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J Biol Chem, Vol. 274, Issue 44, 31641-31647, October 29, 1999

Reciprocal Signaling between Heterotrimeric G Proteins and the p21-stimulated Protein Kinase^*

Jun Wang, Jeffrey A. Frost, Melanie H. Cobb, and Elliott M. Ross^§

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p21-activated protein kinase (PAK)-1 phosphorylated G_z, a member of the G_i family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of G_z in vitro. In transfected cells, G_z 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 G subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated G_z was resistant both to RGSZ1, a G_z-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G_i and G_q families of G proteins. Phosphorylation of Ser²⁷ by PKC did not alter sensitivity to either GAP. The previously described inhibition of G_z GAPs by PKC is therefore mediated by phosphorylation of Ser¹⁶. Phosphorylation of either Ser¹⁶ by PAK1 or Ser²⁷ by PKC decreased the affinity of G_z for G; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates G_z function by attenuating the inhibitory effects of both GAPs and G. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by G, suggesting that PAK1 acts as a G-regulated effector protein. This inhibition of mammalian PAK1 by G contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the G homolog Ste4p/Ste18p.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 G subunits (Ste4p/Ste18p) in response to mating pheromones (1, 2), and in Schizosaccharomyces pombe, the G subunit (Gpa1p) is the signal transducer to the Ste20p homolog Shk1p (3). 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 (4). The PAKs also respond to heterotrimeric G proteins through pathways that include regulation of both GDP/GTP exchange factors and GAPs for Rac and Cdc42 (5-7).

Conversely, protein kinases modulate upstream signaling by heterotrimeric G proteins. Receptors are subject to feedback regulation by second messenger-activated kinases and G protein-coupled receptor kinases (8); G protein-regulated effectors are modulated by phosphorylation (9-12); and in a few cases, G proteins are themselves phosphorylated (13-16). G_z, a sparsely expressed member of the G_i family, is phosphorylated by PKC both in platelets and in cells where it has been expressed artificially (13, 17-19). PKC-catalyzed phosphorylation decreases the affinity of G_z for G subunits, potentially sensitizing G_z to activation because G inhibits GDP/GTP exchange. Phosphorylation by PKC also desensitizes G_z to the GAP activity of RGS proteins, which are widely thought to inhibit G protein signaling (20). PKC may potentiate G_z signaling through either of these mechanisms.

G_z 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 (21-25). 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 Fc receptor-mediated respiratory burst and cytokine secretion (26). 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 G_z is phosphorylated specifically at Ser¹⁶ by PAK1, thus inhibiting its interaction with both G 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 G, which stimulates the PAK homolog Ste20p in Saccharomyces, inhibits the activity of mammalian PAK1 toward both G_z and other substrates.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids and cDNA-- Mammalian expression vectors for full-length PAK1, the constitutively active mutant PAK1-(165-544) (N-terminal truncation leaving residues 165-544) (27), and G12V Rac1 were prepared in pCMV5M (pCMV5 modified to include a Myc epitope tag (27)) as described previously (27). 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 G_z and its S16A, S25A, S27A, and S16A,S25A mutants were constructed in pDP5 and were gifts from D. Manning (University of Pennsylvania) (17). The S16A,S25A,S27P triple mutant was prepared using the QuikChange kit with the S16A,S27A construct as template. Recombinant baculoviruses expressing the G_z mutants were prepared as described previously for wild-type G_z (28).

Protein Expression and Purification-- Wild-type and mutant G and G subunits, other than G_i1, were expressed in Sf9 cells and purified as described (28, 29). G_i1 was expressed in Escherichia coli with or without yeast protein N-myristoyltransferase (30) and purified as described (31). G_q and G_s were gifts from S. Mukhopadhyay and T. Kozasa (this department). Wild-type PAK1 and PAK1-(232-544) were expressed in E. coli as glutathione S-transferase fusion proteins and purified by glutathione-agarose affinity chromatography (27). 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 (32) was a gift from K. Berman (this department), and PKC was a gift from T. Kozasa (this department). Purified phosducin (33) was a gift from R. Gaudet and P. Sigler (Yale University).

Protein Kinase Assays and Protein Phosphorylation-- Phosphorylation of G_z by PKC was performed exactly as described (28, 29). G 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 G_z were performed as described (34). Partial tryptic proteolysis after protection of phosphorylated G_z by GTPS was performed exactly as described (35). Under these conditions, trypsin cleaves G_z after Arg²⁹ (35). Protein kinase assays using MBP as substrate were performed as described (36).

