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Volume 270, Number 39, Issue of September 29, pp. 22731-22737, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Mouse p21 Activated Kinase (*)

(Received for publication, May 4, 1995; and in revised form, July 27, 1995)

Shubha Bagrodia (1),  Stephen J. Taylor (2),  Caretha L. Creasy (3),  Jonathan Chernoff (3),  Richard A. Cerione (1)

From the  (1)Departments of Pharmacology and (2)Biochemistry, Section of Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 and the (3)Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a novel member of the mammalian PAK (p21 activated kinase) and yeast Ste20 serine/threonine kinase family from a mouse fibroblast cDNA library, designated mPAK-3. Expression of mPAK-3 in Saccharomyces cerevisiae partially restores mating function in ste20 null cells. Like other PAKs, mPAK-3 contains a putative Cdc42Hs/Rac binding sequence and when transiently expressed in COS cells, full-length mPAK-3 binds activated (GTPS (guanosine 5`-3-O-(thiotriphosphate)-bound) glutathione S-transferase (GST)-Cdc42Hs and GST-Rac1 but not GST-RhoA. As expected for a putative target molecule, mPAK-3 does not bind to an effector domain mutant of Cdc42Hs. Furthermore, activated His-tagged Cdc42Hs and His-tagged Rac stimulate mPAK-3 autophosphorylation and phosphorylation of myelin basic protein by mPAK-3 in vitro. Interestingly, the amino-terminal region of mPAK-3 contains potential SH3-binding sites and we find that mPAK-3, expressed in vitro and in vivo, shows highly specific binding to the SH3 domain of phospholipase C- and at least one SH3 domain in the adapter protein Nck. These results raise the possibility of an additional level of regulation of the PAK family in vivo.

INTRODUCTION

The cellular functions of various members of the Rho subclass of GTP-binding proteins, including RhoA, Rac1, and Cdc42Hs, have recently received a great deal of attention and are thought to be essential for a number of changes in the actin cytoskeleton (Kozma et al., 1995; Nobes and Hall, 1995) (for reviews, see Hall(1994) and Chant and Stowers(1995)). Recent reports suggest that these three GTP-binding proteins participate in a hierarchical series of cytoskeletal-mediated events, starting with the generation of filopodia by activated Cdc42Hs and followed by the successive appearance of lamellipodia, stimulated by activated Rac1, and actin stress fibers, stimulated by activated RhoA (Kozma et al., 1995; Nobes and Hall, 1995). Despite the indication that these different events are coordinated and potentially the outcome of a single signaling pathway (Cdc42Hs-Rac1-RhoA), it also has been shown that different extracellular signals promote the individual activation of Cdc42Hs, Rac1, and RhoA. In the case of Swiss 3T3 fibroblasts, these signals are bradykinin, platelet-derived growth factor, and lysophosphatidic acid, respectively. The challenging problems that remain are to understand the detailed biochemical events that give rise to these cytoskeletal changes and to determine how different inputs can elicit specific effects through the Cdc42Hs, Rac1, and RhoA GTP-binding proteins.

A logical starting place for obtaining biochemical and mechanistic information would be studies of the regulation of the GTP-binding/GTPase cycles of these Rho subclass proteins. In fact, a significant amount of information is available regarding the actions of three classes of regulators, the GDP dissociation inhibitors, the GEFs (^1)(guanine nucleotide-exchange factors), and the GAPs (GTPase-activating proteins). The GEFs and GAPs have been especially interesting because they represent families of molecules that have in one way or another been implicated in cell growth regulation. For example, the prototype Rho subclass GEF is the oncoprotein Dbl, which contains a region of 250 amino acids that is essential both for its transforming and guanine nucleotide exchange activity (Hart et al., 1994). This region, designated the Dbl homology domain, is found in a number of other proteins including the Vav, Ect2, and Ost oncoproteins (Katzav et al., 1989; Miki et al., 1993; Horii et al., 1994), as well as in the Tiam-1 protein that has been implicated in metastasis (Habets et al., 1994) and the Fgd-1 protein that is involved in the faciogenital dysplasia developmental disorder (Pasteris et al., 1994). Likewise, the Cdc42Hs GAP shares a region of homology with a number of potential growth regulatory proteins including Bcr (Diekmann et al., 1991), the Ras GAP-associated protein p190 (Settleman et al., 1992), the Abl SH3-binding protein 3BP-1 (Cicchetti et al., 1992), and the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (Otsu et al., 1991). The interactions of Dbl and these different GAPs with Cdc42Hs and related GTP-binding proteins have been studied in detail and point to a very tight regulation of the GTP binding/GTPase cycles of the Rho subclass proteins in order to ensure normal cell growth and developmental processes.

