(Received for publication, June 12, 1995; and in revised form, August 14, 1995)
From the
A number of ``target'' proteins for the Rho family of
small GTP-binding proteins have now been identified, including the
protein kinases ACK and p65 (Manser, E., Leung, T.,
Salihuddin, H., Zhao, Z.-S., and Lim, L.(1994) Nature 367,
40-46). The purified serine/threonine kinase p65
has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42.
Here we report the cDNA sequence encoding a new brain-enriched PAK
isoform
-PAK, which shares 79% amino acid identity with the
previously described
-isoform. Their mRNAs are differentially
expressed in the brain, with
-PAK mRNA being particularly abundant
in motor-associated regions. In vitro translation products of
the
- and
-PAK cDNAs exhibited relative molecular masses of
68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A
specific
-PAK peptide sequence was obtained from rat
brain-purified p65
. Recombinant
- and
-PAKs
exhibited an increase in kinase activity mediated by GTP-p21 induced
autophosphorylation. Cdc42 was a more potent activator in vitro of
-PAK kinase, and the fully activated enzyme is 300 times
more active than the unphosphorylated form. Interestingly the
down-regulation in the binding of p21s to recombinant
-PAK and
brain p65
, which is observed upon kinase activation does
not occur with recombinant
-PAK.
Morphological roles for the most common members of the mammalian
Rho family of small GTP-binding proteins, Rac1, RhoA, and Cdc42, have
been established in fibroblasts (Ridley et al., 1992; Ridley
and Hall, 1992; Kozma et al., 1995). Cdc42 in Saccharomyces cerevisiae is required for cell budding and may
provide the polarization signal at this site (Ziman et al.,
1993); in fibroblasts filopodial formation is dependent on the closely
related mammalian homologue of Cdc42 (Kozma et al., 1995;
Nobes and Hall, 1995). Although an increasing number of p21 Rho
GTPase-activating proteins (GAPs) ()have been identified
(for review, see Lamarche and Hall(1994)), there is as yet no evidence
that they are able to exhibit effector function. These proteins can be
identified by sequence homology to the GAP domain and by their activity
in overlay assays (Manser et al., 1992). The most closely
related RhoGAPs comprise the chimaerin family (Hall et al.,
1993; Leung et al., 1993, 1994), acting on Rac and whose
activity is regulated through a protein kinase C-like cysteine-rich
domain (Ahmed et al., 1993). Although many RhoGAPs are
somewhat promiscuous in vitro, they appear to show distinct
p21 specificities in vivo (Ridley et al., 1993).
The prototype small GTP-binding protein p21-Ras is an oncogene that
has effector targets which include Raf kinases (Vojtek et al.,
1993; Warne et al., 1993; Zhang et al., 1993)
phosphatidylinositol 3-kinase (Rodriguez-Viciana et al., 1994)
and RasGAP itself (Schweighoffer et al., 1992). The use of the
p21 GTP/GDP cycle is exemplified by the role of Ras in growth factor
signal transduction, where GTP-Ras functions to activate proteins of
the ``mitogen-activated protein kinase cascade'' through the
serine/threonine kinase Raf (Warne et al., 1993), and MEK
kinase (Lange-Carter and Johnson, 1994). It seems probable that part of
p21 Rho family signaling also occurs through associated kinases for
which the prototypes are the activated Cdc42-associated tyrosine kinase
p120-ACK (Manser et al., 1993) and a Cdc42- and Rac1-activated
kinase p65-PAK (Manser et al., 1994). Both ACK and PAK inhibit
intrinsic as well as GAP-stimulated GTPase activity of the p21s. PAK
belongs to a family of kinases that includes the S. cerevisiae
STE20 gene product (Leberer et al., 1992; Ramer and
Davis, 1993) which acts upstream of the pheromone response
mitogen-activated protein kinase cascade (Ammerer, 1994). Two other
related S. cerevisiae kinases Cla4p ()and a
putative gene product present in the yeast genome we designate as
Sc-PAK show homology to PAK in their putative kinase and Cdc42-binding
domains.
