THE JOURNAL OF BIOLOGICAL CHEMISTRY
4066-4075, 1995
© 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmental Regulation of Expression and Activity of Multiple
Forms of the Drosophila RAC Protein Kinase*
(Received for publication, September 9, 1994, and in revised form, November 22, 1994)
Mirjana Andjelkovic:j:, Pamela F. Jones:j:§, Ueli Grossniklausllll, Peter Cronj,
Alexander F. Schier1l**, Mathias Dickt :j::j:, Graeme Bilbe§§, and Brian A. Hemmingstjj
From the :j:Friedrich Miescher-Institut, P.O. Box 2543, CH-4002, Basel, Switzerland, lIBiozentrum der Unioersittit Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland, and §§Biotechnology, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland
We have characterized the Drosophila homologue of
the proto-oncogenic RAC protein kinase (DRAC·PK).
The DRAC-PK gene gives rise to two transcripts with the
same coding potential, generated by the use of two dif-
ferent polyadenylation signals. Each transcript encodes
two polypeptides because of the presence of a weaker
initiator ACG codon, upstream from the major AUG,
such that the larger protein contains an N·terminal ex-
tension. Like the human isoforms, DRAC-PKs possess a
novel signaling region, the pleckstrin homology domain.
DRAC-PK proteins have a similar expression pattern,
being regulated both maternally and zygotically, and
are expressed throughout Drosophila development.
Antisera specific for recombinant DRAC-PK and for its
C terminus detected two polypeptides of 66 and 85 kDa
in Drosophila extracts. The antirecombinant antisera
also recognized a polypeptide of 120 kDa from Drosoph-
ila, which apparently shared an epitope related to
DRAC-PK sequences. The role of p120 appears to be
restricted compared with that of DRAC-PK, since it was
not detected in larvae or adult flies. There was no spa-
tial restriction of DRAC-PK expression during embryo-
genesis, suggesting that localized activation might be a
regulatory mechanism for its function. DRAC-PK pos-
sesses an intrinsic kinase activity that is -8-fold higher
in adult flies than in 0-3·h embryos undergoing rapid
mitotic cycles.
Protein phosphorylation is involved in many developmental
processes in Drosophila melanogaster (1, 2). To date, at least 13
tyrosine-specific (3) and 25 serine/threonine-specific protein
kinases (3-11) have been identified in the genome of the fruit
fly. The fusion of both molecular biological and classical genetic
approaches affords a powerful method for the determination of
the developmental function of a given gene product. Some
kinases have been shown to have a very specific function in
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
The nucleotide sequencers) reported in this paper has been submitted
to the GenBank™ / EMBL Data Bank with accession numberis) X8351O.
§ Present address: Regeneron Pharmaceuticals, Inc., 777 Old Saw
Mill River Rd., Tarrytown, NY 10591.
11 Present address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold
Spring Harbor, NY 11724.
** Present address: Massachusetts General Hospital, Harvard
Medical School, East 4 13th Street, Bldg. 149, Charlestown, MA 02129.
11 Present address: Central Laboratory, Blood Transfusion Service,
Swiss Red Cross, and Institute of Biochemistry, University of Bern,
Freiestrasse 3, CH-3012 Bern, Switzerland.
1111 To whom correspondence should be addressed. Tel.: 061-697-40-
46; Fax: 061-697-39-76; E-mail: hemmings@fmi.ch.
4066
This is an Open Access article under the CC BY license.
Vol. 270, No.8, Issue of February 24, pp.
Printed in U.S.A.
Drosophila, such as sevenless, which plays a role in the differ-
entiation of the R7 photoreceptor cell during the development
of the eye (12), and torso, which is important in the terminal
system (2). In contrast, some display pleiotropic effects, since
they lie on signaling pathways that are active during different
developmental processes. Most of these are serine/threonine
protein kinases, for example fused (13), shaggy (14, 15), D-raf
(16), pelle (7), and cAMP-dependent protein kinase (17).
We previously reported the molecular cloning of a new sub-
family of the serine/threonine protein kinases, termed RAC
protein kinase (RAC-PK)l (18). Two closely related forms, RAC-
PKa and RAC-PKf3, have been identified (19), and phylogeneti-
cally they appear to represent the midpoint between protein
kinase C and cAMP-dependent protein kinase (protein kinase
A). Although the catalytic domain of RAC-PKs displays the
highest level of homology to the second messenger-regulated
kinases, they seem to have a different mode of regulation.
Members of the RAC-PK family contain a pleckstrin homology
(PH) domain whose role is probably similar to those of 8H2 and
8H3 domains, i.e. interaction with other signaling or cytoskel-
etal molecules (20-23). Unlike its closest relatives, protein
kinase C and protein kinase A, which are both non-oncogenic
protein kinases, RAC-PKa was identified as the cellular homo-
logue ofv-akt, an oncogene encoding a 105-kDa phosphoprotein
(24).
In order to gain insight into the possible functions of RAC-
PK, we have isolated and characterized the Drosophila homo-
logue of the RAC-PK gene, termed DRAC-PK. In our study we
demonstrate that the DRAC-PK gene encodes two polypeptides
whose expression is under both maternal and zygotic control.
Both proteins contain a PH domain and possess kinase activity.
During the preparation of this manuscript Franke et al. (25)
published a report on the molecular cloning of the Drosophila
homologue of RAC-PK t.DaktL).
MATERIALS AND METHODS
Isolation of eDNA and Genomic Clones Encoding DRAC-PK-A Dro-
sophila adult eDNA library in Agtll (kindly provided by Dr. B.
Hovemann of the Center for Molecular Biology, Heidelberg) and a
2-14-h embryo library in the Uni-ZAPl'M XR vector (Stratagene) were
screened as described previously (26). The EcoRI inserts obtained from
positive Agtll clones were subcloned into the pBluescript vector (Strat-
agene) for further analysis, whereas pBluescript plasmids were excised
from the AZAPII phage using R408 helper phage according to the
manufacturer's protocols.
1 The abbreviations used are: RAC-PK, RAC protein kinase; PH,
pleckstrin homology; DRAC-PK, Drosophila RAC protein kinase; bp,
base pair; SSC, standard saline citrate; PBS, phosphate-buffered saline;
TBS, Tris-buffered saline; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride;
MBP, myelin basic protein; PIPES, 1,4-piperazinediethanesulfonic
acid; kb, kilobase pairts).
 Drosophila Homologue of RAe Protein Kinase
Genomic clones were isolated from a Drosophila library (27) con-
structed in AEMBL4. Several clones were isolated and characterized by
restriction enzyme analysis. One of the clones, SDGL-DRAC 10 with a
12-kb-Iong EcoRI insert, contained the entire DRAC-PK gene. Frag-
ments of the SDGL-DRAC 10 were subcloned into the pBluescript
vector following digestion with EcoRI, BamHI, and cua. The DRAC-PK
gene was sequenced on both strands using overlapping restriction
subclones.
DNA Sequencing-----<:DNAs and genomic subclones were sequenced by
the dideoxynucleotide chain termination method (28) using Sequenase
version 2.0 (D. S. Biochemical Corp.). Sequencing reactions were
primed with either the universal or reverse sequencing primers or
specific oligonucleotides. DNA sequence analysis was performed using
the D niversity of Wisconsin Genetics Computer Group (GCG) software
package (29). Data base (GenBank™, EMBL, Swissprot, and GP)
searches were performed with Fasta and Tfasta programs (30).
Northern Blot Analysis-Total RNA was isolated from staged em-
bryos, larvae, and adult flies using the method of Chomczynski and
Sacchi (31). For Northern analysis 20 p.g of total RNA was fractionated
on a 0.8% formaldehyde/agarose gel and transferred to a nylon mem-
brane (Zeta-probe, Bio-Rad) according to the manufacturer's protocols.
Hybridization was performed overnight at 42°C in a solution contain-
ing 50% formamide, 5 x SSC, 5 x Denhardt's solution, 2% SDS, 0.05%
PP;, and 0.25 mg/ml herring sperm DNA, with probes that were radio-
labeled by random priming (32), followed by three 30-min washes at
60°C in 1 x SSC. Autoradiography was at -70°C with two intensifying
screens. Two probes were used for Northern analysis, the DRAC 7
eDNA and a specific probe located between the two putative polyade-
nylation sites at 4374-4379 and 5013-5018 that was prepared by PCR
amplification of this region using two oligonucleotide primers, 5'GG-
GAATTCTTATACCATGATAACGAAGG (EcoRI site underlined) and 3'
CCTCTAGACATAAAGCACAGCACATAGAG (XbaI site underlined).
Primer Extension-To map the 5'-end of the DRAC-PK gene two
antisense 27-base pair oligonucleotides (positions 551-577 and 2079-
2105) were used with polyt.A)" RNA prepared from an overnight Dro-
sophila embryo collection and from adult females. Primers were end-
labeled with [-y_ 32P1ATP (7000 Ci/mmol, ICN) by T4 polynucleotide
kinase. Labeled primer (2.5 X 105 cprn) was hybridized with 10 p.g of
polytA)" RNA at 65°C for 75 minutes, in 50 p.1 of a buffer containing 40
mM PIPES, pH 6.4, 400 mM NaCI, and 1 mM EDTA. After precipitation
with 0.3 M sodium acetate/ethanol, samples were resuspended in 20 p.1
of reverse transcriptase buffer (50 mM Tris-HCI, pH 8.5, 8 mM MgCI 2, 30
mv KCI, 2.5 mM dithiothreitol, 1 mM of each dNTP, 20 units of RNA sin,
50 ug/ml of actinomycin D, and 25 units of avian reverse transcriptase)
and incubated at 42°C for 2 h. Reactions were terminated by precipi-
tating with 0.3 M ammonium acetate/ethanol and analyzed on a se-
quencing gel. The size markers were sequencing reactions of the
DRAC-PK genomic subclone containing the promoter region, primed
with the same oligonucleotides.
In Situ Hybridization to Whole-mount Embryos and Ovaries-The
DRAC 7 eDNA was labeled with digoxygenin-dDTP (Boehringher
Manheim) by random priming (32). Embryos were prepared according
to the modified method of Tautz and Pfeifle (33). Fixation was per-
formed in 10% formaldehyde made in PBS (8 mM Na2HPO., 1.5 mM
NaH 2PO., pH 7.5, 140 mM NaCI, and 2.5 mM KC!) containing 50 mM
EGTA and an equal volume of heptane. After a 20-min incubation the
aqueous phase was removed, an equal volume of methanol was added,
and embryos were devitelinized by vigorous shaking. Ovaries were
dissected and fixed as described (34). The peritoneal sheaths were
removed from the ovaries for better accessibility to the ovarioles. Sub-
sequent steps were the same for both embryos and ovaries. They were
rehydrated in methanollPBST (PBS and 0.1 % Tween-20). Prehybridiza-
tion, hybridization, and detection were as described (33).
In Situ Hybridization to Polytene Chromosomes-To determine the
localization of the DRAC-PK gene on polytene chromosome squashes,
the DRAC 7 cDNA probe was biotinylated by nick-translation (35).
Hybridization was detected using the Vectastain Elite ABC kit (Vector
Laboratories) according to the manufacturer's protocols.
Preparation ofRecombinant DRAC-PK66 and DRAC-PK85-For the
preparation of the DRAC-PK66 expression construct an NdeI site was
introduced at the initiator ATG by PCR. Amplification was performed
using the 5'-oligonucleotide CCCATATGTCAATAAACACAACTTTCG
(NdeI site underlined) and a vector primer. The amplified product was
subcloned into pRK172 (36) as an NdeI-EcoRI fragment. To prepare
recombinant DRAC-PK85 the initiator ACG in the SDE-RAC 109 eDNA
was mutated into an ATG by PCR with the 5'-oligonucleotide
CCCATATGAATTACCTACAGTTTGTTC (NdeI site underlined) and a
vector primer and subcloned into the pRSET-A vector (Invitrogen) as an
4067
NdeI-XhoI fragment. The recombinant DRAC-PK proteins were ex-
pressed in Escherichia coli JM109 (DE3) (Promega),
Generation and Purification ofAntisera Specific for the Recombinant
Protein-Recombinant DRAC-PK66 was partially purified by sedimen-
tation and washing of inclusion bodies, followed by anion exchange
chromatography in the presence of 8 M urea. Initially rabbits were
