Specificity Profiling of Pak Kinases Allows Identification of Novel Phosphorylation Sites
2007; Elsevier BV; Volume: 282; Issue: 21 Linguagem: Inglês
10.1074/jbc.m700253200
ISSN1083-351X
AutoresUlrike Rennefahrt, Sean Deacon, Sirlester A. Parker, Karthik Devarajan, Alexander Beeser, Jonathan Chernoff, Stefan Knapp, Benjamin E. Turk, Jeffrey R. Peterson,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoThe p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks. The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks. The p21-activated kinases (Paks) 4The abbreviations used are: Pak, p21-activated kinase; GST, glutathione S-transferase; PSSM, position-specific scoring matrix; OPS, optimal Pak substrate; PKA, protein kinase A; ROC, receiver operating characteristic; AUC, area under the ROC curve; GTPγS, guanosine 5′-3-O-(thio)triphosphate.4The abbreviations used are: Pak, p21-activated kinase; GST, glutathione S-transferase; PSSM, position-specific scoring matrix; OPS, optimal Pak substrate; PKA, protein kinase A; ROC, receiver operating characteristic; AUC, area under the ROC curve; GTPγS, guanosine 5′-3-O-(thio)triphosphate. interact with active forms of the 21-kDa molecular mass Rho-related GTPases Rac and Cdc42. Paks can be divided into Group I (Pak1 to -3) and the more recently discovered Group II (Pak4 to -6), based on sequence similarity and regulatory mechanism (1Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (877) Google Scholar, 2Jaffer Z.M. Chernoff J. Int. J. Biochem. Cell Biol. 2002; 34: 713-717Crossref PubMed Scopus (312) Google Scholar). Whereas the catalytic activity of the Group I Paks is markedly up-regulated upon binding to GTP-bound Rac or Cdc42, the Group II Paks interact with but are not demonstrably activated by Rac or Cdc42. Substantial data link the expression and hyperactivity of Paks to tumorigenesis and metastasis (1Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (877) Google Scholar, 3Kumar R. Gururaj A.E. Barnes C.J. Nat. Rev. Cancer. 2006; 6: 459-471Crossref PubMed Scopus (493) Google Scholar). Elevated Pak1 is associated with cancer of the breast (4Holm C. Rayala S. Jirstrom K. Stal O. Kumar R. Landberg G. J. Natl. Cancer Inst. 2006; 98: 671-680Crossref PubMed Scopus (165) Google Scholar), colon (5Carter J.H. Douglass L.E. Deddens J.A. Colligan B.M. Bhatt T.R. Pemberton J.O. Konicek S. Hom J. Marshall M. Graff J.R. Clin. Cancer Res. 2004; 10: 3448-3456Crossref PubMed Scopus (149) Google Scholar), and ovary (6Schraml P. Schwerdtfeger G. Burkhalter F. Raggi A. Schmidt D. Ruffalo T. King W. Wilber K. Mihatsch M.J. Moch H. Am. J. Pathol. 2003; 163: 985-992Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Transgenic expression of a constitutively active allele of Pak1 in murine mammary glands is sufficient to induce tumors (7Wang R.A. Zhang H. Balasenthil S. Medina D. Kumar R. Oncogene. 2006; 25: 2931-2936Crossref PubMed Scopus (106) Google Scholar), and the expression of a dominant negative form of Pak1 inhibits invasiveness of a breast cancer cell line (8Adam L. Vadlamudi R. Mandal M. Chernoff J. Kumar R. J. Biol. Chem. 2000; 275: 12041-12050Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). These observations have led to a great interest in understanding the effector pathways downstream of Paks that mediate tumorigenesis and metastasis. Over 30 direct substrates of Group I Paks have been identified, outlining major functional roles in cytoskeletal regulation, survival signaling, cell cycle progression, and mitogen-activated protein kinase pathway activation (reviewed in Ref. 1Bokoch G.M. Annu. Rev. Biochem. 