(Received for publication, September 9, 1994; and in revised form, November 22, 1994)
From the
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 different 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 extension. 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 Drosophila, 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 spatial
restriction of DRAC-PK expression during embryogenesis, suggesting that
localized activation might be a regulatory mechanism for its function.
DRAC-PK possesses 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, 4, 5, 6, 7, 8, 9, 10, 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 Drosophila, such as sevenless, which plays a role in the differentiation 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 subfamily of the serine/threonine
protein kinases, termed RAC protein kinase
(RAC-PK)()(18) . Two closely related forms,
RAC-PK
and RAC-PK
, have been identified(19) , and
phylogenetically 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 SH2 and SH3 domains, i.e. interaction with other signaling
or cytoskeletal
molecules(20, 21, 22, 23) . Unlike
its closest relatives, protein kinase C and protein kinase A, which are
both non-oncogenic protein kinases, RAC-PK
was identified as the
cellular homologue of v-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 homologue 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 (Dakt1).
Genomic clones were isolated
from a Drosophila library (27) constructed in
EMBL4. Several clones were isolated and characterized by
restriction enzyme analysis. One of the clones, SDGL-DRAC 10 with a
12-kb-long EcoRI insert, contained the entire DRAC-PK gene.
Fragments of the SDGL-DRAC 10 were subcloned into the pBluescript
vector following digestion with EcoRI, BamHI, and ClaI. The DRAC-PK gene was sequenced on both strands using
overlapping restriction subclones.
Figure 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 cDNA SDE-RAC 105 contains at the 5`-end 45 nucleotides derived from the intronic sequence. E, EcoRI; B, BamHI; C, Cla; S, SacII.
Figure 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 cDNA SDE-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 (ACG at 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 mRNA destabilization signals in the 3`-untranslated region are underlined, and the two polyadenylation signals are double-underlined.
Figure 3:
Mapping the 5`-end of the DRAC-PK gene by
primer extension. An oligonucleotide that is complementary to the upper
part of the first exon (positions 551-577) was hybridized either
with yeast tRNA as a control or with poly(A) RNA from Drosophila embryos (E) and adult females (A). A sequencing reaction where the same unlabeled
oligonucleotide was used to sequence the DRAC-PK genomic subclone that
contains the promoter region is shown to the left. The 5`-end
of the SDE-RAC 109 cDNA is indicated by an arrow. Each of the
four putative transcription initiation sites is marked by an asterisk.
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 cDNA (which contains exons 2-7) has at its 5`-end 45 nucleotides derived from the 3`-end of the 1.2-kb-long 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 signals. The
same AUUUA sequences are present in the 3`-untranslated region of both
human RAC-PKs, at positions 1709 and 1849 in the
isoform(18) , and at position 1800 in the
isoform
sequence(19) .
A putative initiation codon (AACCATG) in the
correct context 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-PK and -
,
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 cDNA 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 cDNA sequences suggested that the putative
initiator codon was an ACG preceded by CAAC, a sequence compatible with
the consensus for translation initiation in Drosophila (C/A)AA(C/A)(45) ). The open reading frame that starts
from this upstream initiation codon translates into a 611amino
acid-long polypeptide, with a predicted molecular mass 68.5 kDa and an
isoelectric point of 6.2. The apparent molecular 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 apparent
molecular masses on SDS-PAGE.
Figure 7: Identification of DRAC-PK protein in transfected COS-1 cells and in Drosophila extracts by Western blot analysis. COS-1 cells were transfected with the following constructs: pECE. DRAC 7 antisense (DRAC7a) and pECE.DRAC 7 sense (DRAC7s), pECE.SDE-RAC 105 (RAC105), pECE.SDE-RAC 109 (RAC109), and pECE.SDE-RAC 109.ATG (109.ATG). 25 µg of COS-1 cell extracts were subjected to 7.5% SDS-PAGE. Proteins were transferred to PVDF membrane, and specific bands were detected using affinity-purified anti-DRAC-PK antibody No. 36 (A), No. 41 (B), or No. 46 (C). 20 and 40 µg of protein from Drosophila 0-3-h-old embryo, adult, and S2 cell extracts were analyzed by Western blotting with the No. 36 antisera (D). Molecular size markers in kDa are shown.
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 specificity, were found at amino acids 389-394 and 426-434, respectively.
Figure 4:
Homology between predicted amino acid
sequences of DRAC-PK and human RAC-PK and
isoforms. The
sequences were aligned using the PileUp program based on the
progressive alignment method (73) and then optimized by eye.