immunized subcutaneously with -600 p.g of purified recombinant
DRAC-PK66 in complete Freund's adjuvant, followed by booster injec-
tions at 4-week intervals. Antisera (No. 36) was purified by protein A
chromatography (37), followed by "negative" purification on a column
prepared from bacterial extracts coupled to Affi-Gel 10/15 agarose (Bio-
Rad). Antirecombinant antisera were finally affinity-purified on poly-
vinylidene difluoride (PVDF) strips with bound recombinant DRAC-
PK66 protein. After overnight incubation at 4°C, the strips were
washed with a buffer containing 1 x TBS (50 mM Tris-HCI, pH 7.6, and
150 mM NaC!) 1% Triton X-100, and 0.5% Tween-20, followed by washes
in 1 X TBS and H 20 . Specific antibodies were eluted in a buffer
containing 50 mv glycine, pH 2.3, 500 mM NaCI, 0.02% bovine serum
albumin, and 0.2% Nonidet P-40, immediately neutralized with 100 mM
Tris-HCI, pH 8.0, dialyzed against 1 x TBS, and concentrated 5-fold.
Generation and Purification of Antisera Specific for the C-terminal
peptide of DRAC-PK85/66 and for the N-terminal Peptide of DRAC-
PK85-- Peptides No. 239 (602STSTSLASMQ611 with 4 alanine residues
introduced as a spacer at the N terminus) and No. 334 (3YLPFV-
LQRRSTVVASA 1 8 containing an alanine residue at the N terminus)
were coupled to keyhole limpet hemocyanin (38) and used to immunize
rabbits. Anti-C-terminal antisera (No. 41) were purified by precipita-
tion with 50% ammonium sulfate, followed by affinity chromatography
on a column prepared from peptide 239 coupled to Affi-Ge115 (Bio-Rad)
according to the manufacturer's instructions. Anti-N-terminal antisera
(No. 46) were negatively purified on PVDF strips containing bacterial
extracts, followed by affinity purification with recombinant DRAC-
PK85 immobilized on PVDF strips.
Transient Transfection of COS-l Cells-The eDNA clone DRAC 7
was subcloned in both orientations into the EcoRI site of the mamma-
lian expression vector pECE (39). Constructs were designated as pECE.
DRAC 7a (for the antisense orientation) and pECE.DRAC 7s (for the
sense orientation). The cDNAs SDE-RAC 109 and SDE-RAC 105 were
released from pBluescript as EcoRI-XhoI fragments and ligated into the
pECE vector digested with EcoRI and XbaI together with an oligonu-
cleotide adapter containing XhoI-EcoRI-XbaI restriction sites. The
pECE.SDE-RAC 109.ATG construct was prepared by mutating the
initiator ACG into an ATG by PCR, using the 5'-oligonucleotide
CCCAAGCTTCAACATGAATTACCTACCGTTTGT (HindIII site under-
lined) and a vector primer, which allowed subcloning into the pECE
vector following digestion with HindIII and Kpn!. COS-1 cells were
maintained in 10-cm dishes in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.), at
37°C, and in an atmosphere with 6% CO 2, COS-1 cells at 70-80%
confluency were transfected 24-48 h after plating by
a DEAE-dextran method (40). Cells were harvested 48-60 h after
transfection.
Western Blot Analysis-Whole cells and Drosophila adults and em-
bryos were lysed by 15 strokes in a Dounce homogenizer in a buffer
containing 50 mM Tris-HCI, pH 7.6, 120 mM NaCI, 1% Nonidet P-40, 1
mM EDTA, 1 mM EGTA, 25 mM NaF, 25 mM l3-glycerol phosphate, 15
mM PP" 30 mM p-nitrophenyl phosphate, 1 mM sodium vanadate, 2
ug/ml leupoptin, 2 p.g/ml aprotinin, 2 ug/ml pepstatin A, 1 mM phenyl-
methylsulfonyl fluoride, and 1 mM benzamidine. Homogenates were
centrifuged at 4 "C once (for cell extracts) or twice (for Drosophila
extracts) for 15 min at 15,000 x g. Proteins were separated by 7.5%
SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to
PVDF-Immobilon membranes (Millipore Corp.). The filters were
blocked for 1 h with 5% non-fat dry milk in 1 X TBS, 1% Triton X-100,
and 0.5% Tween-20 and then incubated for 2 h with affinity-purified
anti-DRAC-PK antibodies diluted 10-fold in the same blocking solution.
After three washes in blocking buffer membranes were incubated for 1
h with 0.1 p.Ci/ml of F 25-labeled anti-rabbit IgGs from donkey (Amer-
sham Corp.). Specific bands were detected by autoradiography after
exposure at -70°C with two intensifying screens. For quantification
blots were exposed to a PhosphorImager screen (Molecular Dynamics).
Apparent molecular masses were determined using molecular size stand-
ards from Bio-Rad.
Immunoprecipitation and in Vitro Kinase Assay-Freshly prepared
Drosophila extracts (see above) were used to immunoprecipitate active
DRAC-PK. Protein concentration was adjusted to 1 mg/ml. Extracts
were precleared twice with Y20 volume Pansorbin (Calbiochem) and VIo
volume Sepharose 4B (Pharrnacia Biotech Inc.), Protein A-Sepharose,
4068 Drosophila Homologue of RAe Protein Kinase
DRAC7
SDE-RAC 109
SDE-RAC 105
lkb
FIG. 1. Structure of the DRAC-PK gene. The restriction map of
the genomic clone SDGL-DRAC 10 is presented, and the DRAC-PK
gene is shown as a thick bar. Exons are represented as boxes. Filled and
open areas are coding and noncoding sequences, respectively. Initiator
codons are shown as filled inverted triangles for the ATG, and open
triangles for the ACG. The two polyadenylation signals are indicated by
arrows. The cDNAs DRAC 7 (from an adult fly library) and SDE-RAC
109 and 105 (from a Drosophila embryo library) are drawn below. Note
that the eDNA SDE-RAC 105 contains at the 5'-end 45 nucleotides
derived from the intronic sequence. E, EcoRI; B, Bam HI; C, Cla; S,
Saell.
washed extensively with lysis buffer, was incubated with affinity-purified
anti-DRAC-PK antibody No. 36 overnight at 4 DC and then washed ex-
tensively. Precleared extracts were added to antibody-saturated protein
A-Sepharose and incubated for 3 h on ice with occasional mixing. Pelleted
beads were washed extensively with lysis buffer (containing 1 M Nafll),
followed by a wash with kinase buffer (50 mM Tris-HCl, pH 7.5, and 1 mM
dithiothreitol) and resuspended in 30 ,.,J of the same buffer. In vitro kinase
assays were performed for 30 min at 30 DC in a reaction volume of 50 ,.,J
containing 30 J.Ll of immunoprecipitate, 0.5 mg/ml myelin basic protein
(MBP) (Sigma), 10 mM MgC12' 1 i.tM protein kinase A inhibitor peptide
(Peninsula Laboratories, Inc.), and 50 i.tM [y-32PlATP (1600 cpm/pmol).
The reaction was stopped by the addition of SDS sample buffer. Samples
were resolved by 13.5% SDS-PAGE, and phosphorylated MBP was visu-
alized by autoradiography. To determine the DRAC-PK-specific activity,
phosphorylated bands were excised, and incorporated phosphate was
measured by scintillation spectrophotometry.
Immunolocalization Studies-Drosophila embryos were fixed and
devitelinized as described above. After rehydration they were blocked
for 30 min with 1% bovine serum albumin in PBST and incubated
overnight at 4 DC with affinity-purified antibody No. 36. Biotinylated
anti-rabbit IgGs (Vector Laboratories) were preadsorbed with fixed
embryos to reduce the background. Detection was performed with the
Vectastain Elite ABC kit (Vector Laboratories) according to the manu-
facturer's protocols.
RESULTS
Structure of the Drosophila RAC-PK Gene-Initially we used
the RAC-PKa cDNA (18) to isolate clones that encode the
Drosophila homologue. Subsequently DRAC-PK cDNAs were
used to screen adult and embryo eDNA libraries. Several
cDNAs were isolated and partially sequenced (Fig. 1). The
DRAC 7 and the SDE-RAC 109 clones were sequenced com-
pletely. In parallel we isolated a genomic clone, SDGL-DRAC
10, that contains the entire DRAC-PK gene. Restriction, South-
ern blot, and sequence analyses showed that the DRAC-PK
gene spans a ~6-kb region and has seven exons (Fig. 1).
Sequence Analysis ofDRAC-PK-The nucleotide sequence of
the DRAC-PK gene and predicted amino acid sequence are
presented in Fig. 2. A sequence ATCAGTT, which fits well to
the consensus for transcription initiation in Drosophila (AT-
CA(GIT)T(CIT) (41» was found in the promoter region, about 20
nucleotides from the 5' -end of eDNA SDE-RAC 109 (nucleotide
389). However, this sequence is not preceded by a typical TATA
box, and the first one is present -260 nucleotides upstream
from the putative transcription initiation site. Analysis of the
promoter region by primer extension using an oligonucleotide
that hybridizes -185 nucleotides downstream of the 5' -end of
the SDE-RAC 109 eDNA revealed that transcription of the
DRAC-PK gene initiates at four major sites (nucleotides 367,
378, 417, and 432) that all mapped in close proximity to the
SDE-RAC 109 start site (Fig. 3). The same pattern was ob-
served with polyt.A)" RNA from both embryos and adult fe-
males, whereas the signal could not be detected in a control
reaction in which tRNA was used (Fig. 3). A similar result was
obtained upon extension of a primer that hybridizes at the
5'-end of the second exon (data not shown).
The first, noncoding exon, which is located 1.2 kb upstream
of exon 2, contains opa repeats, which are also present in
several developmentally regulated Drosophila genes (42). The
remaining six exons are coding and are separated by five small
introns (60-70 base pairs long). The SDE-RAC 105 eDNA
(which contains exons 2-7) has at its 5'-end 45 nucleotides
derived from the 3'-end of the 1.2-kb-Iong intron and probably
represents a splicing intermediate (see Fig. 1).
The last exon encodes multiple polyadenylation signals.
Analysis of the 3'-end of DRAC-PK cDNAs isolated from a
Drosophila embryo library revealed that two of these are used
(at positions 4374-4379 and 5590-5595). Also, four potential
mRNA AUUUA destabilization signals (43, 44) are found in the
3'-untranslated region between the two polyadenylation sig-
nals. The same AUUUA sequences are present in the 3'-un-
translated region of both human RAC-PKs, at positions 1709
and 1849 in the a isoform (18), and at position 1800 in the f3
isoform sequence (19).
A putative initiation codon (AACCATG) in the correct con-
text for translation initiation (45) was found in the second exon.
The predicted open reading frame encodes a 530-amino acid
polypeptide highly homologous to human RAC-PKa and -{3,
with a predicted molecular mass of 59.9 kDa and an isoelectric
point of 5.7. This reading frame remained open almost to the
5'-end of the second exon, but no upstream in-frame initiator
codons could be found. Expression of the eDNA DRAC 7 in
COS-1 cells produced a protein of the expected size (-66 kDa),
but expression of the longer cDNAs SDE-RAC 109 and 105
revealed the presence of a higher molecular weight form ( ~85
kDa) besides the major 66-kDa protein (see Fig. 7A). These
data suggested the existence of an upstream weaker initiation
codon near the 5'-end of the second exon. Analysis of the
genomic and eDNA sequences suggested that the putative ini-
tiator codon was an ACG preceded by CAAC, a sequence com-
patible with the consensus for translation initiation in Dro-
sophila (C/A)AA(C/A)(45». The open reading frame that starts
from this upstream initiation codon translates into a 611-
amino acid-long polypeptide, with a predicted molecular mass
68.5 kDa and an isoelectric point of 6.2. The apparent molecu-
lar mass on SDS-PAGE (~85 kDa) is higher than the predicted
molecular mass, which could be explained by a high proline
content (11%) in the N-terminal extension of the larger
DRAC-PK polypeptide. The two protein forms were therefore
termed DRAC-PK66 and DRAC-PK85, according to their ap-
parent molecular masses on SDS-PAGE.
The deduced amino acid sequences of the DRAC-PK proteins
possess all conserved motifs of serine/threonine protein kinases
(46). The motif -Gly-X-Gly-X-X-Gly-, residues 273-278 in the
DRAC-PK85 sequence (Fig. 4), with a Lys residue at position
295 conformed exactly to a consensus ATP binding motif
(47). Two motifs, -Asp-Leu-Lys-Leu-Glu-Asn- and -Gly-Thr-
Pro-Glu-Tyr-Leu-Ala-Pro-Glu-, which confer serine/threonine
 Drosophila Homologue of RAe Protein Kinase 4069
2971 TGCTCGGCMGGGCACC'I"I"i'GatAAGG'M'ATM"I'G'l'GTCGcGACAAcocTACCGCCAAcCrcrACGCGATcAAAA'M'CtTAAAMGGAAG
272 LGKGTFGKVILCREKATAKLYAIKILKKEV

