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Identification of a Central Phosphorylation Site in p21-activated Kinase Regulating Autoinhibition and Kinase Activity

1999; Elsevier BV; Volume: 274; Issue: 46 Linguagem: Inglês

10.1074/jbc.274.46.32565

1083-351X

Frank T. Zenke, Charles C. King, Benjamin P. Bohl, Gary Bokoch,

Protein Tyrosine Phosphatases

p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates. The mechanism of Pak activation by these agents remains unclear. We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation. We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67–150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited intrans by recombinant ID peptide, indicating similarities in their mode of activation. However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide. We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity. Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation. p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates. The mechanism of Pak activation by these agents remains unclear. We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation. We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67–150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited intrans by recombinant ID peptide, indicating similarities in their mode of activation. However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide. We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity. Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation. p21-activated kinase glutathione S-transferase inhibitory domain myelin basic protein p21-binding domain polyacrylamide gel eletrophoresis dithiothreitol guanosine 5′-3-O-(thio)triphosphate protein kinase C polymerase chain reaction amino acid(s) Localized regulation of protein kinase activity is an essential means to ensure spatial and temporal control of signaling events in a cellular environment. Hormonal or other stimuli are usually necessary to switch a kinase into a catalytically competent state, allowing phosphorylation of substrates to take place. An emerging regulatory theme is that inhibitory mechanisms exist to keep protein kinases in an inactive state (1Taylor S.S. Radzio-Andzelm E. Curr. Opin. Chem. Biol. 1997; 1: 219-226Crossref PubMed Scopus (44) Google Scholar, 2Kemp B.E. Pearson R.B. Biochim. Biophys. Acta. 1991; 1094: 67-76Crossref PubMed Scopus (123) Google Scholar), and that relief of such inhibition allows activation to occur. Kinases often act autocatalytically to phosphorylate key amino acid residues that relieve autoinhibition and enhance catalytic efficiency. Alternatively, exogenous kinases may also serve this role. However, activation must be reversed in the absence of the stimulus, and dephosphorylation by protein phosphatases is thought to mediate switching the active kinase back to an inactive or basally activated state. p21-activated kinases (Paks)1belong to a growing family of serine/threonine kinases involved in the control of various cellular processes, including the cell cycle, dynamics of the cytoskeleton, apoptosis, and transcription (3Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar). Pak kinase activity is regulated by members of the Rho family of GTPases, specifically Cdc42 and Rac. These GTPases bind to Pak kinase solely in their active forms, i.e. the GTP-bound state, resulting in stimulation of the kinase activity both in vitro andin vivo. The molecular details of how the GTPases exert their effect on the kinase to induce its activation remain unclear, however. Several lines of evidence suggested that the amino-terminal non-kinase region of Pak, in which the Cdc42/Rac-binding site is located, is crucial for the regulation of kinase activity. It has been shown by several groups that removal of the NH2-terminal portion of Pak by protease digestion leads to activation of the kinase fragment (4Benner G.E. Dennis P.B. Masaracchia R.A. J. Biol. Chem. 1995; 270: 21121-21128Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 5Yu J.S. Chen W.J. Ni M.H. Chan W.H. Yang S.D. Biochem. J. 1998; 334: 121-131Crossref PubMed Scopus (44) Google Scholar). A physiologically relevant example is known for the 62-kDa isoform Pak2, which has been shown to be cleaved and activated by the cysteine protease caspase-3 in response to apoptosis-inducing stimuli (6Lee N. MacDonald H. Reinhard C. Halenbeck R. Roulston A. Shi T. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13642-13647Crossref PubMed Scopus (175) Google Scholar, 7Rudel T. Bokoch G.M. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (605) Google Scholar, 8Walter B.N. Huang Z. Jakobi R. Tuazon P.T. Alnemri E.S. Litwack G. Traugh J.A. J. Biol. Chem. 1998; 273: 28733-28739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Zhao et al. (9Zhao Z.S. Manser E. Chen Z.Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar) have recently used mutational analysis to characterize a region in the NH2-terminal regulatory domain of Pak adjacent to the p21-binding domain that is important for the inhibition of kinase activity. Using a plasmid injection approach, they showed that cellular effects that depend on Pak kinase activity, including dissolution of actin stress fibers and focal adhesions, can be blocked by coexpression of the autoinhibitory region. Similar conclusions were reached using a genetic analysis ofSchizosaccharomyces pombe Pak1 (10Tu H. Wigler M. Mol. Cell. Biol. 1999; 19: 602-611Crossref PubMed Scopus (80) Google Scholar). We undertook a biochemical approach to characterize an interaction between the Pak1 kinase domain and the regulatory amino terminus. We localized the interacting site in the NH2 terminus to the same area as the autoinhibitory domain (ID) characterized by Zhao et al.(9Zhao Z.S. Manser E. Chen Z.Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar). Our data suggest that the interaction observed is responsible for maintaining the kinase in an inactive state. Cdc42 was able to disrupt the interaction, but only in the GTP-bound active form. Using characterized mutations in Pak1 that abolish GTPase binding or inactivate the inhibitory function, we could show that the p21-binding and autoinhibitory domains are separable but overlapping. Recently, we have demonstrated that sphingosine is an activator of human Pak1in vitro and in vivo (11Bokoch G.M. Reilly A.M. Daniels R.H. King C.C. Olivera A. Spiegel S. Knaus U.G. J. Biol. Chem. 1998; 273: 8137-8144Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), although the activation by the lipid in vivo is controversial (12Lian J.P. Huang R. Robinson D. Badwey J.A. J. Immunol. 1998; 161: 4375-4381PubMed Google Scholar). We observed that, at non-saturating concentrations, sphingosine-induced Pak1 activation was also sensitive to the autoinhibitory peptide, suggesting that the lipid mediates activation of Pak1 via a similar mechanism, i.e. relief of autoinhibition by the NH2-terminal ID domain. We demonstrate that one major phosphorylation event on residue threonine 423 within the COOH-terminal kinase domain renders the kinase activity independent of the autoinhibitory module and simultaneously increases its specific activity. Both Cdc42 and sphingosine act on Pak1 kinase to expose threonine 423, which in turn becomes accessible for cross-phosphorylation. Cell culture medium, fetal bovine serum and supplements were from Life Technologies, Inc. [γ-32P]ATP (specific activity 4500 mCi/mmol) was from ICN, Costa Mesa CA. Plasmids for transfection were purified using the Qiafilter purification system of Qiagen, Chatsworth CA. The T7-tag monoclonal antibody was purchased from Novagen, Madison WI. Thrombin and sphingosine were purchased from Sigma; GTPγS and [35S]GTPγS were from NEN Life Science Products. For polymerase chain reactions, the Expand High Fidelity PCR system from Roche Molecular Biochemicals was used. The PKCβII antibody (originally raised against a subdomain VIII phosphopeptide of the protein kinase