits p70 S6 kinase
activity and oncogenesis [28, 45, 83, 88, 96, 116]. These findings apply to a
broad range of tumor types, including tumors of the immune system
(multiple myeloma), common solid organ tumors (breast, prostate), and less
common but devastating solid organ tumors (glioblastoma). There are a
variety of phase II and phase III trials that are ongoing now, and the years
ahead hold excitement and promise of a significant impact for rapamycin in
a multitude of diverse clinical applications.
Much of our current understanding of rapamycin targets and mechanism
of action stems directly from genetic and molecular studies conducted in the
model yeast S. cerevisiae. In summary, these contributions include:
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Chapter 14
USE OF YEAST AS A MODEL SYSTEM
FOR IDENTIFYING AND STUDYING
ANTICANCER DRUGS
1 INTRODUCTION
375
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 375–391.
© 2007 Springer.
376 J. O. Liu and J. A. Simon
microarrays that allow for the monitoring of the expression of the complete
yeast genome. In this chapter, we will cover three different ways yeast has
been used to facilitate the identification, characterization, and mechanistic
studies of anticancer drugs. These include (1) the use of yeast as a surrogate
system to identify and study anticancer drugs; (2) the use of yeast as self-
contained microvessles to perform large-scale parallel analysis of interactions
between proteins and small ligands; and (3) the use of the yeast transcriptional
profiling for validating the molecular target for given drugs. For the most part,
we have selected examples from our own laboratories. We thus have
attempted to be more illustrative than comprehensive in this chapter.
from fumagillin. The target protein was isolated using affinity chro-
matography and identified by mass spectrometry as the MetAP2 [16, 41].
Figure 2. Schematic comparison of the domain structures of eukaryotic MetAP1 and MetAP2.
The five amino acid residues in the catalytic domain are shown with single letter code.
Since the MetAP2 was isolated from mouse embryo extract via affinity
chromatography using the fumagillin or ovalicin as an affinity ligand, a
major question was the specificity of fumagillin for MetAP2, given the
extremely high sequence similarity between MetAP1 and MetAP2 [1, 28].
Using the wild type and map1 and map2 mutant strain of yeast, it was found
that only the map1 mutant strain of yeast is sensitive to inhibition by
fumagillin or ovalicin, providing strong evidence that the drugs are highly
specific for MetAP2 (Figure 3) [16, 41]. In addition to demonstrating the
extraordinary specificity of fumagillin for MetAP2, these studies also raised
the interesting question of how inhibition of MetAP2 alone led to cell cycle
inhibition of human endothelial cells. Although the map1 mutant has not
been further exploited, it is apparent that the mutant can be used for
screening of small molecule libraries to identify novel inhibitors of MetAP2.
Figure 3. Specific inhibition of MetAP2 by TNP-470 in yeast. The wild type, map1 (yeast
MetAP1) and map2 (yeast MetAP2) mutant strains were plated on YEPD plates containing
either dimethylsulfoxide as a control or 50 nM of either TNP-470 or ovalicin. The plates were
incubated at 30ºC for 4 days before they were photographed.
Chapter 14: Use of Yeast as a Model System 379
Figure 4. Structure of splitomicin (A) and its effect on yeast responsiveness to α factor (B).
The helo of cells indicates that splitomicin induced loss of sensitivity of yeast to α factor-
induced silencing in the mating type loci.