Despite the increased understanding of the mechanisms underlying the regulation of their GTPase cycles, it is not clear how Cdc42Hs, Rac1, or RhoA mediate the observed cytoskeletal changes. Clearly, an important step will be to identify and characterize the effector proteins that are directly responsible for mediating the actions of the GTP-bound forms of Cdc42Hs, Rac1, and RhoA. Thus far, two putative targets have been identified for the Cdc42Hs and Rac1 proteins. One is the 85-kDa regulatory subunit of the PI 3-kinase (Zheng et al., 1994). The GTP-bound forms of the Cdc42Hs and Rac1 proteins have been shown to bind to the GAP homology domain of p85 and to elicit a 4-5-fold stimulation of PI 3-kinase activity. The GTP-bound form of Ras also binds to the PI 3-kinase, through a direct interaction with its 110-kDa catalytic domain (Rodriguez-Viciana et al., 1994). Thus, the PI 3-kinase may serve as a point of convergence for Cdc42Hs or Rac1 and Ras, enabling these different GTP-binding proteins to cooperate in the stimulation of cytoskeletal changes that accompany growth factor binding to receptors. A second, recently identified potential target for the Cdc42Hs and Rac1 proteins is a 65-kDa serine/threonine protein kinase called PAK (p21 activated kinase) that was reported to be the mammalian homolog of the Saccharomyces cerevisiae Ste20 kinase (Manser et al., 1994). The involvement of Ste20 in the pheromone/mating factor pathway in yeast has been well documented, and in fact the complete signaling pathway starting with the mating factor receptor and continuing through a protein kinase cascade to the nucleus has been elucidated (see Herskowitz(1995) for review). However, at the present time, much less is known about the actions of the mammalian PAK or the effects of Cdc42Hs (or Rac1) stimulation of this kinase and how this stimulation impacts on the cytoskeleton. It now seems likely that a family of mammalian PAKs exist, and in the present report we describe a new member of this kinase family that was initially identified using a Cdc42Hs-GTPS fusion protein as an affinity reagent for detection of cellular targets. This serine/threonine kinase is 80% identical to other PAK/Ste20 kinases and is designated mPAK-3. We show that mPAK-3 can complement deletions of the Ste20 gene product in S. cerevisiae and that its kinase activity toward exogenous phosphosubstrates is strongly stimulated by GTP-bound Cdc42Hs and Rac1. Moreover, mPAK-3 shows a high degree of binding specificity for a particular group of SH3 domains, which may be involved in the regulation and localization of this new protein kinase.

EXPERIMENTAL PROCEDURES

Cloning of mPAK-3 cDNA

Restriction sites at the end of oligonucleotides were introduced to facilitate easy cloning. Oligonucleotides 5`-GCTCTAGACAGGAGGCTGCCATTAAA-3` and 5`-GCTCTAGAACTTCAGGTGCCATCCA-3` (the underlined residues indicate XbaI restriction endonuclease site) corresponding to QEVAIK and WMAPEV, respectively, encoded by rat p65PAK were used to amplify a 405-bp PCR product using Taq DNA polymerase (Promega) from a Zap mouse cDNA library (Stratagene). The PCR product was subcloned into pBluescript II SK- (Stratagene), and partial sequence analysis by the dideoxynucleotide chain termination method (Sequenase version 2; U. S. Biochemical Corp.) revealed a rat p65PAK coding sequence. 750,000 plaques from a Zap mouse cDNA library were screened by using the randomly primed [alpha-P]dCTP-labeled 405-bp PCR fragment (Prime-gene kit, Stratagene). Nylon transfer membranes (0.45 µm, Micron Separation Inc.) were hybridized and washed at 68 °C according to standard protocol. Ten potentially positive plaques were purified, and the excised cDNA inserts were partially sequenced. Two clones contained rat p65PAK-related sequences; one was a partial clone, and the other was a full-length clone. Since the mouse clone is the third mammalian PAK/Ste20 family member, we designate it as mPAK-3. The full-length pBSmPAK-3 was sequenced using an automated sequencer (Applied Biosystems Inc.)

Plasmids

All PCR-generated fragments were sequenced to confirm that no errors were introduced during PCR. In this section, underlined residues indicate a BamHI restriction endonuclease site. Hemagglutinin (HA)-tagged plasmid J3H (^2)and plasmids expressing GST, GST-Cdc42Hs, GST-Rac1, and GST-RhoA have been described previously (Hart et al., 1994). To ligate mPAK-3 in frame with the HA-tagged vector, a 400-bp BamHI -HincII fragment (beginning at the ATG start site of mPAK-3) was amplified from plasmid pBSmPAK-3 with oligonucleotides 5`-GCGGATCCATGTCTGACAGCTTGG-3` and 5`-GGTTGTTGACCGTTTCT-3` by PCR using Pfu DNA polymerase (Stratagene). Plasmid J3HmPAK-3 expressing mouse mPAK-3 under the control of the SV40 promoter was constructed by three fragment ligation containing a 400-bp BamHI- HincII PCR product, a 1300-bp HincII-BamHI fragment from pBSmPAK-3 encoding a hemaglutinin-tagged mPAK-3, and a 3500-bp BamHI-linearized vector J3H. Residues 65-137 from mPAK-3 containing the putative Rac1/Cdc42Hs binding domain were obtained by PCR using oligonucleotides 5`-CGGGATCCAAAGAGCGCCCAGAGATC-3` and 5`-CGGGATCCTATTTCTGGTTGTTGACCG-3`. The PCR product was ligated into the BamHI site of plasmid pGEX-KG. For in vitro transcription/translation driven by SP6 polymerase, the 1700-bp BamHI fragment from plasmid J3HmPAK-3 was ligated into the BamHI site of plasmid pGEM4Z (Promega). The cDNAs coding for GST-AblSH3, GST-Crk(N)SH3, GST-spectrinSH3, GST-PLCSH3, GST-NCKSH3 and GST-FgrSH3 were a gift from Dr. Mordechai Anafi.

Protein Expression

GST fusion proteins were expressed in Escherichia coli and purified by glutathione-Sepharose affinity chromatography (Hart et al., 1994, Zheng et al., 1994; Taylor et al., 1995). mPAK-3 was expressed in vitro using a coupled transcription-translation reticulocyte lysate system in the presence of [S]methionine as described (Taylor et al., 1995). Reaction products were separated by SDS-PAGE and autoradiographed.