3061 TI'ATTATCciAAAGGACGAGGTGoccCATACCCTGACCcAGAGTC~GTCciCAAATCATCCG'M'CCTCATI-gtaagtattg
302 IIQKDEVAHTLTESRVLKSTNHPFLI

)151 c tagtaa ta t.e t ee t t taeg t taaaaaaetaa tatll tgt~t teee tg t t t tgcagTCACTcAAATATTCGTTrcAAACCAACGACCGCCT

271 CGCG'M'GTACAGCTCTl'.v.ATAA~~CCGTAACAGTCocCrAACTA'I"CGCrMMCAooTccc.v.CGCOO 328 SLKYSFQTNDRL

J61
• . • .'09• . . . * ..* ..
ATCGAATCAGT.n\TCGAGCCAG'I"I'ATCGAAAAAAAG'I'GAAGCGI'AAAAACTAAACGGTACTTGGAAAA'ITI'AC'I"M'GCCTGTGTGCGTGC 3241 G'I'G'I"ITI'G'l'GATGCAGTACGTGAACGGTGGccAGC'l'CT'l"i-rcTITAAGCCACGUCGcATATITAciGAGGATCGAACACG'l"M'CTA
340 CFVMQYVNGGELFWHLSHERIFTEDRTRFY

3331 TGGGGCAGAGATCA~TA'I'CTGCATIci.CAGGGCATAiTTTATCGCciCTI'AAAc>cTGcAAAA~AcAi
370 GAEl I SALG'fLHSQGI IYRDLKLENLLLDK

3421 AGATGGTCACATAAAGGTCGcAGAC'I"I'C~GcAGGACATCACCTACOOCCGcACAACGAAiACCTI'C'l'GCGcTACACCcci
631 .CAr.r.GCA9:.r.r•• ceAC.ArA'TA1'Gi.Aoc.nCA.CeAC!.CACACGcACAATAATMGM.GGC~CGTrGTCGCTGCCGTCA 400 DGHIKVADFGLCKEDITYGRTTKTFCGTPE

721 CCATCTMAGc.u.uTACCMCGAGTAGt:w.AACAAACcGCAGTGCAGrAGGGAAAGC.v..v.GTC~ACAAAACTAc9 3511 ATACCTAceTccGGAAGTATtAGATGAcMTGATTA'l'GGCcMGCGG'l'GGAC'l'GG'l'GGGGcACTGGC~TGTACGAGATGgtgag

430 Y LAP E V L D D N 0 Y G Q A V D W W G T G V V M Y E M
811 tgggt tet tgta t t t t tCIl~C4gCIl t t t C~IlCallt t tcc~t t tccacgll~gtgat t tga~a tgt tgcgctgc taggtC!;I'~Cgctgccca~
3601
458
901 Ilcacactcg~llcactcggtggttcactca~tC6gccact~llcaCCCaCIl~gccllgIlcll~tcacacgcll~IlCgqCcagll~llctcacacat
3691 TCATGATGtGcTcn-rACATrcA'I'ITI'GG'i'GGAGGAGGTAA.uTTCCccCcTAATA'I'CACAGATGAGGCGAAAAA'I'C'J'G'i.rAGCAGGCCT
991 ecectcececccet.ececct t tgt~cag~cllcgcgtillc tgtc tgtctc~ctccccct.cacecccecect c tggcctca~tcgcact t tg 469 HDVLFTLILVEEVKFPRNITDEAKNLLAGL