The yeast system not only enabled the identification of hits, but also
facilitated the subsequent verification of splitomicin as a relatively selective
inhibitor for yeast Sir2p and identification of residues in Sir2p that are
critical for its binding to splitomicin [2]. Yeast contains four SIR2 homologs,
HST1-4 (HST stands for Homologue of Sir Two). To determine whether
splitomicin is specific for Sir2p, the transcriptional profiles of wild- type
yeast cells treated with splitomicin and the isogenic yeast strains with
deletion of SIR2 and each of its four homologs were obtained and compared
to each other. It was found that the majority of the changes (88%) in
transcription as a consequence of splitomicin treatment are seen in the sir2
mutant yeast strain. Interestingly, a small number of changes in transcription
(9%) were identical to those seen in the hst1 mutant strain. In contrast, there
was no overlap in transcriptional profile changes between splitomicin-treated
yeast cells and the hst2-4 mutant strains. These results clearly indicated that
splitomicin is selective toward Sir2p and Hst1p, but does not interact with
the other members of the Sir2p family of proteins. PCR-mediated mutagenesis
of the SIR2 open reading frame was used to identify three drug-resistant
mutants of Sir2p using similar reporter screens in yeast. The drug-resistant
mutations all occurred in a region of the protein that is part of the putative
active site for Sir2 and is highly conserved between yeast Sir2p and Hst1p,
suggesting that splitomicin is likely to interact with the active sites of both
Sir2p and Hst1p.
In an independent study, a similar approach was used to screen for the
yeast Sir2p inhibitors with the yeast strain containing a telomeric copy of
URA3 gene [17]. Three small molecule inhibitors of Sir2p were identified,
which also inhibited the human SIRT2 protein with similar potencies.
It is noteworthy that the screens employed in the studies would not have
been possible without the use of yeast. Even though the human SIRT2 is
known, its celluar function is largely unknown and it is much more difficult
Chapter 14: Use of Yeast as a Model System 381
to devise a similar reporter system that would allow for a phenotypic screen
for the human SIRT2 inhibitors directly.
The ability of yeast cells to take up one or more plasmids and express the
specific proteins encoded by the plasmids makes it possible to use individual
yeast cells as independent “containers” for analyzing the interaction between
the proteins expressed in the yeast and the small molecules administered.
Given the ease of culturing yeast cells and the ease with which exogenous
plasmids can be introduced into yeast, large-scale parallel analyses are
feasible. Thus, each yeast cell can be turned into a self-contained reaction
vessel to assay the interaction between a ligand and its target.
One of the major challenges is to develop a suitable readout that allows
for quick and easy detection of the binding between a drug and its protein
target in yeast cells. One type of reporter system couples a ligand-binding
event with the transcription of a specific reporter gene introduced into the
yeast. Another reporter system links the ligand–protein binding event with
the reconstitution of an enzyme that can be easily detected. In this chapter,
we have chosen a few systems that have the potential of being adapted for
such an application or have been used to assess drug-protein interactions.
Since its development, the yeast two-hybrid system has become one of the
most powerful tools for uncovering new protein–protein interactions [6, 8].
The yeast two-hybrid system exploits an intrinsic property of eukaryotic
transcription factors to function with two separable and portable domains, a
DNA-binding domain and a transcriptional activation domain. By splitting a
transcription factor into two separate proteins and fusing into each two
interacting proteins, it is possible to reconstitute the transcription activity.
In theory, the yeast two-hybrid system is ideal for identifying inhibitors
for protein–protein interactions. Unfortunately, due to the relatively large
binding interface often associated with protein–protein interactions [32], it is
in general difficult to discover small molecules that directly compete for a
382 J. O. Liu and J. A. Simon
Figure 5. The basic components of yeast three-hybrid system. The triangle and semicircle
represent two different chemical ligands. UAS, upstreat activating sequences.
Figure 6. Summary of the yeast three-hybrid screen for target proteins for FK506.
The yeast three-hybrid system does have its limitations, including the
requirement of the protein receptors to exist as a fusion protein with either
the DNA-binding domain or transcription activation domain of a trans-
cription factor. The ability of hybrid ligands to penetrate the yeast
membranes is another barrier to its general application. Solutions to these
potential problems are beginning to be addressed.
The three-hybrid system based on the glucocorticoid receptor–
dexamethasone pair has been shown to work when the other ligand–receptor
pair is wild-type FKBP and FK506. It has been found that when the affinity
between FKBP and FK506 is further decreased by replacing FKBP12 with
other members of the FKBP family, such as FKBP13 and FKB P25 or by
generating FKBP12 mutants that have decreased affinity for FK506, the
reporter gene activation can no longer be detected (E. Griffith and J. O. Liu,
unpublished results). This is understandable as the three-hybrid system
involves the formation of a ternary complex from three partners, the two
hybrid fusion proteins and the hybrid ligand, in contrast to the two individual
fusion proteins required for two-hybrid system.
