(Received for publication, July 5, 1995; and in revised form, August 23, 1995)
From the
The brain-enriched p21-activated
serine/threonine kinase, p65
, was identified and purified
on the basis of overlays with [
-
P]GTP-Cdc42
onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H.,
Zhao, Z.-S., and Lim, L.(1994) Nature 367, 40-46). In
this study, the ubiquitously expressed p21
binding protein with relative molecular weight of 62,000 was
purified from rat testes and shown to contain peptides related to PAK.
It has thus been designated as the
-PAK isoform (
- and
-isoforms being brain enriched). Isolation of
-PAK cDNAs show
that the kinase is highly conserved with
-PAK in both the p21
binding and kinase domains. The purified protein exhibited kinase
activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro.
In platelets, a p62 in situ renaturable kinase was recognized
by antibodies raised against
-PAK. This thrombin-activated protein
kinase appears to coprecipitate with another kinase of M
86,000, suggesting that PAK may be part of a thrombin-responsive
signaling complex.
The mammalian p21-related Rho subfamily
of GTP binding proteins, including Rho, Rac, and Cdc42, are involved in
regulating morphology. Microinjection studies with Swiss 3T3
fibroblasts have shown that RhoA plays a role in stress fiber
formation, mediating their formation by growth factors such as
platelet-derived growth factor(1) . Activation of Rac1 induces
membrane ruffling and a subsequent activation of Rho(2) .
Cdc42, originally identified as being required for development of
polarity in Saccharomyces cerevisiae(3) , has two
mammalian homologues(4, 5) . Injection of mammalian
Cdc42Hs causes filopodia formation in Swiss 3T3
cells(6, 7) . Cdc42 is required for bradykinin-induced
morphological changes(6) .
GTPase-activating proteins
(GAPs), ()which stimulate hydrolysis of the GTP bound to Rho
subfamily members include the sequence-related Bcr, chimaerin, and
RhoGAP(8) . These down-regulatory GAPs are generally
multi-functional; for example, Bcr has serine/threonine kinase (9) and dbl homology domains(10) , while
2-chimaerin contains SH2 and phorbol ester binding domains (11) . An overlay method that detects GAPs and Rho binding
proteins (12) has facilitated the cloning and purification of
GAPs including the Bcr-related Abr (13) and
-chimaerins (14, 15) as well as putative target proteins,
including the activated Cdc42-associated tyrosine kinase, p120
(16) and the p21
-activated
kinase (p65
) (17) . Brain-enriched p65
is a serine/threonine kinase, activated by GTP-bound forms of
Cdc42 and Rac and is homologous to the yeast kinase Ste20p, which forms
part of the pheromone-induced kinase cascade pathway in S.
cerevisiae(18) . Another class of Rac targets is
p67
, which regulates the NADPH oxidase activity
in phagocytes(19) .
The overlay method also disclosed a
ubiquitous 62-kDa protein binding Cdc42/Rac(12, 17) .
Here, we show that this p62 represents a PAK isoform. The p62 was purified from rat testes and subsequently cloned from a rat
brain cDNA library. The cDNA encoded a protein designated as
-PAK,
being highly homologous to
- and
-PAK (20) in both
the p21 binding and kinase domains. The purified native p62
exhibited kinase activity that was stimulated by GTP-Cdc42 and
GTP-Rac in vitro.
-PAK is widely expressed in all the rat
tissues tested, unlike brain-enriched
- and
-PAKs(20) .
Thrombin stimulation of human platelets
activates several signaling molecules including GTP binding proteins
and phospholipase C-, leading to aggregation, cell adhesion, and
secretion(21) . Phospholipase C-
hydrolysis of inositides
results in a transient increase of intracellular calcium and
diacylglycerol, which subsequently activates protein kinase
C(22) . Other serine/threonine protein kinases activated by
thrombin have been detected by a renaturable in situ kinase
assay(23) . One of these platelet protein kinases, a 62-kDa
(p62) serine/threonine kinase, is recognized by antibodies to
recombinant
-PAK.