2003; 72: 743-781Crossref PubMed Scopus (877) Google Scholar; see also supplemental Table 2). Pak1 and -2 are the most distantly related Group I Paks yet share 91% sequence identity within their kinase domains, suggesting that they may recognize similar substrates. Indeed, Pak1 and -2 phosphorylate a number of common substrates, including Bad, Raf, Mek, and Merlin (9Beeser A. Jaffer Z.M. Hofmann C. Chernoff J. J. Biol. Chem. 2005; 280: 36609-36615Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Kissil J.L. Wilker E.W. Johnson K.C. Eckman M.S. Yaffe M.B. Jacks T. Mol. Cell. 2003; 12: 841-849Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 11Schurmann A. Mooney A.F. Sanders L.C. Sells M.A. Wang H.G. Reed J.C. Bokoch G.M. Mol. Cell Biol. 2000; 20: 453-461Crossref PubMed Scopus (306) Google Scholar, 12Xiao G.H. Beeser A. Chernoff J. Testa J.R. J. Biol. Chem. 2002; 277: 883-886Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 13Zang M. Hayne C. Luo Z. J. Biol. Chem. 2002; 277: 4395-4405Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Both Pak1 and Pak2 are expressed in many tissue types, but only Pak2 is essential for viability in mice (14Hofmann C. Shepelev M. Chernoff J. J. Cell Sci. 2004; 117: 4343-4354Crossref PubMed Scopus (189) Google Scholar). For the Group II Paks, much less is known regarding regulation of kinase activity, identity of substrates, or function. Similar to Group I Paks, studies have implicated Group II in survival signaling, mitogen signaling, and cell motility (15Cammarano M.S. Nekrasova T. Noel B. Minden A. Mol. Cell Biol. 2005; 25: 9532-9542Crossref PubMed Scopus (65) Google Scholar, 16Dan C. Kelly A. Bernard O. Minden A. J. Biol. Chem. 2001; 276: 32115-32121Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 17Gnesutta N. Minden A. Mol. Cell Biol. 2003; 23: 7838-7848Crossref PubMed Scopus (88) Google Scholar, 18Gnesutta N. Qu J. Minden A. J. Biol. Chem. 2001; 276: 14414-14419Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 19Gringel A. Walz D. Rosenberger G. Minden A. Kutsche K. Kopp P. Linder S. J. Cell Physiol. 2006; 209: 568-579Crossref PubMed Scopus (56) Google Scholar, 20Kaur R. Liu X. Gjoerup O. Zhang A. Yuan X. Balk S.P. Schneider M.C. Lu M.L. J. Biol. Chem. 2005; 280: 3323-3330Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 21Li X. Minden A. J. Biol. Chem. 2005; 280: 41192-41200Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 22Lu Y. Pan Z.Z. Devaux Y. Ray P. J. Biol. Chem. 2003; 278: 10374-10380Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 23Qu J. Cammarano M.S. Shi Q. Ha K.C. de Lanerolle P. Minden A. Mol. Cell Biol. 2001; 21: 3523-3533Crossref PubMed Scopus (141) Google Scholar, 24Qu J. Li X. Novitch B.G. Zheng Y. Kohn M. Xie J.M. Kozinn S. Bronson R. Beg A.A. Minden A. Mol. Cell Biol. 2003; 23: 7122-7133Crossref PubMed Scopus (125) Google Scholar). The best studied member, Pak4, is widely expressed and is an essential gene in mice (24Qu J. Li X. Novitch B.G. Zheng Y. Kohn M. Xie J.M. Kozinn S. Bronson R. Beg A.A. Minden A. Mol. Cell Biol. 2003; 23: 7122-7133Crossref PubMed Scopus (125) Google Scholar). The kinase domain of Pak4 shares only 53-55% sequence identity with Group I Paks. The sequence divergence between the kinase domains of the two Pak groups suggests that they may recognize distinct substrates and serve at least partially divergent functions (2Jaffer Z.M. Chernoff J. Int. J. Biochem. Cell Biol. 2002; 34: 713-717Crossref PubMed Scopus (312) Google Scholar). Nevertheless, reported substrates for Pak4 are limited to Raf (15Cammarano M.S. Nekrasova T. Noel B. Minden A. Mol. Cell Biol. 2005; 25: 9532-9542Crossref PubMed Scopus (65) Google Scholar), Bad (17Gnesutta N. Minden A. Mol. Cell Biol. 2003; 23: 7838-7848Crossref PubMed Scopus (88) Google Scholar), Lim domain kinase 1 (16Dan C. Kelly A. Bernard O. Minden A. J. Biol. Chem. 2001; 276: 32115-32121Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar), and guanine nucleotide exchange factor-H1 (25Callow M.G. Zozulya S. Gishizky M.L. Jallal B. Smeal T. J. Cell Sci. 