From the Departments of Immunology and
Cell
Biology, The Scripps Research Institute,
La Jolla, California 92037
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ABSTRACT |
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Activation of p21-activated kinases (Paks) is
achieved through binding of the GTPases Rac or Cdc42 to a conserved
domain in the N-terminal regulatory region of Pak. Additional signaling components are also likely to be important in regulating Pak
activation. Recently, a family of Pak-interacting guanine nucleotide
exchange factors (Pix) have been identified and which are good
candidates for regulating Pak activity. Using an active, truncated form
of Ras-related small GTPases have been the subject of intense
investigation over recent years. These molecular switches cycle between
GTP-bound "on" and GDP-bound "off" states. In their GTP-bound form they are able to specifically interact with and modulate the
activity of effector molecules (1). The nucleotide state of low
molecular weight GTPases is thus a key factor influencing their ability
to effectively transduce signals, and it is therefore a tightly
regulated process. The proteins that regulate the nucleotide bound
state of small GTPases have been relatively well characterized, and
they fall into three main categories: those that accelerate the
intrinsic hydrolysis of GTP to GDP, hence switching off the G-protein
(GTPase activating proteins, GAPs); those that stabilize the protein in
the GDP-bound form in the cytoplasm awaiting the appropriate signals
(GDP dissociation inhibitors, GDIs); and finally, guanine nucleotide
exchange factors (GEFs).1
GEFs catalyze the exchange of GDP for GTP on the GTPase, thus switching
the molecule to the "on" state (2).
Rho-family GTPases are among the best characterized members of the Ras
superfamily and have been shown to regulate dynamic changes in the
actin cytoskeleton that occur during many different cellular processes
and in response to a wide variety of extracellular signals (3-5).
Members of the Rho GTPase family (Rho, Rac, and Cdc42) interact with a
diverse array of downstream effectors that may regulate specific
processes depending on the extracellular environment or stimuli. It is
still not clear what mechanisms are responsible for determining how
Rac, Rho, or Cdc42 selectively interact with their different effectors
and hence trigger specific intracellular pathways. Recent studies have
suggested that guanine nucleotide exchange factors may be important
determinants of the selectivity of small G-protein signaling (6). This
concept has arisen for several reasons. First, the number of Rho-family GEFs (~40) already exceeds that of the Rho family members (~10), thus increasing the potential diversity of responses. Second, many of
the GEFs identified contain multiple protein-protein interaction domains and are therefore good candidates for enabling coordinated, compartmentalized signaling events to occur (7-9). Finally, and most
intriguingly, several recently identified GEFs for small GTPases have
been shown to bind both the GTPase and its downstream effector, thus
bringing the two key signaling elements in close proximity and
suggesting a possible mechanism through which GEFs may generate the
selectivity of GTPase-effector interaction (10-11).
p21-activated kinases (Paks) were first identified as targets of Rac
and Cdc42 and have subsequently been implicated in various cellular
processes regulated by Rac and Cdc42 (12-15). Studies in our
laboratory and others have suggested that Pak may play a key role in
regulating cytoskeletal dynamics (16-18). Recently a guanine
nucleotide exchange factor termed Pix
(p21-interacting exchange factor)
that binds specifically to Pak via interaction of a Pix-SH3 domain with
the fourth proline-rich region in Pak was identified (11). This region
has also been implicated as playing a role in Pak-dependent
regulation of neurite outgrowth and cytoskeletal activity (17, 19).
Because of the reported exchange factor specificity of Pix toward Rac
and Cdc42, it was suggested Pix might regulate GTPase-mediated
activation of Pak (11). However, the only study to specifically address
this possibility to date demonstrated that one isoform of Pix, We demonstrate here that a functionally active form of Plasmids--
cDNA expression plasmids containing
full-length Pak1 and its various mutants in the pCMV6M vector (CMV
promoter, N-terminal myc tag) have been described elsewhere
(14). Pak1 R193A and P194A double mutations were made in full-length
WT-Pak1 by overlapping PCR. Universal primers PT637-5' and PT638-3'
were used as outer boundary primers, and we used overlapping primer
pairs to introduce the desired mutations (forward primer
5'-GTGATTGCTCCAGCCGCAGAGCACACA-3' and reverse primer
5'-TGTGTGCTCTGCGGCTGGAGCAATCAC-3'). The Pak1A193,194 was
then inserted into pCMV6M at the BamHI/EcoRI site.
