Cellular Signalling 12 (2000) 515524

http://www.elsevier.com/locate/cellsig

Small G-protein networks

Their crosstalk and signal cascades
Takashi Matozaki, Hiroyuki Nakanishi, Yoshimi Takai*
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan
Received 26 March 2000; accepted 9 May 2000

Abstract
Small GTP-binding proteins (G-proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of
more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho/Rac/Cdc42, Rab, Sar1/
Arf, and Ran families. They play key roles not only in temporal but also in spatial determination of specific cell functions. It
has become clear that multiple small G-proteins form signalling cascades that are involved in various cellular functions, such as
budding processes of the yeast and regulation of the actin cytoskeleton in fibroblasts. In addition, two distinct small G-proteins
regulate specific cellular functions in a cooperative or antagonistic manner. A single small G-protein exerts various biological
responses through different downstream effectors. Moreover, some of these downstream effectors sequentially activate further
downstream effector proteins. Thus, small G-proteins appear to exert their functions through their mutual crosstalk and multiple
downstream effectors in a variety of cellular functions. 2000 Elsevier Science Inc. All rights reserved.
Keywords: Small G-proteins; Ras; Rho; Rab

1. Introduction
Small GTP-binding proteins (G-proteins) are monomeric G proteins with molecular masses of 2030 kDa.
The Ha-Ras and Ki-Ras genes were first discovered
as the v-Ha-Ras and v-Ki-Ras oncogenes of sarcoma
viruses around 1980 [1,2]. Their cellular oncogenes were
then identified in human and their mutations were furthermore found in some human carcinomas [38]. Now,
more than 100 small G-proteins have been identified in
eukaryotes from yeast to human and they comprise a
superfamily [9,10]. The members of this superfamily are
structurally classified into at least five families: the Ras,
Rho, Rab, Sar1/Arf, and Ran families (Table 1). The
functions of many small G-proteins have recently been
elucidated: the Ras subfamily members (Ras proteins)
of the Ras family mainly regulate gene expression; the
Rho/Rac/Cdc42 subfamily members (Rho/Rac/Cdc42
proteins) of the Rho family regulate both cytoskeletal
reorganization and gene expression; the Rab and Sar1/
Arf family members (Rab and Sar1/Arf proteins) regulate intracellular vesicle trafficking; and the Ran family
* Corresponding author. Tel.: 81-6-6879-3410; fax: 81-6-68793419.
E-mail address: ytakai@molbio.med.osaka-u.ac.jp (Y. Takai)

member(s) (Ran) regulates nucleocytoplasmic transport during the G1, S, and G2 phases of the cell cycle
and microtubule organization during the M phase.
According to the structures of small G-proteins, they
have two interconvertible forms: GDP-bound inactive
and GTP-bound active forms [9,10] (Fig. 1). An upstream signal stimulates the dissociation of GDP from
the GDP-bound form, which is followed by the binding
of GTP, eventually leading to the conformational change
of the downstream effector-binding region so that this
region interacts with the downstream effector(s). This
interaction causes the change of the functions of the
downstream effector(s). The GTP-bound form is converted by the action of the intrinsic GTPase activity to
the GDP-bound form which then releases the bound
downstream effector(s). Thus, small G-proteins regulate
a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions
and determine the periods of time for the continuation
of the specific cell functions (Fig. 2).
The rate-limiting step of the GDP/GTP exchange
reaction is the dissociation of GDP from the GDP-bound
form. This reaction is extremely slow and therefore stimulated by a regulator, named guanine nucleotide exchange
proteins (GEPs), [also called guanine nucleotide ex-

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Table 1
The small G-protein superfamily
Ras
family
Mammal

Yeast

Ha-Ras
Ki-Ras
N-Ras
R-Ras
M-Ras
RaIA
RaIB
Rap1A
Rap1B
Rap2A
Rap2B
Tc21
Rit
Rin
Rad
Kir/Gem
Ras1
Ras2
Rsr1
Ycr7

