Reciprocal Signaling between Heterotrimeric G Proteins and the p21-stimulated Protein Kinase
1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês
10.1074/jbc.274.44.31641
ISSN1083-351X
AutoresJun Wang, Jeffrey A. Frost, Melanie H. Cobb, Elliott M. Ross,
Tópico(s)Pancreatic function and diabetes
Resumop21-activated protein kinase (PAK)-1 phosphorylated Gαz, a member of the Gαi family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Gαz in vitro. In transfected cells, Gαz was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser16, one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Gα subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Gαz was resistant both to RGSZ1, a Gz-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the Gi and Gqfamilies of G proteins. Phosphorylation of Ser27 by PKC did not alter sensitivity to either GAP. The previously described inhibition of Gz GAPs by PKC is therefore mediated by phosphorylation of Ser16. Phosphorylation of either Ser16 by PAK1 or Ser27 by PKC decreased the affinity of Gαz for Gβγ; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Gαz function by attenuating the inhibitory effects of both GAPs and Gβγ. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gβγ, suggesting that PAK1 acts as a Gβγ-regulated effector protein. This inhibition of mammalian PAK1 by Gβγ contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gβγ homolog Ste4p/Ste18p. p21-activated protein kinase (PAK)-1 phosphorylated Gαz, a member of the Gαi family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Gαz in vitro. In transfected cells, Gαz was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser16, one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Gα subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Gαz was resistant both to RGSZ1, a Gz-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the Gi and Gqfamilies of G proteins. Phosphorylation of Ser27 by PKC did not alter sensitivity to either GAP. The previously described inhibition of Gz GAPs by PKC is therefore mediated by phosphorylation of Ser16. Phosphorylation of either Ser16 by PAK1 or Ser27 by PKC decreased the affinity of Gαz for Gβγ; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Gαz function by attenuating the inhibitory effects of both GAPs and Gβγ. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gβγ, suggesting that PAK1 acts as a Gβγ-regulated effector protein. This inhibition of mammalian PAK1 by Gβγ contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gβγ homolog Ste4p/Ste18p. protein kinase C p21-activated protein kinase GTPase-activating protein glutathione S-transferase guanosine 5′-O-thiotriphosphate myelin basic protein mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Protein kinases are the eventual downstream mediators of most signals initiated by G protein-coupled receptors. Mechanisms of kinase activation are diverse, however. They include both direct stimulation of cyclic AMP-dependent protein kinase and PKC1 by second messenger products of G protein-regulated effectors and less direct activation of tyrosine kinase and mitogen-activated protein kinase cascades. In yeast, heterotrimeric G proteins regulate members of the p21-activated protein kinase (PAK) family. In Saccharomyces cerevisiae, Ste20p is stimulated by Gβγ subunits (Ste4p/Ste18p) in response to mating pheromones (1Leberer E. Wu C. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar, 2Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-195Crossref PubMed Scopus (183) Google Scholar), and in Schizosaccharomyces pombe, the Gα subunit (Gpa1p) is the signal transducer to the Ste20p homolog Shk1p (3Xu H.P. White M. Marcus S. Wigler M. Mol. Cell. Biol. 1994; 14: 50-58Crossref PubMed Google Scholar). The PAKs are mammalian homologs of Ste20p and Shk1p that were initially recognized as kinases that are activated by Rac and Cdc42, members of the Rho family of monomeric GTP-binding proteins (4Lim L. Manser E. Leung T. Hall C. Eur. J. Biochem. 1996; 242: 171-185Crossref PubMed Scopus (273) Google Scholar). 