INTRODUCTION

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The Rho family GTPases Rac1 and Cdc42 mediate diverse biological events including changes in the cytoskeletal architecture (1-3), stimulation of DNA synthesis (4), cellular transformation (5-8), and signaling to the nucleus (9-14). Many of the signaling pathways leading to the execution of these events involve the p21-activated kinases, Paks1-3,¹ which are direct effectors of Rac1 and Cdc42 (15-17). Binding of these GTPases to a conserved p21-binding domain (PBD, also known as CRIB for Cdc42/Rac1 interactive binding) stimulates their serine/threonine kinase activities by a mechanism involving autophosphorylation (15, 18). The important roles that Paks play as effectors of Cdc42/Rac1 signaling have been established from genetic and biochemical studies in yeast and mammalian cells. The budding yeast homolog of mammalian Paks, Ste20, acts in concert with Cdc42 in the pheromone response to activate a MAP kinase cascade leading to transcription of genes required for cell cycle arrest. The same protein functions as an effector of Cdc42 to activate a different MAP kinase cascade leading to filamentous growth in response to nitrogen starvation (19). The yeast model in which Pak/Ste20 links Cdc42 to transcriptional and cytoskeletal events is paralleled in mammalian cells; constitutively activated Pak mutants can activate the c-Jun N-terminal kinase MAP kinase cascade leading to transcriptional control (12, 13) and can mimic some, although not all, of the effects of Rac1 or Cdc42 on cytoskeletal organization (18, 20).

The apparent multiplicity of Pak-mediated signaling pathways suggests that Pak activity must be tightly regulated. This has been made all the more clear from the observations that Pak1 function is required for cellular transformation by Ras (8) and that Pak2 activation is involved in Fas-mediated apoptosis (21, 22). Here we describe the identification of two isoforms of a novel Pak-binding protein, probably resulting from alternative splicing of the same message, one of which is able to suppress Pak activation by upstream regulators.

	EXPERIMENTAL PROCEDURES

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Yeast Two-hybrid Screen-- To identify proteins that interact with Pak3, the yeast strain L40 was co-transformed with Pak3 fused to the LexA DNA-binding domain, which regulates expression of both his3 and lacZ, and a HeLa cDNA library was fused with the Gal4 activation domain (23). Clones positive for -galactosidase activity were rescued, and several clones that provided bait-dependent his3 and lacZ gene activation were sequenced. A 1-kilobase (EcoRI-XhoI) fragment of clone Y107 was used to screen an oligo(dT)-primed, size fractionated (4-9 kilobases) U937 library (a gift from J. Burrows, Massachusetts Institute of Technology, Boston, MA) and a HeLa cDNA library (Stratagene). The U937 library yielded a full-length clone (A6) that was predicted to encode a protein of 436 amino acids, p50^Cool-1. Several partial clones were recovered from the HeLa library that appeared to represent alternatively spliced forms of Y107. One of these (clone 12a) was identical to a recently cloned cDNA, p85^SPR, but lacked the 3' end encoding its C-terminal 31 amino acids. The p85^Cool-1 cDNA was generated by fusing this 3' end, derived from the 3'-untranslated region of the U937 clone A6, to clone 12a (p85^Cool-1 is therefore identical to p85^SPR).

Plasmid Construction-- The coding sequence of Pak3 was excised as a 1700-bp BamHI fragment from plasmid pJ3HmPak3 (12) and subcloned into pLexA (23). A BamHI site (GGA frame) was engineered in Cool-1 in front of the initiation methionine, and the BamHI-XhoI (1-730 bp) and XhoI-BglII (670 bp) fragments, which include the stop codon, were ligated into the BamHI site of pBS SK (Stratagene) to generate plasmid pBSA6Cool. The BamHI-EcoRI fragment from pBSA6Cool, encompassing the entire coding sequence of p50^Cool-1, was subcloned into Myc-tagged eukaryotic expression plasmid CMV6M to express p50^Cool-1. pCMV6M(p50^Cool-1)W43K was constructed by three fragment ligation containing the 420-bp BamHI-XbaI polymerase chain reaction product, the 971-bp XbaI-EcoRI fragment from pBSA6Cool, and the 5000-bp BamHI-EcoRI fragment from the CMV6M vector. Plasmid CMV6Mp85Cool (to express Myc-tagged p85^Cool-1) was generated by ligating a BamHI-BsgI 1610-bp fragment (from clone 12a) and a polymerase chain reaction generated 340-bp BsgI-EcoRI fragment (from the 3'-untranslated region of U937 clone A6) into the BamHI-EcoRI site of the CMV6M vector. Plasmid pCMV6Dbl was generated by ligating a BamHI fragment encoding oncogenic Dbl from plasmid pc11dbl (a gift from Dr. Sandra Eva, Giannina Galini Institute, Geneva, Italy) into the BamHI site of pCMV6. pCMV6HA-Cdc42 (HA-tagged Cdc42), pGEX-PBD (GST-PBD), and J3HmPak3 (HA-Pak3) have been previously described (12).