Rho
family
Rheb
B-Ras1
B-Ras2

RhoA
RhoB
RhoC
RhoD
RhoE/
Rnd3/
Rho8
RhoG
RhoH/
TTF
Rac1
Rac2
Rac3
Cdc42
Rnd1/
Rho6
Rho1
Rho2
Rho3
Rho4
Cdc42
Yns0

Rab
family
Rnd2/
Rho7
Tc10

change factor (GEF) or guanine nucleotide releasing

factor (GNRF1)], of which activity is often regulated
by an upstream signal. GEP first interacts with the GDPbound form and releases bound GDP to form a binary
complex of a small G-protein and GEP. Then, GEP in
this complex is replaced by GTP to form the GTPbound form. The GDP/GTP exchange reactions of Rho/
Rac/Cdc42 and Rab proteins are furthermore regulated
by another type of regulator, named Rho GDP dissociation inhibitors (GDI) and Rab GDI, respectively [11
13]. This type of regulator inhibits both the basal and
GEP-stimulated dissociation of GDP from the GDPbound form and keeps the small G-protein in the GDPbound form. It has also been proposed that Rho GDI
and Rab GDI are involved not only in the regulation
of their activation but also in their translocation between

Fig. 1. Regulation of small G-protein activity.

Rab1A
Rab1B
Rab2
Rab3A
Rab3B
Rab3C
Rab3D
Rab4
Rab5A
Rab5B
Rab5C
Rab6
Rab7
Rab8
Rab9
Rab10
Ypt1
Sec4
Ypt31/
Ypt8
Ypt32/
Ypt9
Ypt51/
Vps21

Rab11A
Rab11B
Rab12
Rab13
Rab14
Rab15
Rab16
Rab17
Rab18
Rab19
Rab20
Rab21
Rab22
Rab23
Rab24
Rab25
Ypt52
Ypt53
Ypt6
Ypt7
Ypt10
Ypt11

Rab26
Rab27A
Rab27B
Rab28
Rab29
Rab30
Rab31
Rab32
Rab33A
Rab33B

Sar1/Arf
family

Ran
family

Arf1
Arf2
Arf3
Arf4
Arf5
Arf6
Sar1a
Sar1b
Arl1
Arl2
Arl3
Arl4
Arl5
Arl6
Arl7
Ard1
Arf1
Arf2
Arf3
Sar1
Arl1
Arl2
Cin4

Ran

Gsp1
Gsp2

the cytosol and the membrane [1416]. Rho GDI and

Rab GDI show wider substrate specificity than GEPs
and GTPase-activating proteins (GAPs) and are active
on all Rho/Rac/Cdc42 and Rab proteins, respectively
[12,13,17,18]. Thus, the activation of Rho/Rac/Cdc42
and Rab proteins are regulated by positive and negative
regulators. Recently, Ran GDI, p10/NTF2, has also
been reported [19,20], but GDIs have not been identified for other small G-proteins. The GTPase activity of
each small G-protein is variable but relatively very slow
and is stimulated by GAPs. Most GAPs, such as Ras
GAP and Rab3 GAP, are specific for each member or
subfamily of small G-proteins [21], but some GAPs,
such as p190, a GAP active on Rho/Rac/Cdc42 proteins,
show wider substrate specificity [22].
In addition to the regulatory mechanisms for the activation or inactivation of small G-proteins, much atten-

Fig. 2. A role of small G-proteins as biotimers rahter than as molecular

switches. G, small G-protein.

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517

Fig. 3. Small G-protein cascade in control of polarized cell growth in the yeast S. cerevisiae.

tion has recently been paid to the crosstalk between

distinct small G-proteins and their downstream pathways. It has become clear that multiple small G-proteins
form a cascade which is involved in budding processes
of the yeast Saccharomyces cerevisiae (S. cerevisiae) and
in regulation of the actin cytoskeleton in fibroblasts.
Moreover, two distinct small G-proteins regulate specific cellular functions in either a cooperative or antagonistic manner. Finally, small G-proteins have recently been
shown to have multiple downstream effectors, some of
which transmit their signals through the cascades consisting of multiple proteins, such as protein kinases, to
elicit biological responses. In this review article, therefore,
we summarize the recent progress on crosstalk between
various small G-proteins and their signal cascades.