1 081 calltgcagc~c tgtc es ec tgactgcctcgg tg tg-eg IIg~gcctgtcCg"~t ta tgcctgtg tg ec tat t t totega t t t t ttl. t t t ta tgt 3781 TCTAGCTAAGc.ATCCCAAAi.AOCG'ITI'GGGAGGTGGAAAGcA.TGA'l'G'Ici.AGGAGATAcAAGCACATCCAT'I'CT'M'GCcAGTA'M'AACTG
499 L A II. 0 P K K R L G G G K D D V K E I Q A H P F F A SIN W

1171 acat.t, tgt. t lllCllgtt t.tll~gatllgatag~gacatttgt~cagcggcgg~cat(;cgtcgt

t gacatt t t~gcaaattgt~aatgaatatg · . . . . . . . .
3 871 GACAGATCTAGTGT'l'GAAAAAAgtagggeaeagt eeet; c tgt cagt t.aectat tcg ta t ata t t aa tcgaa t t taaaa tge t taccgtag
529 T D L V L K K
1261 ct t t tgct t t tgcag t tgtgccagtg tgag tgcg agta~agtgcgag tgeegce tgeg~gteeeetgt~tg tg t aea t~ tg ta ta tgt~
3961 ATACCGCCGCC'ITI'CAAocCrcAGGTGAcATCCGATACOOATACAAGGTACTTCGACAAGGAG'M'TACcGcAGAGAG'J'G'ioGGAG'I'I'GACG
13 51 tgta t t.e tat atgca tata~ataagtt eatgu.eagteg~cgeegtega~t tgt t t tga t t t.ee t t teg~ge t aaa tge~ategegaac~ 536 I P P P F K P Q' V T S O T 0 T R Y F D K E F T G E S V E L T

· . . . . . - . .
4 051 CCGCCGGATCCCACTGGCCCG'M'GGGCTCCATAGCCGAAGAGCCGCTI"I"l'CCCGCAGgtaagateta'lt tgaaaaeat t taaagtggggt
566 P PDP T G P L G S I A E E P L F P Q
1531 eetaaatetgeatgtggge~agtgaetaagaateeaate~eataceatg~eaeaeeata~eaaeeeate~aattcgata~aatcagaat~
4141 tctg t a tgetea t.ceee t t t t tgg t t tca~egecacagM-cAGCTACCAiGGAGACATGGcCTCCACG'I"i'GoocACCTCGrcGCACATI'A
595 F S Y Q G D MAS T L G T S S HIS
16 21 acagtcctc~a teeaat eeeeeccce t tetggegaegga~atgccgeaa~ tgt t t tgca~eage tgetg~cgeaagege~aaetgeat t t
· . . . . . . . .
4231 GTACTTCCACAAGTCTCGCATCGATGCAATAGAACMGTTTACAAAC'lTM'ACCAGCACACATCATGT'l'CTGC'M'CATCACCTA'M'ACTA
1711 gtcataaae~t!ileaaaatg~eaa taacaa t tgt t act at ~agcgtgagataggcgaatgtgggaagtat~t
a tc tgga t~gacttggcag 603 T S T S LAS M Q

· . . . . . . . .
1801 cc t t t tagtg t ctgt t tceeet t t t t tg a tagctagetea t t tgggaae teca t t tgtecaet t tgaetagt t tatgtcgaeagt ecsec

· . . . . . . . .
1 B91 gat eag t ta~ ta te tagta~atg t tg aa t ccc tgaaggt~aaggtg t ta~ t agaegaae~e t etgc eeeeccc t t t t tct t t tcatcae t 4 411 AAAGGGTT'M'CTTGCTAG'M"l'ATATACACCAATAA TAAGAGAATTGAATTGA TAGTAACTTAT'l'TTTGTATAATGAGAAAGGGAACG'M'G

· . . . . . . . .
1981 egea t tet t ~g t t taagta t t tcaaaete~gea tgaa tataaagg at tc~ctgtaeteetet taca~CCTCA'M'CGCTrGTCCCTAG 4501 AACGAAAGGGCAACAGCCGAAGCCGAGGAAATA.U:.I.:I:ACTAAATATGATTATACACATATATATAAATATATATATATATATATATMTT

· . . . . . . . .
2071 GG'I"I'AG'I'GAi.ACAGCCTCAiCAcGAATTACCTAccG'M"l'GTTc'M'CAocGocGATCGAcTaToo-rrocCAGCGCCCCAGCGCCAGGATeT 4591 ATATATACAACTAAATATMCACTTGGGGCATAGCGCT'I'GAAG'I'GATTAGTfAA'M'TTAMCGGAAGGAAAACAI:l:rAACCMGCGACTT
1 KNYLPFVLQRRSTVVASAPAPGS
· . . . . . . . .
AGAGCGAC'ITAGTGCTAGGATAAAGCATGCATCATCGGATCGCATCCTCTAGGGGTCGCTI'AAGCTGA'I"rGA'M'ATACAGATTA'M'CACG
2161 GCCTCTAccATCCCGGAATCcccCACCACCACAGGGAocM.CATCATTAACATCATCTACAGCCAATCci.CCCATCCAAACAGCAGCCCC
24 A SRI PES P T T T G S N I I N I I Y S Q S T H P N SSP · . . . . . . . .
4771 CAGACAGCACTCACACAGCCACTTAGCATATAGCGAACATGAAG'M'TAGTTMC'I'TGACAGCAACAAATCGAAATACACATAT'I"CAACAA

2251 ACCAGCGGci.GTGCAGAeW'M'CAocroGcAGCAA'l'CciGcccAAOCAGGACCTCCACGccCCCTACACACGA'M'CCGGAACCA'l'GTCA · . . . . . . . .
54 T S G S A E K F S W Q Q S W P S R T S T A P T H D S G T )I S 4861 CCGCCCCCACATGCAACACTCGTATATATAAATTA:I:I::I:A.TACTCCAAGAGCAAAAAGAAA,CAGAACAATTACAAGTTATATATAATGTI'A

· . . . . . . . . · . . . . . . . -
2341 ATAAACACAAC'M"l'CGACCTCAGCTCGCCCAGCGTTACATCGGGTCATGCGC'M'ACGGAACAGACTCAGGTCGTAAAGGAGGGGTGGCTG 4 9 51 AATGCTCTATGTGC'l'GTGCT'M'ATGCAAAACTACAACAACAGAAACAAACAAACTAATAG'M'AATAAACGAGGGAAACATACATCAATGC
84 I N T T F D L SSP S V T S G HAL T E Q T Q V V KEG W L
· . . . . . . . .
5041 AGTTTATAGGCGCCAATAATTAGCAAGCAACTAAAAGTTATATTCAGGCAGAAACATAT'M'CGAAACAATAACGGCAACAGCAAAATGAT
2431 ATGAAGAGGGccGAGCACATAAAGAAC'I'GGcGCCAGCacTAC'M'CGTGciccACTCCGATcaAAGACTGATGGGCTACCGcAOCAAGCCG
114 M K R G E H I K N W R Q R Y F V L H S D G R L M G Y R S K P · . . . . . . . .
5131 TACAA'ITGACGCCA'ITIT1'TTATTATAA'M"ITITI'AGCAAAAATATATATTTGTAAATAmA,TAAAAAAAAAAGCGCAAAAAAAAAAAA
· . . . . . . . .
2521 GCAGATAGTGCCAGCACGCCATCGGAC'M'CCTGCTCAACAAC'M"I'ACGGTGCGCGGCTGCCAGA'I'CATGACCGI'CGATCGGCCCAAGCCA · . . . . . . . .
144 ADS A 5 T P S D F L L N N F T V R G C Q I M T V D R P K P 5221 CAAAGGATGTATGTGTCGTGTGGAAAATGGAAG'I'CACCAT'I"I'GAGGCCACAGAAAAGGTITAACC'ITI'GACACMTAMAAATGTATCTI'

· . . . . . . . . · . . . . . . . .
2611 'M'CACCTI'CATl::ATCCGCGGCCTGCAATGGACCACGGTAATCGAAAGGACATTTGCCGTCGAATCAGAATTAGAGCOCCAGCAGTGGACG 5311 TGTCATTI"CA'l'TT'T'I'GGCA'M'GAAAAGTTCATAACAAGTTTTGTATACTTAGAGCAAGTCAGCGAAAMTATATATAAATCAACCAACAC
114 F T F I I R G L Q W T T V I E RTF A V ESE L E R Q Q W T

5401
· . . . . ..
AGACTTACAAATAG'!'TCCAAAAGGAAGA'I'GTAAAAATTATATAAACAGAAAAAAAACCTAAAAGAAAATAAAGAA'M'TTl'GATATTI"ITA
. .
2701 GAGGCCA-rrCGCAACGTATCCAGCCGGC'I'CATAGA'I'G'I'GGcTGAGGTGGCTATGACoccATCTGAACAcACAGACATGACAGACGTGGAC
204 E A I R N V S S R L I D V G E V A M T P SEQ T D M T D V D · . . . . . . . .
5491 TATGCGCAATAAAAACAAAAATCAAGAATAAAACGAAACAAAAAGTTA'MTl'ATA'M"I'CGTAAAGAAAACAAAATAAATATI'GTAMTAA

2791 ATGGCCAccA'I"l'GCCGAooACGAGC'l'I"I'CCGAGCAG'I"I'CTcCGTACAooG.uCGAC'J"l'G'i.AATAGCAGCGGcG'I'TAAGAM.GTGgtaagt
234 MAT I A E DEL SEQ F S V Q G T T C N SSG V K K V AAATTATT~TCMGTTCGGGAATrrcMC'I'GAiAACAAAGAAiTTGAAAGC

2881
· . . . . .
aeeactcacctg'lteattgaeeaagggcgtgateaagetgactccatcttateteett'ltagACT'M'GGAGAA'I'T'M'GAG'M'CCTCAAGG
. . .
262 T LEN F E F L K V