In Vivo Phosphorylation-- 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 (27). 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 G_z, 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]P_i (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 P_i (pH 7.2), 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 10 µg/ml aprotinin, 1% sodium deoxycholate, and 1% Nonidet P-40) that contained 0.2% SDS, 80 mM -glycerophosphate, 0.5 mM Na₃VO₄, 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-G_z 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 (37). Proteins were transferred to Schleicher & Schüll BA85 nitrocellulose membranes for autoradiography or immunoblotting with anti-G_z antibody. Autoradiographs and Western blot images were quantitated with a Molecular Dynamics densitometer and appropriate standards.

Other Methods-- G_z GAP activity was assayed at 15 °C as described (28). [³⁵S]GTPS binding was measured at 30 °C as described. GTPS-bound G_z was partially proteolyzed with trypsin as described (35). Antibody against G_z 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PAK1 Phosphorylates G_z Selectively at Ser¹⁶-- The protein kinase PAK1 phosphorylated G_z in vitro, but did not phosphorylate several other G subunits tested (Fig. 1). PAK1-catalyzed phosphorylation of G_z was efficient relative to that catalyzed by PKC, and the truncated protein PAK1-(232-544) displayed activity similar to that of the wild-type kinase. We were unable to detect phosphorylation of G_z by either TAO1 or protein kinase A (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of G subunits by PAK1. Purified recombinant G protein subunits Gs, Gi1 (non-myristoylated), Gi1 (myristoylated; i1m), Gi2, Gi3, Go, and Gq (1.25 pmol each) and Gz (0.75 pmol) were incubated with partially purified wild-type GST-PAK1 (~0.1 pmol) and [-32P]ATP (3 cpm/fmol) for 60 min at 30 °C as described under "Experimental Procedures." All G subunits were expressed in Sf9 cells except for Gi1, which was expressed in E. coli either with (i1m) or without (i1) coexpression of N-myristoyltransferase. Samples were resolved by SDS-polyacrylamide gel electrophoresis, silver-stained (Ag; lower panel), and exposed to x-ray film (32P; upper panel). Molecular mass markers (in kilodaltons) are shown on the left. This complete experiment and a similar experiment using ~0.5 pmol of PAK1-(232-544) yielded identical results.

PAK1 phosphorylated G_z within the first 29 amino acid residues because all ³²P was removed by limited tryptic proteolysis (Fig. 2A), which cleaves after Arg²⁹ (35). Phosphoamino acid analysis of phosphorylated G_z 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. 2A, lane 12). We then used serine mutants of G_z to determine the site of PAK1-catalyzed phosphorylation near the N terminus. PAK1 catalyzed the phosphorylation of G_z to ~1 mol of phosphate/mol of G_z, 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 G_z (data not shown). PAK1 thus selectively phosphorylates Ser¹⁶ of G_z.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Determination of phosphorylation sites on Gz. A, tryptic mapping. Gz (20 pmol) was phosphorylated by full-length PAK1 or PKC as described in the legend to Fig. 1. After adjusting free [Mg2+] to 1 µM with EDTA, the samples were incubated for 80 min at 30 °C in binding buffer with or without 0.5 mM GTPS. Samples were then incubated with or without L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (1%, w/w) for 40 min at 30 °C. Samples (5 pmol for samples incubated without trypsin and 12.5 pmol with trypsin) were analyzed by SDS gel electrophoresis, followed by Coomassie Blue staining (CB; lower panel) and autoradiography (upper panel). B, phosphorylation of Gz Ser mutants. Purified wild-type (WT; 5 pmol) or mutant (10 pmol) Gz was incubated with either PAK1 or PKC for 60 min at 30 °C as described in the legend to Fig. 1. Samples were analyzed as described for A. These experiments (A and B) were performed at least twice with similar results. C, phosphorylation of wild-type and Ser mutants of Gz. Aliquots (10 pmol) of wild-type (, ), S16A (, ), or S27A (, ) Gz were incubated at 30 °C with 1 pmol of PAK1 (, , ) or 1 pmol of PKC (, , ) and [-32P]ATP (3000 cpm/pmol) as described under "Experimental Procedures." Samples were resolved by SDS gel electrophoresis, and incorporation of 32P was determined by scintillation counting of Coomassie Blue-stained protein bands. Each point represents the mean of at least three experiments where S.D. was <15% of the mean.