2. Small G-protein cascades

Multiple small G-proteins form a signal cascade and
thereby transduce their signals to the downstream effectors. For instance, in the yeast S. cerevisiae, Rsr1, a
member of the Ras family, is thought to be first activated
by an unknown cue that is produced by the previous budding site [23,24] (Fig. 3). In the next step, GTP-Rsr1
binds to Cdc24, a GEP for Cdc42, which in turn binds
GDP-Cdc42 to activate it [25]. The activation of Cdc42
induces not only reorganization of the actin cytoskeleton but also the recruitment of multiple small G-proteins, including Rho1 and Sec4, to the future budding
site. Sec4 would supply the bud with vesicles necessary
for bud enlargement [26], while Rho1 would induce
synthesis of the new cell wall component, 1,3--glucan,
by directly activating 1,3--glucan synthase and stimulating expression of the genes necessary for this synthesis [27,28]. Rho1 would also regulate reorganization of
the actin cytoskeleton necessary for the budding processes [2932].

Another typical example of small G-protein cascade

comprises Rho/Rac/Cdc42 proteins in mammalian cells
[33] (Fig. 4). Rho proteins regulate formation of stress
fibres and focal adhesions [34,35]; Rac proteins regulate
formation of lamellipodia and membrane ruffles [36];
and Cdc42 regulates formation of filopodia [36,37]. The
sequential activation of these three small G-proteins
by extracellular agonists has been best characterized in
quiescent Swiss 3T3 fibroblasts [36,38]. Agonists like
bradykinin activate Cdc42 in these cells to produce filopodia or microspikes. Activation of Cdc42 leads to localized activation of Rac proteins, hence filopodia are often
associated with lamellipodia, that is produced by Rac
proteins. In fact, it is hard to see filopodium formation
unless Rac activity is first inhibited. Quiescent Swiss
3T3 cells have no detectable stress fibres, and activation
of Rac proteins under these conditions leads to weak
and delayed activation of Rho proteins that produces
stress fibres. This typical cascade of Rho/Rac/Cdc42 proteins observed in Swiss 3T3 fibroblasts has not always
been seen in other cell lines. For instance, it has also
been shown that Rho proteins are inhibited by Rac/Cdc42
proteins in cultured cells such as neuroblastoma cells,

Fig. 4. Small G-protein cascade in control of cytoskeletal reorganization in Swiss 3T3 fibroblasts. Rho, Rho proteins; Rac, Rac proteins;
LPA, lysophosphatidic acid.

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N1E-115 cells, and NIH3T3 cells [39,40]. In addition,

Rho proteins may in turn inhibit the activity of Rac/
Cdc42 proteins in N1E-115 cells [41].
3. Crosstalk between small G-proteins
In addition to the sequential cascade of multiple small
G-proteins, a distinct family of small G-proteins regulates various cellular functions in a cooperative or antagonistic manner. Although a dominant active mutant of
Rho proteins does not cause transformation of fibroblasts, dominant negative mutants of Rho proteins inhibit the Ras protein-induced transformation [42]. Similarly, a dominant negative mutant of Rac/Cdc42 proteins
inhibits the Ras protein-induced transformation, while
a dominant active mutant of these Rac/Cdc42 proteins
enhances oncogenic Ras protein-triggered morphologic
transformation [43,44]. Thus, Rho/Rac/Cdc42 proteins
are involved in the Ras protein-induced transformation
in a cooperative manner.
Moreover, it has recently been clarified that Rho/
Rab proteins coordinately regulate cell adhesion and
migration of cultured MDCK cells [45]. 12-O-Tetradecanoylphorbol-13-acetate (TPA), that induces cell scattering of MDCK cells, induces early disassembly of
stress fibres and focal adhesions followed by their reassembly in MDCK cells. Expression of a dominant active
mutant of RhoA inhibits the TPA-induced disassembly
of stress fibres and focal adhesions, while microinjection
of C3, an exoenzyme of Clostridium botulinum, blocks
their reassembly. In addition, microinjection of C3 or a
dominant active mutant of RhoA inhibits the TPAinduced cell scattering. In contrast, expression of Rab
GDI or that of a dominant negative mutant of Rab5
attenuates either the TPA-induced reassembly of stress
fibres and focal adhesions and subsequent cell scattering
of MDCK cells [45]. Expression of a dominant active
mutant of Rac1 or Cdc42 inhibits the hepatocyte growth
factor (HGF)/scatter factor (SF)- or TPA-induced cell
scattering [46,47]. Therefore, during cell migration at
least Rho/Rac/Cdc42 proteins may regulate the reorganization of the actin cytoskeleton, while at least Rab5
may control the recycling of adhesion molecules such
as integrins by intracellular vesicle trafficking. The temporal and spatial activation and inactivation of these
two families of small G-proteins appear to be required
for reorganization of the actin cytoskeleton and cell
migration.
Substantial evidence has accumulated that most Rab
proteins regulate the targeting/docking/fusion processes, but that some of them regulate the budding process, which is mainly regulated by Sar1/Arf proteins
[4852] (Fig. 5). Depletion of Ypt31 and Ypt32 in the
yeast S. cerevisiae accumulates stacks of membranes that
resemble the typical Golgi cisternae of mammalian cells,
suggesting a role for these proteins in intra-Golgi trans-