Lipofectamine-mediated transient transfections of COS cells were performed according to the manufacturer's protocol (Life Technologies, Inc.). Briefly 2-3 times 10^5 COS cells were plated in 35-mm dishes 18-24 h prior to transfection. 0.5 µg of J3HmPAK-3 DNA and 5 µl of Lipofectamine reagent were added to each plate in 1 ml of Dulbecco's modified Eagle's medium (DMEM) in the absence of serum. After 5 h, 1 ml of DMEM containing 20% fetal calf serum (Life Technologies, Inc.) was added; after 14-18 h, the medium was replaced with fresh DMEM containing 10% fetal calf serum. Cells were lysed 48-72 h after addition of DNA.

Cell Culture

NIH 3T3 cells were grown in DMEM supplemented with 5% calf serum (Sigma) at 37 °C in 5% CO(2). Monkey kidney COS-7 cells were cultured in DMEM containing 10% fetal calf serum (Life Technologies, Inc.)

Cell Lysis, Immunoprecipitation, and Immunoblotting

NIH 3T3 cells, untransfected, and HA-mPAK-3-transfected COS cells were washed with phosphate-buffered saline and lysed in buffer A (40 mM Hepes, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 150 mM NaCl, 50 mM NaF, 1 mM Na(3)VO(4), 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 20 min at 4 °C. Lysates were precleared by centrifugation at 12,000 times g for 25 min at 4 °C in JA20.1 rotor (Beckman). Prior to immunoprecipitation, lysates were precleared with protein A-Sepharose (Sigma) for 30 min at 4 °C. HA-tagged mPAK-3 was immunoprecipitated with anti-HA (mAb1 2CA5) (Berkeley Antibody Co.) primary antibody for 1 h, followed by incubation with protein A-Sepharose coated with rabbit anti-mouse IgG for 1 h. Precipitates were washed three times with buffer A, and proteins were eluted with SDS sample buffer and boiled for 3-5 min. SDS-polyacrylamide gel electrophoresis was then performed on 7.5%, 10%, and 12.5% gels. For subsequent Western blot analysis, proteins were transferred to Immobilon P membrane (Millipore), blocked in buffer with bovine serum albumin (BSA), and incubated with primary antibody anti-HA mAb 12CA5. The primary antibody was detected with horseradish peroxidase-coupled sheep anti-mouse antibody using the chemiluminescence reagent ECL (Amersham Corp.).

Detection of Kinase Activity Associated with GST-Cdc42Hs Precipitates

NIH 3T3 cells were lysed and precleared by centrifugation as described above. Lysates were additionally precleared with immobilized GST protein for 30 min at 4 °C. GST-Cdc42Hs (50 µg) was incubated with buffer A containing 10 mM EDTA for 15 min at room temperature to release prebound nucleotide, washed with buffer A, and then incubated with 0.2-1 mM GDP or GTPS in buffer B (buffer A plus 10 mM MgCl(2)) for 25 min at room temperature, and then washed with buffer B to remove unbound nucleotides. Immobilized GST alone or GST-Cdc42Hs bound to GDP or GTPS was then incubated with NIH 3T3 cell lysates supplemented with 10 mM MgCl(2) for 1 h at 4 °C. Protein precipitates were washed twice in buffer B and once in 2 times phosphorylation buffer (40 mM Hepes, pH 7.4, and 10 mM MgCl(2)) and divided into four equal aliquots. Aliquots were mixed with 5 µg each of myelin basic protein (MBP), alpha-casein (Sigma), or histone H1 (Sigma) or with 5 µl of water, and then the kinase assay was initiated by the addition of [-P]ATP (3000 Ci/mmol) and 10 µM ATP in a 30-µl reaction volume for 5 min at room temperature. Reactions were stopped by the addition of 2 times SDS sample buffer. Proteins were eluted by boiling for 3-5 min and separated by 12.5% SDS-PAGE. Gels were stained with Coomassie Brilliant Blue before autoradiography.

Kinase Assay of Proteins on Immobilon P

The procedures used for assaying the kinase activity of proteins after transfer to Immobilon P were as described previously (Ferrell and Martin, 1991). Briefly, proteins associated with GDP or GTPS-bound (immobilized) GST-Cdc42Hs from NIH 3T3 cell lysates were transferred to Immobilon P membranes as described above, and the blot was incubated in denaturation buffer (7 M guanine hydrochloride, 50 mM Tris base, 50 mM dithiothreitol, 2 mM EDTA, adjusted to pH 8.3) for 1 h at room temperature and then in renaturation buffer (140 mM NaCl, 10 mM Tris, pH 7.4, 2 mM dithiothreitol, 2 mM EDTA, 1% BSA, and 0.1% Nonidet P-40) for 16 h at 4 °C. An additional incubation was performed with blocking buffer (30 mM Tris, pH 7.4, and 5% BSA) for 16 h at 4 °C, and finally a kinase assay was performed in 40 mM Hepes, pH 7.4, 10 mM MgCl(2), 2 mM MnCl(2), and 100 µCi/ml [-P]ATP (3000 Ci/mmol) for 30 min at room temperature. The blots were sequentially washed with Tris-HCl at pH 7.4, Tris-HCl plus 0.05% Nonidet P-40, Tris-HCl (pH 7.4), 1 M KOH and Tris-HCl at pH 7.4, and autoradiographed.