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Chem. 1989; 264: 13298-13305Abstract Full Text PDF PubMed Google Scholar). PKC-catalyzed phosphorylation decreases the affinity of Gαz for Gβγ subunits, potentially sensitizing Gz to activation because Gβγ inhibits GDP/GTP exchange. Phosphorylation by PKC also desensitizes Gz to the GAP activity of RGS proteins, which are widely thought to inhibit G protein signaling (20Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). PKC may potentiate Gz signaling through either of these mechanisms. Gz is found primarily in neurons, platelets, and adrenal chromaffin cells, and its intracellular localization suggests that it may regulate formation, transport, or release of secretory granules (21Matsuoka M. Itoh H. Kozasa T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5384-5388Crossref PubMed Scopus (154) Google Scholar, 22Fong H.K.W. Yoshimoto K.K. Eversole-Cire P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3066-3070Crossref PubMed Scopus (185) Google Scholar, 23Garibay J.L.R. Kozasa T. Itoh H. Tsukamoto T. Matsuoka M. Kaziro Y. Biochim. Biophys. Acta. 1991; 1094: 193-199Crossref PubMed Scopus (42) Google Scholar, 24Casey P.J. Fong H.K.W. Simon M.I. Gilman A.G. J. Biol. Chem. 1990; 265: 2383-2390Abstract Full Text PDF PubMed Google Scholar, 25Hinton D.R. Blanks J.C. Fong H.K.W. Casey P.J. Hildebrandt E. Simons M.I. J. Neurosci. 1990; 10: 2763-2770Crossref PubMed Google Scholar). The ability of PAK1 to cause remodeling of cytoskeletal structures points to a role in regulating processes such as cell motility and secretion, and PAK1 has been implicated directly in the Fcγ receptor-mediated respiratory burst and cytokine secretion (26Izadi K.D. Erdreich-Epstein A. Liu Y. Durden D.L. Exper. Cell Res. 1998; 245: 330-342Crossref PubMed Scopus (24) Google Scholar). Few natural PAK substrates are known, however; but PAK1 probably phosphorylates proteins that regulate either cytoskeletal disassembly or the cytoskeletal elements themselves. We report here that Gαz is phosphorylated specifically at Ser16 by PAK1, thus inhibiting its interaction with both Gβγ and RGS proteins. We have used this specificity to distinguish and delineate the functional consequences of phosphorylation at Ser27 and Ser16, which we show to be the two principal substrate sites for PKC. An unexpected outcome is the finding that Gβγ, which stimulates the PAK homolog Ste20p inSaccharomyces, inhibits the activity of mammalian PAK1 toward both Gαz and other substrates. Mammalian expression vectors for full-length PAK1, the constitutively active mutant PAK1-(165–544) (N-terminal truncation leaving residues 165–544) (27Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar), and G12V Rac1 were prepared in pCMV5M (pCMV5 modified to include a Myc epitope tag (27Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar)) as described previously (27Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). The G12V Rac1 mutation was prepared using a QuikChange mutagenesis kit (Stratagene), and the cDNA was inserted into pCMV5. Mammalian expression vectors for wild-type Gαz and its S16A, S25A, S27A, and S16A,S25A mutants were constructed in pDP5 and were gifts from D. Manning (University of Pennsylvania) (17Lounsbury K.M. Schlegel B. Poncz M. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 268: 3494-3498Abstract Full Text PDF PubMed Google Scholar). The S16A,S25A,S27P triple mutant was prepared using the QuikChange kit with the S16A,S27A construct as template. Recombinant baculoviruses expressing the Gαz mutants were prepared as described previously for wild-type Gαz(28Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Wild-type and mutant Gα and Gβγ subunits, other than Gαi1, were expressed in Sf9 cells and purified as described (28Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 29Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Gαi1 was expressed in Escherichia coli with or without yeast protein N-myristoyltransferase (30Mumby S.M. Linder M.E. Methods Enzymol. 1994; 237: 254-268Crossref PubMed Scopus (112) Google Scholar) and purified as described (31Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Gαq and Gαswere gifts from S. Mukhopadhyay and T. Kozasa (this department). Wild-type PAK1 and PAK1-(232–544) were expressed in E. colias glutathione S-transferase fusion proteins and purified by glutathione-agarose affinity chromatography (27Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). To prevent proteolysis, wild-type GST-PAK1 was purified in the presence of 20 μg/ml aprotinin, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. Purified PAK1 and PAK1-(232–544) were dialyzed against 20 mm Tris-Cl (pH 8.0), 100 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA, and 1 mm benzamidine and stored