Figure 3:
Isolation, nucleotide, and predicted amino
acid sequence of -PAK. A, schematic diagram showing the
cloning strategy for
-PAK cDNA. PCR was performed by using
degenerate primers corresponding to peptides IVSIGDP and WMAPEV shown
in Fig. 3B to amplify rat testis cDNA. An amplified
600-bp PCR product (probe A) was sequenced and used to screen
a rat brain cDNA library. Three phage clones with insert sizes of 4,
1.5, and 2.5 kb (clones A1, A2, and A3,
respectively) contained overlapping C-terminal sequences. The longest
clone (A1) was truncated at the 5`-end, and an upstream probe (probe B) of 400 bp from clone A1 was used to rescreen the
same library. Four clones of sizes 4, 0.9, 3, and 2 kb (clones
B1, B2, B3, and B4, respectively) were
obtained. Clone B1 was identical to clone A1, and clones B2 and B3
contained overlapping N-terminal sequences. The 2-kb clone (B4)
contained the entire
-PAK cDNA sequence including the
translational start site. The restriction map for clone B4 is also
shown. Bottom, a schematic diagram of the B4 open reading
frame and the relative positions of the p21 binding and kinase domains,
and untranslated regions. B, the cDNA and predicted amino acid
sequences of
-PAK. The five peptide sequences derived from
purified p62
are underlined.
Figure 1:
Purification of the ubiquitous
p62 binding protein. A, a rat
testes supernatant fraction was loaded onto an S-Sepharose column. The
flow-through (lane 1) was collected, and proteins were eluted
with 0.1, 0.2, 0.3, and 0.4 M NaCl (lanes 2, 3, 4, and 5, respectively). The p62-enriched
fractions (*) were pooled and loaded onto a Q-Sepharose column. The
flow-through (lane 6) and bound proteins were eluted with 0.1,
0.2, 0.3, and 0.4 M NaCl (lanes 7-10). The
p62-containing fractions were pooled (lane 11) and loaded onto
a zinc-chelating column. The flow-through was collected (lane
12), and proteins were eluted at pH 7.5, 6.0, and 5.0 (lanes
13-15). The p62-containing fraction (lane 16) was
next loaded onto a GTP
S-Cdc42 affinity column (B). The
flow-through (lane 17), pH 6.0 wash (lanes
18-20), and pH 8.8 eluted fractions (lanes
21-23) were collected. The GST/Cdc42 protein was eluted with
glutathione (lane 24). The protein fractions were
electrophoresed on 9% SDS gels, transferred onto nitrocellulose
filters, and subjected to [
-
P]GTP-Cdc42
binding assay(17) . A Coomassie-stained gel corresponding to lanes 23 and 24 shows the purified p62 and p60
proteins and the GST/Cdc42 protein eluted from the affinity
column.
Figure 2:
Activation of p62 kinase
activity. A, Cdc42 and Rac stimulates phosphorylating
activities of p62
. Purified p62
, incubated
with MBP (5 µg) and 20 µM [
-
P]ATP alone (lane 1) and
with either GTP
S-Cdc42 (lane 2) or GTP
S-Rac (lane 3) was separated on 9% SDS gels, stained with Coomassie
Blue, and put to film. B, p62
is
autophosphorylated in vitro on serine and threonine residues.
The phosphorylation of p62
and MBP was stimulated with
GTP
S-Cdc42, and bands corresponding to
P-labeled
p62
and MBP were subjected to phosphoamino acid
analysis(17) . The positions of the ninhydrin-stained
phosphorylated standards are phosphoserine (PS),
phosphothreonine (PT), and phosphotyrosine (PY). C, Cdc42 and Rac1 induce a mobility shift of
p62
. Purified p62
(1 µg, lane
3) or p62
incubated with either 2.5 µg
GTP
S-Cdc42 (lane 4) or GTP
S-Rac (lane 5)
were assayed for [
-
P]GTP-Cdc42 binding as
described in Fig. 1. Binding of
[
-
P]GTP-Cdc42 to rat brain cytosol (lane 1) and testis p62
(lane 2) are
also shown for comparison.
