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

Abstract

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 was using and the and the of The NH2-terminal of acid and derivative were in The wild and of were by PCR from the using and with and and inserted into The p21-binding domain wild fragment acid was by PCR using and with and and inserted into The the or the mutations were from the and inserted by into and mutations in the PCR was using and as and to the mutations and for and the The COOH-terminal kinase domains of human Pak1 was by PCR for The fragment was with and inserted into The protein was in and purified to the of for purification contained from the the The was cleaved the Cdc42 with at a of glutathione Thrombin was by and Cdc42 against was and the purified protein was by proteins in were in The recombinant kinase fragment was purified the purification The and were purified to the purification by the proteins were against of and at Pak1 kinase fragment to glutathione S-transferase and an NH2-terminal of human Pak1 were purified and used for in vitro binding The Pak1 COOH-terminal kinase domain in amino acid from the human of human Pak1 that amino acid in human Pak1 is and as in the data of kinase domain was with of NH2-terminal protein in binding in a of the in binding were to the reaction and for to at were with of binding were in and on for or Cdc42 the binding were with binding to and Cdc42 was to the for on or for at as in the The was with binding and for or were on cell culture at and using Technologies, as a transfection of plasmid and of were used and the transfection was to the Technologies, the were with Technologies, and in of The of proteins in the was by For of were with protein and monoclonal antibody for at or at The was with at or used for kinase in kinase the was in kinase Cdc42 was with GTPγS the of Cdc42 were in and GTPγS for at in a of The reaction was by of at was in at a of and at were and in at of sphingosine to the kinase the lipid was for with purified recombinant or Pak were in kinase in a of with for the shown in was used. was used at The were for at and by of basic protein was used as a at Pak to a kinase the protein was with and an activator of Cdc42 or for at The were with and with kinase and used in kinase as described Pak kinases of a NH2-terminal regulatory domain and a COOH-terminal kinase domain of the of known serine/threonine Several lines of evidence have suggested that the NH2-terminal region the enzymatic activity of the COOH-terminal We we could detect a interaction between these domains that be responsible for Pak kinase activity. in the purified fragment with the COOH-terminal the kinase domain. was specific to the the amino terminus was with the terminus with Cdc42 the of the in the was specific for as a or a Cdc42 was able to of Cdc42 in a A fragment in which the site was as as the wild fragment to the kinase domain and However, in this the Cdc42 was able to disrupt the interaction, indicating that binding of the GTPase to the NH2-terminal fragment is for the observed vitro kinase showed is to kinase activity of the NH2 autophosphorylation and phosphorylation of myelin basic protein were However, was a for and to it to the Cdc42 Pak1 in a region of amino D. A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). However, amino this to the 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 on the Cdc42 that the domain interacting with the kinase be in or with the Cdc42 binding the carboxyl-terminal interacting and autoinhibitory domain in the NH2 we of human Pak1 as recombinant proteins in and these in with on We a interaction with a of the wild The amino-terminal fragment bind to the protein the of this amino-terminal Pak and were by the COOH-terminal fragment The region amino 67–150 the characterized domain and that the interaction site is in to the binding domain. the interacting we of and However, we could detect an interaction of these with the Pak1 kinase domain using the We characterized mutations within to the of this region. of both residues to has been shown to disrupt the interaction with small GTPases, by a 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, R.H. Bokoch G.