Kinase Assays, Affinity Precipitation, and Immunoprecipitations-- Kinase reactions were initiated by the addition of 2× kinase buffer (40 mM Hepes, pH 7.4, 20 mM MgCl₂, and 4 mM MnCl₂) and 20 µM [-³²P]ATP (3000 Ci/mmol) for 3.5 min at room temperature. Reactions were stopped by the addition of 2× SDS sample buffer containing 20 mM EDTA. Affinity precipitation with GST-PBD was as described (24) except that COS cells were lysed in 25 mM Hepes, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 10 mM MgCl₂, 1 mM EDTA, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates containing equal amounts of Pak3 were immunoprecipitated with anti-Pak3 primary antibody (a gift from Dr. S. Pelech, Kinetek Biotechnology Corporation, Vancouver, Canada)

	RESULTS AND DISCUSSION

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To identify potential Pak-binding partners we used the yeast two-hybrid screen. One positive clone was used to screen cDNA libraries and data bases (see "Experimental Procedures") to yield two Pak3-binding proteins, p50 and p85^Cool-1. They share an N-terminal SH3 domain (amino acids 7-65), followed by a Dbl homology (DH) domain (amino acids 100-279) and an adjacent pleckstrin homology (PH) domain (amino acids 295-400) (Fig. 1A). Because p50 and p85^Cool-1 are identical at the nucleotide level over amino acids 1-418, it is likely that they arise from alternative splicing of the same message. p85^Cool-1 is identical to two recently cloned proteins, p85^SPR (25) and -PIX (26). The DH domain of the Cool proteins shows the highest sequence identity to the DH domains of Dbl (33%), Scd1 (30%), Dbs (29%), Tiam-1 (29%), Still Life2 (29%), Cdc24 (26%), and Vav2 (24%). In addition to the genetic screen, we used recombinant GST-Pak3 (amino acids 148-239) to purify Pak-binding proteins from Src(Y527F)-transformed NIH 3T3 cells. Sequence obtained from an ~85-kDa Pak-binding protein was identical to portions of p50 and p85^Cool-1 (data not shown). We also identified a closely related cDNA from the data bases, and we have found that its product also interacts with Pak3.² This protein, Cool-2, is identical to the recently described -PIX (26).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Cool-1 is a Pak3-binding protein. A, schematic representation of Cool-1. The coding region of p50Cool-1 containing the SH3, DH, and PH domains is identical to that of p85Cool-1, which additionally contains a C terminus with potential SH3-binding sites (PXXP) denoted by vertical lines. B, COS cells were co-transfected with the plasmids (1 µg of each) expressing the indicated HA- or Myc-tagged proteins. Anti-Myc (9E10) (lanes 6-10) and anti-HA (12CA5) (lanes 11-14) immunoprecipitates were Western blotted. The top (>~60 KDa) and bottom (<~60 KDa) parts of the blots were probed with anti-HA and anti-Myc antibodies, respectively. Lanes 1-5 represent 10% of the whole cell lysates (WCL) used in the immunoprecipitation (IP) reactions. C, endogenous proteins were immunoprecipitated from NIH 3T3 cell lysates (WCL, lanes 1) using anti-Pak3 (NT) polyclonal antibody (lanes 2, Pak3 IP), anti-Cool-1 polyclonal antibody (lanes 3, Cool-1 IP), or rabbit preimmune serum (lanes 4, Pre). Proteins were Western blotted, and blots were probed with anti-Pak3 or anti-Cool-1. MW, molecular weight markers.