The use of a [-
P]GTP-p21
overlay technique has allowed us to identify at least eight mammalian
candidate target proteins for Rac1, Cdc42, and RhoA (Manser et
al., 1994). The brain-enriched p65
co-purified with
a number of kinases of similar size also identified in
[
-
P]GTP-Cdc42 overlays. A human PAK
designated hPAK65 has been reported to be ubiquitously expressed
(Martin et al., 1995) and probably represents the human
homologue of the ubiquitous rat p62 Cdc42/Rac1 binding protein. Thus
although PAK kinases are most abundant in the brain, they appear to be
a common target for Cdc42 and Rac ``molecular switches'' in
all mammalian cells. Two mammalian Cdc42 isoforms have been identified
(Munemitsu et al., 1990; Shinjo et al., 1990).
Here we describe a novel PAK cDNA (designated -PAK) encoding a
protein which is closely related to our previously published sequence
(now termed
-PAK) and also to the hPAK65 cDNA (Martin et
al., 1995). The putative protein products exhibit remarkable
conservation of amino acid residues in their kinase and p21-binding
domains. Despite this similarity it has been possible to establish the
relationship between the
- and
-cDNAs and PAK species found
in the brain based on differences in their biochemical properties. In vitro translated
- and
-PAK exhibit relative
molecular masses of 68,000 and 65,000 Da, respectively. A peptide
sequence derived from purified p65
has been found to be
specific for the
-PAK isoform. We show that, in a manner similar
to purified p65
, binding of Cdc42 to recombinant
-PAK kinase, but not to
-PAK, is down-regulated upon kinase
activation.
Figure 1:
Nucleotide sequence of rat -PAK.
Numerals on the left and right side of the sequence
indicate the nucleotide and predicted amino acid positions,
respectively. Regions encompassing the p21-binding and kinase domains
have been marked in bold. The N-terminal sequence of a 34-kDa
cyanogen bromide-generated peptide derived from purified p65
is underlined.
Figure 2:
Alignment of PAK-related protein kinases.
Predicted amino acid sequences of mammalian -PAK (Manser et
al., 1994),
-PAK (Fig. 1), and hPAK65 (Martin et
al., 1995), and the S. cerevisiae sequences of Ste20p
(GenBank(TM) accession number M94719), Cla4p (GenBank(TM)
accession number X82499) and a putative open reading frame encoding a
related kinase present in the yeast genomic DNA (GenBank(TM)
accession number Z48149) we designate as Sc-PAK, previously assigned as
a 36-kDa kinase (GenBank(TM) accession number X69322) were aligned
using the clustal method (DNAStar).
For immunoprecipitation the rabbit reticulocyte lysate was diluted to 4 mg/ml in tissue extraction buffer. Extracts (100 µl) were incubated with 10 µl of affinity-purified antibody for 1 h at 4 °C collected on 50 µl of protein A-Sepharose (Sigma), washed with 200 µl of extraction buffer, then 400 µl of phosphate-buffered saline + 1% Triton X-100 and eluted in SDS sample buffer.
Figure 3:
Northern analysis of PAK mRNAs. P-Labeled cDNAs derived from the more divergent regions of
- and
-PAK (see ``Materials and Methods'') were
used to probe Northern blots containing 20 µg/lane of total RNA
from rat tissues. The
- and
-PAK mRNAs were sized relative to
RNA markers (Life Technologies, Inc.). The lanes are marked as follows: Th, thymus; Sp, spleen; Lu, lung; T, testis; Br, brain; Ki, kidney; Li, liver; H, heart.
We
performed in situ hybridization with specific oligonucleotides
to determine the regional expression pattern of the two kinases (Fig. 4). -PAK mRNA was more abundant than
-PAK mRNA,
and exposure times were chosen to determine brain regional distribution
of
- and
-mRNAs, rather than to compare their absolute
levels. Equivalent sections hybridized to
- and
-specific
probes are shown side by side (Fig. 4). In both cases sense
oligonucleotide probes were used to determine ``background''
signals (data not shown).
Figure 4:
In situ localization of -
and
-PAK mRNAs. Progressive rostral to caudal coronal sections of
adult rat brain (A-E) were hybridized with
P-labeled
- or
-PAK-specific oligonucleotides (left- and right-hand panels, respectively). A, note high expression of
-PAK mRNA in cerebral cortex (Cx) and piriform cortex (Pir);
-PAK mRNA in
medial preoptic nucleus (MPO) and piriform cortex. B,
in the thalamus,
-PAK mRNA was highly expressed in certain
subdivisions e.g. lateral dorsal (LD) and ventral (V) thalamic nuclei.