In addition to the dexamethasone-FK506 as the hybrid ligand, two alter-
native hybrid ligands were recently developed and tested in a three-hybrid
386 J. O. Liu and J. A. Simon
setting, which further validated the concept of the three-hybrid system. Both
groups employed dihydrofolate reductase and its potent inhibitor metho-
trexate as one receptor–ligand pair to replace FKBP12-FK506 [22, 30].
Cornish and colleagues used the LexA DNA-binding domain and B42
transactivation domain [30] while Henthorn and coworkers employed
the Gal4 DNA-binding domain and Gal4 transactivation domain [22]. In
both cases, robust LacZ reporter gene activity was observed in yeast expres-
sing appropriate fusion proteins in the presence of the dexamethasone-
methotrexate hybrid ligands. Cornish and colleagues found that at the same
concentration of the hybrid ligand, the reporter gene activity was about
150- fold higher for the dexamethasone-methotrexate system than for the
dexamethasone-FK506-based system [30]. Assuming that the reporter gene
activation is proportional to the overall affinity of the hybrid ligand for
the two fusion receptors, this is expected as the affinity of methotrexate for
dihydrofolate reductase is in the picomolar range [38] while the affinity of
FK506 for FKBP12 is in the low nanomolar range [20, 40]. Using the Gal4-
based system, Henthorn and coworkers further demonstrated the utility of
the system to screen a cDNA library to identify clones encoding dihydro-
folate reductase [22]. Similar to the three-hybrid screen for FKBPs, no false
positive clones were found after their elimination using the competition
assay with excess amount of free methotrexate. It will be interesting to see
whether there is a significant improvement in sensitivity of the three-hybrid
system when the dihydrofolate reductase–methotrexate pair is used to
replace the glucocorticoid receptor–dexamethasone pair.
Figure 7. Correlation coefficients for gene expression profiles as a result of the different types
of perturbation of yeast using microarray.
It is worth noting, however, this approach, while powerful, does have its
limitations. The major limitation lies at the limited size of the yeast genome
in comparison to that of humans. As a consequence, many genes present in
mammalian cells, including those involved in cell–cell communications and
intracelluar signaling such as protein tyrosine kinases, are absent in yeast.
The collection of the compendium of the yeast mutants is not applicable to
studying drugs that interact with those proteins. Another limitation will be
those genes that are essential to yeast growth. More sophisticated methods of
downregulating those genes such as reduction in the expression level without
complete knockout will be important for studying drugs that affect those
genes. Nevertheless, the existing mutants and their signatures should take us
a long way toward facilitating the study of many types of drugs already.
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Chapter 15
GENETIC ANALYSIS OF CISPLATIN
RESISTANCE IN YEAST AND MAMMALS
*
Deceased
393
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 393–408.
© 2007 Springer.
394 S. Ishida and I. Herskowitz
Work from many laboratories has implicated DNA as a critical target for
cisplatin cytotoxicity, the most revealing evidence being the hypersensitivity
to cisplatin of both prokaryotic and eukaryotic cells deficient in DNA repair
[3, 20, 45]. The most prevalent cisplatin-induced DNA adduct is the
intrastrand cross-link in which the platinum is covalently bound to the N7
positions of the imidazole ring of two adjacent guanines [15]. The
intrastrand cross-links are repaired by the nucleotide excision repair pathway
[4]. Unrepaired DNA damage triggers cell cycle arrest, and cells eventually
undergo programmed cell death (apoptosis) [13].