Fig. 5. Intracellular vesicle trafficking. Rab, Rab proteins; Arf, Arf

proteins.

port and in the formation of transport vesicles at the exit

point of the Golgi apparatus [53]. Dominant negative
mutants of Rab1 and Rab9 appear to block the budding
of the COPI-coated vesicles from the endoplasmic reticulum and that of the clathrin-coated vesicles from the
late endosomes, respectively [54,55]. A complex of Rab5
and Rab GDI appears to be required for coat invagination and receptor sequestration in an in vitro assay
for clathrin-coated pit assembly [56]. Similarly, clathrincoated vesicles can recruit Rab5 [57]. However, it has
not been demonstrated that Rab proteins regulate the
assembly of the coat proteins. Currently, the possibility
can not be excluded that the effects of Rab proteins in
the budding process may be their unknown secondary
effect in the targeting/docking processes, because after
or during the bud formation, coat proteins are disassembled to produce uncoated vesicles. Before, during, or
after this uncoating process Rab proteins may be associated with the vesicles (Fig. 5). Consistently, Rab11BP/
Rabphilin-11 [58,59], a downstream effector of Rab11
mainly implicated in vesicle recycling [60,61], directly
interacts with mammalian Sec13 [62], of which yeast
counterpart is involved in the Sar1-induced vesicle coat
assembly in the yeast [63] (see below). This result indicates that Sar1/Arf proteins and Rab proteins cooperatively regulate budding/targeting/docking/fusion processes.
Arf6 functions either downstream of or in concert with
Rac1 to mediate cytoskeletal reorganization. EFA6, a
low molecular weight Arf GEP, induces cytoskeletal remodelling that is blocked by coexpression of a dominant
negative mutant of Arf6 or that of Rac1 [64]. In addition,
a dominant negative mutant of Arf6 inhibits growth
factor- and Rac1-mediated membrane ruffling [65].
Moreover, a deletion mutant of POR1/arfaptin-2, which
interacts with both Rac and Arf proteins, but not a
dominant-negative mutant of Rac1, inhibits Arf6-mediated cytoskeletal rearrangements [66]. An effector of
Arf6 that has been implicated in membrane ruffling is
phosphatidylinositol 4-phophate 5-kinase [67]. It translocates to ruffling membranes and produces phosphatidylinositol 4,5-bisphosphate (PIP2) synergistically by
Arf6 and phosphatidic acid, the production of which is

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519

Fig. 6. Dowstream cascades of small G-proteins. (a) Downstream cascade of Ras proteins; (b) downstream cascade of Rho proteins; (c) downstream
cascade of Cdc42. Ras, Ras proteins; Rho, Rho proteins.

