COMMUNICATION
alpha Pix Stimulates p21-activated Kinase Activity through Exchange Factor-dependent and -independent Mechanisms*

R. Hugh DanielsDagger §, Frank T. ZenkeDagger , and Gary M. BokochDagger parallel

From the Departments of Dagger  Immunology and parallel  Cell Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
References

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 alpha Pix (amino acids 155-545), we observe stimulation of Pak1 kinase activity when alpha 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 alpha Pix. The activation of wild-type Pak1 by alpha Pix155-545 also requires that alpha 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 alpha Pix155-545 and by a mutant of alpha Pix155-545 that no longer has exchange factor activity. Pak1 activity stimulated in vitro using GTPgamma S-loaded Cdc42 was also enhanced by recombinant alpha Pix155-545 in a binding-dependent manner. These data suggest that Pak activity can be modulated by physical interaction with alpha Pix and that this specific effect involves both exchange factor-dependent and -independent mechanisms.

    INTRODUCTION
Top
Abstract
Introduction
References

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, alpha Pix, appears to down-regulate Pak activation by upstream signals (20). The effect of the alpha 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.

We demonstrate here that a functionally active form of alpha 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 alpha Pix Dbl domain and the binding of alpha Pix to Pak1. Additionally, we report an exchange factor-independent enhancement of Pak1 activity induced by the binding of alpha Pix.

    EXPERIMENTAL PROCEDURES

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 alpha Pix (cDNA accession number D25304) was a gift of N. Nomura (Kazusa DNA Research Institute, Japan). Truncated alpha Pix155-545 was amplified from the full-length alpha 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, alpha 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 alpha Pix155-545 point mutations in the Dbl homology domain ((DH-)alpha 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.

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 -80 °C until required.

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 GTPgamma 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.

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.

    RESULTS AND DISCUSSION

We initiated studies to evaluate the effects of the recently described Pak-interacting protein alpha Pix on kinase activity of Pak1. Co-expression of full-length alpha Pix with Pak1 and WT-Cdc42 had no effect on Pak1 kinase activity in vivo (Fig. 1). Indeed, overexpression of full-length alpha 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 alpha 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 alpha 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 alpha Pix and cloned it into mammalian and bacterial expression vectors (Fig. 1A). Co-expression of either full-length alpha Pix or alpha 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 alpha 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 alpha Pix155-545 or when full-length alpha 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 alpha Pix, and WT-Cdc42 caused no significant substrate phosphorylation (Fig. 1B). These data suggested that the truncated active version of alpha Pix was able to promote nucleotide exchange on WT-Cdc42 in vivo and hence stimulate Pak kinase activity (see Fig. 3 below).


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Fig. 1.   alpha Pix155-545 enhances Cdc42-stimulated Pak1 kinase activity in vivo. A, schematic representation of full-length alpha Pix and alpha 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) alpha Pix and alpha Pix155-545 as indicated. Note the slower migrating Pak1 species in lane 3 where Pak1 and Cdc42 were co-transfected with alpha 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 alpha Pix155-545 was dependent on an intact DH domain in alpha 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 alpha Pix abolished GEF activity (11). We mutated the corresponding residues in alpha 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-) alpha 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 alpha 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 alpha 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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Fig. 2.   Enhancement of Pak1 kinase activity by alpha Pix155-545 requires a functional DH domain and Pak binding. A, Pak1 immunoprecipitated from COS-1 cells transfected with 2.5 µg of the indicated constructs (total 7.5 µg each of DNA) was subjected to a kinase assay in the presence of histone H4. The numbers under each lane represent the -fold activation compared with lane 1, which was assigned a value of 1. B, COS-1 lysates expressing myc-tagged Pak1 constructs (either WT-Pak1, Pak1H83, 86L, or Pak1A193, 194) were mixed with recombinant GST-alpha Pix155-545 on glutathione beads for 1 h at 4 °C. The beads were sedimented and washed vigorously prior to being solubilized in sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was then probed with an anti-myc antibody to detect any Pak1 associated with GST-alpha Pix155-545. No Pak1A193,194 was pulled down with the alpha Pix construct. C, left panel, histone H4 phosphorylation induced by Pak1A193,194 and WT-Pak1 immunoprecipitated from COS-1 cells transfected with the indicated constructs. The level of H4 phosphorylation as determined by PhosphorImager analysis is shown below each lane with phosphorylation of H4 by Pak1A193,194 alone assigned a value of 1. Right panel, Cdc42-GTPgamma S-induced phosphorylation of WT-Pak1 and Pak1A193,194 immunoprecipitated from COS-1 cells. These data are representative of at least three independent experiments.