Figure 4:
Comparison of PAK sequences. Alignment of
amino acid sequences of -PAK(17) ,
-PAK, and
hPAK65(25) , including the p21 binding domain
(
-PAK
) and the kinase domain
(
-PAK
), is shown. Sequences were aligned
using the Clustal method (DNASTAR), and shaded areas represent
regions of identity of the PAK isoforms.
Figure 5:
Analysis and distribution of -PAK
mRNA. A, Northern analysis of
-PAK mRNA. A Northern blot
(Clonetech) containing
2 µg of poly(A
) RNA
from various rat tissues was probed with a 800-bp BamHI-HpaI fragment of clone B4 (see Fig. 3A), labeled with
[
-
P]dCTP, and then reprobed with
-actin cDNA, similarly labeled. The tissue samples are as follows:
brain (B), spleen (S), lung (Lu), liver (Li), skeletal muscle (SM), kidney (K), and
testes (T). B, comigration of p62
and in vitro translated p62 product. Soluble proteins from tissues
(150 µg/lane) were assayed for
[
-
P]GTP-Cdc42 binding as described in Fig. 1. The tissue samples are as follows: brain (B),
heart (H), kidney (K), liver (Li), lung (Lu), spleen (S), testis (Te), and thymus (Thy). An in vitro translation reaction with B4 cDNA
was performed using a TNT-coupled reticulocyte lysate system according
to manufacturer's protocol (Promega). The
-PAK translated
products (5 and 10 µl of lysate, lanes 1 and 2)
were separated on 9% SDS gel and exposed to Hyperfilm
(Amersham).
Figure 6:
Comparison of p21 binding between
-PAK and
-PAK. A,
-PAK and
-PAK proteins
bind GTP-Cdc42 with similar affinity. Various concentrations
(0.05-1 µg) of the GST/
-PAK
and
GST/
-PAK
fusion proteins were assayed for
[
-
P]GTP-Cdc42 binding as described in Fig. 1. The amount of [
-
P]GTP-Cdc42
bound to the immobilized fusion proteins (
-PAK (
) or
-PAK (
)) was quantified with a phosphoimager (Molecular
Dynamics), as shown in the graph. The results from duplicate
experiments are indicated by empty or filled symbols. B, inhibition of Cdc42 GTPase activity by GST/
-PAK and
GST/
-PAK. 1 µg of [
-
P]GTP-Cdc42
was incubated at 20 °C in the absence (
) or presence of
10-fold excess purified GST (
), GST/
-PAK
(
), full-length GST/
-PAK (
]), or
GST/
-PAK
(
). At each time point, two
10-µl aliquots were absorbed onto nitrocellulose, washed, and
assessed for bound radioactivity.
Figure 7:
Thrombin stimulation of renaturable
kinases in platelets. A, platelets were incubated with 1
unit/ml of thrombin at 37 °C for 0, 0.5, 1, and 3 min as indicated.
The platelet lysates were separated by 9% SDS gels, transferred onto
PVDF membranes, and assayed for kinase activity in situ. B, The p62 kinase comigrated with the major
[-
P]GTP-Cdc42 binding activity in these
extracts.
Figure 8:
Identification of Cdc42 GAPs and binding
proteins in platelets. A, rat brain cytosol (lanes 1 and 4), purified testis p62 (lanes 2 and 5), and thrombin-activated platelet cytosol (lanes 3 and 6) were separated on 9% SDS gels,
transferred onto PVDF membrane, and assayed for
[
-
P]GTP-Cdc42 binding (lanes
1-3) or for Cdc42-GAPs (lanes 4-6) by the
overlay assay(12) . B, rat brain cytosol (lane
1) and platelet cytosol (lane 2) were separated on 9% SDS
gels, transferred onto nitrocellulose, and incubated with either
anti-Abr antibody (13) or anti-p85 antibody
(UBI).
However, as we observed strong GAP activity in
p85 and p50 in platelets even in the binding assays (Fig. 7B, seen as white bands), we subjected
platelets and rat tissues to the GAP overlay technique(12) .