-PAK mRNA was high in the CA1
pyramidal cell layer of the hippocampal formation (CA1), low
in dentate gyrus (dg), where
-PAK mRNA levels were high.
The medial nucleus of the amygdala (Me) and the ventromedial
nucleus of the hypothalamus (VMH) showed enhanced
-PAK
mRNA expression. C, in midbrain, highest expression of
-PAK mRNA was in the subiculum (S) and of
-PAK mRNA
in the dorsal raphe nucleus (DR). D, the cerebellum (Cb), pontine nucleus (Pn), and reticulotegmental
nucleus (RtTg) of the pons showed high levels of
-PAK
mRNA but not of
-PAK mRNA. E, in the medulla
-PAK
mRNA was high in the lateral reticular nucleus (LRt). F, in situ hybridization to rat embryo sections;
-PAK mRNA (13.5 day embryo) was high in neural structures
including brain (Br), spinal cord (SCrd), dorsal root
ganglia (DRG), and olfactory epithelium (Olf).
Similarly
-PAK mRNA (20 day embryo) was high in the brain, spinal
cord, and dorsal root ganglia. Magnification: bar equals 2.4
mm (A-E) and 0.45 mm (F).
-PAK mRNA was expressed in the cortex
with highest levels of hybridization over cell layers IV and V, in
limbic regions of the cortex, and in piriform cortex (Fig. 4A).
-PAK mRNA was also relatively high in
piriform cortex while in the cortex enriched in layers II-III and
V. Both mRNAs were expressed in the hippocampal formation (Fig. 4B) in both the CA1 and dentate gyrus.
-PAK
mRNA was highly expressed in subiculum (Fig. 4C) and
showed some of its highest expression in the ventral tier nuclei of the
thalamus. Here
-PAK mRNA exhibited only low expression, but showed
greater enrichment in the hypothalamus; in medial preoptic (Fig. 4A), ventromedial and arcuate nuclei (Fig. 4B). Relatively high levels of
-PAK mRNA
were present in the monoaminergic dorsal raphe nucleus (Fig. 4C) and locus coeruleus.
Interestingly,
-PAK mRNA was highly expressed in a number of neuronal groups
associated with motor function, including the pontine nucleus,
reticulotegmental, external cuneate, and lateral reticular nuclei,
which send mossy fiber input to the cerebellum (Fig. 4, D and E).
-PAK mRNA was also highly expressed in
scattered neurons in the pontine and medullary reticular formation, and
in patches of cells comprising the linear nucleus of the medulla, with
moderate expression in the inferior olivary nucleus which provides
climbing fiber input to the cerebellum (see Fig. 5for detailed
comparisons with protein expression). Both
- and
-PAK mRNAs
were highly enriched in the embryonic central nervous system (Fig. 4F) with relatively little expression elsewhere,
confirming the specificity of the probes.
Figure 5:
Co-localization of -PAK mRNA and
immunoreactivity in neurons of the medulla. A, in situ hybridization for
-PAK mRNA in the mid-portion of the medulla
(and cerebellum, Cb) shows labeling of the inferior olivary
nucleus (IO), external cuneate (ECu), and in patches
of cells (arrows) which comprise the linear nucleus of the
medulla. B, anti-PAK immunoreactivity in a similar section of
medulla in which the same regions are labeled. C, shows a high
power photomicrograph of cells labeled by in situ hybridization for
-PAK mRNA. Silver grains were concentrated
over large reticular neurons in the linear nucleus of the medulla (arrows) which were adjacent to smaller unlabeled cells (arrowheads); sections were counter-stained with methyl
green/pyronine red. D, shows anti-PAK immunoreactivity in
perikarya and processes (but not nucleus, arrow) of large
neurons in the pontine reticular formation. Magnification: bar equals 1 mm in A and B and 10 µm in C and D.