Chapter 15: Genetic Analysis of Cisplatin Resistance in Yeast and Mammals 395
treated DNA after incubation with nuclear extract [16]. Human cancer cell
lines deficient in mismatch repair due to mutations in hMSH2 or hMLH1 are
more resistant to cisplatin treatment than cells whose repair deficiency is
complemented by introduction of chromosome 3 (containing hMSH2) or
chromosome 2 (containing hMLH1) [1]. No difference is observed in the
level of cisplatin bound to DNA or in the rate of removal of cisplatin-DNA
adducts between these mismatch repair-deficient and -proficient cells [9].
Furthermore, cell lines deleted for the MSH2 gene demonstrate increased
resistance to cisplatin compared to wild-type cell lines in vitro and when
implanted in nude mice [17].
It is hypothesized that mismatch repair proteins sense the cisplatin-induced
damage and initiate a series of events that results in cell cycle arrest and death.
Two models have been proposed to explain the connection between the
mismatch repair pathway and sensitivity to DNA damage caused by anticancer
drugs such as cisplatin. In one model, DNA lesions are recognized and
processed by the mismatch repair pathway, but because mismatch repair
excises a tract from the newly incorporated strand, damage in the parental
strand is not removed. Repeated attempts at processing are proposed to create
persistent gaps that trigger cell cycle arrest or cell death. This model is
consistent with the observations that mismatch repair-proficient cells show a
reduced level of nascent DNA synthesis as measured by incorporation of
radiolabeled nucleotides into chromosomal DNA after treatment of cells with
cisplatin [56] or into cisplatinated plasmids after incubation in vitro with cell
extracts [14]. In addition, mismatch repair-proficient cells are less efficient
in reactivation of cisplatin-damaged plasmid DNA as measured by luciferase
activity from a cisplatin-damaged reporter plasmid [9]. It is possible that a
more direct signaling pathway links the mismatch repair pathway and
cisplatin cytotoxicity. Assembly of mismatch repair proteins on cisplatin-
DNA adducts, whose DNA structure is different from that of unpaired or
mispaired bases [55], could initiate a signaling cascade that leads to cell
death.
398 S. Ishida and I. Herskowitz
3 UNDERSTANDING MECHANISMS OF
CISPLATIN RESISTANCE IN THE BUDDING
YEAST SACCHAROMYCES CEREVISIAE
Three methods have been undertaken to identify yeast genes which could
potentially govern cisplatin resistance – searching for genes whose products
bind to platinated DNA and genes whose overexpression or loss of function
allows cells to grow in the presence of a toxic level of cisplatin (Table 1).
Chapter 15: Genetic Analysis of Cisplatin Resistance in Yeast and Mammals 399
both of which are not observed in the ctr1∆ mutant, indicating that the
effects of copper on reducing cisplatin uptake are mediated by the copper
transporter Ctr1p [26]. Interestingly, cisplatin treatment results in
delocalization and degradation of Ctr1p [26]. Taken together, these findings
support a hypothesis that the copper transporter Ctr1p mediates uptake of
cisplatin in yeast.
Mammals contain functional homologs of the yeast CTR1 gene, hCTR1
in humans and mCTR1 in mice, which complement a yeast ctr1 mutant for
intracellular copper deficiency [41, 58]. Mouse cell lines that are wild-type,
heterozygous, or homozygous for a knockout allele of mCTR1 display a
graded increase in cisplatin resistance and decrease in cisplatin accumulation
[26]: heterozygous cells are fourfold more resistant to cisplatin and exhibit a
35% decrease in cisplatin accumulation compared to wild-type cells;
homozygous cells show an eightfold increase in resistance and a 70%
decrease in drug accumulation. These observations in mice suggest that
hCtr1 in humans, which is 92% identical to mCtr1, may play a key role in
cisplatin uptake and resistance.