at −80 °C. The protein kinase TAO1 (32Hutchison M. Berman K.S. Cobb M.H. J. Biol. Chem. 1998; 273: 28625-28632Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) was a gift from K. Berman (this department), and PKCα was a gift from T. Kozasa (this department). Purified phosducin (33Gaudet R. Bohm A. Sigler P.B. Cell. 1996; 87: 577-588Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) was a gift from R. Gaudet and P. Sigler (Yale University). Phosphorylation of Gαz by PKCα was performed exactly as described (28Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 29Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Gα subunits were phosphorylated by PAK1 at 30 °C for 60 min or the times indicated in 50 mm Hepes (pH 8.0), 10 mm MgCl2, 1 mm dithiothreitol, and 0.5 mm ATP. Phosphoamino acid analysis and tryptic phosphopeptide mapping of Gαz were performed as described (34Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Partial tryptic proteolysis after protection of phosphorylated Gαz by GTPγS was performed exactly as described (35Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Under these conditions, trypsin cleaves Gαz after Arg29(35Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Protein kinase assays using MBP as substrate were performed as described (36Frost J.A. Khokhlatchev A. Stippec S. White M.A. Cobb M.H. J. Biol. Chem. 1998; 273: 28191-28198Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Human embryonic kidney fibroblasts (HEK-293 cells) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For transfection, cells were grown in 60-mm culture dishes to ∼70% confluence and then transfected by calcium phosphate precipitation (27Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). Twenty hours after transfection, the medium was replaced by Dulbecco's modified Eagle's medium without serum, and the cells were incubated for another 24 h. For determination of in vivo phosphorylation of Gαz, transfected cells were washed once with phosphate-free Dulbecco's modified Eagle's medium and incubated for 2–3 h in phosphate-free Dulbecco's modified Eagle's medium plus [32P]Pi (0.5 mCi/ml). For harvesting, cells were washed once with phosphate-buffered saline and scraped into 0.5 ml of radioimmune precipitation assay buffer (50 mm sodium Pi (pH 7.2), 150 mm NaCl, 2 mmEDTA, 1 mm dithiothreitol, 10 μg/ml aprotinin, 1% sodium deoxycholate, and 1% Nonidet P-40) that contained 0.2% SDS, 80 mm β-glycerophosphate, 0.5 mmNa3VO4, 50 mm NaF, 20 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin A. Lysates were stored at −20 °C. For immunoprecipitation, lysates were sonicated at 0 °C for 10 s and centrifuged at 10,000 ×g for 5 min. The supernatants were diluted to 800 μl with radioimmune precipitation assay buffer that contained 0.2% SDS and 0.2 mm phenylmethylsulfonyl fluoride and precleared by passage through a 0.2-ml column of Sephadex G-25. The precleared lysates were incubated with 20 μl of protein A-agarose and 10 μg of purified anti-Gαz antibody for 15 h at 4 °C. After extensive washing with radioimmune precipitation assay buffer that contained 0.2% SDS, precipitates were solubilized with SDS sample buffer and analyzed by electrophoresis on 10% polyacrylamide gels (37Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). Proteins were transferred to Schleicher & Schüll BA85 nitrocellulose membranes for autoradiography or immunoblotting with anti-Gαz antibody. Autoradiographs and Western blot images were quantitated with a Molecular Dynamics densitometer and appropriate standards. Gz GAP activity was assayed at 15 °C as described (28Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). [35S]GTPγS binding was measured at 30 °C as described. GTPγS-bound Gαz was partially proteolyzed with trypsin as described (35Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Antibody against Gαz was raised by injecting rabbits with the N-terminal peptide GCRQSSEEKEAARRSRR conjugated to hemocyanin. Antibody was purified from serum by precipitation with (NH4)2SO4 and, following dialysis, immunoaffinity chromatography on a column of the immunizing peptide coupled to CNBr-activated Sepharose CL-4B. The protein kinase PAK1 phosphorylated Gαz in vitro, but did not phosphorylate several other Gα subunits tested (Fig.1). PAK1-catalyzed phosphorylation of Gαz was efficient relative to that catalyzed by PKCα, and the truncated protein PAK1-(232–544) displayed activity