FIG. 2. Nucleotide sequence of the DRAC-PK gene and predicted amino acid sequence. The promoter and exon sequences are in
uppercase letters; intron sequences are in lowercase letters. The putative transcription initiation site (found at positions 366-372) is in italic letters,
and the start of the cDNASDE-RAC 109 is indicated by an arrow. Each of the four transcription initiation sites are indicated by an asterisk. opa
repeats in the first exon are underlined. The two initiator codons and the corresponding methionine residues are in boldface letters. Translation
from the first initiator codon (ACGat position 2092 in the genomic sequence) would give a 611-amino acid-long polypeptide, whereas usage of the
second one (at 2335) would produce a 580-amino acid-long polypeptide. Four putative mRNAdestabilization signals in the 3' -untranslated region
are underlined, and the two polyadenylation signals are double-underlined.

specificity, were found at amino acids 389-394 and 426-434, ll-amino acid insertion (positions 224-234), which is not pres-
respectively. ent in the human isoforms.
Comparison of the predicted Drosophila and human peptide DRAC-PKs have an extension at the amino-terminal region
sequences (Fig. 4) showed the conserved nature of RAC-PKs. in comparison with human homologues. DRAC-PK85 possesses
Overall, the degree of homology between RAC-PKll' and an additional 81 amino acid residues at its N terminus that did
DRAC-PK was 76% (62% identity), and homology between not show any significant homology to sequences in the data
RAC-PKJ3 and DRAC-PK was 75% (61% identity). The catalytic bases (GenBank™, EMBL, Swissprot, and GP). The predicted
domain of DRAC-PK exhibited a high degree of homology to extension of DRAC-PK85 has an isoelectric point of 10.7 and is
those of the human ll' and J3 isoforms (86 and 87%, respectively). rich in serine/threonine residues (highlighted in Fig. 4). At the
As in the human isoforms, DRAC-PKs also possess a PH do- C terminus, both DRAC-PK forms have an 18-amino acid ex-
main that is located N-terminal to the catalytic domain (20) tension, which, like the similar extension found in human
and is 71% homologous to the PH domain of human RAC-PKs. RAC-PKJ3 (19), has a high serine/threonine content (high-
This region has been identified in 71 signaling and cytoskeletal lighted in Fig. 4).
molecules, indicating its involvement in signal transduction, Homology to Other Protein Kinases-The DRAC-PK catalytic
possibly by interacting with GTP binding proteins (22, 23). domain showed the highest degree of homology to serum and
Between the PH and catalytic domains DRAC-PKs have an glucocorticoid-regulated kinase, sgk (72% homology, 57% iden-
4070 Drosophila Homolog ue of RAe Protein Kinase
< CRA C-PKSS lolN"it. PF'VL.
. .. .
RR STVVAS A P APGSASR I PES PT TTGSN I I N I I Y 5 S T H p r:

GA TC ~E A D RA C -P KS S 50 1 55 PTSGSAEK rsw S:.tPSRTST APTHDSGT HS I ~J T T F D L S S P S V T S GHA

D R AC- P K 66 1 MS I N T T f D L S S P S V T S C HA

DR AC -PK8S / 66 15 1 5DfLLN Nf'TVRCC I HT VD RP KP FTF IIRCL WT T V I £ R T F A V E S £ L E R

hRA C -PK Q SO A P • • - - - -S - A - -L -KTE--R-N--- - - C - - - - - - - - - --H--T PE··£
hFUC- PK ll so PP •• •• - - S- A E- - 1.. K1'£ - - R -N - - v- - c - - _. -. - - - - FH- 0 - Po- - E

OR AC - P K 8 5 1 6 6 2 01 O:.l T EA I RN ] .
SSRL I OVGEVA MT PSE TO MTOV OM ATI A E D E L S E Q F S V Q G
hRAC ·P KU 98 E -- - -- QT - AOG - I<K . • EEEE M .• . . • • • • • • • D FRSGSPSD NSG AEE M
hR AC · PK P 98 E - MR - -O M- A N S - K R APG EDP M . . . . •. •• ••• OY KCGS PSOSS TT EE H

OR A C Pl<85 /66 251 ~FEFL KVLG KCTFGKVILCRE ;C AT A KLY A IKIL I< KE

hR AC -P Ka 135 EVSL AKP KHR-- H E--Y --L - - - - - - - - - - -V K - - ·-CRY - - H - - - - · -
hRA C -PK P 13 7 £V AVS KAR AKVT HI D - DY · -L - - · - - - - - - - -V - - - -·CR Y · - H ·--R--

OR AC - P K 8 5/ 6 6 301
hR A C -P KU 185
hRAC-P KP 187

D R A C - P K 8 5/ 6 6 351 L F WHLSH ER I ~· T E D R T R F Y GA E I I SALeY LH SO. c I I YRDL K LE NLL LD K

hR C A - P KU 235 - - f'- - - R - -V -S -D - A - - - -- - - V- - - 0- - - - E KNVV - - - - •• - · - M· --
hR A C -P K P 237 - -F - - -R - -V -- -E -.\ - - - - -- V--- E---- . R D V V··- I · - · - · M·_·

..-
-
O R .\ C - P K 8 5 / 6 6 400 De ll I XVA O FG LC K ED I TYGRTT KTFCGTPE Y L A P EVLDO NDYGQ .A.VD WWG
hR.\ C - PK (I 285 - - - - - IT- - - - - - - - - K O- A - M--- - - - - - . - - - - - - E--· · - R- · - -- •
hRA C · PK P 286 - - . - - IT- -- - - - -G- SO - A- M - -. - . - - - . - - . -- - E - - -- - R- - - - - -

DRA C - PK 8 5 /6 6 " 50 TGVV H Y E M ICGRL PFY NRDIfDVL~'TL I LVEEV K F PRN I TDE AKN LL AGL L
hRAC ·PK a 33 5 L- · · · - - - M · · · - · · · · - -E K - -E · - - H--IR ---TLGP ---S- -S- - L
hR ,\ C -P K P 336 L· · · · · · M H - - · - - - - N -- f. R- - E--- H - - I R--- T L S P E - - S- · A · - -

O R AC· P K 85/6 6 500 .A.KD P K K R L C C CK D D V K E I A H P F F A S · ~ WT D LV L K K I P P P F K P

V T S DT D
hRAC -PK (I 38 5 K - - - - Q - - - - - S E -A - - - MQ - - - - - c V - Q B VYE·-LS- - - - - - -- -E - -
hR AC ·PK P 386 K - · - - O - · · · - P S · A - · · ME - R · - L - N- -VV - - L L - - - - - - - - · E-·

ORA C' -P K85 / 66 550 TRYFDKEFTeESVELTPPOPTCPLCSIAEE • • PL FP FSY GD lo!ASTLCT

hRA C' -P KO 435 · - - · · E--- A Q H I T I - - - - ODS MECV OS£RR-H - - - · - ·S ASSTA
hRA C - PK !l " 36 - - - . - D - · - 1. 0- ITI - •• - R YOS - - L L E LD Q R T H - - - - - . SAFRf.EKDLL

DRAC · P K 9 S 16 6 S99 S5H I STSTSL AS H O

hR AC · P Kp .:.86 M · LFVSL I L FSDFSSL KS HS FSSN F I LLSFSSL K K

F IG. 4. Homology b e t we en predicted a m ino acid seq uences of

DRAC-PK a n d human RAC-PKa a n d fJ is oforms. Th e sequences
wer e a ligned usin g th e PileU p progr am based on the pr ogr essive align-
ment meth od (73) a nd then opti mized by eye. Ga ps (.) were int rodu ced
for maximu m align me nt. -, ide ntical a mino acids . Th e beginning a nd
end of th e PH doma in ar e indica ted by vertical bars. Th e catalytic
domain is boxed, with asterisks indica t ing consensus residu es of th e
ATP binding domain a nd se rine/threonine-specific pr otein kinases. Ser-
ine a nd thr eoni ne residues at th e N-ter mina l exte ns ion of DRAC-PK85
and a t th e C terminus are highlighted .