Association of HA-mPAK-3 with GST-GTP-binding Proteins

COS cell lysates transiently expressing HA-mPAK-3 were prepared as described above. Immobilized GST-Cdc42Hs, GST-Rac1, and GST-RhoA were loaded with GDP or GTPS as described above and incubated with COS cell lysates supplemented with 10 mM MgCl(2) for 1-2 h at 4 °C. Precipitates were washed three times with buffer B, and bound proteins were eluted in SDS sample buffer subjected to 10% SDS-PAGE, Western blotted, and probed with anti-HA mAb 12CA5.

mPAK-3 Kinase Assay

mPAK-3 immunoprecipitates (as described above) from HA-mPAK-3-transfected COS cell lysates were washed in 2 times phosphorylation buffer (40 mM Hepes, pH 7.4, and 10 mM MgCl(2)) and divided into equal aliquots. One aliquot was subjected to Western blot analysis. The remaining aliquots were incubated with soluble GDP or GTPS bound His-tagged Cdc42Hs and His-tagged Rac1 (5 µg of protein) and 5 µg of MBP (Sigma) for 5 min on ice. Kinase assays were initiated by the addition of 5 µCi of [-P]ATP (3000 Ci/mmol) and 10 µM ATP in a 30-µl reaction volume for 5 min at room temperature. Reactions were stopped by the addition of 2 times SDS sample buffer. Subsequent procedures were similar to those described above.

Potato Acid Phosphatase Treatment

COS cells transiently expressing HA-mPAK-3 were lysed as described above and incubated with specified immobilized GST fusion proteins. Protein precipitates were washed twice with buffer B, once with phosphatase buffer (20 mM Hepes pH 7.0, 5 mM MgCl(2), 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and dephosphorylated with 2 µg potato acid phosphatase (PAP) in 20 µl phosphatase buffer for 30 min at 30 °C. PAP stock solution was prepared as described previously (Shenoy et al., 1989).

Phosphoamino Acid Analysis

Procedures were as described previously (Shenoy et al., 1989) except that proteins were transferred to Immobilon PVDF membranes and hydrolyzed with 6 N HCl at 110 °C for 1 h.

Mating Assays

S. cerevisiae strains bearing a deletion in either STE20 (YEL-33-7-3B; MATa,ade2, his3, leu2, trp1, ura3, can1, ste20::TRP1; Leberer et al., 1992), STE5 (betaSTE5 Delta; MATa,ura3-52, lys2, leu2, trp1-289, his3-Delta200, met, GAL, ste5::HIS3; Pearlman et al., 1993) or STE11 (E929-6C-20; MATa, ste11-6, cyc1, CYC7-H2, can1, leu2-3,112, trp1Delta1, ura3-52; Rhodes et al., 1990) were transformed by a lithium acetate procedure (Gietz et al., 1992) with a galactose-inducible expression vector, pYES2 (Invitrogen), bearing either no insert or a cDNA encoding full-length mPAK-3, or a plasmid bearing the appropriate wild type yeast gene (STE20-pFLC-1 (Ramer and Davis, 1993), STE5-p2-1PN (Perlman et al., 1993), or STE11-pNC192 (Rhodes et al., 1990)). Transformants were selected for growth on minimal medium lacking uracil. To perform quantitative mating assays four isolates from each transformation were grown to mid-log phase with galactose as the carbon source, a defined number of cells were mixed with mating strain, RSY16 (MATalpha, ade2-1, leu1-2, lys2-1, trp5-20, ura1; a gift from R. Strich, Fox Chase Cancer Center), and allowed to mate on rich medium containing galactose for 7 h. Cells were collected and serial deletions plated onto minimal medium lacking leucine. Mating efficiency was scored the following day (Sprague, 1991).

RESULTS

Serine/Threonine Protein Kinase Activity Associated with GTPS-bound Cdc42Hs

Immobilized GST, GST-Cdc42Hs-GDP, and GST-Cdc42Hs-GTPS were used in a binding assay to identify protein kinases and associated substrates from NIH 3T3 cells that might serve as targets for Cdc42Hs. The underlying strategy was that a target would bind specifically to the GTPS-bound form of Cdc42Hs, show little or no binding to Cdc42Hs-GDP, and no binding to immobilized GST alone. The presence of associated protein kinase activity was detected by incubating the different glutathione-agarose-precipitated Cdc42Hs proteins with [-P]ATP, followed by SDS-PAGE and autoradiography. As shown in Fig. 1A, protein kinase activity was precipitated by GTPS-bound (lane3) but not by GDP-bound GST-Cdc42Hs (lane2). This Cdc42Hs-associated kinase activity preferentially phosphorylated MBP, compared to alphacasein and histone H1. An in vitro phosphorylated protein of apparent molecular mass of 66 kDa was found to associate specifically with Cdc42Hs-GTPS (lanes3, 6, 9, and 12). The phosphorylated 45-kDa band is GSTCdc42, which is phosphorylated in the absence of an exogenous substrate, such as MBP (compare lane6 with lanes 3, 9, and 12). Two other proteins of molecular mass 96 and 80 kDa were also phosphorylated but to a lesser extent (lanes3, 6, 9, and 12). The 66- and the 96-kDa proteins were found to be phosphorylated on serine and threonine residues, based on phosphoamino acid analysis (data not shown). Aside from phosphorylated GST-Cdc42 (molecular mass 45 kDa), there were no other phosphoproteins detected below 50 kDa. To determine whether the observed phosphoproteins were substrates for a Cdc42Hs-associated protein kinase(s) or were themselves kinases capable of autophosphorylation, affinity precipitates were transferred to a polyvinylidene difluoride membrane, denatured, renatured, and incubated with [-P]ATP. In this assay, only proteins capable of autophosphorylation should be detected. As shown in Fig. 1B, the 66-kDa protein, but not the 96- or 80-kDa proteins, incorporated P (lane5) predominantly on serine. These results indicated that the Cdc42Hs-GTPS-associated 66-kDa protein was a serine/threonine kinase capable of autophosphorylation on a serine residue(s).