similar to that of the wild-type kinase. We were unable to detect phosphorylation of Gαz by either TAO1 or protein kinase A (data not shown). PAK1 phosphorylated Gαz within the first 29 amino acid residues because all 32P was removed by limited tryptic proteolysis (Fig. 2 A), which cleaves after Arg29 (35Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). Phosphoamino acid analysis of phosphorylated Gαz detected only phosphoserine (data not shown), consistent with the absence of Thr residues in this region. In contrast to the site specificity displayed by PAK1, PKC phosphorylated at least one additional site C-terminal to Gln30, usually accounting for ∼10% of the total incorporation of phosphate (Fig.2 A, lane 12). We then used serine mutants of Gαz to determine the site of PAK1-catalyzed phosphorylation near the N terminus. PAK1 catalyzed the phosphorylation of Gαz to ∼1 mol of phosphate/mol of Gαz, and phosphorylation was blocked completely by mutation of Ser16 to Ala. Phosphorylation was not diminished by mutation of either Ser25 or Ser27 (Fig. 2,B and C). Longer incubation with PAK1 or the use of more PAK1 did not cause further phosphorylation of Gαz(data not shown). PAK1 thus selectively phosphorylates Ser16 of Gαz. In contrast to PAK1, PKC catalyzed the addition of 2 mol of phosphate/mol of Gαz, and phosphorylation was decreased by about half when either Ser16 or Ser27 was mutated (Fig. 2, B and C). Both residues are thus PKC substrate sites. The time courses of phosphorylation of S16A and S27A Gαz suggest that Ser27 is the kinetically preferred PKC substrate (Fig. 2 C). Such preference agrees with the conclusion of Lounsbury et al. (17Lounsbury K.M. Schlegel B. Poncz M. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 268: 3494-3498Abstract Full Text PDF PubMed Google Scholar) that Ser27 is the major phosphorylation site in transfected 293 cells, although cellular phosphorylation might also be influenced by selectivity of whatever protein phosphatases naturally dephosphorylate Gαz. We have not attempted to map the minor, more C-terminal PKC phosphorylation site on Gαz. To determine whether PAK1 also phosphorylates Gαz in vivo, we expressed Gαz in HEK-293 cells and measured its differential steady-state phosphorylation upon coexpression with PAK1, with or without the PAK activator G12V Rac1 (Fig. 3). Gαz was phosphorylated by endogenous HEK-293 cell kinases during the 3-h incubation with [32P]Pi, primarily at one of the Ser residues near the N terminus. Phosphorylation was slightly increased by coexpression of wild-type PAK1 and was further increased when both PAK1 and the constitutively activated G12V mutant of Rac1 were present. It was difficult to quantitate the in vivophosphorylation of Gαz by PAK1 because an unknown fraction of Ser16 may already be phosphorylated prior to addition of 32P and because considerable phosphorylation by other kinases occurred at Ser27 (Fig. 3 A,lanes 6–8). However, phosphorylation of the S25A,S27P Gαz mutant showed that wild-type PAK1 plus G12V Rac1 can at least double the incorporation of 32P into Ser16 of Gαz and that the activated mutant PAK1-(165–544) can increase labeling by 50%. For reference, phosphorylation of Gαz by endogenous PKC was monitored by stimulating the cells with phorbol ester, which increased phosphorylation of both wild-type and S16A Gαz by ∼2–2.6-fold (Fig. 3) (17Lounsbury K.M. Schlegel B. Poncz M. Brass L.F. Manning D.R. J. Biol. Chem. 1993; 268: 3494-3498Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of Gαz at sites C-terminal to Arg29 was relatively minor (Fig.3 B, lanes 8–14). Activation of Gαz by AlF4− had no effect on its phosphorylation by either PAK1 or PKC (Fig.4). Activation by GTPγS was also without effect (data not shown). Conversely, neither PAK1-catalyzed phosphorylation of MBP or PAK1 autophosphorylation was altered by Gαz when bound to GDP/AlF4− (Fig. 4), GDP, or GTPγS (data not shown). To determine whether PAK1-catalyzed phosphorylation of Gαz decreases its affinity for Gβγ, as is true for PKC (18Fields T.A. Casey P.J. J. Biol. Chem. 1995; 270: 23119-23125Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), we monitored the effect of phosphorylation on the concentration dependence with which Gβγ inhibits GDP/GTPγS exchange. As shown in Fig.5 A, phosphorylation of Gαz by PAK1 markedly attenuated the ability of Gβγ to inhibit nucleotide exchange on Gαz, but had no effect on the