of embryos a nd larva e a nd from adult femal e flies using DRAC

FIG. 3. Mapping the 5 ' -end of the DRA C-PK gene b y primer
e xte nsio n. An oligonucleotide that is compleme nta ry to th e upp er part
of the first exon (positions 55 1-577) was hybridized eithe r with yea st
tR NA as a control or with polytA)" RNA from Drosophila embryos (E)
an d adu lt fem ales (A ). A sequencing reacti on where th e sa me unlabeled
oligonucleotide was used to seq uence the DRAC-PK genomic subclone
that conta ins th e prom oter region is shown to the left . Th e 5'-end of th e
S DE-RAC 109 cDNA is indi cat ed by a n arrow . Each of th e four putati ve
t ra nscri ptio n initia tio n sites is marked by a n asterisk ,
tity (48» , protein kinase CTj (76% homology, 56% identity (49» ,
mitogen- stimulated ribo somal 86 kin ase (69% homol ogy (50» ,
a nd t he a catalyt ic subunit of bovin e pr otein kin ase A (65%
homology (5 1» . Th ese were simi lar to the homologies see n with
RAC-PK a a nd -f3 (19). DRAC-P Ks ha ve slig htly lower hom ology
to t he Drosophila homologu es of pr otein kinase C and protein
kinase A. Of t he three known Drosophila pr otein kinase C
ge nes, DRAC-PK shows 72% homology to 98F (52) and 62% to
t he Drosoph ila protein kin ase A catalyt ic subunit DCO (53).
Expression of DRA C-PK Transcript s- We analyzed the tem-
poral a nd spa tial expression of the DRAC-PK transcripts dur-
ing developm ent. For Northern blot a nalysis two pr obes wer e
used: th e cDNA DRAC 7 a nd a pr obe from th e 3'-untranslated
region located betw een th e first polyadenylation sign al (n ucle-
otides 4374-4379 ) a nd a polyadenylation signa l at nu cleotides
5013-50 18 in the DRAC-PK genomic seque nce. Whereas DRAC
7 recogni zes a ll classes of transcripts, the 3'-en d probe would
detect only those that use t he second polyadenylation signa l.
Nor th ern a na lysis of total RNA isolated from staged collections
7 as a prob e revealed th e pr esence of two major transcripts of
2.7 and 3.9 kb (Fig. 5, A an d B ). The 2.7-kb transcript was
expre ssed at high level s in 0-3-h embryos an d wa s a lso ex-
pressed in a dult fem ales, ind ica ting t hat t his is a maternally
regu lated mRNA . The 3.9-kb transcript was expressed
t hroughout embryogenesis a nd lar va l stages (Fig. 5, A a nd B)
and wa s a lso detected in ea rly a nd late pupal stages (data not
shown). Thi s transcript was also expressed in the a dult fem ale
flies, indicating both maternal a nd zygotic regulation of gen e
expression. When th e 3' -pr obe was used , only th e larger tran -
script could be detected (Fig. 5C ), suggesting that th e 2.7-kb
tran script was generate d by use of t he first polyaden yla tion
signa l. Thi s indicates th at th e cDNA 8DE-RAC 109 was de-
riv ed from th e 2.7-kb maternal t ra nsc ript.
In sit u hybridization to wh ole-mount embryos and dissect ed
ova ries, usin g DRAC 7 as a prob e, dem onstrated that mater-
nally pr ovided DRAC-PK tran scripts were synt hesized in th e
nu rse cells of th e ova ries (Fig. 6A ). During oocyte ma turation
the re wa s no apparen t localization of th e mRNA (Fig. 6B ). No
va ria tion in expression could be det ected throughout embryo-
genesis. Before cellularization (Fig. 6C), a fter cellulari zation
(Fig. 6D ), a nd throughout gas t rulation, ger m band extension,
and retraction (Fig. 6E ) DRAC-PK tran scripts remained uni -
form ly distributed a nd were expressed at a high level.
Chromosomal Locali zation- In situ hybridization showed
that the DRAC-PK gen e is localized at the 89B4-1O region of
polyt ene ch romo somes, which agrees wit h the find ing of
Franke et al. (25), who mapped it to 89BC . Only one sig na l was
detected. Two recessive embryonic mutations map to thi s locus:
serpent and pannier (54). In situ hybridization to polyt ene
 Drosophila H om olog ue of RAC Protein Kina se
A Embryo Larva
3.9-
2.7-
B c Embryo
3.9 - ' 3.9-
2.7-
FIG. 5. Northern blot analy si s of DRAC·PK transcript. Tot al
RNA (20 p.g ) isolated from the ind icat ed developm ental stages was
fractiona te d on a 0.8% agaroseJforma ldehy de ge l and t ran sferred to a
nylon membrane. Blots were hybridized either with cDNA DRAC 7 (A
and B) or a 3' -end pr obe det ectin g on ly transcri pts th at use th e second
polyad en ylati on signal (C). After hyb ridiza tio n blots wer e wash ed at
60 "C in 1 x SSC for 3 x 30 min , a nd exposed at - 70 °C between two
inte nsify ing scree ns.
chromosomes of Drosophila st rains ca rryi ng deficiencies in this
region, Df(3R )sbd 4 5 a nd Df(3R)sbd 10 5 , sugges ted th at th e
DRAC-PK gene might corresp ond to the pannier locus, but our
attempt s to rescue the pannier ph enotyp e were un su ccessful,
an d subsequently t he pa nnier locus wa s shown to encode a
GATA transcription factor (55, 56). In order to achiev e fine
mapping of the DRAC-PK gene, we isolated genomic clones
spanning a - 70-kb region by bidirectiona l chromosom al walk-
ing . We were t he re fore abl e to es tablis h th at th e DRAC-PK
gene is - 30 kb pr oxim al to the Stubble gene (57). Th is
finding plac es th e DRAC-PK gene at 89B9, bet ween th e pan-
nier a nd th e Stubble genes, wit h it s 5' -end closest to th e
centromere.
Identification of the DRA C-PK Protein-In order to gain in-
sight into the differen t form s of the prote in a nd their functions
in vivo, we gene rated a ntisera specific for t he bacteria lly ex-
pr essed recombi nant DRAC-P K66 prote in (No. 36), for th e C
terminus of the DRAC-PKs (No. 41 ), and for the N terminus of
DRAC-PK85 (No. 46 ). Specificity and sens it ivity of t he antire-
combinant a ntise ra were tested by West ern blotting usin g se-
rial dilutions of th e partially purified recomb inant protein s. To
further confirm specificity of th e an ti sera , we a na lyzed protein
pro ducts of COS-1 cell s t ra ns fecte d with pE CE .DRAC 7a (an-
tisense) a nd pE CE.DRAC 7s (se nse ) constructs. Western blot
analysis revealed a specific signal in COS- 1 cells tran sfected
wit h the sense, but not wit h t he an ti sen se construct (Fig. 7A ,
lanes 1 and 2 ). The polyp eptide expressed in COS-1 cells had an
apparent molecula r mass of - 66 kDa, which ag rees wit h the
predicted molecula r mass (60 kDa ). We a lso expressed cDNAs
SDE -RAC 109 an d 105 in COS-1 cells (Fig. 7A, lan es 3 and 4).
4071
E
FIG. 6. III sit u locali zation o f DRA C-PK t ranscript durin g
oogenesis a n d e m bryogenesis . Whole-moun t embryos an d ovaries
were hybridized with cDNA DRAC 7 a s a probe. A , stage 10 oocyte; B,
stage 12 oocyte; C, syncytial embryo before nuclea r migration; D, em-
bryo at cellula r blastoderm; E , dorsal view of em bryo aft er germ ba nd
re tract ion. The a nterior is to t he left .
We det ected two molecula r size form s, of 66 and 85 kDa, with
t he lower molecular ma ss form a pproxi mately 6 tim es more
abundant. Th is implied th at both for ms could be pr oduced from
a single tran scrip t, To furth er confirm that DRAC-PK85 ini-
ti ates from a weak er initiator codon, the ACG in the SDE-RAC
109 cDNA was mu tat ed into a n ATG by PCR, a nd t he res ulting
plasmid (pECE. SDE-RAC 109.ATG) was expressed in COS-1
cells . Th is pr odu ced a major 85-kDa form an d a less a bunda nt
66-kDa form , converting the DRAC-PK66 ;DRAC-PK85 ra tio
from - 6:1 to - 1:3 (Fig. 7A, lane 5). Both DRAC-PK pr otein s
could a lso be det ected by the No. 4 1 a ntise ra (Fig. 7B ). As
expecte d, only th e larger form was detected by t he a nt isera (No.
46 ) specific for the N terminus of DRAC-PK85 (Fig. 7C ).
To ana lyze DRAC-PK expression in flies, we exa mined th e
protein produc ts in ea rly embryos (0-3 hours), ad ult flies a nd
S2 cells (which are of lat e embry onic origin). Western blot
a nalysis reveal ed th e pr esen ce of three distinct molecular size
forms with apparent molecular masses of 66, 85, a nd 120 kDa
that were differen tially expressed (Fig. 7D ). Th ese bands were
not detect ed by th e pr eimmune a ntise ra or a fte r competition
with th e DRAC-PK recombinan t protein (data not shown). Th e
two lower molecul ar size forms (DRAC-PK66 and -85) were
present in a ll ext ra cts exa mine d, wherea s t he 120-kDa form
(p120) wa s detected only in emb ryos a nd S2 cells (see below).
The major for m wa s DRAC-PK66 t ha t comigrated with protein
product from COS-1 cells expressing th e DRAC 7 eDNA. Al-
th ough t he expression of DRAC-PK66 a nd -85 was about 2-fold
high er in embryos a nd S2 cells , t hey a ppea re d with a consta nt
3:1 ra tio in Drosophil a extracts, which is slightly lower th an in
COS- 1 cells expressing th e SDE-RAC 109 a nd 105 cDNAs.
In order to better cha racterize p120, we perform ed Western
blot a na lysis of Drosophila embryos wit h an a nt i-peptide a nti -
body No. 41 , specific for t he C terminu s of DRAC-PK. Only two
bands were det ect ed , DRAC-PK66 a nd DRAC-PK85, th at could
be compet ed with t he a ntigenic peptide (da ta not shown). We
also perfor med Western blot analysis of la te muta nt embryos
homozygous for th e deficiency Df(3R)shd'lf' (t he DRAC-PK gene
is delet ed in such flies ). Th e signa l for DRAC-PK66 was - 10-
4072 Drosophila Homologue of RAC Protein Kina se

~ Embryo Larva Pupa Adult

"- or.
A r:.:: r-- 0
0\
0
u u <
< < u U
D::: D::: < < 0'
0
c Cl D::: D:::

--
2()()-
116.2 _
97.4 - p120 -
66.2-
45 -
DRAC-PK85 -
DRAC-PK66 - e4••~. -
FIG. 8. De ve lo p m e n t al t im e course of DRAC-PK protein ex-
pression. Prot ein ext ra cts wer e mad e from Drosophi la at the indicate d
stages. a nd 20 Ilg was a na lyzed by West ern blotting using a ffinity-
Vl Q Vl Q purified a nti-DRAC-PK a nt ibody No. 36.
B r-- :-
U
<
C ti I-
~
< 0: <
0::: 0:
D::: 0
a 0
c Adult