Wild-type full-length
The glutathione S-transferase (GST) was fused to amino acids
67 to 150 of human Pak1 as follows. The WT-Pak1 region was amplified by
PCR using primers OP1/67-5'
(5'-CGCGGATCCAAGAAAGAGAAAGAGCGG-3') and OP1/150-3'
(AAGGAAAAAAGCGGCCGCGTCGACTCAAGCTGACTTATCTGTAAAGCT-3') using pCMV6M/Pak1 as a template. The
BamHI/SalI cut PCR product was inserted into
BamHI/SalI cut pGEX-4T3 to give
pGEX-hpak1-(67-150).
Transfection of COS-1 Cells--
COS-1 cells grown to 75%
confluence on 10-cm tissue culture dishes were transiently transfected
using the LipofectAMINE transfection protocol (Life Technologies, Inc.)
with a total of 7.5 µg of either pRK5M or pCMV6M expression vectors
containing various myc-tagged constructs. 2.5 µg of each
construct was used, and if the total number of constructs added was
less than 7.5 µg, the concentration of DNA was normalized by adding
the appropriate amount of empty vector. The cells were allowed to
express the protein for 40 h post-transfection and were then
washed in phosphate-buffered saline and scraped into 250 µl of lysis
buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 5 mM
MgCl2, 1 mM DTT, 5% glycerol, 1% Nonidet
P-40) at 4 °C. Lysates were passed four times through a 21-gauge
needle and then clarified by centrifuging for 5 min at 7,000 rpm in a
bench-top Eppendorf microfuge to remove unbroken cells and large
cellular debris. Supernatants were decanted, and 30 µl of each lysate
was run on SDS-PAGE gels and transferred to nitrocellulose prior to
probing for the expression of myc-tagged protein using an
anti-myc antibody (9E10); the remainder was immediately frozen at Kinase Assays--
Cell lysates (normalized for protein
expression levels) were subjected to immunoprecipitation using the
anti-Myc antibody and protein G-Sepharose beads, and the
immunoprecipitated proteins were analyzed using an in vitro
kinase assay as described previously (21). Briefly, after washing five
times, the immunoprecipitates (20 µl of beads) were resuspended in 40 µl of kinase buffer containing 50 mM Hepes, pH 7.5, 10 mM MgCl2, 2 mM MnCl2,
0.2 mM DTT with 1 µCi of [32P]ATP, 100 µM ATP, and 1 µg of histone H4 (or myelin basic
protein) in each incubation. Samples were then incubated for 20 min at 30 °C, and the reaction was stopped by adding 20 µl of 4× Laemmli sample buffer. The samples were boiled, and the beads were pelleted by
centrifugation. 40 µl of each reaction was separated by SDS-PAGE, and
the gel was dried and exposed to autoradiography film (Kodak, X-OMAT-AR) or PhosphorImager screen (Molecular Dynamics). For experiments where GTP GTPase Activation Assay--
Formation of Cdc42-GTP was measured
using an assay based upon specific interaction with a GST fusion of the
Pak1 p21-binding domain (PBD), as
described.2 COS-1 lysates
normalized for Cdc42 expression were mixed with 8 µg GST-PBD attached
to glutathione-Sepharose beads (Amersham Pharmacia Biotech), adjusted
to 400 µl with binding buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 0.5% Nonidet P-40), and incubated for 1 h at
4 °C. The bead pellet was then washed three times with 25 mM Tris-HCl, pH 7.5, 1 mM DTT, 30 mM MgCl2, 40 mM NaCl, 1% Nonidet
P-40 and twice with the same buffer without Nonidet P-40. The bead
pellet was finally suspended in 40 µl of Laemmli sample buffer.
Proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose
membrane, and blotted with anti-myc 9E10 antibody.
We initiated studies to evaluate the effects of the recently
described Pak-interacting protein Pix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when
Pix155-545 is co-expressed
with Cdc42 and wild-type Pak1 in COS-1 cells. This activation does not
occur when we co-express a Pak1 mutant unable to bind
Pix. The
activation of wild-type Pak1 by
Pix155-545 also
requires that
Pix155-545 retain functional exchange
factor activity. However, the Pak1H83,86L mutant that does
not bind Rac or Cdc42 is activated in the absence of GTPase by
Pix155-545 and by a mutant of
Pix155-545 that no longer has exchange factor activity.