Figure 6:
The -PAK cDNA encodes
p65
. A,
[
-
P]GTP-Cdc42 overlay of rat brain extract
(at concentrations indicated, lanes 1 and 2)
predominantly detects three protein bands, the central band appears to
migrate at the same position as purified rat p65
(lanes 3 and 4). B, supercoiled cDNA
(1 µg, pBluescript SK-) plasmid was used to generate
[
S]methionine-labeled
- and
-PAK
proteins by coupled in vitro transcription/translation with T3
polymerase (50 µl, Promega; C is control luciferase). Lanes 1-3 show 10 µl of total translation mix
resolved on a 9% SDS-polyacrylamide gel. Antibodies (
4 µg)
purified with recombinant
-PAK
were then
used to immunoprecipitate 40 µl of each translation mix (IP
+, lanes 4, 6, 8). These were run after heat treatment in SDS
sample buffer to disrupt complexes. As controls 10 µl of
translation mix (lanes 5 and 7) were heated to 90
°C for 3 min in SDS sample buffer showing the same mobility as
unheated samples (lanes 1 and 2). C, rat
brain PAK protein was purified as described previously (Manser et
al., 1994). The purified material was precipitated with 2 volumes
of acetone and the pellet dissolved in 100 µl of 70% formic acid. Lane 3 shows 10 µg of this material after lyophilization.
Cynaogen bromide was added to 10 mg/ml, and the solution was degassed
and left overnight under nitrogen. The digest was then lyophilized and
dissolved in SDS sample buffer. Lanes 1 and 2 show 45
and 5 µg of material run on 12% polyacrylamide gels and transferred
to PVDF membranes. Following Coomassie Blue staining (left-hand
panel) the membrane was overlaid with
[
-
P]GTP-Cdc42 to detect peptides containing
the Cdc42 binding domain.
Tryptic peptides obtained previously
from within the kinase domain of affinity-purified bovine PAK
corresponded to regions of - and
-PAK which are identical in
sequence. However confirmatory evidence for the presence of
-PAK
in affinity-purified rat brain PAK (Fig. 6C, lane 3)
was obtained from a cyanogen bromide digest of the protein. The
peptides were subjected to SDS-PAGE, transferred to PVDF membrane, and
overlaid with [
-
P]GTP-Cdc42. Lanes 1 and 2, with 45 and 5 µg of digested protein(s),
contains a Cdc42-binding polypeptide with apparent mass of 34 kDa which
was subjected to N-terminal amino acid sequencing. The obtained
sequence (M)APEEXNXXAxLXXIFPGGG was not
informative at every position but corresponds to the predicted
-PAK product following cleavage at M37 (underlined in Fig. 1). Since this region of
-PAK is divergent from both
-PAK and hPAK65 sequences, it is highly likely that the peptide is
derived from the
-PAK gene product. Although a mass of >30 kDa
for a CNBr-generated peptide of
-PAK is not consistent with its C
terminus being derived from cleavage at
-M138, there are many
documented cases of methionines which show inefficient CNBr cleavage,
and lower bands were also detected by
[
-
P]GTP-Cdc42 (Fig. 6C). We
cannot estimate the stoichiometry of
- and
-PAK by CNBr
digestion and overlay because the
-PAK p21-binding domain is
probably destroyed by cleavage at at an internal methionine
(
-M99), a position occupied by a conservative isoleucine in
-PAK(I94).
Figure 7:
Tissue distribution of -PAK by
Western analysis. Soluble proteins from tissues (150 µg/lane) were
run on 9% SDS-polyacrylamide gels and transferred to PVDF membranes.
Identical filters were probed with affinity purified
-PAK
antibodies followed by horseradish peroxidase-coupled second antibody
with luminol detection for 30 s (A) and 2 min (B) or
analyzed for [
-
P]GTP-Cdc42 binding (C). Tissues are as follows: b, brain; h,
heart; k, kidney; li, liver; lu, lung; s, spleen; and te,
testis.
Figure 8:
Recombinant PAK kinases are p21-activated. A, kinase activity assayed in vitro with myelin basic
protein (MBP) in the absence (-) or presence (+) of
GTPS/Cdc42. Each lane contains 1 µg of GST/PAK and 10 µg
of MBP ± 2 µg GTP
S-GST/Cdc42 and the reaction carried
out by incubating the proteins in kinase buffer at 30 °C for 10 min
with 20 µM [
-
P]ATP. The gel
stained with Coomassie Blue shows the positions of GST/PAK, GST/Cdc42,
and MBP. The autoradiograph shows
P-phosphorylated
proteins. B, effect of various p21s on GST/
-PAK activity;
Rho-p21s (1 µg) were preloaded with the appropriate nucleotide (0.5
mM) and incubated with 0.5 µg of GST/
-PAK and 5
µg of MBP at 30 °C. Activity incorporated into MBP was
quantified using a PhosphorImager (Molecular Dynamics). C,
Cdc42 and Rac1 activation of of GST/
-PAK with 0.25 mM unlabeled ATP. Left-hand panel shows Coomassie
Blue-stained kinase; in the right-hand panel the proteins were
resolved on a 9% polyacrylamide gel co-polymerized with 0.1 mg/ml MBP.