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to on the other leads to activation of Pak1, and activity is independent of GTPases L. M. J. S. J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), binding is We how these mutations the interaction with the in the and and as to the kinase domain as the wild peptide. the binding to the kinase domain. The purified to kinase The wild was in kinase activity, the was in its inhibitory and the was in kinase activity. The were phosphorylated We the region Using kinase to the inhibitory of the region the that the terminus is of However, we activity could also be inhibited by the purified a binding site is contained within the it was that inhibition of kinase activity could in be to of the The of the to bind activated Cdc42 was the wild fragment and the could bind Cdc42 in although the with of wild of and either or to a of binding to kinase inhibition by these Cdc42 be to of We were able to show that only the but also the were able to activity. both autophosphorylation and phosphorylation are by The wild and kinase activity. The inhibition by the is to of the activated the GTPase the was in its of position the inhibitory effect to a similar as the All showed inhibitory We the to the inhibitory using a of with Cdc42 The purified were phosphorylated and with We the as for phosphorylation the inhibition data with p21-activated Pak1 with using these data demonstrate that the domains for binding and autoinhibition of kinase activity in within the Both be from that the and the ID have for Pak kinases stimulation with activated Cdc42 and remain activated removal of the GTPase G. A. J. 1995; PubMed Scopus Google Scholar). of this could that phosphorylation events the inhibitory effect of the ID domain by its interaction with the kinase domain. was activated by autophosphorylation for in the of and these were by shown in a kinase reaction with the of into was to an of in the was highly active of activated The wild and also the phosphorylation activity of the autophosphorylated Pak1 at to of activated GTPase to the sensitivity inhibition We also observed that the kinase domain as a protein in E. is autophosphorylated and also inhibited by of the that phosphorylation renders the kinase domain independent of a (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 activated by at phospho-acceptor site threonine 423 in the activation loop, is also inhibition by the this both autophosphorylation and phosphorylation are by the peptide. that the phosphorylation state of threonine 423 is a critical determinant for kinase inhibition by the ID domain. by the threonine 423 to acid in the phosphorylation of the activation loop to be of for the autoinhibitory this in more we a threonine 423 to alanine that at this and the activity of this was with the wild protein vitro kinase of from wild and The a activity exogenous from to of wild activity was and in the of of wild Pak1. The activity was sensitive to inhibition by the of in the of Cdc42 and to a in the of into the protein in a kinase The its activity but this activity was sensitive of fragment It was of to activation of is sensitive to the domain which is one of the more to (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 in the absence and of the The used in the in a highly activated in which and phosphorylation was to the these activation was insensitive to the wild However, we observed that activation of with non-saturating of the lipid was by the wild of in the of sphingosine shown for activation by to an activity in the absence of the autophosphorylation is to Pak1 activity. this lipid was the the autophosphorylated activated could be shown to be to of the purified fragment of by sphingosine or GTPases results in kinase activity that is in the absence of the raised the phosphorylation of by active Pak1 could be for activation in the absence of We used recombinant kinase to phosphorylate wild human Pak1. The kinase fragment phosphorylated the of in the recombinant kinase fragment was highly active substrates we were able to it to autophosphorylation However, intermolecular phosphorylation of and be activation evidence suggested that the key phosphorylation site threonine 423 is from intermolecular phosphorylation Pak1 is in an inactive A specifically the threonine 423 phosphorylated of the subdomain VIII activation loop showed that threonine 423 was phosphorylated by the recombinant kinase this the threonine of wild with Cdc42 or sphingosine and this the was used to the of activator binding on threonine 423 phosphorylation Cdc42 or sphingosine were with the recombinant kinase to threonine 423 was These data suggest that the of Pak1 the of threonine 423 for intermolecular phosphorylation. the of this residue is of the Pak1 by these and leads to exposure of threonine 423 for phosphorylation. The that both the same results that sphingosine on the Pak1 is to that of The data suggest the for the regulation of Pak kinase activity A key in the control of Pak enzymatic activity is a regulated interaction between an amino-terminal inhibitory domain and the catalytic kinase domain. Cdc42 and Rac GTPases that are in the active bind to the amino-terminal and disrupt this interaction, inhibition of the of the GTPase to Pak is a the GTPase the inhibitory mechanism can and the kinase as as autophosphorylation Phosphorylation of the kinase domain at threonine 423 in the activation loop in subdomain VIII renders the