2005; 118: 1861-1872Crossref PubMed Scopus (135) Google Scholar), proteins also phosphorylated by Pak1 (11Schurmann A. Mooney A.F. Sanders L.C. Sells M.A. Wang H.G. Reed J.C. Bokoch G.M. Mol. Cell Biol. 2000; 20: 453-461Crossref PubMed Scopus (306) Google Scholar, 26Chaudhary A. King W.G. Mattaliano M.D. Frost J.A. Diaz B. Morrison D.K. Cobb M.H. Marshall M.S. Brugge J.S. Curr. Biol. 2000; 10: 551-554Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 27Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (840) Google Scholar, 28Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (359) Google Scholar, 29Jin S. Zhuo Y. Guo W. Field J. J. Biol. Chem. 2005; 280: 24698-24705Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 30Zenke F.T. Krendel M. DerMardirossian C. King C.C. Bohl B.P. Bokoch G.M. J. Biol. Chem. 2004; 279: 18392-18400Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Thus, an important outstanding question is to what degree these two groups recognize similar substrates and are functionally redundant. Previous studies have generally investigated Pak kinase substrate specificity in an ad hoc manner based on testing the impact of amino acid substitutions within known Pak protein and peptide substrates. Such studies, however, are generally not comprehensive and can incur bias from the particular sequence context. For example, using mutagenesis, King et al. (31King A.J. Wireman R.S. Hamilton M. Marshall M.S. FEBS Lett. 2001; 497: 6-14Crossref PubMed Scopus (35) Google Scholar) identified important sequence determinants for Pak3 phosphorylation of Raf-1 at Ser338, but the importance of these specific residues in other sequence contexts is unknown. Similar work analyzing Pak2 phosphorylation of synthetic peptides derived from the Rous sarcoma virus nucleocapsid NC protein has elucidated recognition determinants in this context (32Tuazon P.T. Spanos W.C. Gump E.L. Monnig C.A. Traugh J.A. Biochemistry. 1997; 36: 16059-16064Crossref PubMed Scopus (59) Google Scholar). Unfortunately, whereas the Raf-1 study focused primarily on amino acids C-terminal to the targeted serine, the NC study mainly investigated residues N-terminal to the phosphorylation site, making it difficult to determine if the features identified in each study are context-dependent or independent. However, both studies did identify an arginine residue two amino acids N-terminal to the phosphoacceptor site (the -2-position) as a critical determinant for Pak recognition. Recently Shaw and co-workers (33Zhu G. Fujii K. Liu Y. Codrea V. Herrero J. Shaw S. J. Biol. Chem. 2005; 280: 36372-36379Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) confirmed this finding using an alternative approach employing degenerate peptide mixtures and observed a strong bias for peptides containing arginine at the -2-position and lesser preferences for arginine at the -3- and -4-positions. We report here the application of a recently described positional scanning peptide library approach to determine the complete, context-independent substrate sequence preferences of members of both the Group I and Group II Paks and apply that information to identify a novel Pak1 phosphorylation site in Pix. Although several serine/threonine kinases have been analyzed with this method (34Bullock A.N. Debreczeni J. Amos A.L. Knapp S. Turk B.E. J. Biol. Chem. 2005; 280: 41675-41682Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 35Fu Z. Schroeder M.J. Shabanowitz J. Kaldis P. Togawa K. Rustgi A.K. Hunt D.F. Sturgill T.W. Mol. Cell Biol. 2005; 25: 6047-6064Crossref PubMed Scopus (54) Google Scholar, 36Hutti J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Methods. 