-
Embryo
11 6.2 -
97.4 -
66.2 - -..
116.2 -
97.4 -
66.2- - In vitro
kinase assay
- MBP

D Embryo Adult S2
I I I I
20 40 20 40 20 40
200-
11 6.2 -
97.4 -
66.2 - -
-
-~
-- --
45-
FIG. 7. Identification of DRAC· P K protein in transfectcd
COS· I cells and in D rosophila extracts b y Western b lot a nalysis.
COS- I cells wer e tran sfected with the following construct s: pECE.
DRAC 7 an tise nse (DRAC7a ) a nd pE CE .DRAC 7 se nse (DRAC7s ).
pECE.SD E-RAC 105 (RAC I05), pECE .SDE -RAC 109 (RAC109), and
pECE.SDE-RAC 109.ATG (l 09.ATG ). 25 Ilg of COS-I cell extra cts were
subjecte d to 7.5% SDS -PAGE . Proteins were transferre d to PVDF mem-
brane, a nd specific ban ds wer e detected using a ffinity-puri fied a nti-
DRAC-PK antibody No. 36 (A). No. 4 1 (B ). 01' No. 46 (e). 20 a nd 40 Ilg
of prote in from Drosophi la 0 - 3-h-old embryo, adult , a nd S2 cell extra cts
were a nalyzed by West ern blotting with t he No. 36 antiser a (D ). Mo-
locular size ma rk ers in kDa a re shown.
fold decreased , prob ably represen tin g a ca r ry-over from the
moth er , a nd DRAC-PK 85 could not be det ected . In contrast ,
p120 levels remained un chan ged . Th er efore, we concluded th at
p120 is not a pr odu ct of a DRAC-PK tra nscript .
Western blot a na lysis wit h t he No. 46 a ntise ra specific for
t he N-te r minal pep tid e of DRAC-PK 85 detected only t he 85-
kDa form in Drosoph ila extracts . This ba nd comigrated with
DRAC-PK 85 t hat was expressed in COS-I cells t ra ns fected
with the pECE .SDE-RAC 109.ATG constr uct (data not shown ).
Developmental Regulation of the DRAC-PK Gene Protein
Products-Northern a na lysis demonstra ted that t he DRAC-PK
gene expression was both ma tern all y an d zygot ica lly regu lated
(see Fig. 5). Also Western blot a na lysis (Fig. 7D) revealed
differ ences in DRAC-PK protein form s a nd a bunda nce between
embryos a nd a dult flies. Th us, we decided to a na lyze the ex-
pr ession of the DRAC-PK gene pr otein pr odu cts du ring the
Drosophila life cycle (Fig. 8). Th e majo r for m detected through-
out developm en t was DRAC-PK 66. Th e highest level of expr es-
sion (a rbitra rily tak en as 100%) was found in 0-3-h embryos
a nd subse que ntly declin ed to 60% du ring late embryogenesis. A
further declin e in DRAC-PK 66 levels was observed during la r-
va l developm ent (20% in t he th ird insta l' larvae), followed by a
sha r p increa se in th e ea rly pup ae (80%). Th e protein levels
were - 30% in adult flies. DRAC-PK 85 followed t he expression
of DRAC-PK66, except in th e t hre e larval stages where it could
not be detected . Th ere was no difference in th e expression of
\ \ \ I//
ug
Western blot
116.2-
97.4-
66.2 -
-
-
DRAC-PK85
DRAC-PK66
FIG. 9. Demons tra ti o n of t h e DRA C·PK activity. Pr oteins wer e
immunopreci pita te d from 0.3 a nd 0.6 mg of 0 - 3-hour-old embryos a nd
adult flies usin g affini ty-purified antibody No. 36. which wa s omitte d in
a control experi ment. III vitro kin ase assays were perform ed as de-
scribed und er "Ma terials a nd Met hods," a nd 50% of the ass ay mixt ur e
wa s a na lyzed by SDS-PAGE. Phosph orylat ed MBP was vis ua lized by
a utoradiogra phy. The DRAC-PK protein was detected in im rnunopre -
cipita tes by Western blot a na lysis, using the sa me a nt ibody.
DRAC-PK66 a nd -85 bet ween fem al e a nd male flies. p120 was
detect ed during embryogenesis, wh ere its expres sion followed
th e pattern of DRAC-PK66 a nd -85. It was found to be signif-
icantly higher in ea rly th an in late pup a e, wh ich was not t he
case wit h DRAC-PK66 and -85. Th ese resu lts sugges t th e in-
volvem entofDRAC-PK a nd th e p120 protein in embryogenes is,
as well as post embryonically.
Th e spatial express ion of th e DRAC-PK pr otein s during em-
bryogen esis was a na lyzed using th e a ffinity-purified a ntire-
combina nt a ntibody No. 36. We found th at the protein s were
uniforml y distributed in all embryonic stages, whi ch is in good
correlation with th e tra nscript localiz ation (da ta not shown).
Also, du rin g t he syncyt ia l blastoderm stage, specific sta ining
was detected in th e cyto plasm sur rounding dividi ng nuclei and
was a lways excluded from t he chromatin .
DRA C-PK Possesses an Intrinsic Kinase Acti vity-Since
DRAC-PK is highl y homologous to the hu man RAC-PK (~ , wh ich
shows t he high est specific activi ty towa rd MBP (18), we de-
cided to use thi s subst ra te to assay kin ase activity in irnm uno-
pr ecipitates pr epared from Drosophila ext ra cts . Th e DRAC-PK
protein immunopr ecipitated wit h a nti recombinant a ntibo dy
No. 36 from 0 - 3-h embryos a nd a du lt flies (in t he pr esen ce of
phosphatase inhibito rs ) ph osph oryla ted MBP in vitro (Fig. 9).
Only DRAC-PK66 an d -85 wer e det ected in t he immunoprecipi-
tates, wher eas p120 could not be detected (Fig. 9). Th e
DRAC-PK pr otein a nd th e associa te d kinase activ ity could not
be det ect ed in a cont rol experime nt, when th e a ntibody was
eit he r omitted (Fig. 9) or pr eincub a ted with th e recombinant
protein immobilized on PVDF st rips.
Th e specific acti vity of DRAC-PK immunoprecipitated from
ea rly embryos was - 0.04 pmoVmin/mg, whereas th at from
 Drosophila Homologue of RAe Protein Kinase 4073
II III IV V VI

DRAC-PK VKEGWLMKRGE. HI .•• KNWRQRYFVLHSIlGRLMGYRSKPAD •...• SASTPSDFLLNNFTVRGCQIMTVDRPKP .•••••••...••.•..•.••. FTFIIRGLQWTTVIERTFAVESELERQQWTEAIRNV

hRAK-PKa VKEGWLHKRGE. YI. .. KTWRPRYFLLKNIlGTFIGYKERPQD •••.• VDQREAP•. LNNFSVAQCQLMKTERPRP •...•••••.••......••.• NTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTV
hRAC-PK~ IKEGWLHKRGE. YI. •• KTWRPRYFLLKSIlGSFIGYKERPEA.•••• PDQTLPP •• LNNFSVAECQLMKTERPRP ..•••..•••..•••••.•... NTFVIRCLQWTTVIERTFHVDSPDEREEWMRAIQMV
celRAC-PK GI ESWLHKKGE. HI. •• RNWRPRYFILFRIlGTLLGFRSKPKE.••.• DQPLPEP •• LNNFMIRDAATVCLDKPRP •••.••.•.••••.•....... NMFIVRCLQWTTVIERTFYADSADFRQMWIEAIQAV

h~ark IMHGYMSKMGNPFL ••• TQWQRRYFYLFPN .. RLEWRGEGE .•..•• APQSLLT . MEEIQSVEETQI .• KER•.•.••••..•....••........ KCLLLKI RGGK .•• QFILQCDSDPELVQWKKELRDA
rpark IMHGYMLKLGNPFL .•• TQWQRRYFYLFPN .. RLEWRGEGE .•.... SRQNLLT• MEQIMSVEETQI. . KDR••••......••...•........• KCI LLRVKGGK ••• QFVLQCESDPEFAQWLKELTCT
GPRKI I LHGYIKKLGGSFA.•. SLWQTKYAKLYPN .. RLELHSESGN..••. NKPELIF • MDQVEDI SSDFILHKNE ..•••••••.••••.•••....... NCIQIRINDGTRIlGRIILTNSDEIGLKEWSSSLRSA

nrkA THRGHVNKLGGGNG .•• KSWKPRFLQIVRG•• QLILTDDEEG ..... NNPKGLN • LEQVQGACPVPHSTAKRD .•••••...•..••.••..•.•.. FVFALNTVGGK ••• GMWFQAVSHGDMEMWVHAIQRG

atk LESI FLKRSQQKKKT SPLNFKKRLFLLTVH•. KLSYYEYDFERGRRGSKKGSID. VEKI TCVETWPEKNPPPERQI PRRGEESSEMEQISI I ERFPYPFQVVYDEG P •••• LYVFSPTEELRKRWIHQLKNV
T8k LEEQLIKKSQQKRRTSPSNFKVRFFVLTKA•. SLAYFEDRHGKKR .. TLKGSIE. LSRIKCVEIVKSD •.....•.•.•••.••....• lSI PCHYKYPFQWHDNYL •.•. LYVFAPDCESRQRWVLTLKEE