Figure 1: A protein kinase activity associates with GST-Cdc42-GTPS. A, NIH 3T3 cell lysates were incubated with immobilized GST (lanes1, 4, 7, and 10), GST-Cdc42Hs-GDP (lanes2, 5, 8, and 11), and GST-Cdc42Hs-GTPS (lanes3, 6, 9, and 12) for 1 h at 4 °C. Bound proteins were washed and subjected to an in vitro kinase assay in the absence (lanes 1-3) or presence of exogenous substrates, i.e. myelin basic protein (lanes 4-6), alpha-casein (C, lanes7-9) and histone H1 (H, lanes 10-12). The band at 45 kDa corresponds to GST-Cdc42, which is phosphorylated in the absence of an exogenous substrate. B, NIH 3T3 cell lysates were incubated with immobilized GST, GST-Cdc42Hs-GDP, and GST-Cdc42Hs-GTPS (lanes3, 4, and 5, respectively) for 1 h at 4 °C. Lane1 contains 5.7% of the whole cell lysate (WCL) used in the binding reactions. Bound proteins were washed, separated on SDS-PAGE, and transferred to an Immobilon P membrane. The bottom part of the gel (<50 kDa) was stained with Coomassie Blue to ascertain that each lane contained equal amounts of fusion protein. The proteins on the membrane were subjected to a kinase assay using [-P]ATP and MnCl(2) as described under ``Experimental Procedures.'' Autoradiography was for 14 h at room temperature. E. coli-expressed, immobilized GST-Cdc42Hs-GDP was incubated with lysis buffer (LB) as a negative control in lane2. Phosphoamino acid analysis was performed on the band in lane5 as described under ``Experimental Procedures.'' PS, PT, and PY indicate the position of nonradioactive phosphoserine, phosphothreonine, and phosphotyrosine markers.


Molecular Cloning of mPAK-3

A 65-kDa serine/threonine kinase, p65PAK, originally isolated from rat brain, has been reported to bind specifically to the GTPS-bound states of Cdc42Hs and Rac1 (Manser et al., 1994). It therefore seemed plausible that the 66-kDa serine/threonine kinase that we detected by binding to GST-Cdc42Hs-GTPS complexes represented the mouse homolog of p65PAK or a related protein kinase. Using primers based on conserved sequences within the kinase domain of rat p65PAK encoding residues QEVAIK in subdomain II and WMAPEV in subdomain VII of rat p65PAK (Manser et al., 1994), a partial cDNA fragment was generated by PCR amplification from a NIH 3T3 mouse fibroblast cDNA library. The PCR fragment (405 bp) was highly homologous to the cDNA for rat p65PAK and was used to identify two positive clones from the mouse fibroblast cDNA library. One of these was full-length, and the other was a partial clone that lacked the first 165 bp of the full-length clone. The longest open reading frame of the full-length clone encodes a protein of 543 amino acids. Fig. 2shows the amino acid comparison between rat p65PAK and mPAK-3.

Figure 2: Amino acid sequence comparison of rat p65PAK and mPAK-3. The amino acid sequence of mPAK-3 was deduced from the sequence of a cDNA clone isolated from a mouse fibroblast cDNA library. Protein sequences are presented in single-letter code. The sequences were aligned using the GAP program. Underlined residues in mPAK-3 indicate putative SH3-binding regions. mPAK-3 shows 81% identity and 89% similarity to rat p65PAK. Romannumerals indicate conserved kinase subdomains. Dashes between amino acids represent identical sequences, doubledots signify conservative changes, and singledots denote less conservative changes. A stretch of acidic residues is highlighted in bold. The putative Cdc42 and Rac binding domain of mPAK-3 (amino acids 65-128) shares similarity with rat p65 PAK.


There are at least three mammalian PAK family members. Two human PAK family members have recently been identified. One, designated hPAK-1, is 98% identical to the rat p65PAK and is the human homolog of rat p65PAK, while the second, hPAK-2, is 78% identical to rat p65PAK.^2 The mouse homolog that we have identified is 81% identical to hPAK-1 and 76% identical to hPAK-2, and so we believe it represents a third mammalian form and have designated it mPAK-3. The kinase domains and the putative Cdc42Hs/Rac binding domains are highly conserved between the three PAKs. However, the amino terminus of mPAK-3 diverges from that of p65PAK and the two human proteins. In addition, mPAK-3 contains four distinct proline-rich sequences that represent potential binding sites for SH3 domain-containing proteins (Feng et al., 1994), which are also present in hPAK-1, and a stretch of acidic amino acids (residues 173-185), which is not fully conserved in other members of the family.

mPAK-3 Complements Ste20 Defects in S. cerevisiae

The PAK family is significantly related to Ste20 of S. cerevisiae, a protein kinase involved in transmission of the pheromone mating response. Since a family of PAK proteins is emerging, we examined whether mPAK-3 was capable of suppressing a mating defect in S. cerevisiae caused by a deletion of ste20. To do this, a ste20 null strain was transformed with a galactose-inducible expression vector that contained either no insert, or the cDNA encoding Ste20, or the cDNA encoding mPAK-3. As shown in Fig. 3and Table 1, mPAK-3 was able to suppress the mating defect caused by the lack of Ste20 expression and allowed mating at a level that was 3-4% that of Ste20. However, mPAK-3 did not complement the mating defect in strains lacking ste5 or ste11, which act downstream of Ste20 (Fig. 3), indicating that mouse Pak-3 is a functional homolog of yeast Ste20.