2004; 1: 27-29Crossref PubMed Scopus (280) Google Scholar), it has not yet been applied to a member of the Ste20-related kinase family, and no comprehensive peptide library analysis has yet been conducted for a Pak kinase. Cell Culture—HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mm l-glutamine, and 100 units/ml penicillin/streptomycin. Antibodies—A phosphopeptide and corresponding unphosphorylated peptide were synthesized according to the sequence surrounding βPix serine 340 (acetyl-CLSASPRMS(PO3)GFI-CONH2; Synpep Corp.). The anti-phosphoserine 340 βPix antibody was generated by immunizing rabbits with the phosphorylated peptide (Covance). Phosphospecific Pix antibodies were isolated in our laboratory by negative selection on immobilized, unphosphorylated peptide followed by positive affinity selection on immobilized immunogen. Antibodies eluting in 100 mm glycine, pH 2.5, were recovered and dialyzed against phosphate-buffered saline. Bovine serum albumin (2 mg/ml final concentration) and glycerol (50% final concentration) were added, and the purified antibodies were stored at -20 °C. The remaining antibodies used were anti-βPix (polyclonal; Chemicon), phospho-PAK1/2 (Thr423/Thr402) (polyclonal; Cell Signaling), and anti-Myc 9B11 antibody (monoclonal; Cell Signaling). Plasmids—pcDNA3-Myc-βPix was obtained from R. Cerione (Cornell). pJ3H-Pak1 K299R and pJ3H-Pak1 T423E were prepared by subcloning Pak1 from pJ3M-Pak1 using BamHI/EcoRI restriction sites. Pak2 was cloned as a BamHI/XhoI fragment into pET28. A cDNA for human αPix obtained from the Kazusa DNA Research Institute (KIAA0006) was used as a PCR template to generate a BamHI/EcoRI fragment encoding amino acids 155-546 of human αPix (MTEN... LNRL) that was cloned into pGEX-6P-1 (GE Healthcare) for expression as a GST fusion protein. The GST-Pak4 (amino acids 209-501) expression construct was previously reported (37Eswaran J. Lee W.H. Debreczeni J.E. Filippakopoulos P. Turnbull A. Federov O. Deacon S.W. Peterson J.R. Knapp S. Structure. 2007; 15: 201-213Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Mutagenesis was conducted using the QuikChange site-directed mutagenesis kit (Stratagene) using the following primers (mutated nucleotides in boldface type): βPix S340A, forward (5′-CTG CCA GTC CTA GGA TGGCTG GCT TTA TCT ATC AGG-3′) and reverse (5′-CCT GAT AGA TAA AGC CAGCCA TCC TAG GAC TGG CAG-3′); αPix S488A, forward (5′-CTG CAA GTC CTC GGA TGGCTG GCT TTA TCT ATC AGG G-3′) and reverse (5′-CCC TGA TAG ATA AAG CCA GCC ATC CGA GGA CTT GCA G-3′); Pak2-QR, forward (5′-CAG AAA CAG CAAAGG AAG GAG CTC ATC ATT AAC G-3′) and reverse (5′-CGT TAA TGA TGAGCT CCT TCC TTT GCT GTT TCT G-3′); Pak4-PK, forward (5′-GCG CAA GCA GCC GAA GCG CGA GCT CCT CTT CAA CG-3′) and reverse (5′-CGT TGA AGA GGA GCT CGC GCT TCG GCT GCT TGC GC-3′). All mutant constructs were fully sequenced. For expression in Escherichia coli, GST-βPix wild type and GST-βPix S340A were subcloned from pcDNA3-Myc-βPix into pGEX-6P-1 using BamHI/EcoRI. Sequence Logos—Position-specific scoring matrix (PSSM) sequence logos were generated manually in Adobe Illustrator using the Arial black font and scaling letters by the absolute value of the log2 of the raw selectivity score. Determination of Pak Phosphorylation Specificity—Peptide library screens were carried out as described previously with minor modifications (36Hutti J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Methods. 2004; 1: 27-29Crossref PubMed Scopus (280) Google Scholar). Briefly, a series of 198 partially degenerate peptides with the general sequence YAXXXXX(S/T)XXXXAGKK(biotin) was employed, in which S/T indicates an even mixture of Ser and Thr, and all positions X except one are a degenerate mixture of the 17 amino acid residues excluding Ser, Thr, and Cys. Each individual peptide bears one of 22 amino acid residues (all unmodified residues, plus phosphothreonine and phosphotyrosine) fixed at one of the X positions. Reactions were carried out in sealed multiwell plates for 2 h at 30 °C in a buffer containing 50 mm HEPES, pH 7.4, 12.5 mm NaCl, 1.5 mm MgCl2, 1.5 mm MnCl2, 0.1% Tween 20 with 50 μm [γ-32P]ATP or [33P]ATP at 0.3 μCi/μl, 50 μm peptide substrate, and the kinase of interest. Pak1 reactions also included 1.3 μm GTPγS-charged Cdc42. Aliquots of the reactions were spotted onto streptavidin membranes and washed as described previously (38Turk B.E. Hutti J.E. Cantley L.C. Nat. Protocols. 