To determine whether the Cool proteins bind to Pak3 in mammalian cells, we transiently co-expressed Myc-tagged p50^Cool-1 and HA-tagged Pak3 in COS cells and assayed for complex formation by immunoprecipitation and Western blot analysis (Fig. 1B). HA-Pak3 was detected in anti-Myc immunoprecipitates (Fig. 1B, lane 8, upper panel) and Myc-p50^Cool-1 was detected in anti-HA immunoprecipitates (Fig. 1B, lane 13, lower panel). Mutation of a conserved tryptophan residue within the SH3 domain (W43K) eliminated the ability of p50^Cool-1 to associate with Pak3 (Fig. 1B, lane 10, upper panel, and lane 14, lower panel), indicating that the SH3 domain of p50^Cool-1 binds Pak3. The Src SH3-binding protein, Sam68, did not co-immunoprecipitate with Myc-p50^Cool-1 (Fig. 1B, lane 9, upper panel), although it bound to the SH3 domain of Cool-1 in vitro,³ and SH3 domains from the Dbl family proteins Dbs and Vav did not bind Pak3 (data not shown), showing specificity of the Cool-1/Pak3 association in vivo. Pak3 contains four conventional (PXXP motif) SH3-binding sites (P1-P4); Pak3 containing Pro to Ala mutations in the P1-P4 sites, alone or in combination, were used to establish that these sites do not mediate Pak3-Cool-1 interactions (data not shown). While this manuscript was in preparation, Manser et al. reported binding of -PIX and -PIX (which correspond to Cool-2 and p85^Cool-1) to residues 182-203 of Pak1, confirming the atypical (non-PXXP motif) nature of Cool binding to Paks (26). Using a rabbit antiserum raised against full-length p50^Cool-1, we detected two predominant proteins (from NIH 3T3 fibroblast lysates) that migrated at ~85 and ~78 kDa on a SDS-polyacrylamide gel (Fig. 1C, right panel, lane 1). Both of these proteins were detected in anti-Cool-1 immunoprecipitates (Fig. 1C, right panel, lane 3) as well as in anti-Pak3 immunoprecipitates (Fig. 1C, right panel, lane 2), demonstrating an interaction between endogenous Cool proteins and Pak3 in NIH 3T3 cells. The less reactive band at ~50 kDa recognized by the anti-Cool-1 antibody (Fig. 1C, right panel, lane 1) may represent p50^Cool-1; however, we were unable to determine whether p50^Cool-1 was present in the immunoprecipitates because of the overlapping signal for IgG.

The presence of a DH-PH tandem motif in Cool-1, a hallmark of Dbl family exchange factors (27), initially led us to consider the possibility that Cool-1 might activate Cdc42 or Rac. However, we were unable to detect stimulation of [³H]GDP dissociation from Cdc42 or Rac by recombinant p50^Cool-1 purified from Escherichia coli or insect cells under conditions where the Dbl oncoprotein strongly stimulated GDP dissociation from Cdc42 or RhoA (data not shown).