Figure 3: mPAK-3 can complement a Ste20 defect in yeast. S. cerevisiae strains (described under ``Experimental Procedures'') bearing a deletion in ste20, ste5, or ste11 were transformed with vector alone (pYES2), mPAK-3, ste5, ste11, and ste20 plasmids, as indicated, were grown on minimal medium lacking uracil with galactose as the carbon source followed by patch mating to strain RSY16. Diploids were selected for growth on minimal medium lacking leucine.


mPAK-3 Associates with Cdc42Hs and Rac1

Since mPAK-3 contains a putative Cdc42/Rac-binding domain (Manser et al., 1994), we examined its interaction with different GTP-binding proteins. A HA-tagged mPAK-3 protein was transiently expressed in COS cells, and lysates from these cells were incubated with immobilized GST-Cdc42Hs, GST-Rac1, and GST-RhoA in different guanine nucleotide-bound states. Fig. 4shows that HA-tagged mPAK-3 bound to Cdc42Hs-GTPS (lane4) and Rac1-GTPS (lane6) but not to GTPS-bound RhoA (lane8). The GDP-bound forms of Cdc42Hs and Rac1 were ineffective in binding mPAK-3, as was a putative effector domain mutant, Cdc42Hs (T35A) (lane10) by analogy with a known effector domain mutant of Ras (Marshall, 1993). A GTPase-defective mutant, Cdc42Hs (Q61L), bound to mPAK-3 as well as wild type (lanes4 and 11).

Figure 4: mPAK-3 specifically binds Cdc42Hs and Rac1. COS cells were transiently transfected with a plasmid encoding NH(2)-terminal HA-tagged mPAK-3 using lipofectamine. After 48 h, cells were lysed and lysates incubated with immobilized GST, wild type GST-Cdc42Hs-GDP, GST-Cdc42Hs-GTPS, GST-Rac1-GDP, GST-Rac1-GTPS, GST-RhoA-GDP, and GST-RhoA-GTPS, a putative effector domain mutant GST-Cdc42HsT35A-GDP, GST-Cdc42HsT35A-GTPS, and a GTPase-defective mutant GST-Cdc42HsQ61L-GTPS in lanes3-11, respectively, for 1 h at 4 °C. Lane1 represents 19% of the whole cell lysate (WCL) used in the binding reaction. Bound proteins were Western-blotted and probed with anti-HA (mAb 12CA5) to detect HA-mPAK-3. The higher molecular weight band seen in lane1 (here and in Fig. 6B) is a nonspecific band that cross-reacts with the anti-HA antibody and is also observed in untransfected COS cells.


Figure 6: mPAK-3 binds to the PLC- and Nck SH3 domains. A, in vitro translated [S]methionine-labeled mPAK-3 was incubated with approximately equal amounts of immobilized GST(-) or GST fusion proteins (GST-SH3 affinity precipitates (AP)) as indicated. Lane1 represents 20% of the reticulocyte lysate (input). After washing resin-bound proteins were analyzed by SDS-PAGE and fluorographed. B, HA-tagged mPAK-3 from transiently transfected COS cell lysates were incubated with immobilized GST(-) or GST fusion proteins as indicated. Lane1 represents 10% of the whole cell lysate (wcl) used in the binding reactions. After washing, resin-bound proteins were Western blotted and probed with anti-HA antibody.


Treatment of affinity-precipitated Cdc42Hs-GTPS/mPAK-3 complexes with potato acid phosphatase eliminated the retarded electrophoretic mobility band observed in Fig. 4, and a band appeared that had faster mobility than mPAK-3 in lysates (data not shown). These results suggest that mPAK-3 has a certain basal level of phosphorylation in cell lysates and that binding to GTP-bound forms of Cdc42Hs further increases the level of phosphorylation, for example by stimulating the autophosphorylation activity of the kinase and causing the electrophoretic mobility shift.

Activation of mPAK-3 by Cdc42Hs and Rac1

We set out to determine whether Cdc42Hs and Rac1 stimulate autophosphorylation of mPAK-3 and increase its kinase activity toward exogenous substrates. Anti-HA immunoprecipitates from COS cell lysates containing HA-tagged mPAK-3 were incubated with GDP- or GTPS-bound Cdc42Hs or Rac1 fused to either GST or a hexahistidine (His) tag. The kinase activity of mPAK-3 was measured by assaying the phosphorylation of MBP (Fig. 5). mPAK-3 kinase activity toward MBP and mPAK-3 auto-kinase activity were strongly stimulated by the His- or GST-tagged Cdc42Hs-GTPS complex (lanes2 and 9 in Fig. 5A, also Fig. 5B) and His-Rac1-GTPS (lane4 in Fig. 5A, also Fig. 5B). Although the GTPS-bound GST-tagged Cdc42Hs stimulated mPAK-3 kinase activity (lane9 in Fig. 5A), the GTPS-bound GST-tagged Rac1 did not stimulate mPAK-3 kinase activity (lane7 in Fig. 5A) in four independent experiments.