2006; 1: 375-379Crossref PubMed Scopus (58) Google Scholar). Incorporation of radioactivity into peptides was quantified by exposure to a phosphor screen and analysis using ImageQuant software. PSSMs were generated from background-subtracted data that were normalized as described (36Hutti J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Methods. 2004; 1: 27-29Crossref PubMed Scopus (280) Google Scholar). Data reflecting the average selectivity values from at least two separate runs are shown. To determine the phosphoacceptor residue specificity, similar peptides bearing the sequence YAXXXXXZXXXXAGKK(biotin) were used in which all X positions were degenerate, and the Z residue was either Ser, Thr, or Tyr. Reactions were carried out in microcentrifuge tubes as described above for the peptide library screens. Aliquots (2 μl) were spotted onto the streptavidin membrane, which was washed and quantified as above. In Vitro Kinase Assays with Optimal Pak Substrates (OPS)—Kinase assays were performed at 30 °C in 1× phospho buffer (50 mm HEPES, pH 7.5, 12.5 mm NaCl, 0.625 mm MgCl2, 0.625 mm MnCl2). For reaction rate determinations, Pak2 (10 nm final concentration) or Pak4 kinase domain (50 nm final concentration) was mixed with 10 μm OPS I or II and pre-equilibrated to 30 °C. Reactions were started by the addition of 100 μm ATP containing [γ-32P]ATP. Mixtures were incubated for 2, 5, or 10 min and stopped on dry ice, followed by incubation at 95 °C for 10 min. Reactions were then spotted onto P81 cation exchange paper (Whatman), washed extensively in 0.1% phosphoric acid, and analyzed by scintillation counting on a Beckman LS 6000 SC instrument. Under these conditions, phosphoryl transfer remained linear over the time course, without substantial substrate depletion. Phosphate incorporation was calculated using counts obtained from ATP standard solutions. Substrate titration reactions were carried out as described above in the presence of increasing amounts of either OPS-I or II (0-50 μm final concentration) at 30 °C for 10 min. OPS-I and -II were obtained as crude synthetic products (Biosynthesis, Inc.) and were purified by high pressure liquid chromatography to >95% purity. Recombinant Protein Expression and Purification—GST-βPix wild type and GST-βPix S340A were purified from Rosetta (DE3) pLysS bacteria (Novagen) as described (39ten Klooster J.P. Jaffer Z.M. Chernoff J. Hordijk P.L. J. Cell Biol. 2006; 172: 759-769Crossref PubMed Scopus (212) Google Scholar). Full-length wild type Pak2 was expressed as a His-tagged fusion protein as previously reported (40Pirruccello M. Sondermann H. Pelton J.G. Pellicena P. Hoelz A. Chernoff J. Wemmer D.E. Kuriyan J. J. Mol. Biol. 2006; 361: 312-326Crossref PubMed Scopus (68) Google Scholar). As reported (40Pirruccello M. Sondermann H. Pelton J.G. Pellicena P. Hoelz A. Chernoff J. Wemmer D.E. Kuriyan J. J. Mol. Biol. 2006; 361: 312-326Crossref PubMed Scopus (68) Google Scholar), this protein is constitutively active as purified and is not further stimulated by Cdc42. The wild type Pak4 kinase domain (37Eswaran J. Lee W.H. Debreczeni J.E. Filippakopoulos P. Turnbull A. Federov O. Deacon S.W. Peterson J.R. Knapp S. Structure. 2007; 15: 201-213Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and mutant were expressed and purified as GST-tagged fusion proteins essentially as reported (37Eswaran J. Lee W.H. Debreczeni J.E. Filippakopoulos P. Turnbull A. Federov O. Deacon S.W. Peterson J.R. Knapp S. Structure. 