C isoform βII, kindly provided by Erica Dutil and Alexandra Newton, University of California, San Diego, CA) was used to detect the threonine 423 phosphorylated subdomain VIII activation loop of Pak1. pGEX-KG/rpak1 (233–544) was kindly provided by Melanie H. Cobb (University of Texas Southwestern Medical Center, Dallas, TX). pCMV6M is a myc tag-containing derivative of pCMV5 and has been described elsewhere (13Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). All Pak1 variants (human Pak1 wild type, Pak1-(T423E), Pak1-(T423A), Pak1-(H83L,H86L), Pak1-(L107F), Pak1-(K299A) were inserted into pCMV6M and contain a myc epitope at the amino terminus for detection. The threonine to alanine exchange at position 423 in hPak1 was introduced using oligonucleotides (5′-CAGAGCAAACGGAGCgCCATGGTAGGAACCCC-3′ and the complementary oligonucleotide) and the Quikchange Site-directed mutagenesis kit of Stratagene, La Jolla. The NH2-terminal fragments of hpak1 (amino acid 1–234: hpak1-(1–234), aa 67–150: hpak1-(67–150) and mutated derivative H83L,H86L) were subcloned in pET28a (Novagen, Madison). The wild type and H83L,H86L mutation of hpak1-(1–234) were amplified by PCR from the corresponding pCMV6M/hPak1 derivatives using primers pT637 and OP1/234–3′ (5′-CCG GAATTC TTA AGC ATC TGG TGG AGT GGT-3′), cut with BamHI and EcoRI, and inserted into BamHI-EcoRI-cut pET28a. The p21-binding domain (PBD)/ID wild type fragment (amino acid 67–150) was produced by PCR using primers OP1/67–5′ (5′-CGC GGATCC AAG AAA GAG AAA GAG CGG-3′) and OP1/150–3′ (AAG GAA AAA AGC GGC CGC GTCGAC TCA AGC TGA CTT ATC TGT AAA GCT-3′), cut with BamHI and EcoRI, and inserted into pET28a. The PBD/ID fragments containing the H83L,H86L or the L107F mutations were amplified from the corresponding full-length hPak1 cDNA clones and inserted by BamHI andEcoRI restriction into pET28a. To produce H83L and H86L single mutations in the PBD/ID fragment, overlapping PCR was performed using OP1/67–5′ and OP1/150–3′ as outer boundary primers and overlapping primer pairs to introduce the desired mutations (for H83L: forward primer 5′-TCA GAT TTT GAg CtC ACA ATT CAT-3′ and reverse primer 5′-ATG AAT TGT GaG cTC AAA ATC TGA-3′; for H86L: forward primer 5′-GAA CAC ACA Ata Cta GTC GGT TTT-3′ and reverse primer 5′-AAA ACC GAC taG tAT TGT GTG TTC-3′; lowercase letters indicate the introduced base mutations). The COOH-terminal kinase domains of human Pak1 (amino acids 232–545) was amplified by PCR (primers for hpak1-(232–545): 5′-GCGGATCC CCA GAT GCT TTG ACC CGG-3′, 5′-G CCGGTCGAC TTA GTG ATT GTT CTT TG-3′). The hpak1 fragment was cut with BamHI-SalI and inserted into BamHI-SalI-cut pET28a. The GST-rpak1-(233–544) protein was expressed in DH10B cells and purified according to the standard protocol of Amersham Pharmacia Biotech, Uppsala, Sweden (GST gene fusion system, 3rd edition). Buffers for GST-Cdc42 purification contained 1 μm GDP starting from the lysis, excluding the dialysis buffer. The GST moiety was cleaved off the Cdc42 with thrombin at a final concentration of 10 units/ml glutathione beads. Thrombin was removed by incubation withp-aminobenzamidine beads (Sigma) and Cdc42 dialyzed four times against buffer (25 mm Tris/HCl, pH 7.5, 1 mm EDTA, 5 mm MgCl2, 1 mm DTT, 0.1 mm phenylmethylsulfonyl fluoride). After dialysis, 1 μm GDP was added again and the purified protein was concentrated by ultrafiltration. His-tagged fusion proteins in vector pET28a were expressed in BL21/DE3 (pLysS). The recombinant kinase fragment (hpak1-(232–545)) was purified following the standard batch purification protocol under native conditions (Qiagen QIAexpressionist, March 1997). The histidine-tagged fusion proteins, hpak1-(1–234) and hpak1-(67–150), were purified under denaturing conditions according to the batch purification method by Qiagen. After elution the proteins were dialyzed twice against 2 liters of buffer (50 mm Hepes/NaOH, pH 7.5, 100 mm NaCl, 10 mm MgCl2, 5% glycerol) and stored frozen at −70 °C. Rat Pak1 kinase fragment (amino acids 233–544) fused to glutathione S-transferase (GST-rpak1-(233–544)) and an NH2-terminal His6/T7-tagged fusion of human Pak1 hpak1-(1–235) were purified and used for in vitro binding studies. The rat Pak1 COOH-terminal kinase domain does not differ in amino acid sequence from the human homologue (sequencing of human Pak1 revealed that amino acid 503 in human Pak1 is glutamic acid, codon GAG, and not aspartic acid, codon GAT, as in the GenBank data base sequence). 