intrinsic nucleotide exchange rate. This attenuation reflected a 10–20-fold decrease in the affinity of phospho-Gαz for Gβγ (Fig. 5, B and D). PAK1 and PKC inhibited Gβγ binding equally. Furthermore, phosphorylation of Ser16 by PAK1 alone (Fig. 5, B and D) or of Ser27 by PKC in the S16A mutant (Fig. 5 C) decreased Gαz-Gβγ affinity equally. Phosphorylation of both residues by PKC had no greater effect than phosphorylation of only one or the other. Although mutation of either residue to Ala itself decreased affinity for Gβγ somewhat (IC50shifted from ∼1 μm to ∼2.5 μm in this and other experiments), the additional decrease caused by phosphorylation was much greater, ∼12-fold in Fig. 5 B and ∼20-fold in Fig. 5 (C and D) and similar experiments. Phosphorylation of Gαz by PKC blocks the GAP activity of RGS proteins (29Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar,38Glick J.L. Meigs T.E. Miron A. Casey P.J. J. Biol. Chem. 1998; 273: 26008-26013Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Selective phosphorylation of Ser16 by PAK1 also substantially inhibited the GAP activities of both RGSZ1 and RGS4 (Fig.6 and TableI). RGS4 was inhibited by >96%. RGSZ1 was inhibited by ∼60–80% under the conditions shown. GzGAP purified from bovine brain, which contains RGSZ1 and at least one other member of the RGSZ subfamily (29Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), was inhibited to the same extent as was recombinant RGSZ1 (data not shown). Inhibition of GAP activity by Gαz phosphorylation is caused by an increase in K m , which probably indicates a decrease in affinity (29Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Fractional inhibition will therefore vary with the concentration of phospho-Gαz relative to itsK m , and the greater fractional inhibition of RGS4 thus reflects its lower affinity for GTP-bound Gαz.Table IModulation of the Gz GAP activity of RGS proteins by Gβγ and by PAK1- or PKC-catalyzed phosphorylation of GαzHydrolysis rate constantBasalGβγPAK1PAK1, GβγPKCαPKCα, Gβγmin−1Wild typeNo GAP0.0135 ± 0.0010.0119 ± 0.0010.0134 ± 0.0010.0134 ± 0.0010.0134 ± 0.0010.0134 ± 0.001 RGSZ10.0583 ± 0.0020.0176 ± 0.0020.0257 ± 0.0020.0214 ± 0.0020.0214 ± 0.0010.0219 ± 0.001 RGS40.0580 ± 0.0020.0122 ± 0.0010.0146 ± 0.0010.0138 ± 0.0010.0139 ± 0.0010.0136 ± 0.001S16ANo GAP0.0112 ± 0.0010.0113 ± 0.0010.0127 ± 0.0010.0128 ± 0.001RGSZ10.0226 ± 0.0020.0181 ± 0.0010.0338 ± 0.0020.0265 ± 0.001RGS40.0153 ± 0.0020.0119 ± 0.0010.0143 ± 0.0010.0134 ± 0.001S27ANo GAP0.0104 ± 0.0010.0103 ± 0.0010.0117 ± 0.0010.0107 ± 0.001RGSZ10.0266 ± 0.0020.0212 ± 0.0020.0248 ± 0.0020.0155 ± 0.001RGS40.0117 ± 0.0020.0110 ± 0.0010.0139 ± 0.0010.0119 ± 0.001The rates of hydrolysis of wild-type and mutant GTP-bound Gαz(∼2 nm in all experiments) were measured either without GAP or in the presence of 50 pm RGSZ1 or 750 pmRGS4. Gαz was phosphorylated by PAK1 or PKCα as shown. Assays also contained 2.5 μm Gβ1γ2where shown. Observed hydrolysis rate constants,k app (28Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), are means ± S.D. from 4 to 20 determinations. Open table in a new tab The rates of hydrolysis of wild-type and mutant GTP-bound Gαz(∼2 nm in all experiments) were measured either without GAP or in the presence of 50 pm RGSZ1 or 750 pmRGS4. Gαz was phosphorylated by PAK1 or PKCα as shown. Assays also contained 2.5 μm Gβ1γ2where shown. Observed hydrolysis rate constants,k app (28Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), are means ± S.D. from 4 to 20 determinations. Because PAK1 phosphorylates Ser16 exclusively and PAK-catalyzed phosphorylation inhibits responses to GAPs as much as does phosphorylation of both Ser16 and Ser27 by PKC, phosphorylation of Ser16 can account for the inhibitory effect of PKC on GAP activity. Phosphorylation of Ser27 in S16A Gαz had no effect on its sensitivity to either GAP. Thus, PAK1 will be just as efficacious an inhibitor of the RGSZ family of Gz GAPs as is PKC. Addition of Gβγ to phosphorylated Gαz had little further inhibitory effect on GAP activity (Fig. 5). The slight inhibition that was observed occurred primarily below 200 nm Gβγ, well below the K d of phospho-Gαz for Gβγ. Residual inhibition by Gβγ probably reflects effects on the small fraction of Gαzthat was not phosphorylated during incubation with kinase. In addition to blocking phosphorylation, mutation of either Ser16 or Ser27 to Ala also inhibited the intrinsic