enzymatic activity insensitive to the autoinhibitory the of the inhibitory module to with the kinase domain. this the of the GTPase from Pak is to enzymatic dephosphorylation has to to Pak to and the inhibitory However, data that phosphorylation of threonine 423 a as it is also to kinase activity. to be similar to the mechanism described for protein kinase in which phosphorylation of the threonine the activation loop to in the between the of the R.A. H. Mol. Cell. Biol. PubMed Google Scholar). GTPase binding with the intramolecular interaction within the Pak we could the interacting and autoinhibitory domain in the amino terminus to amino 67–150 of human Pak1. region also the p21-binding domain. The for GTPase binding that has been by al. D. A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) amino of human Pak1. However, it has been shown that amino both and carboxyl-terminal of the site enhance the binding to the GTPases (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 using characterized amino acid in Pak1, that both domains but are at to in a of this inhibited Pak1 kinase activity. the other of to demonstrated to Pak1 binding to GTPases but interaction with the kinase domain. is a key residue for both GTPase binding and this to the interaction with the kinase as by a important for inhibition of the Pak kinase an site the of the GTPase binding domain and the of the autoinhibitory domain. The between the and autoinhibitory domains a for the of the Pak of the GTPase to the with important of the autoinhibitory module and the intramolecular We that lipids, sphingosine, induce Pak kinase activation in a similar as small (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) suggested that the lipid interaction domain is also in within the characterized region. on leads to phosphorylation of threonine 423, as activated We that sphingosine with the autoinhibitory module to of the Pak We have been to show that sphingosine the interaction in to the to to binding of the amino-terminal to The of the lipid necessary for sphingosine to act 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 described an autoinhibitory region by in vitro kinase in Pak, which is the of human Pak1. and human Pak1 are more and only at amino acid in the NH2-terminal regulatory region. with the autoinhibitory we characterized the inhibitory domain in biochemical interaction using recombinant 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) identified an autoinhibitory region from amino acid to that is contained within inhibitory region amino 67–150 of amino between Pak and human Pak1 is to amino acid human Pak1 an acid Using they identified amino in essential for the inhibitory These amino are within the COOH-terminal portion of the region acid and acid we demonstrated for these mutations disrupt GTPase binding but abolish the inhibitory function, the that both have within this region. A more approach using the system was used to demonstrate that the regulatory and kinase domains of the S. pombe Pak1 in a (10Tu H. Wigler M. Mol. Cell. Biol. 1999; 19: 602-611Crossref PubMed Scopus (80) Google Scholar). of the in to the GTPase binding in the carboxyl-terminal region for the autoinhibitory module are located, suggesting that the described mechanism of kinase is within the Pak kinase a recently and characterized Pak kinase activity, independent of the binding of a p21-binding but in the carboxyl-terminal portion of this region for the inhibitory domain are A. J. C. A. A. J. 1998; PubMed Scopus Google Scholar). Phosphorylation of threonine 423 to be a key event for activation of Pak1 and maintaining the kinase in a catalytically competent state. on (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) suggested that autophosphorylation of the kinase domain results from and intramolecular phosphorylation For protein kinase it has been that phosphorylation within the activation loop an intermolecular mechanism R.A. H. Mol. Cell. Biol. PubMed Google Scholar). to its position and to the catalytic we it that threonine 423 can be phosphorylated in we this we were able to show that threonine 423 is accessible for intermolecular phosphorylation. Our intermolecular phosphorylation studies showed that threonine 423 is accessible in the Pak 423 was only phosphorylated in Pak1-(K299A) activated GTPase or sphingosine were indicating that of the Pak is to threonine 423 accessible for intermolecular phosphorylation. the autoinhibitory module threonine 423 from phosphorylation or it a in the kinase domain as in the in is phosphorylation only the Pak are to We this as a mechanism to Pak activation the for example to GTPases are Pak interacting with be activated by intermolecular an chain reaction of Pak activation. We Erica M. Dutil and Alexandra C. for their and Melanie H. Cobb for the pGEX-KG/rpak1 We for