Figure 5: In vitro activation of mPAK-3. A, COS cells were transfected with HA-mPAK-3 and lysed as described under ``Experimental Procedures.'' Anti-HA immunoprecipitates were subjected to an in vitro kinase assay using [-P]ATP and 5 mM MgCl(2) in the presence of GDP- or GTPS-bound Cdc42Hs (lanes1, 2, 8, and 9) or GDP- or GTPS-bound Rac1 (lanes3, 4, 6, and 7), fused to either GST or a hexahistidine (His) tag. Reactions were stopped after 5 min with 2 times SDS sample buffer. B, procedures were as described in A except that units corresponding to equivalent amounts of GTPS-bound His-Cdc42Hs or His-Rac1 were added in each lane as indicated. A unit is determined by [S]GTPS counts bound to the G-protein. His-Cdc42Hs-GDP was used in lane 12 (control).(-) indicates immunoprecipitates alone.


mPAK-3 Is an SH3 Domain-binding Protein

The amino-terminal proline-rich sequences in mPAK-3 (underlinedresidues, Fig. 2) contain the PXXP motif (X is any amino acid) that has been shown to represent a minimal unit for binding to SH3 domains. We examined the ability of mPAK-3 to bind to a number of different SH3 domains, prepared as recombinant GST fusion proteins. In vitro translated, S-labeled mPAK-3 bound to the SH3 domain of phospholipase C- (PLC-), but not to the SH3 domains of Src, Abl, Crk (NH(2)-terminal), the p85 subunit of phosphoinositide 3-kinase, spectrin, or Fgr (Fig. 6A). HA-tagged mPAK-3, from transiently transfected COS cell lysates, bound to the SH3 domain of PLC- and also to a GST fusion protein containing the three SH3 domains of Nck (Fig. 6B, lanes4 and 6). Transiently expressed HA-mPAK-3 did not bind the Src or Grb2 (COOH-terminal) SH3 domains (lanes3 and 5).

DISCUSSION

In this report we describe a third member of the mammalian family of PAK serine/threonine kinases. This protein, mPAK-3, is 80% identical to both rat PAK (i.e. the first member of the family identified by Manser and colleagues (Manser et al., 1994)) and the human PAK-1,^2 and is 76% identical to a second human homolog, hPAK-2.^2 The mPAK-3 also is 70% identical to the catalytic domain of S. cerevisiae protein, Ste20, and we show here that mPAK-3 will compensate for mating defects caused by the deletion of ste20. It has been well established that the Ste20 kinase is a key participant in the pheromone mating factor pathway. Specifically, in response to pheromone, Ste20 initiates a protein kinase cascade that includes Ste11 (the functional homolog of mammalian mitogen-activated protein (MAP) kinase kinase kinases or MEKKs (Lange-Carter et al., 1993)), Ste7 (the homolog of mammalian MAP kinase kinases or MEKs (Crews et al., 1992; Ashworth et al., 1992)), and FUS3/KSS1 (the homolog of mammalian MAP/ERK kinases). (For recent reviews on the MAP kinase cascade, see Herskowitz(1995), Marshall(1994), Johnson and Vaillancourt(1994), and Errede and Levin(1993).) Given this role of Ste20 in yeast, it will be important to see if the different mammalian PAKs initiate kinase cascades involving MEKKs or MEK proteins in response to extracellular signals. In particular, since mPAK-3 is activated by activated Rac1 and Cdc42Hs, it may initiate kinase cascades leading to mitogenic responses to growth factors. Extracellular signals, including bradykinin, PDGF, and lysophosphatidic acid, have indirectly been shown to activate Cdc42Hs and Rac1 (Kozma et al., 1995; Ridley et al., 1992; Ridley and Hall, 1992). In addition, Rac1 has recently been shown to act downstream of Ras during Ras-induced cellular transformation and to possess growth-stimulatory properties (Qiu et al., 1995). Alternatively, the activation of MAP kinases by mPAK-3 could mediate the characteristic cytoskeletal re-arrangements induced by Cdc42Hs, Rac1, and their respective extracellular stimuli.

The serine/threonine kinase activity of mPAK-3 is strongly stimulated by both the Cdc42Hs and Rac1 proteins, but not by RhoA. This is similar to what was observed for the rat p65PAK (Manser et al., 1994). The mechanism that underlies the stimulation of kinase activity by these GTP-binding proteins is not yet known, although it is likely that the binding of the GTP-binding protein to a specific region within the amino-terminal half of the kinase releases a negative constraint. De-repression of PAK activity by Cdc42Hs and Rac1 would therefore represent another example of the common mechanism of activation of several protein kinases. Such an activation mechanism would predict that the binding of a GTP-binding protein to a PAK would simultaneously result in the stimulation of the kinase activity. However, interestingly, we have found that while GST-Rac1, when in the GTPS-bound state, will bind specifically to mPAK-3, it shows no detectable stimulation of the mPAK-3 kinase activity. Thus, this represents an example where specific binding to mPAK-3 by a GTP-binding protein is uncoupled from the stimulation of kinase activity and suggests that the activation mechanism entails more than simply a single (specific) binding event. Another Rac1 fusion protein, that contains 20 amino acids upstream from the start site for Rac1 (i.e. a His-tagged Rac1), exhibits both specific binding and stimulation of kinase activity. At present, we are trying to understand the molecular mechanism that underlies the striking differences observed with the different Rac fusion proteins, since the results indicate that the presence of the GST moiety interferes with a second type of interaction between the GTP-binding protein (Rac) and the kinase that is necessary for the stimulation of kinase activity. Apparently, the presence of the GST moiety does not interfere with this second stimulatory interaction between Cdc42Hs and mPAK-3, since GST-Cdc42Hs both specifically binds to and stimulates mPAK-3.