2007; 15: 201-213Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Human Pak1 was subcloned using BamHI/EcoRI sites into pFastBac HTB (Invitrogen), and baculovirus expressing recombinant His-Pak1 was prepared according to the manufacturer's protocol. Serum-free adapted Sf9 cells where grown in suspension (in SFM-900) to a density of 1 × 106 cells/ml and infected for 50-60 h with a 25-fold dilution of the viral stock. His-Pak1 was purified from cell pellets by sonication in 50 mm sodium phosphate, pH 8.0, 0.5 m NaCl, 5 mm imidazole, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each of chymostatin, leupeptin, and pepstatin. 20,000 × g supernatants of the resulting extract were applied to nickel-nitrilotriacetic acid beads. Beads were washed in 50 mm sodium phosphate, pH 8.0, 0.5 m NaCl, 20 mm imidazole, and Pak1 was eluted by wash buffer containing 250 mm imidazole. Pak1 was dialyzed against 50 mm Tris, pH 7.5, 100 mm NaCl, 5% glycerol, 1 mm dithiothreitol and stored at -80 °C. Expression, purification, and charging of Cdc42 were performed as described (41Peterson J.R. Lokey R.S. Mitchison T.J. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10624-10629Crossref PubMed Scopus (82) Google Scholar). In Vitro βPix Phosphorylation—Recombinant Pak1 was activated by incubation with GTPγS-charged Cdc42 for 30 min at 30 °C in phospho buffer containing 1 mm ATP. Activated Pak1 was then incubated with 2.5 μg of either wild type βPix or S340A βPix in the presence of 1.3 mm ATP in phospho buffer for 30 min at 30 °C. Reactions were stopped by the addition of 2× sample buffer (125 mm Tris, pH 6.8, 4% SDS, 10% glycerol, 200 mm dithiothreitol, 0.02% bromphenol blue). Reaction products were analyzed by SDS-PAGE and Western blotting using the indicated antibodies. In Vitro αPix Phosphorylation—∼1.4 μg each of wild type or S340A αPix was incubated with recombinant His-Pak2 for 30 min at 30 °C in phospho buffer containing 20 μm ATP and ∼0.5 μCi of [γ-32P]ATP. Reactions were stopped by the addition of 2× sample buffer and analyzed by SDS-PAGE and autoradiography. βPix/Pak Expression in Cells—HEK293 cells were seeded at 6 × 106 cells/well of a 6-well plate and grown for 24 h before transfection using Lipofectamine 2000 (Invitrogen). 36 h later, cells were washed once with phosphate-buffered saline and lysed in radioimmune precipitation buffer (25 mm Tris-HCl, pH 8, 137 mm NaCl, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1% Nonidet P-40, 2 mm EDTA, 1 mm sodium ortho-vanadate, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml chymostatin, leupeptin, and pepstatin). 15,000 × g (10 min) supernatants were prepared, and Myc-tagged βPix was immunoprecipitated from equal amounts of total protein from each lysate with anti-Myc antibody and protein A-agarose (Pierce). Immunoprecipitations were washed twice in radioimmune precipitation buffer without phosphatase inhibitors and washed twice in 1× dephosphorylation buffer (50 mm Tris, pH 8.5, 0.1 mm EDTA) and were then incubated for 90 min at 30 °C in the presence or absence of 80 units of calf intestinal phosphatase (Invitrogen). Samples were then analyzed by SDS-PAGE and Western blotting with the indicated antibodies. The Substrate Specificity of Group I and II Paks—In order to determine the relative preference of Paks for phosphorylation of serine, threonine, and tyrosine independent of sequence context, we incubated full-length, recombinant Pak2 (40Pirruccello M. Sondermann H. Pelton J.G. Pellicena P. Hoelz A. Chernoff J. Wemmer D.E. Kuriyan J. J. Mol. Biol. 2006; 361: 312-326Crossref PubMed Scopus (68) Google Scholar) or Pak4 kinase domain (37Eswaran J. Lee W.H. Debreczeni J.E. Filippakopoulos P. Turnbull A. Federov O. Deacon S.W. Peterson J.R. Knapp S. Structure. 