The mechanism of cisplatin uptake has been unclear. Inability to saturate
the rate of cisplatin uptake supports a simple diffusion model, whereas the
existence of agents and conditions that modulate cisplatin accumulation,
such as pH, osmolarity, sodium, potassium, protein kinase C (PKC), protein
kinase A (PKA), and the calcium/calmodulin pathway, suggests that some
component of cisplatin uptake is mediated by a transport mechanism [22]. It
has been proposed that about 50% of the intial rate of uptake is due to
passive diffusion and that the remaining 50% is due to facilitated diffusion
through an as yet unidentified gated channel. Ctr1p might be responsible for
the facilitated uptake of cisplatin. Cisplatin uptake is not completely
abolished in the ctr1∆ mutants in both yeast and mice [26]. The residual
cisplatin uptake observed in the ctr1∆ mutants might be due to diffusion
across the plasma membrane or to additional transport proteins.
Cisplatin is one of the most widely used anticancer drugs effective in the
treatment of a variety of cancers. Intrinsic or acquired resistance to cisplatin
reduces its efficacy, however, which limits its curative potential.
Considerable effort has been made to define the cellular and molecular
mechanisms responsible for cisplatin resistance in mammals. Most of the
mammalian genes governing cisplatin resistance were identified due to their
biochemical properties – cMOAT/MRP2 and ATP7B are involved in efflux
of drugs and metals, γ-glutamylcysteine synthetase, and metallothionein
increase intracellular thiols that react with cisplatin, and mismatch repair
proteins and HMG domain proteins bind to platinated DNA.
Genetic selections in yeast have identified a number of genes that affect
cisplatin resistance, most of which either have mammalian homologs or are
involved in functions that are also present in mammals. Genetic analyses of
404 S. Ishida and I. Herskowitz
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Chapter 15: Genetic Analysis of Cisplatin Resistance in Yeast and Mammals 407
1 INTRODUCTION
409
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 409–427.
© 2007 Springer.
410 A. T. Rogojina, Z. Li, K. C. Nitiss and J. L. Nitiss
2 HOW TOPOISOMERASE-TARGETING
DRUGS KILL CELLS: USING YEAST
FOR MECHANISM-BASED STUDIES
OF DRUG ACTION AND DRUG SPECIFICITY
3 RESISTANCE TO TOPOISOMERASE-
TARGETING DRUGS: IDENTIFYING CHANGES
IN TOPOISOMERASE PROTEINS THAT ALTER
DRUG SENSITIVITY
agents in yeast has made genome-wide screens more difficult for this class
of drugs. These two approaches are clearly complementary, and both
approaches have led to the identification of important pathways for repairing
topoisomerase-mediated DNA damage. This section will first discuss some
of the targeted approaches, and then summarize some of the interesting
results from genome-wide approaches.
The repair of topoisomerase-mediated DNA damage requires the
collaboration of several distinct DNA repair pathways. As shown in Figure
1, replication in the presence of Top1 poisons leads directly to double-strand
breaks. As expected, cells lacking double-strand break repair pathways such
as homologous recombination are hypersensitive to camptothecin [39]. The
repair of camptothecin-induced double-strand breaks includes recruitment
of the histone variant H2AX to the site of double-strand breaks, with
subsequent recruitment of other repair proteins [7, 53, 57]. Since the
generation of double-strand breaks following collisions with topoisomerase
II may require additional processing steps, the role of H2AX and other
signaling processes are poorly understood with topoisomerase II poisons.
Nonetheless, double-strand break repair pathways also play essential roles in
cell survival following exposure to topoisomerase II poisons. Interestingly,
the nonhomologous end-joining pathway of double-strand break repair is
important for cell survival following exposure to the clinically important
Top2 poison etoposide [31], which may relate to the genesis of translocations
leading to secondary cancers. By contrast, yeast cells lacking nonhomologous
end-joining are not hypersensitive to camptothecin (unpublished data),
consistent with results obtained with DT-40 (chicken) cells [1].
In addition for the need to repair double-strand breaks, there are other
aspects of topoisomerase/DNA covalent complexes that require other DNA
repair and stress tolerance pathways. For example, in addition to double-
strand break repair, recombination pathways and replication functions are
also required for restarting of blocked or collapsed replication forks [12, 37].