1–5 μg of GST-rpak1-(233–544) kinase domain was incubated with 1–3 μg of NH2-terminal protein fragments in binding buffer (50 mm Hepes/NaOH, pH 7.5, 100 mm NaCl, 10 mm MgCl2, 1 mm DTT, 1% Nonidet P-40) in a volume of 200–500 μl. To immobilize the GST protein, glutathione-agarose beads equilibrated in binding buffer were added to the reaction and incubated for 30 min to 1 h at 4 °C under constant agitation. Binding reactions were washed four times with 1 ml of binding buffer each. Samples were boiled in SDS-sample buffer and separated on SDS-PAGE gels for immunoblotting or Coomassie staining. In Cdc42 competition experiments, the binding reactions were washed twice with binding buffer to remove unbound hpak1-(1–234) and Cdc42 was added to the complex for 15 min on ice or for 15 min at 30 °C as indicated in the figure legends. The bead fraction was washed again four times with binding buffer and prepared for SDS-PAGE. Cos-1 or HeLa cells were seeded on 10 cm cell culture dishes at 50–70% confluency and transfected using LipofectAMINE (Life Technologies, Inc.) as a transfection agent. 5 μg of plasmid DNA and 15 μl of LipofectAMINE were used per dish, and the transfection protocol was essentially followed according to the manufacturer's guidelines (Life Technologies, Inc.). After 48 h the dishes were washed once with 1× Hanks' buffered saline solution (Life Technologies, Inc.) and lysed in 0.5 ml of lysis buffer (25 mm Tris/HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1 mmEGTA, 1 mm DTT, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride). The expression of proteins in the lysates was analyzed by immunoblotting. For precipitations of myc-tagged hPak1, lysates were incubated with equilibrated protein G beads and monoclonal anti-myc antibody (9E10) for at least 3 h or overnight at 4 °C. The bead fraction was washed four times with lysis buffer, stored frozen at −70 °C, or directly used for kinase assays. To use immunoprecipitated hPak1 in kinase assays, the bead fraction was washed twice in kinase buffer. Cdc42 was loaded with GTPγS under the following conditions. 5–20 μg of Cdc42 were incubated in 25 mm Hepes/NaOH, pH 7.5, containing 20 mm EDTA and 1 mm GTPγS for 10 min at 30 °C in a total volume of 25–100 μl. The reaction was stopped by addition of MgCl2 at 100 mm final concentration. Sphingosine was dissolved in 95% ethanol at a concentration of 15 mm and stored at −20 °C. Aliquots were dried under nitrogen gas and dissolved in 25 mm Tris/HCl, pH 7.5, at 3–4 mm concentration. Before addition of sphingosine to the kinase reaction, the lipid solution was sonicated for 30–60 s. Kinase reactions with purified recombinant or immunoprecipitated Pak were performed in kinase buffer (50 mm Hepes/NaOH, pH 7.5, 10 mmMgCl2, 2 mm MnCl2, 0.2 mm DTT) in a volume of 60 μl with 250 μmATP except for the experiment shown in Fig. 1 D, where 50 μm ATP was used. Radiolabeled ATP was used at 10 μCi/reaction. The reactions were incubated for 30 min at 30 °C and stopped by addition of sample buffer. Myelin basic protein (MBP) was used as a substrate at 2–4 μg/reaction. To prephosphorylate Pak prior to a kinase reaction, the protein G-bound hPak1 was incubated with 250 μm unlabeled ATP and an activator (0.5–2 μg of GTPγS-loaded Cdc42 or 0.4 mmsphingosine) for 30 min at 30 °C. The reactions were washed three times with lysis buffer and twice with kinase buffer and directly used in kinase assays as described above. Pak kinases consist of a NH2-terminal