2007; 15: 201-213Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) with radiolabeled ATP and three degenerate peptide mixtures containing either serine, threonine, or tyrosine as the phosphoacceptor. Quantitation of the reaction products revealed a substantial preference of both Paks for serine over threonine that was more pronounced for Pak4 (Fig. 1). As expected, no significant phosphorylation of tyrosine-containing peptides was observed for either Pak. We assume that the substrate specificity of the isolated kinase domain of Pak4 toward short peptides reflects that of the full-length protein. We next characterized the overall phosphorylation specificity of two Group I Paks (Pak1 and Pak2) and one Group II Pak (Pak4) in radiolabel kinase assays using mixtures of partially degenerate peptides as previously described (36Hutti J.E. Jarrell E.T. Chang J.D. Abbott D.W. Storz P. Toker A. Cantley L.C. Turk B.E. Nat. Methods. 2004; 1: 27-29Crossref PubMed Scopus (280) Google Scholar, 38Turk B.E. Hutti J.E. Cantley L.C. Nat. Protocols. 2006; 1: 375-379Crossref PubMed Scopus (58) Google Scholar). Each Pak was incubated in parallel with an array of 198 peptide substrate mixtures in which each peptide in each mixture contained a fixed, central serine or threonine residue as a phosphoacceptor. In each peptide mixture, one of the 20 amino acids was systematically fixed at one of nine positions surrounding the phosphorylation site (see Fig. 2), and the other eight positions were degenerate. In addition, phosphothreonine and phosphotyrosine were included at the fixed positions to investigate the influence of prior nearby phosphorylation on recognition by Paks. After incubation, each reaction was spotted on a filter membrane, which was then washed to remove unincorporated label, and the membrane was analyzed by phosphorimaging. Those peptide mixtures that include residues favored at a particular position are preferentially phosphorylated by the kinase and thus provide increased signals on the resulting array (e.g. Arg at the -2-position in Fig. 2A). This method allows a complete and quantitative description of the kinase substrate specificity. Quantified data were normalized by dividing the amount of phosphate incorporated into each peptide by the average amount incorporated into all peptides with the same fixed position to generate selectivity scores for each residue. PSSMs that include the complete set of selectivity values for each Pak are presented in supplemental Table 1. To compare the global similarity in the substrate preferences between pairs of Pak kinases, we prepared log score scatter plots (43Fujii K. Zhu G. Liu Y. Hallam J. Chen L. Herrero J. Shaw S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13744-13749Crossref PubMed Scopus (82) Google Scholar) (Fig. 3, A-C). In these plots, each data point corresponds to a particular amino acid residue at a particular position (198 data points in this case). The abscissa reflects the log2 of the selectivity score (log score) for this residue position for one kinase, and the ordinate reflects the log score for the same residue position for the second kinase. Differences in the log scores of the two kinases for a particular residue position, therefore, are reflected by off-diagonal residue position scores. Low log scores (strong negative selection) have inherently greater variability due to the poorer signal/noise ratios of the raw data. Consequently, departures from the central diagonal in the bottom, left-hand quadrant are less likely to reflect true specificity differences. Consistent with their high degree of sequence identity, Pak1 and -2 exhibited highly correlated residue position scores (Fig. 3A). Spearman's rank correlation analysis of the paired kinase log scores ret
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