We then considered the possibility that Cool-1 exchange activity may require cellular co-factors or post-translational modifications as proposed for Vav, Sos, and Tiam-1 (28-31). To measure Rac1 or Cdc42 activation in vivo, we used a modification of a recently described assay for activated, GTP-bound Ras (24). The PBD of Pak3 was expressed as a GST fusion protein and immobilized by binding to glutathione-Sepharose beads. The immobilized GST-PBD was used to precipitate activated Cdc42 or Rac1 from transfected COS cell lysates (Fig. 2). In untreated control cells, relatively low levels (<5%) of HA-Rac1 (upper panel) or HA-Cdc42 (lower panel) were precipitated with GST-PBD (compare lane 1 (cell lysates) and lane 9 (affinity-precipitated GTPase) in both panels). When Dbl was co-expressed with HA-tagged GTPases, there was a marked increase in the amounts of activated HA-Rac1 and HA-Cdc42 that were precipitated with GST-PBD (lane 16). The cellular activation of both Cdc42 and Rac by Dbl is consistent with results from micro-injection studies (32). In contrast, p50^Cool-1 did not detectably activate either HA-Rac1 or HA-Cdc42 (compare lanes 9 and 10), even after exposure to a number of different biological stimuli (lanes 11-15). Activation of Rac1 or Cdc42 by p85^Cool-1 was also not detected, even when co-expressed with Pak3, under conditions where Tiam-1 resulted in the activation of Rac1 but not Cdc42 (data not shown). We have not detected Cool-1-mediated Rac1 activation in cells co-expressing potential activators of Rac including Src(Y527F), Ras(G12V), and Cdc42(Q61L)². We next tested whether p50/p85^Cool-1 could directly modulate Pak activity. Although p85^Cool-1 was without significant effect (Fig. 3A, compare lanes 2 and 4), p50^Cool-1 inhibited Dbl-stimulated Pak3 activity (lanes 2 and 3). p50^Cool-1 (W43K) did not inhibit Dbl-activated Pak3 (lanes 2 and 5), indicating that binding of p50^Cool-1 to Pak3 was required for its inhibitory effect. Immunoblotting confirmed that wild type and mutant Cool-1 proteins were equally expressed and that Dbl expression levels were unaffected by Cool-1 expression (data not shown); immunoprecipitated Pak3 levels were equal (Fig. 3A, lower panel). We also found that purified, recombinant p50^Cool-1 completely blocked Cdc42(Q61L)-stimulated autophosphorylation of Pak3 and strongly inhibited the phosphorylation of myelin basic protein in Pak3 immunoprecipitates (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Cool-1 does not detectably activate Cdc42 or Rac1 in vivo. COS cells were transfected with HA-Rac1 (top) or HA-Cdc42 (bottom) alone (lanes 1 and 9) or co-transfected with Myc-p50Cool-1 (lanes 2-7 and lanes 10-15) or with Dbl (lanes 8 and 16) for 48 h and then serum-starved for another 4 h and treated with 100 ng/ml epidermal growth factor (EGF) plus 25 ng/ml insulin-like growth factor-1 (IGF-1), 40 ng/ml platelet-derived growth factor (PDGF), 20% fetal calf serum, 50 ng/ml interleukin 1 (IL-1) for 10 min, or 200 µM methylmethane sulfonate (MMS) for 1 h as indicated. Cells were lysed and affinity-precipitated with immobilized GST-PBD (GST-PBD AP, lanes 9-16), and bound proteins were Western blotted and probed with anti-HA. Lanes 1-8 represent 5% of the whole cell lysate (WCL) used in the binding reaction.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Cool-1 inhibits Dbl- and Cdc42-stimulated Pak3 activity. A, COS cells were transiently co-transfected with Pak3 (0.5 µg), Myc-p50Cool-1 (1 µg), Myc-p85Cool-1 (1 µg), Myc-p50W43KCool-1 (1 µg), or Dbl (1 µg), and Pak3 kinase activity was measured in Pak3 immunoprecipitates using myelin basic protein (MBP) as a substrate. The top part (bottom panel) of the blot was probed with anti-Pak3, and the bottom part (top panel) of the blot was autoradiographed. Based on Western blot analysis the amount of Dbl was similar in lanes 2-5, the amounts of p50Cool-1 and p50Cool-1(W43K) were also similar in lanes 3 and 5, and p85Cool-1 was present in a slightly larger amount. B, COS cells were transiently transfected with 1 µg each of Myc-Pak3, Myc-p50Cool-1, and Myc-p50W43KCool-1. Cells were lysed and affinity-precipitated with immobilized GST-Cdc42 (lanes 5, 7, 9, and 11) or with constitutively active GST-Cdc42(Q61L) (lanes 6, 8, 10, and 12). Bound proteins were Western blotted and probed with anti-Myc. Lanes 1-4 represent 10% of the whole cell lysate used in the binding reaction. C, COS cells were transiently co-transfected with empty vector (lane 1) or plasmids encoding HA-Pak3 (lanes 2-6, 0.5 µg of DNA), Myc-p50 Cool-1 (lanes 1, 4, and 6, 0.5 µg of DNA, and lane 5, 1.5 µg of DNA), Myc-p85 Cool-1 (lanes 3, 4, and 5, 0.5 µg of DNA, and lane 6, 1.5 µg of DNA). Cells were lysed and affinity-precipitated (AP) with immobilized GST-Cdc42L61 (~12 µg, lanes 7-12). Bound proteins were Western blotted, and the blot was probed with anti-HA and anti-Myc. Lanes 7-12 of the blot were reprobed with anti-Cool-1 to detect p50 Cool-1. Lanes 1-6 represent 10% of the whole cell lysate (10% WCL) used in the binding reaction. Numbers above the lanes denote the ratio of p50 to p85 Myc-tagged Cool-1 cDNA that was transfected.