Gaps (.) were introduced for maximum alignment. -, identical
amino acids. The beginning and end of the PH domain are indicated by vertical bars. The catalytic domain is boxed, with asterisks indicating consensus residues of the ATP binding
domain and serine/threonine-specific protein kinases. Serine and
threonine residues at the N-terminal extension of DRAC-PK85 and at the
C terminus are highlighted.
Comparison of the predicted Drosophila and
human peptide sequences (Fig. 4) showed the conserved nature of
RAC-PKs. Overall, the degree of homology between RAC-PK and
DRAC-PK was 76% (62% identity), and homology between RAC-PK
and
DRAC-PK was 75% (61% identity). The catalytic domain of DRAC-PK
exhibited a high degree of homology to those of the human
and
isoforms (86 and 87%, respectively). As in the human isoforms,
DRAC-PKs also possess a PH domain that is located N-terminal to the
catalytic domain (20) and is 71% homologous to the PH domain of
human RAC-PKs. This region has been identified in 71 signaling and
cytoskeletal molecules, indicating its involvement in signal
transduction, possibly by interacting with GTP binding
proteins(22, 23) . Between the PH and catalytic
domains DRAC-PKs have an 11-amino acid insertion (positions
224-234), which is not present in the human isoforms.
DRAC-PKs
have an extension at the amino-terminal region in comparison with human
homologues. DRAC-PK85 possesses an additional 81 amino acid residues at
its N terminus that did not show any significant homology to sequences
in the data bases (GenBank, EMBL, Swissprot, and GP). The
predicted extension of DRAC-PK85 has an isoelectric point of 10.7 and
is rich in serine/threonine residues (highlighted in Fig. 4). At the C terminus, both DRAC-PK forms have an 18-amino
acid extension, which, like the similar extension found in human
RAC-PK
(19) , has a high serine/threonine content (highlighted in Fig. 4).
Figure 5:
Northern blot analysis of DRAC-PK
transcript. Total RNA (20 µg) isolated from the indicated
developmental stages was fractionated on a 0.8% agarose/formaldehyde
gel and transferred to a nylon membrane. Blots were hybridized either
with cDNA DRAC 7 (A and B) or a 3`-end probe
detecting only transcripts that use the second polyadenylation signal
(C). After hybridization blots were washed at 60 °C in 1
SSC for 3
30 min, and exposed at -70 °C between two
intensifying screens.
In situ hybridization to whole-mount embryos and dissected ovaries, using DRAC 7 as a probe, demonstrated that maternally provided DRAC-PK transcripts were synthesized in the nurse cells of the ovaries (Fig. 6A). During oocyte maturation there was no apparent localization of the mRNA (Fig. 6B). No variation in expression could be detected throughout embryogenesis. Before cellularization (Fig. 6C), after cellularization (Fig. 6D), and throughout gastrulation, germ band extension, and retraction (Fig. 6E) DRAC-PK transcripts remained uniformly distributed and were expressed at a high level.
Figure 6: In situ localization of DRAC-PK transcript during oogenesis and embryogenesis. Whole-mount embryos and ovaries were hybridized with cDNA DRAC 7 as a probe. A, stage 10 oocyte; B, stage 12 oocyte; C, syncytial embryo before nuclear migration; D, embryo at cellular blastoderm; E, dorsal view of embryo after germ band retraction. The anterior is to the left.
To analyze DRAC-PK expression in flies, we examined the protein products in early embryos (0-3 hours), adult flies and S2 cells (which are of late embryonic origin). Western blot analysis revealed the presence of three distinct molecular size forms with apparent molecular masses of 66, 85, and 120 kDa that were differentially expressed (Fig. 7D). These bands were not detected by the preimmune antisera or after competition with the DRAC-PK recombinant protein (data not shown). The two lower molecular size forms (DRAC-PK66 and -85) were present in all extracts examined, whereas the 120-kDa form (p120) was detected only in embryos and S2 cells (see below). The major form was DRAC-PK66 that comigrated with protein product from COS-1 cells expressing the DRAC 7 cDNA. Although the expression of DRAC-PK66 and -85 was about 2-fold higher in embryos and S2 cells, they appeared with a constant 3:1 ratio in Drosophila extracts, which is slightly lower than in COS-1 cells expressing the SDE-RAC 109 and 105 cDNAs.
In order to better characterize p120,
we performed Western blot analysis of Drosophila embryos with
an anti-peptide antibody No. 41, specific for the C terminus of
DRAC-PK. Only two bands were detected, DRAC-PK66 and DRAC-PK85, that
could be competed with the antigenic peptide (data not shown). We also
performed Western blot analysis of late mutant embryos homozygous for
the deficiency Df(3R)sbd (the DRAC-PK gene is
deleted in such flies). The signal for DRAC-PK66 was
10-fold
decreased, probably representing a carry-over from the mother, and
DRAC-PK85 could not be detected. In contrast, p120 levels remained
unchanged. Therefore, we concluded that p120 is not a product of a
DRAC-PK transcript.