catalyzed by phospholipase D. Because phospholipase

D itself is an effector of Arf proteins and PIP2 is an
activator of Arf proteins, it is tempting to speculate that
the local phospholipid metabolism that is regulated by
Arf6 may play a crucial role in membrane ruffling.
4. Downstream signal cascade and multiple effectors
of small G-proteins
Ras mediates its effects on cellular proliferation, at
least in part, by activation of a protein kinases cascade
consisting of Raf protein kinase (c-Raf-1, A-Raf, and
B-Raf), MEK [mitogen-activated protein (MAP) kinase
kinases 1 and 2], and MAP kinase (Fig. 6A). This is the
best example for the downstream cascade of a small
G-protein. A linear pathway where Ras functions downstream of receptor tyrosine kinase and upstream of a cascade of these serine/threonine kinases provides a complete link between the cell surface and the gene. Ras
proteins activate this protein kinase cascade by directly
binding to Raf proteins [68,69]. Raf proteins then phosphorylate and activate MEK [70], which then phosphorylates and activates MAP kinase [71,72]. The activated
MAP kinase translocates to the nucleus, where it phosphorylates and stimulates the activity of various transcription factors, including Elk-1 transcription factor [73].
In addition to Raf proteins, a variety of candidate Ras
protein effectors have been reported. These include
Ral GDS [74,75], RIN1 [76], and phosphatidylinositol
3-kinase (PI 3-kinase) [77]. AF6/Canoe is also suggested
to be a binding partner of Ras proteins [78,79], but this
result is claimed not to be reproduced [80]. It has recently been shown that Rap1 shows a much higher affinity to AF6 than Ras proteins do [81]. p120 Ras GAP may
participate in Ras protein-mediated gene expression,
although it is still unclear whether p120 Ras GAP is a

regulator, an effector, or both for Ras proteins. In contrast, activation of PI 3-kinase by Ras proteins may
promote cell survival [77,82]. However, it has not been
established whether these effector molecules other than
Raf proteins really play a role in the downstream pathway of Ras proteins.
Another example for the downstream cascade of a
small G-proteins is a Rho-ROCK-myosin phosphatase
pathway (Fig. 6B). p160ROCK, also named ROK/Rho
kinase, is a recently identified serine/threonine protein
kinase [8385] as one of the downstream effectors of
Rho proteins. The activity of this protein kinase is stimulated by GTP-Rho proteins. Many substrate proteins of
ROCK/Rho kinase have been identified: these include
the myosin-binding subunit of myosin light chain phosphatase [86], myosin light chain [87], ERM [88], and
cofilin [89]. Of these substrates, myosin light chain phosphatase appears to be a physiological substrate of ROCK/
Rho kinase [86]. GTPS, a non-hydrolyzable GTP analogue, increases the sensitivity of smooth muscle contraction to Ca2 [9092]. This action of GTPS is inhibited by C3 or an exoenzyme of Staphylococcus aureus,
named EDIN, which also inhibits the function of Rho
proteins, and mimicked by GTPS-RhoA, but not by
GDP-RhoA, indicating that Rho proteins are involved
in GTPS-induced Ca2 sensitization of smooth muscle
contraction [93]. Subsequently, myosin light chain phosphatase has been shown to be a downstream effector
of Rho proteins [94,95]. ROCK/Rho kinase has finally
been shown to phosphorylate and to inhibit myosin light
chain phosphatase, causing the sustained smooth muscle
contraction even after decrease in Ca2 concentrations
[86]. In this mode of action of Rho proteins, they do
not induce smooth muscle contraction without Ca2 triggering and they just modify Ca2 sensitivity of smooth
muscle contraction. In this sense, the physiological rele-