sensitivity of Gαz to GAP activity (Fig. 6 and Table I). Sensitivity of non-phosphorylated S16A Gαz to either RGSZ1 or RGS4 was equivalent to that of phospho-Ser16 Gαz and was not further altered by phosphorylation of Ser27. S27A Gαz was more sensitive to GAP activity than was S16A Gαz, but was still a far worse GAP substrate than wild-type Gαz. It was striking that PKC-catalyzed phosphorylation at Ser27 in the S16A mutant reproducibly increased its sensitivity to GAPs. Although this effect was relatively small, it was a consistent finding in multiple experiments. The small inhibitory effect of Gβγ on the sensitivity of mutated or phosphorylated Gαz to GAPs was similarly reproducible. The intrinsic rates at which S16A and S27A Gαz hydrolyzed bound GTP were diminished in comparison to the wild type, by ∼15 and 25% respectively (Table I). Proteolysis of the N-terminal 29 amino acid residues of Gαz provided a control to show that the effects of mutation or phosphorylation of Ser16 and Ser27 are local. Limited tryptic proteolysis increased thek hydrol for each of these proteins to ∼0.022 min−1 (data not shown), the value characteristic of the proteolyzed wild-type, non-phosphorylated protein (35Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar). During the course of the experiments described above, we noticed that Gβγ reproducibly inhibited the protein kinase activity of PAK1-(232–544) (Fig. 7 A). Inhibition was detectable by 80 nm and was half-maximal at ∼200 nm, well within the range of concentrations over which other regulatory actions of Gβγ have been described (39Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar). Similar results were obtained with full-length, wild-type PAK1 both before and after removal of the fused GST domain (data not shown). The buffer used to store Gβγ had no effect on PAK1 activity (Fig. 7 A). Gβγ also inhibited the ability of PAK1 to phosphorylate either MBP (Fig. 7) or MEK1 (data not shown). Gβγ thus appears to inhibit PAK directly rather than simply binding the Gαz substrate and blocking access to the kinase. The inhibitory activity of Gβγ was relatively specific for PAK. It had no effect on the protein kinase activities of either protein kinase A or TAO1 in two separate experiments and inhibited PKC insignificantly (10–15% at the highest concentrations tested). Gβγ slightly but reproducibly stimulated PAK1 autophosphorylation in the presence or absence of added substrate (example in Fig.7 A), indicating that Gβγ binds directly to PAK1 to alter its function. In yeast, Gβγ also binds (and stimulates) the PAK homolog Ste20p directly (1Leberer E. Wu C. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar, 2Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-195Crossref PubMed Scopus (183) Google Scholar), and its stimulatory activity is blocked by binding to Gα. Surprisingly, Gβγ was a potent inhibitor of PAK1 protein kinase activity both when free or when complexed as a heterotrimer with GDP-bound Gαz or Gαi(Fig. 7, A and B) or Gαq (data not shown). Addition of the Gβγ-binding protein phosducin (up to 6.7 μm) also failed to reverse inhibition by Gβγ (Fig.7 B). Mammalian PAKs were discovered as effectors of the small G proteins Rac and Cdc42, although the yeast homolog Ste20p was first identified as a protein kinase activated by Gβγ. Our current findings now indicate that PAKs function both upstream and downstream of heterotrimeric G proteins in animal cell signaling pathways. First, PAK1-catalyzed phosphorylation potentiates Gz activation by inhibiting the GAP activity of RGS proteins, including the RGSZ subfamily of Gz-selective GAPs. Second, phosphorylation of Gαz decreases its affinity for Gβγ subunits and thus attenuates the inhibitory effects of Gβγ on Gzactivation. The net result is a two-pronged potentiation of Gz signaling by PAK. The reduced affinity of phospho-Gαz for Gβγ will promote Gβγ release and might thereby potentiate Gβγ signaling, but Gαz is expressed at such low levels that it may release too little Gβγ to have significant impact on intracellular signaling. Several lines of evidence are consistent with the hypothesis that PAK phosphorylates Gαz under physiological conditions in cells. In vitro, PAK1-phosphorylated purified Gαz to a stoichiometry of 1 mol of phosphate/mol of Gαz. The rate of phosphorylation was also reasonably fast in comparison with PKC, which phosphorylates Gαz in platelets stimulated by either