regulatory domain and a COOH-terminal kinase domain having many of the conserved features of all known serine/threonine kinases. Several lines of evidence have suggested that the NH2-terminal region down-regulates the enzymatic activity of the COOH-terminal kinase. We tested whether we could detect a physical interaction between these domains that might be responsible for inhibiting Pak kinase activity. As seen in Fig. 1 A, the purified hpak1-(1–234) fragment efficiently complexed with the bead-coupled GST-rpak1-(233–544) COOH-terminal piece containing the entire kinase domain. Binding was specific to the GST-rpak1-(233–544) fusion since the amino terminus was not precipitated with GST alone. Incubating the preformed GST-rpak1-(233–544)/NH2 terminus complex with GTPγS-loaded Cdc42 reduced the amount of the hpak1-(1–234) in the pull-down fraction (Fig. 1 A,lane 4). This reduction was specific for GTPγS-loaded Cdc42, as a GDP-loaded or a non-loaded Cdc42 was not able to compete (Fig. 1 B). Addition of GTPγS-loaded Cdc42 decreased complex formation in a concentration-dependent manner (Fig. 1 C). A hpak1-(1–234) fragment in which the Cdc42-binding site was mutated (H83L,H86L) bound as efficiently as the wild type fragment to the kinase domain (Fig. 1 C, comparelanes 2 and 7). However, in this case the GTPγS-loaded Cdc42 was not able to disrupt the interaction, indicating that binding of the GTPase to the NH2-terminal fragment is required for the observed competitive effect. In vitro kinase assays showed hpak1-(1–234) is capable to inhibit GST-rpak1-(233–544) kinase activity (Fig. 1 D,lanes 3–6). With increasing amounts of the NH2 terminus, autophosphorylation and phosphorylation of myelin basic protein were decreased. However, hpak1-(1–234) itself was a good substrate for GST-rpak1-(233–544) (data not shown) and this, due to competitive substrate phosphorylation, made it difficult to determine the K i value. Cdc42 binds Pak1 in a minimal region consisting of amino acids 75–89 (14Burbelo P.D. Dreschsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). However, amino acids surrounding this sequence contribute to the efficiency of the interaction (9Zhao Z.S. Manser E. Chen Z.Q. Chong C. Leung T. Lim L. Mol. Cell. Biol. 1998; 18: 2153-2163Crossref PubMed Google Scholar). We hypothesized, based on the Cdc42 competition experiments (Fig. 1), that the domain interacting with the kinase core might be in close vicinity or overlap with the Cdc42 binding site. To localize the carboxyl-terminal interacting and autoinhibitory domain in the NH2 terminus, we expressed smaller regions of human Pak1 as recombinant His6/T7-tagged proteins in Escherichia coli and tested these in pull-down assays with GST-rpak1-(233–544) immobilized on glutathione-agarose. We detected a very efficient interaction with a His6/T7-tagged fusion of the wild type hpak1-(67–150) peptide (Fig.2 A, lane 4). The amino-terminal fragment did not bind to the GST protein (Fig. 2 A, lane 3), demonstrating the specificity of this interaction. Other amino-terminal Pak fragments (amino acids 1–74 and 174–306) were not pulled down by the COOH-terminal fragment (data not shown). The region encompassing amino acids 67–150 contains the previously characterized GTPase/p21-binding domain and confirmed our hypothesis that the interaction site is in close proximity to the p21 binding domain. To further refine the interacting region, we constructed smaller peptides of hPak1 (aa 67–89, 67–108, and 109–150). However, we could not detect an interaction of these peptides with the Pak1 kinase domain using the pull-down assay (data not shown). We employed already characterized mutations within hpak1-(67–150) to further analyze the function of this region. Mutation of both conserved histidine residues to leucine (H83L,H86L) has been shown to disrupt the interaction with small GTPases, accompanied by a moderate increase in kinase activity (13Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 15Daniels