Topoisomerase: DNA covalent complexes have been shown to impede
replication fork progression, leading to a requirement for functions needed
for fork restart. Mutants have been isolated that are hypersensitive to Top1
poisons and are defective in functions that are important for lagging strand
DNA synthesis [59]. These results indicate that the aberrant DNA replication
that occurs in the presence of Top1 poisons results in an enhanced
requirement for specific DNA replication functions that may play roles in
restarting blocked or broken replication forks. In addition to replication
restart mechanisms, a variety of checkpoint functions are also needed to
ensure that compete and accurate completion of replication occurs. Mutants
defective in both S-phase and DNA damage checkpoints are hypersensitive
to camptothecin [70] (see below).
418 A. T. Rogojina, Z. Li, K. C. Nitiss and J. L. Nitiss
Figure 2. Pathways for the repair of Top2 mediated DNA damage. Following recognition of
Top2 covalent complexes (perhaps by collision with replication forks as shown in Figure 1),
repair can be initiated either by proteolysis or by nucleolytic processing. Proteolysis will not
completely remove the protein, since the phosphotyrosyl linkage to DNA cannot be removed
by proteases. Therefore, after proteolysis, a nucleolytic processing step is still required. The
product of nucleolytic processing is a DNA molecule containing a double-strand break.
ACKNOWLEDGMENTS
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429
430 Index
centromeres 1, 14, 20, 42, 248 etoposide 201, 316, 410, 412,
centromeric 2, 10, 245, 264, 266 415, 417, 419, 422
ceramidases 195, 198 exocyst 107
cerulenenin 293
chemotypes 322, 323 FKBPs 142, 144, 147, 151, 153,
chromatinized 240, 256, 260 154, 161–163, 347–350, 362,
cisplatin 319, 331, 393–404 365, 383–388
cMOAT 395, 399, 403 FOA 213–217, 246, 251, 256,
cohesins 14, 20, 42, 45 257, 265
counterselection 212, 213, 215, Fraumeni 19, 235
246 farnesylated 103, 105–110, 115,
cross-linking 330, 338 116
cyclin 3, 6, 7, 38–40, 42–46, 48, farnesylation 101–109, 115–116
49, 55, 57, 76, 88–92, 161, farnesyltransferase 101–105, 110,
165, 180, 316, 321, 323, 329, 115
330, 341 forkhead 356
cyclohexanone 329 fumonisins 195, 196
cyclophilin 151, 153, 154, 162,
387, 388 GeneChip 241, 249
cyclosome 44, 55, 89 GGTase 102–105
Gleevec 341
DnaJ 107–109, 158 GPCRs 203
DnaK 158 GTPase 46, 60, 80–84, 109, 110,
DSBs 13, 186, 187, 257, 315, 361, 363
319, 320, 322, 326, 327, 332, geldanamycin 143, 148, 164
333, 337, 411, 413, 414, 418, gemciytabine 201
421, 422 geminin 4
dideoxynucleotides 241 geranylgeranylation 102
dihydroceramidase 198 geranylgeranyltransferase
dihydroceramide 195 102
dimer 5, 144, 154, 155, 160, 234, glutamylcysteine 396, 399, 403
237, 239, 259, 358, glutamylcysteinyl 396
382–384, 399, 413 glycerophospholipid 197
dissociators 211–218, 220, 221,
223–226, 256 hyperrecombination 19