Brain cytosol contained GAPs (white bands) of 150, 100,
85, 50, and 45 kDa as previously reported (Fig. 8A, lane 4, marked) with a faint band of 75 kDa (the
white bands of 60-68 kDa result from
[
-
P]GTP-Cdc42 sequestration (binding) by
the PAK isoforms rather than from GAP activity). Platelets contained
Cdc42 GAPs of sizes 85, 75, 50, and 45 kDa (Fig. 8A, lane 6). To determine if these were due to proteins containing
Rho GAP-like domains such as Abr or the p85
regulatory subunit of
phosphatidylinositol 3-kinase, an immunoblot was assayed with
appropriate antibodies. Abr, present in brain, was not detected in
platelets; both tissues, however, apparently contained the p85 subunit (Fig. 8B).
Polyclonal antibodies against
-PAK
, lacking the kinase domain, detected
only the p62
and did not significantly cross-react with
the
- and
- isoforms, as assessed by
[
-
P]GTP Cdc42 binding to immunoprecipitates
from brain extract (Fig. 9C, Cdc42 overlay panel, cf. lanes 1 and 2). The platelet p62 was efficiently
immunoprecipitated, exhibiting both Cdc42 binding and serine/threonine
kinase activity in situ (Fig. 9, A and B, lanes 2). It was not tyrosine phosphorylated (data
not shown). A serine/threonine kinase of about 86 kDa appeared to be
coimmunoprecipitated with p62
and which did not bind
Cdc42 (Fig. 9, A and B, lanes 2).
This p86 was not detected by the
-PAK anti-serum (data not shown).
Preincubation of the antibodies with
-PAK
fusion protein drastically decreased immunoprecipitation of both
p62
and p86 (Fig. 9B, lanes 3).
The p86 kinase was not detected in brain immunoprecipitates (Fig. 9C) (the brain p90 with weak kinase activity was
not affected by the preincubation of the antibodies with
-PAK
fusion proteins, cf. lanes 2 and 3). Another
renaturable immunoprecipitated brain protein kinase (p65) was detected (Fig. 9C, in situ kinase panel, lane
2). Although it did not bind Cdc42 (Fig. 9C, Cdc42 overlay panel, lane 2), its activity was diminished
when the antibodies were preincubated with
-PAK proteins (Fig. 9C, in situ kinase panel, lane
3). The identities of the p62
-associated protein
kinases, p86 and p65, are under study.
Figure 9:
Immunoprecipitated p62 protein exhibits
both Cdc42 binding activity and in situ kinase activity. A, and B, immunoprecipitation of p62 from platelets. Thrombin-activated platelet lysates (lanes
T) were immunoprecipitated with either
-PAK antibodies
(raised to GST/
-PAK
fusion proteins) alone
(lanes IP) or antibodies preincubated with
GST/
-PAK
fusion proteins (lanes
IP+C). The lysates or immunoprecipitates were separated on 9%
SDS gels, transferred, and assayed for
[
-
P]GTP-Cdc42 binding activity or for
kinase activity in situ. Strong signals in lane IP+C of the Cdc42 overlay correspond to the
GST/
-PAK
, which does not include the kinase
domain. C, immunoprecipitation of p62
from brain
supernatant. Rat brain supernatant (lanes T) was
immunoprecipitated with
-PAK antibodies alone (lane IP)
or in the presence of GST/
-PAK
fusion
proteins (lanes IP+C). The purified
GST/
-PAK
protein was also shown as a
reference (lanes C). The brain lysate and immunoprecipitates
were separated on 9% SDS gels, Coomassie stained, or transferred onto
PVDF membranes, and assayed for [
-
P]-Cdc42
binding activity or for kinase activity in
situ.
In this study, we report the purification and subsequent
cloning of a 62-kDa Cdc42/Rac binding protein, which corresponds to an
isoform of PAK, termed -PAK.