FIG. 10. Alignment of the PH domains from serine/threonine and tyrosine protein kinases. Initially, the sequences were aligned using
the PileUp program and then optimized by eye. Accession numbers of the sequences are given in brackets. Human (M77198 and M63167),
Drosophila, and C. elegans RAC-PK (M88917) contain a PH domain at the N terminus (residues 6-106 in human RAC-PKs, 108-209 in
DRAC-PK85, and 27-128 in DRAC-PK66). The {3ARK family members contain a PH domain at the C terminus (residues 560-650 in human {3ARK
(M80776) and rat {3ARK2 (M87855» and residues 559-655 in the Drosophila G protein-coupled receptor kinase 1 (GPRK1 (M80493)). nrkA is a
Trypanosoma brucei serine/threonine protein kinase (L03778) that has a PH domain at the C terminus (residues 333-427). atk (or Btk) is a human
B-cell specific tyrosine kinase (X58957), whereas Tsk (or itk) is a mouse Tvcell-specific tyrosine kinase (L03778). Their PH domains are located at
the N terminus (5-133 in atk, and 6-111 in Tsk) and have a high degree of homology (68.5%). The degree of homology between the RAC-PK family
members and the {3ARK family members is 39-46%, and between RAC-PKs and nrkA it is 41.5-43.6%. Between RAC-PKs and atk, and RAC-PKs
and Tsk, homology is 46-50 and 44-48%, respectively.

adult flies was ~0.1 pmol/min/mg. When the DRAC-PK protein
levels in immunoprecipitates were quantified, the activity of
DRAC-PK from adult flies was ~8-fold higher.
DISCUSSION
In this report we present the characterization of the Dro-
sophila RAC protein kinase gene and protein products. The
DRAC-PK gene consists of seven exons producing two differen-
tially regulated mRNA species via the use of two different
polyadenylation signals. This may represent a regulatory
mechanism for the temporal and spatial expression of the
DRAC-PK gene. This hypothesis is supported by the presence
offour putative AUUUA mRNA destabilization signals (43, 44)
located between the first and the second polyadenylation sites.
The cDNA cloned by Franke et al. (25) is derived from the
zygotic DRAC-PK transcript, since it contains the second polya-
denylation signal. However, in contrast to their observations,
we found that the smaller DRAC-PK transcript represents a
maternally derived mRNA and that DRAC-PK gene expression
is regulated not only at the zygotic level, but also by the
mother.
Heterogeneity of transcripts has been observed in the case of
some other Drosophila genes that encode protein kinases, such
as DCO (53) and shaggy (15). The DCO gene for the catalytic
subunit of protein kinase A contains four polyadenylation sig-
nals that give rise to 4 different transcripts encoding the same
protein, whereas the shaggy gene encodes at least 10 tran-
scripts and five related proteins that differ in their N-terminal
domains and/or the C termini. In the case ofthe DRAC-PK gene
both transcripts encode two protein forms with distinct N ter-
mini. Translation of the larger 61l-amino acid polypeptide
apparently initiates from an ACG codon that is less efficiently
used than the AUG for the shorter, 530-amino acid polypeptide.
Several viral and mammalian mRNAs (Refs. 58-60 and refer-
ences therein) use non-AUG codons for translation initiation.
Some Drosophila proteins also initiate from non-AUG codons
(61-64). Experiments in COS-1 cells (65) have demonstrated
that non-AUG codons can initiate translation when they are in
a good context, but they differ in their initiation potential.
Some of the messages use an upstream non-AUG codon to
produce two or more polypeptides (Refs. 59, 60, 63, and refer-
ences therein), but the abundance of the N-terminally extended
protein depends on the start codon. In all ofthe cases the larger
form is thought to have a regulatory role, since its synthesis is
usually at a lower level. It is possible that DRAC-PK85 has a
similar function. Also, the weaker upstream initiation codon
acts as a negative regulator of DRAC-PK66 expression from the
downstream AUG. In addition, the ~460-nuc1eotide5'-untrans-
lated region attenuates expression of both DRAC-PK forms.
Both protein forms encoded by the DRAC-PK gene have a
common domain structure, except that the larger polypeptide
has an 81-amino acid-long N-terminal extension. The degree of
homology between DRAC-PK and the human isoforms (75-
76%) is comparable with that between Drosophila and mam-
malian homologues of protein kinase C (85% (66)) and protein
kinase A (80% (53)). Such a high level of homology suggests a
functional conservation of RAC-PKs. This is also supported by
the presence of a PH domain in both Drosophila and human
isoforms (20, 67), showing 71% homology/60% identity. How-
ever, it is not known if the N-terminal extension ofDRAC-PK85
would affect the interaction of the PH domain with its putative
partner and thus modify DRAC-PK85 functional characteris-
tics. A similar degree of homology in the PH domain has also
been observed between the human and Caenorhabditis elegans
RAC-PK homologues (68) as well as between the Drosophila
and C. elegans RAC-PK homologues (71 and 69%, respectively).
A high degree of homology (62%) in a PH domain has been
observed between the mammalian and the Drosophila homo-
logues of the f3-adrenergic receptor protein kinase (f3ARK; Refs.
4 and 22), whereas the degree of homology in the PH domains
between the members of the RAC-PK and f3ARK families, and
the other protein kinases containing a PH domain (67) is lower
(ranging from 39 to 50%, see Fig. 10). Such a divergence sug-
gests that different PH domains may interact with similar but
not necessarily identical molecules. The PH domain of f3ARK
was shown to interact with the f3'Y subunits of G-proteins,
which could then lead to translocation of f3ARK to the mem-
brane (22). A similar type of interaction was postulated for the
PH domains of phospholipases (69). There are some other ex-
amples of signaling molecules containing a PH domain that
have been conserved in both mammals and Drosophila. They
include SOS and GAP that are known to interact with small
GTPases, as well as an SH3 binding protein, dynamin, and a
cytoskeletal protein f3-spectrin (22). It is possible that members
of the RAC-PK family also fulfill their function(s) in cell sig-
naling through an interaction with GTP binding proteins.
We detected three distinct protein forms in Drosophila ex-
tracts with apparent molecular sizes of 66, 85, and 120 kDa. By
using three different antibodies, we demonstrated that p66 and
p85 are products of the DRAC-PK gene, whereas p120 is de-
rived from a distinct gene. The fact that it was detected by
antirecombinant antibody (No. 36) and that the signal could be
competed with DRAC-PK recombinant protein suggests that
p120 shares a common strong epitope or epitopes with DRAC-
PK. The expression of all forms is under both maternal and
4074 Drosophila Homologue of RAe Protein Kinase
zygotic control, and levels of DRAC-PK66 and DRAC-PK85
show a good correlation to the expression ofDRAC-PK mRNA.
Although there was no difference in the DRAC-PK protein
between female and male flies, message levels were higher in
females, which could be explained by higher levels of maternal
transcripts in nurse cells and oocytes.
The highest level of expression of DRAC-PK was observed
during embryogenesis, when some other Drosophila serine/
threonine kinases are known to act: pelle is involved in estab-
lishing the dorsoventral polarity in embryos (7), D-raf and Dsor
are transducing signals in the terminal system (70, 8), fused
and shaggy are segment polarity genes (13, 14), and protein
kinase A mediates communication between cells (17). Muta-
tions in these genes show a maternal effect phenotype. The
pattern of DRAC-PK expression suggests it plays a role in
embryogenesis.
There was no spatial restriction in the expression of the
DRAC-PK transcripts and proteins during embryogenesis, thus
resembling protein kinase A (17), D-raf (16, 70), and shaggy
(15). The DRAC-PK gene is also expressed postembryonically.
The level of DRAC-PK protein decreases during the larval
stages and increases again during pupation. Such a pattern of
expression has been observed for the D-raf (70) and the Dsor
messages (8). Mutants of the D-raf gene display abnormal
proliferation of both somatic and germ line cells (71), which is
consistent with the function that Raf-1 plays in mammalian
cells, i.e. transduction of growth-stimulating signals (reviewed
in Ref. 72). Like Raf-1, RAC-PKa is also a proto-oncogene. The
viral oncogene v-akt transforms cells in culture (24), suggesting
that the proto-oncogene RAC-PKa transduces signals that reg-
ulate cell growth. It is possible that DRAC-PK might playa
similar role in Drosophila development.
We demonstrate that the DRAC-PK protein has intrinsic
kinase activity. The specific activity of immunoprecipitated
DRAC-PK using MBP as a substrate was estimated to be in the
range of 0.04 pmol/min/mg for embryos and 0.1 pmol/min/mg
for adult flies. The difference in the DRAC-PK activity between
early embryos and adult flies can be explained by different
activation states in the two stages. It is likely that the
DRAC-PK activity increases after cellularization, when cell
signaling through cell surface receptors is taking place. It has
been shown that human RAC-PKa is heavily phosphorylated in
vivo,2 which is also the case with the v-akt oncogene (24),
implying that phosphorylation regulates RAC-PK activity. It is
therefore possible that a similar mechanism controls DRAC-PK
activity.
In summary, our results demonstrate that expression and
translation of DRAC-PK transcripts is tightly regulated, im-
plying functional significance of the two DRAC-PK protein
forms. Further analysis and isolation of mutant flies will pro-
vide information on the role ofDRAC-PK and its PH domain in
signal transduction.
Acknowledgments-We thank Prof. W. Gehring for his encourage-
ment and generous support throughout the project; Prof. C. Niisslein-
Volhard for fly strains; Prof. J. Fristrom for providing the phage from
the Stubble chromosome walk; G. Halder for help with irnmunolocal-
ization experiments; U. Kloter for performing in situ hybridization to
chromosomal squashes; F. Fischer, G. Aeschbacher, and P. Miiller for
synthesis of the peptides and oligonucleotides; and Prof. R. Franklin,
Dr. E. Ingley, T. Millward, and N. Andjelkovic for critical comments on
this manuscript.
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