We examined the effects of Cool-1 on binding of activated Cdc42 to Pak3 by expressing Myc-tagged Pak3 and/or p50^Cool-1 in COS cells and assaying the binding of Pak3 to GST-Cdc42 by affinity precipitation and anti-Myc-immunoblotting (Fig. 3B). Pak3 was precipitated by immobilized GST-Cdc42(Q61L) (GTPase-defective GTP-bound, lane 6) but not by GST-Cdc42 (GDP-bound, lane 5) and displayed a gel mobility shift due to Cdc42(Q61L)-stimulated autophosphorylation (compare lanes 1 and 6). (Pak3 autophosphorylation is sustained by the presence of Mg²⁺ during affinity precipitation.) Although p50^Cool-1 expression did not affect Pak3 recovery in GST-Cdc42(Q61L) precipitates (compare lanes 1 and 6 with lanes 3 and 10), the precipitated Pak3 did not display the gel shift observed in the absence of p50^Cool-1 or when co-expressed with p50^Cool-1(W43K) (compare lanes 6, 10, and 12). Therefore, p50^Cool-1 binding to Pak3 did not inhibit Cdc42 binding but did inhibit Cdc42-stimulated Pak3 autophosphorylation. Relative to Myc-Pak3, very little Myc-p50^Cool-1 is detected in these precipitates (lane 10), which may be due to dissociation of p50^Cool-1 from Pak3 during the precipitation and washing procedures but may also reflect the relatively poor ability of anti-Myc to detect Myc-tagged p50^Cool-1 co-precipitated with activated Cdc42 and Pak3 (see below).

Unlike p50^Cool-1, expression of p85^Cool-1 did not inhibit Cdc42(Q61L)-stimulated Pak3 autophosphorylation (Fig. 3C, compare lanes 8 and 9), which is consistent with the results shown in Fig. 3A. As expected from the presence of identical SH3 domains, p50 and p85^Cool-1 competed for binding to Pak3 in vitro (lanes 9-12). In these experiments, p50^Cool-1 co-precipitating with Pak3 was not detected with anti-Myc (upper panel, lanes 8 and 10-12) but was readily detectable with an anti-Cool-1 antibody (lower panel, lanes 8 and 10-12). The fact that an ~3-fold excess of either p50^Cool-1 or p85^Cool-1 was able to significantly inhibit the binding of the other to Pak3 suggests that they have similar affinities for Pak3.

Our results suggest that p50^Cool-1, by competing with endogenous p85^Cool-1 or other Cool proteins for binding to Pak3, might sequester Pak3 away from its site(s) of activation. On the other hand p85^Cool-1, which has a permissive effect on the stimulation of Pak activation by Dbl or other Rho family exchange factors, could play an important role in recruiting Pak to its sites of activation, possibly via its C-terminal region, which is not present in p50^Cool-1. In support of this model, our preliminary data indicate that p50^Cool-1 has a diffuse cytoplasmic localization,⁴ whereas p85^Cool-1 is concentrated at focal adhesions (25). Moreover, HeLa cell p85^Cool-1 (-PIX) has recently been shown to localize to focal complexes and appears to mediate Pak1 recruitment to these sites by activated Cdc42 (26). However, this would not account for the ability of p50^Cool-1 to inhibit Cdc42-stimulated Pak3 activity in vitro. Because both p50^Cool-1 and p85^Cool-1 appear to bind to a common site on Pak3, the specific inhibition of Pak3 activity by p50^Cool-1 may be due to the differences in the C-terminal regions of p50 and p85^Cool-1.

Protein kinases are often subject to multiple levels of regulation. The involvement of Paks in cellular signaling pathways leading to changes in gene expression or cytoskeletal architecture and their participation in both Ras-mediated transformation (8) and Fas-mediated apoptosis (21, 22) mandates that their activities be tightly controlled. The ability of p50^Cool-1 to suppress and p85^Cool-1 to permit Pak activity indicates that signaling through Pak-dependent pathways may be regulated by cell type, cell cycle, or developmental-specific expression patterns. It will be important to establish the role of DH and PH domains in Cool function and how differential expression of Cool impacts upon Pak signaling to different effector pathways.