-PAK is ubiquitously expressed
in rat tissues, unlike
- and
-PAK, which are found in few
tissues other than brain(20) . Thus,
-PAK reflects the
widespread distribution of Rac and Cdc42 (28) and is probably a
common target protein for signaling by these proteins. Purified native
p62
activity toward MBP is increased on binding to
GTP-Cdc42 and GTP-Rac in vitro. Unlike purified
p65
, the p62
does not show decreased
binding to Cdc42 upon activation. This is similar to results using
recombinant hPAK65(25) , except that we find PAK to be
phosphorylated in both threonine and serine residues. hPAK65 and rat
-PAK appear to be homologues. The only non-conservative amino acid
difference is at
-PAK
, where hPAK65 contain an
arginine; otherwise, this glycine is present in all mammalian and yeast
PAK-like kinases that are known. Curiously, although the predicted
hPAK65 contains 18 amino acid residues less than
p62
, its reported mobility represents a molecular
mass of 65 kDa. This difference could be due to the presence of 12
amino acids derived from the placental lactogen mRNA sequence. It is
likely that the reported hPAK65 cDNA does not exist as a bona fide mRNA in vivo, although a splicing event between two mRNAs
is possible. The rat
-PAK N terminus shows blocks of homology to
-PAK and has been identified in two independent clones. The use of
a probe derived from the conserved kinase domain of hPAK65 (25) will result in detection of mRNA of various PAK isoforms.
The use of a probe sequence corresponding to the more variable linker
domain allowed us to determine that the 4-kb mRNA is the dominant
-PAK mRNA in rat tissues. Although the
170-kDa Cdc42/Rac
binding protein might be thought to arise by alternate splicing from
the larger 7-kb mRNA, this family of binding proteins is not sequence
related to PAK. (
)
In platelets, the thrombin-activated
p62 kinase (23) probably corresponds to p62,
suggesting the involvement of Rac and Cdc42 in platelet function.
Thrombin also appears to require Rho proteins to mediate cell
aggregation, which can be inhibited by C3 transferase, which
ribosylates Rho(29) . Rho has been reported to activate a
downstream target, p85 phosphatidylinositol 3-kinase, in
platelets(30) . The role of Cdc42 and Rac is likely to involve
the dramatic changes in cell shape that accompany platelet activation,
since mammalian Cdc42 has been associated with cell spreading in
monocytes (3l), and microinjected Cdc42 (6, 7) and Rac (2) proteins can induce filopodia and membrane ruffling,
respectively, in Swiss 3T3 cells. In yeast, the PAK-related kinase
Ste20p is thought to lie at the top of the pheromone-induced
mitogen-activated protein kinase cascade (18) . This kinase has
been implicated as a target for the Ste4/Ste18
subunits and
interacts with Cdc42(32) , suggesting that mammalian Rho p21s
may regulate mitogen-activated kinase cascades.
Since Ras and Rac
can co-operate in transformation (33) and a Ras target,
phosphatidylinositol 3-kinase, has been suggested to mediate
platelet-derived growth factor-induced membrane ruffling in fibroblasts (34) , these p21s may signal both morphological and
transcriptional changes in cells. A candidate Rho target is
phosphatidylinositol 4-phosphate 5-kinase, which may mediate the
effects of Rho on the cytoskeleton in the integrin signaling
pathway(35) . It is likely that Rho family p21s as with Ras
p21s function in part by activating protein and lipid kinases. Although
transforming tyrosine kinases, Ras and its target Raf kinases, are
generally thought to act in cell proliferation and differentiation, it
is noteworthy that proteins such as Src and mitogen-activated protein
kinase are present in high concentrations in
platelets(36, 27) . Platelets undergo extensive
cytoskeletal reorganization during activation(21) ; the
rapidity of -PAK activation suggests a primary role for the kinase
in these events. Additionally in mast cells, Rac and Rho have been
reported to play a role in regulating secretion(37) . The
putative p62
-associated p86 kinase in platelets, which is
also a rapidly activated serine/threonine kinase(23) , may form
a signaling complex perhaps mediated by the heterotrimeric G-proteins
activated by thrombin. We have yet to determine whether this p86 kinase
is responsible for the Cdc42 GAP activity observable in overlay assays.
Further analysis of proteins associated with
-PAK are likely to
throw light on the role of PAK since some of these may well be
downstream targets for the kinase.