Following the denaturation/renaturing steps (see ``Materials and
Methods''), [
-
P]ATP was added to the
gel in kinase buffer to detect MBP kinase activity (2-h exposure). D, activity of inactive and Cdc42-activated
-PAK
(
); 2 µg of recombinant kinase was dialyzed for 2 h against
kinase buffer and incubated at 32 °C with 20 µg of MBP (total
volume, 100 µl) with 50 µM [
-
P]ATP. At the times shown 25-µl
aliquots were removed, quenched by the addition of SDS buffer and
fractionated on a 12% gel. Radioactivity associated with MBP is in
arbitrary units (an averaged value of three determinations is shown);
errors for the unactivated PAK (
) were within the values covered
by the symbol.
Following complete activation (1 h in the
presence of GTPS-Rac or GTP
S-Cdc42),
-PAK exhibited
slower mobility under SDS-polyacrylamide electrophoresis (Fig. 8C), as seen with the purified protein (Manser et al., 1994) and many other protein kinases. The
hyperphosphorylated kinase exhibited some size heterogeneity in its
activated state. After separation from the GTP
S-p21s by SDS-PAGE,
-PAK was still active as determined by an in-gel kinase assay
against MBP (Fig. 8C). No labeling was seen in the
absence of MBP in the gel (data not shown). This supports the model we
presented previously in which the autophosphorylated kinase, after
dissociation of p21, would remain active in the absence of
dephosphorylation. The ability of this active kinase to phosphorylate
MBP was tested (under similar conditions to the co-activation assay in Fig. 8B). Based on the initial rates of phosphorylation (Fig. 8D), the activity of the phosphorylated form was
found to be more than 300 times higher than the unphosphorylated
-PAK.
Figure 9:
Decrease in Cdc42 binding to activated
-PAK. A, full-length GST/PAK was activated in the
presence of 2-fold excess GTP
S-Cdc42 and 0.5 mM ATP for 1
h at 30 °C (+Activation) or in the presence of 1 unit
of alkaline phosphatase (unphosphorylated control). Proteins separated
by SDS-PAGE and stained with Coomassie Blue are shown in the left-hand panel. Indicated amounts of the proteins were also
transferred to PVDF and processed for
[
-
P]GTP-Cdc42 overlay. The filters were
placed against a phosphor screen at -20 °C (output from one
experiment shown) and analyzed using the Imagequant software (Molecular
Dynamics) to determine radioactivity associated with each band. Average
data from three independent experiments are presented. B,
residues corresponding to
-PAK
(BamHI/BglII cDNA fragment) and
-PAK
(SmaI/Sca1 cDNA
fragment) fused to GST were phosphorylated as in A with
full-length GST/
-PAK. The unphosphorylated and phosphorylated
proteins were resolved on a 9% gel, transferred to PVDF, and overlaid
with [
-
P]GTP-Cdc42. C, 2 µg of
fusion protein, as in B, were overlaid and bound counts at the
position of the 60-kDa band were quantified and found to be ±
10% between Rac1 and Cdc42 in two independent
experiments.
The
Rac1/Cdc42-binding domain of PAK shows sequence homology to the
Cdc42-specific binding domain of the tyrosine kinase ACK (Manser et
al., 1993). This region is highly conserved in - and
-PAK (Fig. 2). Although PAK binding to Rac1 appeared
significantly weaker than to Cdc42, their ability to activate the
autophosphorylation and MBP kinase activity of purified p65-PAK
depended on the assay conditions (Manser et al., 1994). As
illustrated in Fig. 9C, by normalizing the labeling of
the p21s using excess (cold) GTP in the ``exchange''
reaction, the amount of [
-
P]GTP-Cdc42 or
[
-
P]GTP-Rac1 bound to the N-terminal region
of
- and
- PAK in overlays was found to be the same
(±10%). Note there is no potential autophosphorylation of the
construct. The labeling of Rac1 with
[
-
P]GTP is normally poor, probably because
of its high intrinsic GTPase activity during the exchange reaction in
low magnesium buffer (Menard et al., 1992).