In contrast to PAK1, PKC catalyzed the addition of 2 mol of phosphate/mol of G_z, 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 G_z suggest that Ser²⁷ is the kinetically preferred PKC substrate (Fig. 2C). Such preference agrees with the conclusion of Lounsbury et al. (17) 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 G_z. We have not attempted to map the minor, more C-terminal PKC phosphorylation site on G_z.

To determine whether PAK1 also phosphorylates G_z in vivo, we expressed G_z 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). G_z was phosphorylated by endogenous HEK-293 cell kinases during the 3-h incubation with [³²P]P_i, 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 vivo phosphorylation of G_z 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. 3A, lanes 6-8). However, phosphorylation of the S25A,S27P G_z mutant showed that wild-type PAK1 plus G12V Rac1 can at least double the incorporation of ³²P into Ser¹⁶ of G_z and that the activated mutant PAK1-(165-544) can increase labeling by 50%. For reference, phosphorylation of G_z by endogenous PKC was monitored by stimulating the cells with phorbol ester, which increased phosphorylation of both wild-type and S16A G_z by ~2-2.6-fold (Fig. 3) (17). Phosphorylation of G_z at sites C-terminal to Arg²⁹ was relatively minor (Fig. 3B, lanes 8-14).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Rac1-stimulated phosphorylation of Gz at Ser16 by PAK1 in HEK-293 cells. HEK-293 cells were transfected with plasmids that encode wild-type or mutant Gz, wild-type (WT) PAK1 or PAK1-(165-544), and G12V Rac1 as shown. [32P]Pi was added to the medium; after 3 h, cells were harvested, and Gz was immunoprecipitated and analyzed by gel electrophoresis as described under "Experimental Procedures." In some cases, 12-O-tetradecanoylphorbol-13-acetate (TPA; 0.1 µM) was added 5 min before harvest to stimulate endogenous PKC. The amount of Gz in each immunoprecipitate was determined by comparing the density of bands in immunoblots of the immunoprecipitates (A, WB) with samples of purified Gz on the same blot (data not shown). Incorporation of [32P]Pi was determined by scintillation counting of radioactivity in each sliced band. Phosphorylation of Gz was quantitated as cpm/pmol of Gz. Similar results were obtained in two other experiments. HEK-293 cells do not express Gz naturally, although a phosphoprotein of slightly smaller size can sometimes be detected in Gz immunoprecipitates (B, lane 1).

Activation of G_z and PAK1-catalyzed Phosphorylation-- Activation of G_z by AlF₄ had no effect on its phosphorylation by either PAK1 or PKC (Fig. 4). Activation by GTPS was also without effect (data not shown). Conversely, neither PAK1-catalyzed phosphorylation of MBP or PAK1 autophosphorylation was altered by G_z when bound to GDP/AlF₄ (Fig. 4), GDP, or GTPS (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of Gz or MBP by PAK1 or PKC is independent of the presence of active or inactive Gz. PKC (0.5 pmol) or PAK1-(232-544) (2 pmol) was incubated for 20 min at 30 °C under standard phosphorylation conditions with Gz (5 pmol) and/or MBP (10 pmol), with or without 10 mM NaF and 30 µM AlCl3 (AMF) as indicated. Samples were analyzed by electrophoresis, Coomassie Blue staining, and autoradiography. Similar results were obtained with Gz activated with GTPS or with full-length PAK (data not shown).