thrombin or phorbol ester (13Lounsbury K.M. Casey P.J. Brass L.F. Manning D.R. J. Biol. Chem. 1991; 266: 22051-22056Abstract Full Text PDF PubMed Google Scholar, 19Carlson K.E. Brass L.F. Manning D.R. J. Biol. Chem. 1989; 264: 13298-13305Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of Gαz in HEK-293 cells was increased by expression of constitutively active PAK1 or of wild-type PAK1 and its activator Rac. PAK1-driven incorporation of 32P into Gαz was of the same order as that catalyzed by PKC in response to 12-O-tetradecanoylphorbol-13-acetate despite the fact that PKC phosphorylates Gαz on two sites rather than one. Finally, phosphorylation of Gαz by PAK is associated with altered function of the protein as discussed more fully below. We conclude that stimulation of PAKs, via the activation of either Rac or Cdc42, provides a novel means of potentiating the cellular function of Gz. PAK1 displays marked selectivity for Gαz relative to other Gα subunits and for Ser16 relative to other potential phosphorylation sites in Gz. Selectivity for Ser16 allowed us to delineate the individual contributions of phosphorylation of Ser16 and Ser27 to regulation of Gαz in a manner not possible using mutagenesis. Phosphorylation of Ser16 was both sufficient and necessary to decrease sensitivity to the GAP activity of RGS proteins (Fig. 6 and Table I). It was also sufficient to decrease affinity for Gβγ. PKC-catalyzed phosphorylation of Ser27 in the S16A mutant also decreased affinity for Gβγ. On the other hand, phosphorylation of both residues in wild-type Gαz had no more effect than phosphorylation of Ser16 alone. These data confirm the idea that the extreme N-terminal helix of Gα subunits is crucial for interaction with RGS proteins (29Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 35Tu Y. Wang J. Ross E.M. Science. 1997; 278: 1132-1135Crossref PubMed Scopus (130) Google Scholar), despite the fact that this interaction was not observed in the crystal structure of the Gαi1-RGS4 complex (40Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). The ability of Gβγ subunits to inhibit the protein kinase activity of PAK1 is provocative for two reasons. First, it suggests that the PAKs may be a new family of heterotrimeric G protein-regulated effectors. Inhibition of PAK1 by Gβγ was nearly complete, was effective with multiple protein substrates, and occurred over a physiological range of Gβγ concentrations. Second, PAK inhibition by Gβγ extends the pattern of G protein regulation of the PAK family that was established in yeast. However, whereas Shk1p is activated, perhaps indirectly, by Gα in S. pombe and Ste20p is directly activated by Gβγ in S. cerevisiae, PAK1 is directly inhibited by mammalian Gβγ. The ability of Gβγ to regulate PAK1-(232–544) also indicates that the Gβγ-binding site on PAK is unrelated to the binding site for Rac and Cdc42, which lies near the PAK N terminus (41Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). A tantalizing possibility is that mammalian G proteins may regulate PAKs through multiple mechanisms. Inhibition of PAK by Gβγ in vitro fulfilled most criteria for physiological validity, so it was initially puzzling that inhibition was not blocked either by GDP-bound Gαi or by phosducin. However, yeast Gβγ binds Ste20p through the N-terminal helix of Gβ (2Leeuw T. Wu C. Schrag J.D. Whiteway M. Thomas D.Y. Leberer E. Nature. 1998; 391: 191-195Crossref PubMed Scopus (183) Google Scholar, 42Leberer E. Dignard D. Hougan L. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4805-4813Crossref PubMed Scopus (76) Google Scholar), which is not occluded in the phosducin-Gβγ complex (33Gaudet R. Bohm A. Sigler P.B. Cell. 1996; 87: 577-588Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Whereas the N-terminal helix of Gβ makes extensive contact with Gα (43Wall M.A. Coleman D.E. Lee E. Iñiguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1014) Google Scholar, 44Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1051) Google Scholar), the face of the helix that binds PAK may remain accessible in some conformations of the Gα-Gβγ heterotrimer. Contact sites for other Gβγ-regulated effectors cluster on the face of the Gβ torus rather than near its N terminus (45Ford C.E. Skiba N.P. Bae H. Daaka Y. Reuveny E. Shekter L.R. Rosal R. Weng G. Yang C.-S. Iyengar R. Miller R.J. Jan L.Y. Lefkowitz R.J. 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