R.H. Zenke F.T. Bokoch G.M. J. Biol. Chem. 1999; 274: 6047-6050Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Mutating leucine 107 to phenylalanine, on the other hand, leads to strong activation of full-length Pak1, and activity is independent of GTPases (16Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. Curr. Biol. 1996; 6: 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), even though binding is maintained. We analyzed how these mutations influence the interaction with the GST-rpak1-(233–544) fusion. As seen in Fig. 2 A(lanes 5–12), the H83L and H86L single and double mutants bound as efficiently to the kinase domain as did the wild type peptide. In contrast, the L107F mutation exhibited drastically reduced binding to the kinase domain. The purified hpak1-(67–150) fragments had different abilities to inhibit GST-rpak1-(233–544) kinase (Fig. 2 B). The wild type hpak1-(67–150) was most potent in inhibiting kinase activity, whereas the L107F mutated peptide was strongly reduced in its inhibitory potency, and the H83L,H86L double mutant was moderately reduced in inhibiting GST-rpak1-(233–544) kinase activity. The hpak1-(67–150) fragments, unlike hpak1-(1–234), were not phosphorylated substantially. We termed the hpak1-(67–150) region PBD/ID (p21 bindingdomain/inhibitory domain). Using GST- rpak1-(233–544) kinase to evaluate the inhibitory potential of the PBD/ID region (aa 67–150) had the advantage that the isolated COOH terminus is constitutively active, independently of GTPases. However, we asked whether Cdc42-stimulated hPak1 activity could also be inhibited by the purified peptides. Since a functional p21 binding site is contained within the PBD/ID fragment, it was expected that inhibition of kinase activity could in part be due to sequestering of the GTPases. The ability of the PBD/ID fragments to bind activated Cdc42 was tested (Fig. 3). Only the wild type fragment and the L107F mutated form could bind Cdc42 in overlay assays, although the latter bound with somewhat reduced affinity (about 50% of wild type). Mutation of histidines 83 and 86 either separately or together led to a complete loss of binding to Cdc42. Any kinase inhibition by these Cdc42 binding-deficient fragments would therefore not be due to titration of GTPases. We were able to show that not only the Cdc42-binding proficient, but also the binding-deficient versions were able to inhibit full-length hPak1 activity. As Fig. 4 Ashows, both autophosphorylation and substrate phosphorylation are reduced by hpak1-(67–150) peptides. The wild type and L107F mutant peptides strongly reduce kinase activity. The inhibition by the L107F peptide is most likely due to sequestration of the activated Cdc42. Of the GTPase binding-deficient versions, the H83L mutant peptide was most potent in its kinase-inhibitory effect. Mutation of position 86 significantly reduced the inhibitory effect to a similar extent as did the H83L,H86L double mutant. All peptides showed concentration-dependent inhibitory effects. We titrated the H83L peptide to determine the half-maximal inhibitory concentration using a constant amount of immunoprecipitated hPak1 stimulated with Cdc42 (Fig. 4 B). The purified peptides were not significantly phosphorylated and therefore did not interfere with theK i determination. We calculated the apparentK i as 1.2 μm for MBP phosphorylation (Fig. 4 C). Overall, the peptide inhibition data obtained with p21-activated Pak1 fit well with those using GST-rpak1-(233–544) (Fig. 2 C). Taken together, these data demonstrate that the functional domains for p21 binding and autoinhibition of kinase activity overlap in part within the PBD/ID. Both activities can, however, be separated from each other, demonstrating that the PBD and the ID have distinct structural determinants for function. Pak kinases autosphosphorylate after stimulation with activated Cdc42 and remain activated after removal of the GTPase (17Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (305) Google