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vance of the ROCK/Rho-kinase-induced direct phosphorylation of the myosin light chain and the subsequent
activation of the myosin ATPase could not be considered [87]. The physiological relevance of the phosphorylation of other substrate proteins, including moesin, is
also controversial. ROCK/Rho kinase is shown to phosphorylate moesin that induces microvilli formation [88],
but another report indicates that ROCK/Rho kinase
has not been involved in this phosphorylation [96].
p140mDia, a mammalian homologue of Bni1 and
Bnr1 in the yeast S. cerevisiae [30,31] (see below) and
that of Drosophila diaphanous [97], has also been implicated in a downstream effector of Rho proteins [98].
mDia has formin homology (FH)1 and FH2 domains.
mDia as well as Bni1 and Bnr1 bind profilin via its FH1
domain to regulate reorganization of the actin cytoskeleton. Overexpression of mDia induces weak formation
of stress fibres without affecting the formation of focal
adhesions [99,100]. ROCK and mDia cooperatively regulate the Rho protein-induced reorganization of the
actin cytoskeleton [99,100] (Fig. 6B).
ROCK comprises another downstream cascade of
Rho proteins, a Rho-ROCK-LIM kinase pathway [89]
(Fig. 6B). ROCK directly phosphorylates LIM-kinase,
that in turn is activated to phosphorylate cofilin. Cofilin
exhibits actin-depolymerizing activity that is inhibited
as a result of its phosphorylation by LIM-kinase [89].
Overexpression of LIM-kinase induces the formation
of actin stress fibres in a ROCK-dependent manner.
Thus, phosphorylation of LIM-kinase by ROCK and
subsequent increased phosphorylation of cofilin by
LIM-kinase contribute to the Rho protein-induced reorganization of the actin cytoskeleton.
In addition to ROCK, a variety of effector proteins
of Rho have been discovered recently. These include
serine/threonine protein kinase PKN [101,102], citron
[103], rhotekin [104], and rhophilin [102]. Citron contains a protein kinase domain that is related to ROCK.
Citron kinase localizes to the cleavage furrow and midbody of cultured cells [105]. Overexpression of citron
mutants results in the production of multinucleate cells
and a kinase-active mutant causes abnormal contraction
during cytokinesis, suggesting that citron kinase is involved in the Rho protein-regulated cytokinesis [105].
In the budding yeast, Pkc1, a yeast homologue of
mammalian protein kinase C, was first elucidated to be
a downstream effector of Rho1 [106,107]. Pkc1 regulates
gene expression through the MAP kinase cascade, consisting of Bck1 (MAP kinase kinase kinase), Mkk1/Mkk2
(MAP kinase kinase), and Mpk1 (MAP kinase) [108,109].
This MAP kinase cascade regulates expression of the
genes necessary for cell wall integrity. Bni1 has been shown
to be another effector of Rho1 [31]. Bni1 has two domains, named FH1 and FH2, which are found in a variety of proteins involved in cell polarity and cytokinesis
through cytoskeletal rearrangement [110]. Bni1 binds

profilin, an actin monomer-binding protein, via its FH1

domain to regulate reorganization of the actin cytoskeleton [31,111]. Furthermore, Bni1 binds Aip3 (Bud6),
another actin-binding protein [112], and elongation factor 2, which is known to stimulate actin polymerization
[32], suggesting that Bni1 is the downstream effector of
Rho1 that directly regulates reorganization of the actin
cytoskeleton. More recently, Bni1 has also been shown
to participate in microtubule function, since disruption
of BNI1 causes defects in spindle orientation [113], Kar9
localization [114], and growth defect together with mutation either PAC1 and NIP100 [115], whose gene products are implicated in microtubule function [116].
1,3--Glucan synthase is the third effector of Rho1
in S. cerevisiae [27,117]. This enzyme synthesizes 1,3-glucan, a major component of the cell wall. This series
of experiments have first established that Rho proteins
regulate complicated cell functions through multiple effectors in a cooperative manner. Another protein having
FH domains has also been found in the yeast and named
Bnr1 [30]. This protein serves as an effector of Rho4
and binds both profilin and Aip3 (Bud6), indicating that
Rho4 regulates the actin cytoskeleton at least though
Bnr1. Rho2 has 53% identity to Rho1, and a rho2 null
mutant does not show any growth defect [118]. Hence
it has been assumed that Rho2 has redundant functions
with Rho1, although several lines of evidence have been
raised to suggest the Rho2-specific functions [119,120].
Rho3 and Rho4 are implicated in polarized growth,
presumably through regulating the actin cytoskeleton
via their interactions with Bni1 and Bnr1.
Most recently, N-WASP, a ubiquitously expressed
Cdc42-interacting protein [121], and the Arp2/3 complex, have been shown to participate in the downstream
cascade of Cdc42 for the Cdc42-induced actin polymerization [122124] (Fig. 6C). Wiskott-Aldrich syndrome
protein (WASP), which is only expressed in hematopoietic cells, was originally identified as a protein mutated
in patients with Wiskott-Aldrich syndrome [125]. WASP
and N-WASP [121] possess a pleckstrin homology (PH)
domain that binds PIP2, and a Cdc42/Rac interactive binding (CRIB) domain. The binding of both GTP-Cdc42
and PIP2 to N-WASP would activate N-WASP by stabilizing the active conformation of this molecule. The
C-terminus of N-WASP thereby binds the Arp2/3 complex, that generates new barbed ends by stimulating nucleation, and consequently stimulates its ability to nucleate actin polymerization in vitro [122,126]. Therefore, the
interaction of N-WASP with the Arp2/3 complex is a core
mechanism that directly connects the Cdc42-mediated
signal transduction pathway to the stimulation of actin
polymerization.
Several other potential effectors of Cdc42 and Rac
proteins have also been identified. Among them, the family of serine/threonine protein kinases known as PAKs
could be the most likely candidate. Homologues of the