Pak1 activity stimulated in vitro using GTP
S-loaded
Cdc42 was also enhanced by recombinant
Pix155-545 in a
binding-dependent manner. These data suggest that Pak
activity can be modulated by physical interaction with
Pix and that
this specific effect involves both exchange
factor-dependent and -independent mechanisms.
INTRODUCTION
Top
Abstract
Introduction
References
Pix,
appears to down-regulate Pak activation by upstream signals (20). The
effect of the
Pix isoform on Pak activity has not yet been
investigated, nor has it been determined whether Pak catalytic activity
can be directly enhanced by any Pix isoform.
Pix is able
to induce Pak1 activation when co-expressed with Pak1 and Cdc42 or Rac
in COS-1 cells. This activation requires both an intact
Pix Dbl
domain and the binding of
Pix to Pak1. Additionally, we report an
exchange factor-independent enhancement of Pak1 activity induced by the
binding of
Pix.
EXPERIMENTAL PROCEDURES
Pix (cDNA accession number D25304) was a
gift of N. Nomura (Kazusa DNA Research Institute, Japan). Truncated
Pix155-545 was amplified from the full-length
Pix
and cloned into pGEX 4T-1 (Amersham Pharmacia Biotech) by engineering
an EcoRI site on the 5'-oligonucleotide
(5'-GCGAATTCATGACGGAAAATGGAAGTCATCAG-3') and an XhoI site on
the 3' oligonucleotide
(5'-CGCGCTCGAGGGCAGGTCCTCTGATCAGTCTGTT-3'). For mammalian
expression,
Pix155-545 was sub-cloned into
pRK5-myc by engineering a HindIII site at the 3'
end and cloning into the BamHI/HindIII sites of
pRK5-myc. The
Pix155-545 point mutations in
the Dbl homology domain ((DH-)
Pix155-545) were
introduced by generating sense and antisense oligonucleotides containing two base pair changes in the codons for amino acids Leu-383
and Leu-384 and utilizing the "Quick Change" kit
(Stratagene). Full-length cDNAs encoding wild-type and mutations of
Cdc42 (WT and Q61L) were subcloned into the pRK5 expression plasmid
containing an N-terminal myc tag via BamHI and
EcoRI sites generated by PCR using oligonucleotides flanking
the cDNA coding sequence.
80 °C until required.
S-loaded Cdc-42 was used to stimulate Pak activity in vitro, 1 µg of preloaded Cdc42 was added to
the above reaction mix prior to initiating the reaction at
30 °C.
RESULTS AND DISCUSSION
Pix on kinase activity of Pak1.
Co-expression of full-length
Pix with Pak1 and WT-Cdc42 had no
effect on Pak1 kinase activity in vivo (Fig.
1). Indeed, overexpression of full-length
Pix with an activated mutant of Cdc42 (Q61L) inhibited the
autophosphorylation of Pak and its ability to phosphorylate exogenous
substrate (data not shown). In a recent study on the
Pix isoform
(COOL1), which itself has two alternatively spliced isoforms, a similar
inhibition of stimulated-Pak3 kinase activity was seen (20). It is
known with other Dbl-homology domain-containing GEFs that the
full-length molecules often do not exhibit effective guanine nucleotide
exchange activity. This probably reflects the requirement for
additional, unidentified signals to allow appropriate conformational
changes in the GEF leading to activation. Truncation of other Dbl
domain-containing Rho family GEFs, such as Ect, Ost, p115 Rho-GEF, and
Dbl, removes internal inhibitory constraints and allows full activity
to be observed (22-25). Manser et al. (11) demonstrated
that effective exchange factor activity was only detected in
vitro when
Pix was truncated to amino acids 155-545,
encompassing the SH3, DH, and PH domains of the protein. Therefore, we
constructed an identical version of
Pix and cloned it into mammalian
and bacterial expression vectors (Fig. 1A). Co-expression of
either full-length
Pix or
Pix155-545 with WT-Pak1
had no effect on Pak activity (data not shown). However, when WT-Cdc42
was overexpressed along with WT-Pak1 and
Pix155-545 in
COS-1 cells, we detected the formation of slower migrating species of
Pak1 indicative of autophosphorylation and activation (Fig.
1B). This activity was not seen in the absence of
Pix155-545 or when full-length
Pix was co-expressed.