The heterogeneity in Cdc42-binding proteins with apparent
molecular mass between 60 and 70 kDa in different tissues suggested
these kinases to be expressed from a number of related genes. The
identity and relationship of two of these kinases (- and
-PAK) have now been established through isolation of their cDNAs.
Amino acid sequence comparison between the two mammalian PAKs described
here and hPAK65 (Martin et al., 1995) reveals functionally
important regions of the protein. In particular the p21-binding domain
showed almost no sequence divergence. PAK kinases also contain
polyacidic and proline-rich sequences between the p21-binding and
kinase domains. The use of purified recombinant proteins has enabled us
to confirm that addition of GTP
S-p21 was sufficient to activate
the kinase in the absence of any factor that might have co-purified
with brain-derived native PAK. This activation by Rac1 or Cdc42 was
achieved through the p21-mediated autophosphorylation of the kinase.
Recombinant (p68)
-PAK showed robust activity toward MBP in the
presence of GTP
S-p21, as we have described for purified
p65
(
-PAK). Here we show with recombinant
-PAK
protein that there is a 300-fold increase in activity following
p21-mediated autophosphorylation.
While -PAK was expressed
predominantly in the brain, both mRNA and protein were detected in the
spleen. Within the immune system it appears that PAK-related kinases
are relatively abundant; the report that neutrophil p62 and p68
Cdc42/Rac-binding proteins are not reactive to antibodies to the
conserved PAK kinase domain (Martin et al., 1995) is in
conflict with our demonstration that antibodies generated against
-PAK recognize the neutrophil p68 protein (Prigmore et
al., 1995). The p65
-PAK appears to be more restricted in its
expression outside of the nervous system. We have been unable to
correlate the PAK expression pattern with any known signaling molecule
or receptor, but the strikingly restricted expression pattern of both
isoforms lends credence to the idea that
- and
-PAKs may be
coupled to specific Rho-p21 pathways in the brain. Another Rho-p21
interacting protein with highly restricted expression is the RacGAP
2-chimaerin, which is only present in cerebellar granule cells
(Leung et al., 1994) where high expression of
-PAK mRNA
is also observed.
The hPAK65 mRNA has been reported to be expressed
in a wide variety of tissues and in cultured cells. We have obtained
peptide sequence data allowing the cloning of the ubiquitous rat p62
Cdc42/Rac binding protein which we designate as -PAK.
Because the published hPAK65 cDNA contains, in the 5`-noncoding
sequence and 38 base pairs of putative open reading frame, sequence
identical to human placental lactogen (somatomammotropin, Seeburg et al., 1977), this region of the hPAK cDNA may have arisen as
a library artifact. The only biochemical property reported for this
recombinant enzyme that differs significantly from those of
brain-purified p65
is that there is no decrease in Cdc42
binding of activated hPAK65 (Martin et al., 1995). Our study
clearly shows that a difference in this respect exists between the
- and
-isoforms in that
-PAK, but not
-PAK, binds
GTP-Cdc42 more weakly after activation of the kinase. This is
consistent with
-PAK cDNA encoding the purified p65
protein which exhibits similar behavior. Autophosphorylation of
-PAK occurs on at least 6 residues in the N-terminal regulatory
region. (
)There is, therefore, potential for similar
phosphorylation events in the
-PAK, perhaps altering the
conformation of the p21-binding domain such that its affinity is
affected. The loss of affinity of
-PAK for GTP-Cdc42 after
activation of the kinase suggests that the p21 in this case could
amplify its signal by activating several kinase molecules, or
alternatively it allows the activated kinase to act at a different
site. In contrast,
-PAK activity may co-localize with GTP-p21 and
remain as part of the signaling complex.
Although functions for PAK
kinases in relation to the roles of Cdc42 and Rac1 in mammalian cells
is yet to be established, we have been able to demonstrate that Ste20p,
a kinase required for the mating response pathway in S. cerevisiae (Leberer et al., 1992; Ramer and Davis, 1993), requires
GTP-Cdc42p for its function. ()As in higher organisms, there
appear to be a family of PAK kinases in yeast (Blumer and Johnson,
1994); the 36-kDa yeast kinase previously thought to only contain a
kinase domain is now confirmed as having an N-terminal p21-binding
domain (Fig. 2). The role of Ste20p as a kinase acting above the
pheromone-responsive mitogen-activated protein kinase cascade may point
to the involvement of mammalian Cdc42 and Rac proteins in cell
proliferation and differentiation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33314[GenBank].