Scholar). In light of our above results, this could indicate that phosphorylation events antagonize the inhibitory effect of the ID domain by decreasing its interaction with the kinase domain. Full-length hPak1 was activated by autophosphorylation for 30 min in the presence of excess unlabeled ATP and Cdc42-GTPγS; these were then removed by washing (Fig.5 A). As shown inlanes 3–7, in a subsequent kinase reaction with labeled ATP the incorporation of label into hPak1 was drastically decreased due to an efficient incorporation of unlabeled ATP in the initial reaction. Autophosphorylated hPak1 was highly active toward substrate even without further addition of activated Cdc42. The hpak1-(67–150) wild type peptide and also the mutated versions (data not shown) did not affect substrate phosphorylation activity of the autophosphorylated Pak1 at levels sufficient to efficiently block Cdc42-stimulated hPak1. Addition of activated GTPase to prephosphorylated hPak1 did not restore the sensitivity toward ID-mediated inhibition (data not shown). We also observed that the hPak1 kinase domain (aa 232–545), when expressed as a His6/T7-tagged fusion protein in E. coli, is autophosphorylated and also not inhibited by excess concentrations of PBD/ID peptide (data not shown), supporting the idea that phosphorylation renders the kinase domain independent of autoinhibition. Interestingly, a hPak1 mutant, hPak1-(T423E) (13Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar), which is constitutively activated by mutation at phospho-acceptor site threonine 423 in the activation loop, is also completely inert toward inhibition by the PBD/ID fragments (Fig. 5 B). In this case, both autophosphorylation and substrate phosphorylation are not affected by the hpak1-(67–150) peptide. This argues that the phosphorylation state of threonine 423 is a critical determinant for kinase inhibition by the ID domain. As indicated by the threonine 423 to glutamic acid mutation in hPak1, the phosphorylation status of the activation loop appears to be of importance for the autoinhibitory mechanism. To analyze this in more detail, we constructed a threonine 423 to alanine mutant that does not autophosphorylate at this residue, and the activity of this mutant was compared with the wild type protein inin vitro kinase assays. Basal activities of immunoprecipitated T423A hPak1 did not differ from wild type (Fig. 6, lanes 1 and 6). The Cdc42-stimulated hPak1 T423A had a decreased activity toward exogenous substrate, ranging from 10 to 30% of wild type activity (Fig. 6, compare lanes 2and 7). Autophosphorylation was less affected and in the range of 40–60% of wild type Pak1. The Cdc42-stimulated hPak1-(T423A) activity was sensitive to inhibition by the H83L hpak1-(67–150) fragment. Prephosphorylation of hPak1-(T423A) in the presence of GTPγS-loaded Cdc42 and excess cold ATP led to a significant decrease in the incorporation of radioactivity into the protein in a subsequent kinase reaction. The prephosphorylated hPak1-(T423A) retained its (reduced) activity toward substrate but surprisingly this activity was still sensitive toward addition of hpak1-(67–150) fragment (Fig. 6,lane 10). It was of interest to analyze if lipid-mediated activation of hPak1 is sensitive to the ID/kinase domain interaction. Sphingosine, which is one of the more effective lipids to activate hPak1 (11Bokoch G.M. Reilly A.M. Daniels R.H. King C.C. Olivera A. Spiegel S. Knaus U.G. J. Biol. Chem. 1998; 273: 8137-8144Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), was used in in vitro kinase assays in the absence and presence of the PBD/ID peptide (Fig.7). The concentration (400 μm) used in the assay resulted in a highly activated hPak1 in which auto- and substrate phosphorylation was comparable to the Cdc42-stimulated enzyme (Fig. 7 A). Under these conditions, sphingosine-mediated activation was completely insensitive to the wild type PBD/ID fragment. However, we observed that activat