The increase in Pak1 activity was confirmed when the transfected
proteins were immunoprecipitated from the lysates and subjected to an
in vitro kinase assay with histone H4 as exogenous substrate
(Fig. 1B). Expression of Pak1 and WT-Cdc42 alone or Pak1,
full-length
Pix, and WT-Cdc42 caused no significant substrate
phosphorylation (Fig. 1B). These data suggested that the
truncated active version of
Pix was able to promote nucleotide
exchange on WT-Cdc42 in vivo and hence stimulate Pak kinase
activity (see Fig. 3 below).
View larger version (31K):
[in a new window]
Fig. 1.
Pix155-545
enhances Cdc42-stimulated Pak1 kinase activity in
vivo. A, schematic representation of
full-length
Pix and
Pix155-545 showing the location
of the relevant domains in each molecule (CHD, calponin
homology domain; SH3, src homology 3 domain; DH,
Dbl homology domain; PH, pleckstrin homology domain).
B, COS-1 cells were transfected with plasmids expressing the
indicated constructs. The left panel shows a Western blot of
lysates probed with an anti-myc antibody to detect
myc-tagged Cdc42, Pak1, full-length (FL)
Pix
and
Pix155-545 as indicated. Note the slower migrating
Pak1 species in lane 3 where Pak1 and Cdc42 were
co-transfected with
Pix155-545. The right hand
panel shows the level of H4 phosphorylation induced by each of the
lysates (normalized for Pak1 expression). The number below each
lane indicates the -fold activation, with Pak1 alone taken as 1. These data are representative of four similar experiments.
We therefore investigated whether increased activation of Pak by Cdc42
in the presence of Pix155-545 was dependent on an
intact DH domain in
Pix. Dbl exchange activity has been shown to be
dependent on two conserved leucine residues in the DH-domain (26).
Mutation of these residues in
Pix abolished GEF activity (11). We
mutated the corresponding residues in
Pix155-545
(L383R, L384S) and observed that when this construct was co-expressed with Pak1 and WT-Cdc42 in COS-1 cells, there was an ~60% decrease in
the enhancement of Pak1 kinase activity observed with the active protein (Fig. 2A). The (DH-)
Pix155-545 bound to Pak to the same extent as the
unmutated construct (data not shown). This strongly implicates GEF
activity in the effects of
Pix155-545 on the
Cdc42-dependent enhancement of Pak kinase activity in vivo. The fact that the enhancement of kinase activity is not completely abolished reflects the retention of some
Pix155-545 exchange activity in the presence of Pak.
This residual activity can be detected when this construct is
co-expressed with Pak1 and then assayed for GEF activity (Fig.
3C).
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Pix and
Pix have been shown to bind specifically to a
nonconventional proline-rich region in Pak1. We tested whether binding of
Pix155-545 to Pak1 was required to induce the
Cdc42-dependent activation of kinase activity. A
full-length Pak1 mutated in the consensus SH3-binding domain critical
for Pak/Pix interaction, Pak1A193,194, was generated. This
construct was no longer able to bind to
Pix155-545
(Fig. 2B) but retained fully the ability to be activated
in vitro by GTP
S-loaded Cdc42, indicating its intrinsic
kinase activity was unaffected by mutating the Pix binding site (Fig.
2C). When Pak1A193,194 was co-transfected with
the truncated
Pix and WT-Cdc42 into COS-1 cells, no enhancement of
Pak kinase activity was observed (Fig. 2C). This indicates
that a direct binding between the exchange factor and Pak was required
to enhance Pak activation in our system and that Pix exchange activity
alone is not sufficient to promote the Cdc42-dependent
activation of Pak1.