Western blot analysis with the No. 46 antisera specific for the N-terminal peptide of DRAC-PK85 detected only the 85-kDa form in Drosophila extracts. This band comigrated with DRAC-PK85 that was expressed in COS-1 cells transfected with the pECE.SDE-RAC 109.ATG construct (data not shown).
Figure 8: Developmental time course of DRAC-PK protein expression. Protein extracts were made from Drosophila at the indicated stages, and 20 µg was analyzed by Western blotting using affinity-purified anti-DRAC-PK antibody No. 36.
The spatial expression of the DRAC-PK proteins during embryogenesis was analyzed using the affinity-purified antirecombinant antibody No. 36. We found that the proteins were uniformly distributed in all embryonic stages, which is in good correlation with the transcript localization (data not shown). Also, during the syncytial blastoderm stage, specific staining was detected in the cytoplasm surrounding dividing nuclei and was always excluded from the chromatin.
Figure 9: Demonstration of the DRAC-PK activity. Proteins were immunoprecipitated from 0.3 and 0.6 mg of 0-3-hour-old embryos and adult flies using affinity-purified antibody No. 36, which was omitted in a control experiment. In vitro kinase assays were performed as described under ``Materials and Methods,'' and 50% of the assay mixture was analyzed by SDS-PAGE. Phosphorylated MBP was visualized by autoradiography. The DRAC-PK protein was detected in immunoprecipitates by Western blot analysis, using the same antibody.
The specific activity of DRAC-PK
immunoprecipitated from early embryos was 0.04 pmol/min/mg,
whereas that from 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.
In this report we present the characterization of the Drosophila RAC protein kinase gene and protein products. The DRAC-PK gene consists of seven exons producing two differentially 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 of four 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 polyadenylation 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
DC0 (53) and shaggy(15) . The DC0 gene for the
catalytic subunit of protein kinase A contains four polyadenylation
signals that give rise to 4 different transcripts encoding the same
protein, whereas the shaggy gene encodes at least 10
transcripts and five related proteins that differ in their N-terminal
domains and/or the C termini. In the case of the DRAC-PK gene both
transcripts encode two protein forms with distinct N termini.
Translation of the larger 611-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 ( (58, 59, 60) and references
therein) use non-AUG codons for translation initiation. Some Drosophila proteins also initiate from non-AUG codons (61, 62, 63, 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 references therein), but the abundance of the N-terminally extended
protein depends on the start codon. In all of the 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-nucleotide 5`-untranslated 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 mammalian 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.
However, it is not known if the N-terminal extension of DRAC-PK85 would
affect the interaction of the PH domain with its putative partner and
thus modify DRAC-PK85 functional characteristics. 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 homologues of the -adrenergic receptor protein
kinase (
ARK; (4) and (22) ), whereas the degree
of homology in the PH domains between the members of the RAC-PK and
ARK families, and the other protein kinases containing a PH domain (67) is lower (ranging from 39 to 50%, see Fig. 10).
Such a divergence suggests that different PH domains may interact with
similar but not necessarily identical molecules. The PH domain of
ARK was shown to interact with the
subunits of
G-proteins, which could then lead to translocation of
ARK to the
membrane(22) . A similar type of interaction was postulated for
the PH domains of phospholipases(69) . There are some other
examples 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
-spectrin(22) . It is possible that members of the RAC-PK
family also fulfill their function(s) in cell signaling through an
interaction with GTP binding proteins.
Figure 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 ARK family members contain a PH
domain at the C terminus (residues 560-650 in human
ARK
(M80776) and rat
ARK2 (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 T-cell-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
ARK 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.
We detected three distinct protein forms in Drosophila extracts 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 derived 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 zygotic control, and levels of DRAC-PK66 and DRAC-PK85 show a good correlation to the expression of DRAC-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 establishing 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) . Mutations 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 (72) ). Like Raf-1,
RAC-PK is also a proto-oncogene. The viral oncogene v-akt transforms cells in culture(24) , suggesting that the
proto-oncogene RAC-PK
transduces signals that regulate cell
growth. It is possible that DRAC-PK might play a 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-PK is
heavily phosphorylated in vivo,(
)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, implying functional significance of the two DRAC-PK protein forms. Further analysis and isolation of mutant flies will provide information on the role of DRAC-PK and its PH domain in signal transduction.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X83510[GenBank].