downregulated 18, 20, 90, 92, 257 helicase 5, 9, 11, 33, 87, 318, 320
downregulation 199 hematopoetic 125
dTOR 363 heterodimers 192, 195, 298, 318,
319
Euplotes 128, 129 hexadecanal 194
Exportin 79–81, 87 hnRNPs 76, 87, 88
ellipticine 336, 339 homodimeric 300, 411
enzymology 9 hTERT 129–132, 149
epipodophyllotoxin 332, 410 hus 183
Index 431
hydroxyurea 8, 10, 182, 183, 317, mAMSA 410, 412, 415, 419,
331 420
hyperacetylation 256 Microsatellite 316, 318
hyperphosphorylated 56, 60, 90 MRP 289, 292–296, 300, 302
hyperrecombinant 11 manumycin 105
mcm 4–6, 9, 12, 15–19, 33
imatinib 341 mec 51, 52
immunophilins 147, 151, 153, metabolomics 304
154, 156, 161, 164, 387 metastatic 20
immunosupressant 192 microarray 299, 376, 386–388
importin/karyopherin 78, 80, 81 microdomains 201
importins 78, 79, 81–83, 87, microsequence 128
89–91, 93, 357 minichromosome 4
inositolphosphoceramides 192, misexpression 91
197, 198 mislocalization 91, 110, 219
interactors 257 mislocalized 220
intercalaters 333 mispaired 396, 397
intercalators 327 mTOR 350, 351, 359–362, 364,
intergenic 2 365, 366
initiation 3, 4, 6–9, 14–16, 18, mulitdrug 114, 289, 340, 395,
33, 34, 39, 41, 47, 233, 399, 409, 410
352–354 multicellular 179, 181, 243
intrastrand 394, 396 multichaperone 156
intronic 239 mutator 12
myriocin 192, 195
KRPs 362
Kss/Fus 105 Nibrin 52, 53
karyopherins 78–83, 86, 87 nitroaromatic 331, 332
kinases 3, 4, 6–8, 10, 11, 14, nitrosoguanidine 183, 318
17–19, 33, 34, 38–40, 43, 44, noncomplementation 350
46, 47, 51–55, 57–64, 77, 78, nucleoporins 76, 77, 81, 85, 86
89–91, 105, 106, 142, 145,
147–149, 152, 159–162, 164, overexpression 18, 58, 114, 145,
165, 182, 184, 185, 194–197, 149, 153, 154, 159, 160, 186,
199, 203, 235, 236, 238, 240, 212, 219, 221, 245, 263, 287,
244, 258, 321–323, 341, 347, 291, 293, 295, 301, 321, 342,
351–354, 358–366, 382, 389, 361, 395, 396, 398, 400, 401,
399, 400, 402 404, 412
kinetochores 13, 42, 60, 61, 321 oncogene 17, 19, 78, 106, 161,
165, 199, 212, 233
LacZ 152, 309, 353, 383 orthologue 45, 53, 55, 56, 63,
LexA 152, 383, 386 165, 289
ligase 9, 12, 18, 44, 89, 183, 238 ovalicin 376–378
lysophospholipids 295 overexpress 321, 341
432 Index
p53 75, 126, 142, 145, 146, 148, RecQ 11, 318, 320
157, 165, 211–226, 233–268, RFC 8, 9, 14, 53, 54, 57, 59,
284–288, 379, 403 182
PDREs 297, 298, 300 Rheb 103, 107, 109, 110–116,
Pdr 289, 291–301, 304, 340 352, 363, 364
Pedicin 328 RhoA 102
PolII 355 Rnr 50, 51, 56, 58
Polymorhphism 286, 295 Rothmund 18, 19, 320
PxxP 236 Rpa 8, 9, 18, 54, 59, 319, 320
palmitaldehyde 194 rad 10, 19, 49–51, 64, 182, 183,
palmitoyltranferase 192 184, 185, 186
pastoris 291, 303 rDNA 10, 379
peptidomimetic 103, 104 recombinogenic 124
perfetto 264, 265 reductase 10, 15, 50, 56, 193,
phosphorylation 3, 4, 6–8, 11, 17, 329, 330, 386
20, 33, 40, 41, 43, 46, 48, reductional 15
51–57, 60, 61, 63, 78, 89–93, reinitiation 8, 47, 62
147, 157, 165, 180, 184, 185, relocalized 89
194, 201, 212, 218, 236, 238, replicative 5, 233