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Fig. 3.   alpha Pix155-545 activates Pak1 independently of exchange factor activity, and Pak1 binding reciprocally influences alpha Pix155-545 exchange activity. A, left panel, Pak1H83,86L immunoprecipitated from COS-1 cells transfected with Pak1H83,86L and the indicated construct of alpha Pix155-545 was incubated with histone H4 in a kinase assay as described under "Experimental Procedures." The number underneath each lane indicates the -fold activation (from PhosphorImager analysis) of H4 phosphorylation. The level of H4 phosphorylation induced by Pak1H83,86L alone was assigned a value of 1. Data shown are representative of three separate experiments. Right panel, histone H4 phosphorylation by immunoprecipitated Pak1H83,86L in the presence or absence of recombinant GST-alpha Pix155-545 as indicated. These data are representative of two independent experiments. B, immunoprecipitated Pak1 or Pak1A193,194 was stimulated with recombinant Cdc42-GTPgamma S in an in vitro kinase assay either in the presence or absence of recombinant GST-alpha Pix155-545. Either histone H4 or myelin basic protein was used as a substrate. The data show the mean (± S.D.) -fold activation as measured by PhosphorImager using Cdc42-GTPgamma S-stimulated Pak1 kinase activity as a value of 1. These data were obtained from four independent experiments. C, COS-1 cells were transfected with Cdc42 and the indicated constructs. WT-Cdc42 converted to the GTP-bound form was precipitated with recombinant GST-p21-binding domain attached to glutathione beads as described under "Experimental Procedures." The left hand lane represents the total amount of myc-tagged Cdc42 present in each of the lysates used. These data are representative of two independent experiments.

alpha Pix and beta Pix have been shown to bind specifically to a nonconventional proline-rich region in Pak1. We tested whether binding of alpha 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 alpha Pix155-545 (Fig. 2B) but retained fully the ability to be activated in vitro by GTPgamma 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 alpha 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 alpha Pix155-545 required direct interaction with Pak, two possibilities were raised: 1) alpha Pix may influence the ability of Pak to be activated independently of its exchange factor function, and 2) efficient exchange factor activity of alpha 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 alpha 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 alpha 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 alpha Pix155-545. This is in contrast to WT-Pak, which was not activated by alpha 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 alpha Pix exerts an exchange factor-independent influence on Pak activity. This was further confirmed when the alpha Pix155-545 mutated at residues Leu-383,384 ((DH-) alpha 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 alpha 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 alpha Pix155-545 on Pak1 kinase activity in a standard in vitro kinase assay using Cdc42 loaded with GTPgamma S. As Fig. 3B shows, Cdc42-GTPgamma S activation of WT-Pak is increased 2- to 3-fold when recombinant GST-alpha 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 alpha Pix.

To test the second possibility, i.e. that alpha 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 alpha Pix155-545 resulted in an increased production of GTP-bound Cdc42 that was dependent on a functional alpha Pix DH domain (Fig. 3C). GTPase activation was substantially enhanced when WT-Pak1 was also expressed with alpha 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 alpha Pix155-545 on Pak kinase activity, Pak can stimulate the exchange factor activity of alpha 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 alpha Pix with membrane-targeted Pak resulted in an enhanced Pix exchange factor activity, suggesting that Pak can also modulate the GEF activity of the alpha Pix isoform.

The data we have presented establish that the Pak interacting exchange factor alpha 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 alpha 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 (alpha 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.

    ACKNOWLEDGEMENTS

We thank Dr. Nobuo Nomura for supplying the alpha 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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

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; GTPgamma S, 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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