Because the enhancement of Cdc42-dependent activation of
Pak kinase activity by Pix155-545 required direct
interaction with Pak, two possibilities were raised: 1)
Pix may
influence the ability of Pak to be activated independently of its
exchange factor function, and 2) efficient exchange factor activity of
Pix155-545 requires Pak binding. To evaluate these two
possibilities, we first tested whether the increased activation of Pak
we observed was a consequence of an exchange factor-independent effect
of
Pix155-545 on Pak catalytic activity. We utilized a
mutant of Pak that no longer binds Rac or Cdc42 but has a low level of
intrinsic kinase activity in the absence of activated GTPase,
Pak1H83,86L (14, 27). Pak1H83,86L was
co-expressed with
Pix155-545 in COS-1 cells,
immunoprecipitated, and subjected to an in vitro kinase
assay. As Fig. 3A shows, the activity of
Pak1H83,86L is significantly increased when co-expressed
with
Pix155-545. This is in contrast to WT-Pak, which
was not activated by
Pix155-545 unless Cdc42 was also
overexpressed in the same cells (Fig. 1B). Because
PakH83,86L does not bind GTPases at all, these data suggest
that
Pix exerts an exchange factor-independent influence on Pak
activity. This was further confirmed when the
Pix155-545 mutated at residues Leu-383,384 ((DH-)
Pix155-545) was also able to enhance the kinase
activity of Pak1H83,86L (Fig. 3A). Additionally,
we established that Pak1H83,86L activity was enhanced
in vitro using a recombinant GST-fusion construct of
Pix155-545 (Fig. 3A). This confirms that the
Pak-Pix interaction alone in the absence of other cellular co-factors
is sufficient to increase Pak activity.
To further demonstrate that Pix utilizes an exchange factor-independent
mechanism to increase Pak1 activity, we measured the effect of
Pix155-545 on Pak1 kinase activity in a standard
in vitro kinase assay using Cdc42 loaded with GTP
S. As
Fig. 3B shows, Cdc42-GTP
S activation of WT-Pak is
increased 2- to 3-fold when recombinant GST-
Pix155-545
is added to the reaction. This effect is consistent and was not seen
when we used the mutant of Pak1 (Pak1A193, 194), unable to
bind to
Pix.
To test the second possibility, i.e. that
Pix155-545 exchange activity was modulated by Pak1
binding, we utilized an assay to determine the level of GTP-bound Cdc42
in vivo. Co-expression of Cdc42 with
Pix155-545 resulted in an increased production of
GTP-bound Cdc42 that was dependent on a functional
Pix DH domain
(Fig. 3C). GTPase activation was substantially enhanced when
WT-Pak1 was also expressed with
Pix155-545 and Cdc42
but not when Pak1A193,194 was expressed instead of WT-Pak1
(Fig. 3C). This suggests that in addition to the effect of
Pix155-545 on Pak kinase activity, Pak can stimulate
the exchange factor activity of
Pix. This effect is entirely
dependent on the binding of Pak to Pix, although the mechanism by which
this occurs is not yet understood at a molecular level. Interestingly,
Manser et al. (11) observed that co-expression of
Pix
with membrane-targeted Pak resulted in an enhanced Pix exchange factor
activity, suggesting that Pak can also modulate the GEF activity of the
Pix isoform.
The data we have presented establish that the Pak interacting exchange
factor Pix can significantly enhance Cdc42-stimulated Pak1 kinase
activity. Additionally, we have observed similar enhancement of
Rac-stimulated Pak1 activity in vitro and in vivo
(data not shown). Both functional exchange factor activity and the
specific binding interaction between Pak1 and
Pix is required for
the optimal enhancement of Pak catalytic activity. Taken together, these results suggest a model in which direct protein-protein interaction between a GTPase effector kinase (Pak) and a GTPase exchange factor (
Pix) can modulate the activities of both proteins. This provides a mechanism by which Pak can be more effectively activated in an environment where Pix is present and able to bind to
Pak. This could achieve specificity in localized signaling and may also
explain differences in Pak activity in various cell types that may
reflect differences in Pix expression. Interactions with Pix may also
account for differences in Pak activation/inactivation kinetics
observed between intact cells versus in vitro. We
are currently investigating these possibilities.
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ACKNOWLEDGEMENTS |
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We thank Dr. Nobuo Nomura for supplying the
Pix full-length cDNA, Dr. Valerie Benard for help and advice
with the PBD pull down assay, and Dr. Jean-Paul Mira for helpful
comments during the preparation of this manuscript. We also thank
Antonette Lestelle for expert secretarial assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR45738 (to R. H. D.) and GM39434 (to G. M. B.). This is manuscript number 12121-IMM of The Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-8392; Fax: 619-784-8218.
¶ Recipient of an EMBO fellowship.
2 V. Benard, B. P. Bohl, and G. M. Bokoch, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
GEF, guanine
nucleotide exchange factor;
DH, Dbl homology domain;
Pak, p21-activated
kinase;
Pix, Pak interacting exchange factor;
SH3, src homology 3 domain;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
WT, wild type;
PCR, polymerase chain reaction;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
PBD, p21-binding domain;
CMV, cytomegalovirus.
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REFERENCES |
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