300, 351–354, 359, 360, 382 replication 7, 33
phytoceramidase 198 reveromycin 292
phytoceramide 195, 198 rhbA 364
polymerase, DNA 7–9, 12, 14, rheostatable 245, 253, 259, 261
15, 19, 47–49, 59, 125, 155, ribonucleotide 10, 15, 50, 56,
184, 185, 341 185, 329, 330
premeiotic 15, 16 rRNAs 86, 87, 355
prenyltransferases 102
preRC 3–5, 7, 8, 18 Schizosaccharomyces pombe
prereplicative 3, 33 1–20, 33, 39, 43, 52, 53, 56, 64,
primase 9, 54, 59 76, 80, 88, 91, 92, 103, 104,
processivity 9, 184 108–116, 154, 155, 157, 165,
proteasome 89, 90, 147, 238, 321 179, 181, 182, 184, 186, 195,
215, 257, 350
Rab 103 ScRheb 111, 113–115
Rac 103 SpRas 109
RAD52 51, 128, 129, 316, 320, SpRheb 111, 113–115
322, 332–339 securins 19, 42, 45, 46, 49, 56, 57
Raf 112, 113, 148, 160, 162 separase 42, 45, 49
Ral 107 separin 56
RanGAP 80, 82–84 sevenless 160
RanGDP 82–84, 86, 87, 90 signaling 46, 53–55, 60, 63, 64, 92,
RanGEF 80, 82, 84 141, 145–149, 151–153, 155,
RanGTP 82–87, 90 158–160, 181, 185, 186, 191,
Rapamycin 347–366 199, 201, 203, 241–243, 299,
Index 433
349, 351, 352, 353, 354–365, 220, 222, 223, 224, 239, 240,
382, 389, 397, 417 244, 250, 256, 261, 284, 286,
snRNPs 87 290, 297, 298, 354, 355, 361,
sphingomyelinases 195, 198 365, 366, 376, 379–383
spingholipid 299 triapine 329
splitomicin 379, 380 tribrid 382
sporulate 15 transactivation 213, 214, 217,
staurosporine 293 218, 222, 234–237, 239–242,
subcomplexes 5 244–253, 255–268, 284–287,
subtelomeric 359 382, 386
sumolation 236, 238, tumourigenesis 20, 124, 127, 129,
supertrans 253, 259, 268 130, 133, 141, 145, 155, 165
synaptobrevin 108 tumourigenic 123, 130, 131, 148,
synthase 195–198 165, 166
syringomycin 193, 195, 351–366 tumours 7, 17–20, 63, 75, 82, 88,
91, 103, 105, 113, 124, 127,
Tor 113, 347, 348, 350, 351, 352, 130, 131, 148, 155, 161, 165,
353, 354, 355, 356, 357, 358, 187, 203, 211, 221, 225, 226,
359, 360, 361, 362, 363, 364, 233–238, 240–245, 247–254,
365, 366 257, 259, 260, 262–264, 266–
TPRs 77, 142, 144, 147, 268, 290, 315, 316, 320, 322,
151–157, 161, 164 323, 329, 338, 340, 341, 363–
TRanscription/EXport 88 365, 379, 396, 404, 410, 422
Transportin 80, 395, 399
Trrap 351 Ub 142
taxanes 328 ubiquitin 4, 43–45, 48, 55, 57,
telangiectasia 18, 19, 53, 186, 351 89, 201, 212, 214–217, 238,
tetramerization 220, 222, 235, 319, 420
236, 255, 262, 263, 287 ubiquitinate 45
tetratricopeptide 144 ubiquitinated 321
thialysine 107, 113, 114 ubiquitination (Ubi) 41, 45, 46,
topoisomerase 315, 332–334, 49, 61, 108, 236, 418
337, 409–414, 416–418, ubiquitylation 202
420–423
topoisomerase I 327, 410–416 Xenopus 4, 6, 9, 38, 43, 44, 48,
topoisomerase II 327, 410, 412, 54, 91, 110, 162, 179, 181
413, 415, 417, 422 XPA 260, 319
tracrolimus 348 XPC 260
transactivate 249, 250, 263 xeroderma 18, 19, 316, 319
transactivational 158
transactivators 356, 358 YACs 124
transactive 250
transcriptional 50, 56, 58, 87, 92, zebrafish 110
127, 153, 155, 202, 217, 218,