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mammalian PAKs have been identified in S. cerevisiae

(Ste20 and Cla4) [127,128], S. pombe (Pak1, also called
Shk1) [129,130], Drosophila (PAK1) [131], and C. elegans
(Ste20) [132]. In the yeast S. cerevisiae, Cdc42 interacts
with Ste20, which in turn associates with Ste5 and Bem1,
both of which interact with actin [25,133]. Drosophila
PAK1 also plays a role in dorsal closure [131]. Thus, it
is likely that PAK proteins are involved in mediating
the effect of Cdc42/Rac proteins on the cytoskeleton.
However, in mammalian cells, the role of PAKs remains
unclear. Expression of a mutant form of Rac proteins
or Cdc42 that is unable to bind and activate PAKs can
still induce the formation of membrane ruffles and lamellipodia [134,135], indicating that PAKs are not essential for the Rac protein-elicited membrane ruffling
and lamellipodium formation or for the Cdc42-triggered
filopodium formation. This does not exclude the possibility, however, that PAKs themselves may play a role
in cytoskeletal rearrangements by inducing actin reorganization independently of Rac/Cdc42 proteins. Alternatively, PAKs may mediate effects on the cytoskeleton
induced by Rac/Cdc42 proteins, that are different from
the immediate actin reorganization [136,137].
In addition to the CRIB-containing proteins such as
PAK and WASP proteins, a 34-kDa protein, POR1 (partner of Rac), that interacts specifically with GTP-Rac proteins, is implicated to mediate the Rac protein-dependent
membrane ruffling [138]. A mutant Rac1 (Rac1V12L37)
that fails to bind POR also fails to induce membrane
ruffling [134]. Another protein with a potential role in
cytoskeletal organization is IQGAP [139,140]. IQGAP
interacts with both GTP-Rac1 and GTP-Cdc42 and localizes to membrane ruffles. IQGAP has been shown
to be localized in cellcell adhesion sites [141]. It has
been suggested that activated Cdc42 blocks the ability
of IQGAP to inhibit assembly of a cadherin-catenin
complex and thereby promotes formation of adherens
junctions [141], but this model has not been substantiated. Although IQGAP contains some interesting motifs found in signalling molecules, such as a WW domain,
a Src homology-3 (SH3) domain, a calmodulin-binding
domain, and, somewhat surprisingly, a Ras GAP-like
motif, its function remains to be established.
Downstream effectors have been isolated and characterized for several Rab proteins. The first effector to be
identified is Rabphilin-3 for the Rab3 subfamily members [142,143]. Rab3A plays a key regulatory role in
Ca2-dependent exocytosis, particularly neurotransmitter release from nerve terminals [144]. Rabphilin-3 is
expressed in neurons and localized on synaptic vesicles
[145]. It has not been established how Rabphilin-3 localizes on synaptic vesicles: one model is that it is recruited
to the vesicles by Rab3A by analogy with the Ras protein-Raf protein kinase system [146]; the other model
is that it is targeted to synaptic vesicles independently
of Rab3 [147149]. The N-terminal region constitutes