When comparing the dose-dependent stimulation of mPAK-3 by the His-tagged Cdc42Hs and Rac1 proteins, we find that these two GTP-binding proteins are approximately equipotent in activating mPAK-3. This then raises the question of GTP-binding protein/target specificity. Do both of these GTP-binding proteins bind to the same target in the cell or do the two GTP-binding proteins in fact regulate different members of the mammalian PAK family? Both Cdc42Hs and Rac1 have been implicated in cytoskeletal organization (Kozma et al., 1995; Nobes and Hall, 1995; Ridley et al., 1992), and recent studies even argue that Cdc42Hs may be functioning upstream from Rac1 in a common pathway that impacts on the cytoskeleton (Kozma et al., 1995; Nobes and Hall, 1995). Thus, one possibility is that Cdc42Hs may initially activate a specific PAK to initiate cytoskeletal alterations. However, this stimulation may be transient and perhaps is replaced by a more persistent stimulation of the same PAK when Rac1 is subsequently activated. Another possibility is that the Cdc42Hs- and Rac1-mediated stimulations of PAKs (either the same PAK family member or distinct members) occur with both spatial and temporal specificity and that this specificity accounts for distinct cytoskeletal events (e.g. filopodia formation in the case of activated Cdc42Hs versus membrane ruffling in the case of activated Rac1). Presumably, such specificity would be mediated by other cellular proteins.

One possibility for additional modes of regulation of PAK activity is via the binding of SH3 domain-containing proteins. mPAK-3 contains four potential (PXXP) SH3 domain-binding motifs within the amino-terminal half of the molecule. Moreover, we have found through in vitro binding assays that mPAK-3 binds with high specificity to the SH3 domains of PLC and Nck. To our knowledge this represents the first example of an identified serine/threonine protein kinase that is able to associate with SH3 domains. An unidentified serine kinase activity has recently been shown to bind to Nck via one of its SH3 domains (Chou and Hanafusa, 1995); based on our results, this could be a PAK family member. The Btk tyrosine kinase has been shown to bind in vitro to the SH3 domains of Fyn, Lyn, and Hck (Cheng et al., 1994). One of the SH3-binding sites within Btk is strikingly similar to one of the potential SH3-binding sites in mPAK-3 (residues 33-41, KPLPXXPEE), raising the possibility that these two otherwise quite distinct protein kinases share common regulatory mechanisms. Interestingly, another target of Cdc42Hs, the 85-kDa regulatory subunit (p85) of the PI 3-kinase, also possesses SH3-binding sites. PLC- has been shown to associate with the actin cytoskeleton in fibroblasts (McBride et al., 1991) and may therefore serve to localize mPAK-3 to sites of Cdc42Hs/Rac1 action. Furthermore, the substrate of PLC-, phosphatidylinositol 4,5-bisphosphate, may play a role in actin polymerization-depolymerization events via an interaction with the actin-binding protein profilin (Goldschmidt-Clermont et al., 1990). Thus, mPAK-3 may serve to connect cytoskeletal interactions involving PLC- and/or phosphatidylinositol 4,5-bisphosphate with Cdc42Hs or Rac1 activation. The adapter protein Nck, which contains one SH2 and three SH3 domains, is recruited to activated PDGF receptors via its SH2 domain (Nishimura et al., 1993). Nck could therefore mediate localization of mPAK-3 to its sites of action in response to growth factors such as PDGF, which may also, through other pathways, activate Cdc42 and/or Rac1. It will be important to determine whether mPAK-3 associates with PLC- or Nck in vivo, and, if so, whether this association is modulated in response to extracellular stimuli. Additional studies will be directed toward determining the specific proline-rich region on mPAK-3 that is responsible for these interactions in order to generate and express mutant mPAK-3 proteins that may be used to uncouple specific cellular events that normally require the convergence of GTP-binding proteins, mPAK-3, and SH3 domain-containing proteins.

FOOTNOTES

*
This was supported by National Institutes of Health Grants GM47458 (to R. A. C.) and CA58836 (to J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: GEF, guanine nucleotide-exchange factor; GAP, GTPase-activating protein; PAK, p21 activated kinase; PCR, polymerase chain reaction; bp, base pair(s); MBP, myelin basic protein; mAb, monoclonal antibody; HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; GTPS, guanosine 5`-3-O-(thio)triphosphate; PLC, phospholipase C; MAP, mitogen-activated protein; MEK, MAP kinase kinase; MEKK, MAP kinase kinase; PDGF, platelet-derived growth factor.

(^2)
M. A. Sells, U. G. Knaus, S. Bagrodia, C. L. Creasy, D. Ambrose, G. Bokoch, and J. Chernoff, submitted for publication.

ACKNOWLEDGEMENTS

We thank Dr. Mordechai Anafi for providing the cDNAs expressing GST-SH3 domains and Dr. Jill Platko for helpful discussions. We also thank Wen Jin Wu and Judith Glaven in our laboratory for providing His-tagged Cdc42Hs and Rac1 proteins. We thank Cindy Westmiller for expert secretarial assistance.

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