521

the Rab3-binding region, while the C-terminal region

contains two C2 domains that bind Ca2 and phospholipid [143,150,151]. Expression or microinjection of either
the N-terminal or the C-terminal region of Rabphilin-33
blocks Ca2-dependent exocytosis in several different
systems [152154]. Recently, it has been shown that
abnormalities of synaptic transmission and synaptic
plasticity, which are observed in Rab3A-deficient mice,
are not observed in Rabphilin-3-deficient mice [155].
This observation suggests that another effector of Rab3
proteins is present and compensates for loss of function
of Rabphilin-3 in the mice. Rim has also been identified
to be an effector of Rab3 proteins [156]. Rim has the
N-terminal Rab3A-binding domain that is homologous
to that of Rabphilin-3, the central PSD-95/Dlg-A/ZO-1
(PDZ) domain, and two C-terminal C2 domains that
are separated by alternatively spliced sequences. In contrast to Rab3A and Rabphilin-3, Rim is clearly absent
from synaptic vesicles but enriched on the presynaptic
plasma membrane, especially at the active zone. Although the precise function of Rim is still obscure, it
has been revealed from overexpression experiments of
its N-terminal fragment that Rim is implicated in neurotransmitter release.
Rabaptin-5 has been found in a yeast two-hybrid
screen using the GTPase-deficient mutant of Rab5 as a
bait [157]. Rabaptin-5 is recruited from the cytosol to
endosomal membranes by GTP-Rab5. In addition, its
overexpression induces the formation of large endosomes, as does overexpression of the GTPase-deficient
mutant of Rab5. Rabaptin-5 has the N-terminal coiledcoil region that serves as a self-interacting determinant
and the C-terminal Rab5-binding domain. Rabaptin-5 is
part of a large complex required for membrane docking/
fusion processes [158]. EEA1, another effector of Rab5,
is also included in this complex [158160]. This protein
may be a core protein of this complex and serve as a
tethering protein, because it alone could replace the
requirement for the cytosol in the early endosome fusion
assay [159]. EEA1 contains two spatially separate Rab5binding domains and the PIP3-binding FYVE finger at
the C-terminus [160]. Rabaptin-5 moreover interacts
with the N-terminal region of Rabphilin-3, and this interaction is inhibited by GTP-Rab3A [161]. Because
endocytosis is often coupled with exocytosis, particularly at nerve terminals, this Rabphilin-3Rabaptin-5
interaction may contribute to the coupling mechanism
between these two events.
Rabkinesin-6 is an effector of Rab6, and a member
of the kinesin family [162]. This protein displays a conventional kinesin structure with the N-terminal motor
domain, followed by a region predicted to form an
-helical coiled-coil stalk and a tail domain. Rab6 may
regulate microtubule-dependent retrograde transport
from the Golgi apparatus through Rabkinesin-6 [163].

522

T. Matozaki et al. / Cellular Signalling 12 (2000) 515524

Rab11BP/Rabphilin-11, an effector of Rab11, is also

involved in microtubule-based vesicle transport [58,59],
although it is not a motor protein. This protein contains
the proline-rich domain at the N-terminal half and the
WD40 repeats, which are important for the protein
protein interactions, at the C-terminal half. Furthermore, Rab11BP/Rabphilin-11 directly interacts with
mammalian Sec13 [62], of which yeast counterpart is
involved in the Sar1-induced vesicle coat assembly in
the yeast [63]. This result provides a physical connection
between a Rab protein and the structural molecule necessary for vesicle budding.

5. Concluding remarks
In the last few years, considerable progress has been
made toward understanding the signalling networks and
downstream pathways of small G-proteins. It has also
been shown that some of these signalling pathways of
small G-proteins are well conserved from yeast to mammals, implying that they play important roles in a variety
of fundamental cellular functions. In particular, many
effectors and downstream cascades of the Rho family
small G-proteins have recently been identified. However, it is largely unknown at molecular levels how extracellular signals activate these Rho family small G-proteins and how crosstalk and signalling in cascades among
small G-proteins are regulated. In addition, in contrast
to the Ras and Rho family small G-proteins, limited
knowledge is yet available regarding the effector proteins for the Rab and other small G-proteins. Further
efforts are clearly required to answer these issues.

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