Phosphorylation of G_z at Either Ser¹⁶ or Ser²⁷ Inhibits Binding to G-- To determine whether PAK1-catalyzed phosphorylation of G_z decreases its affinity for G, as is true for PKC (18), we monitored the effect of phosphorylation on the concentration dependence with which G inhibits GDP/GTPS exchange. As shown in Fig. 5A, phosphorylation of G_z by PAK1 markedly attenuated the ability of G to inhibit nucleotide exchange on G_z, but had no effect on the intrinsic nucleotide exchange rate. This attenuation reflected a 10-20-fold decrease in the affinity of phospho-G_z for G (Fig. 5, B and D). PAK1 and PKC inhibited G binding equally. Furthermore, phosphorylation of Ser¹⁶ by PAK1 alone (Fig. 5, B and D) or of Ser²⁷ by PKC in the S16A mutant (Fig. 5C) decreased G_z-G 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 G somewhat (IC₅₀ shifted 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. 5B and ~20-fold in Fig. 5 (C and D) and similar experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effects of phosphorylation of Gz at Ser16 and/or Ser27 on its interaction with G. Gz was phosphorylated by either PAK1 or PKC by incubation at 30 °C for 90 min as described in the legends to Figs. 1 and 2. After adjusting free [Mg2+] to 1 µM with EDTA, the samples (2.2 pmol/assay point) were preincubated for 10 min at 0 °C in binding buffer with or without G. Binding of [35S]GTPS (20 µM, 1500 cpm/pmol) was then determined as described (28). A, GTPS binding to either PAK1-phosphorylated (, ) or control (, ) wild-type Gz was measured in the presence (, ) or absence (, ) of 100 pmol of G. Each curve was fit to a first-order reaction scheme, and all four curves share the same value of maximal GTPS binding. B-D, the rate constants for GTPS binding to wild-type (WT) Gz, S16A Gz, and S27A Gz, respectively, were determined at increasing concentrations of G (shown as the molar ratio G/Gz). Gz was either phosphorylated (closed symbols) or not (open symbols; no kinase added) as shown. Data are means of duplicate determination in two or three experiments (n = 4 or 6).

Phosphorylation of G_z at Ser¹⁶ Blocks the GAP Activity of RGS Proteins-- Phosphorylation of G_z by PKC blocks the GAP activity of RGS proteins (29, 38). Selective phosphorylation of Ser¹⁶ by PAK1 also substantially inhibited the GAP activities of both RGSZ1 and RGS4 (Fig. 6 and Table I). RGS4 was inhibited by >96%. RGSZ1 was inhibited by ~60-80% under the conditions shown. G_z GAP purified from bovine brain, which contains RGSZ1 and at least one other member of the RGSZ subfamily (29), was inhibited to the same extent as was recombinant RGSZ1 (data not shown). Inhibition of GAP activity by G_z phosphorylation is caused by an increase in K_m, which probably indicates a decrease in affinity (29). Fractional inhibition will therefore vary with the concentration of phospho-G_z relative to its K_m, and the greater fractional inhibition of RGS4 thus reflects its lower affinity for GTP-bound G_z.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Interactive effects of phosphorylation or mutation at Ser16 and Ser27 and of G on GAP-stimulated hydrolysis of Gz-bound GTP. Samples of wild-type (WT) or mutant Gz were phosphorylated by either PAK1 or PKC as described in the legend to Fig. 2C, presumably to nearly stoichiometric incorporation of phosphate at Ser16 and/or Ser27. Control (non-phosphorylated) samples were treated identically, except that protein kinase was omitted. Samples were then bound to [-32P]GTP, and hydrolysis of Gz-bound GTP (2 nM) stimulated by 50 pM RGSZ1 was measured as described (28) at increasing concentrations of G. Data represent increases in the first-order hydrolytic rate constant kapp (28) relative to the basal rate observed in the absence of GAP or G. Data points are means of 20 (, ), 8 (, ), or 4 (, ) determinations in which S.D. was <15% of the mean. Basal GTP hydrolysis rates for each protein and GAP-stimulated rates from similar experiments are summarized in Table I.

View this table: [in this window] [in a new window]   Table I Modulation of the Gz GAP activity of RGS proteins by G and by PAK1- or PKC-catalyzed phosphorylation of Gz The rates of hydrolysis of wild-type and mutant GTP-bound Gz (~2 nM in all experiments) were measured either without GAP or in the presence of 50 pM RGSZ1 or 750 pM RGS4. Gz was phosphorylated by PAK1 or PKC as shown. Assays also contained 2.5 µM G12 where shown. Observed hydrolysis rate constants, kapp (28), 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 G_z had no effect on its sensitivity to either GAP. Thus, PAK1 will be just as efficacious an inhibitor of the RGSZ family of G_z GAPs as is PKC.

Addition of G to phosphorylated G_z had little further inhibitory effect on GAP activity (Fig. 5). The slight inhibition that was observed occurred primarily below 200 nM G, well below the K_d of phospho-G_z for G. Residual inhibition by G probably reflects effects on the small fraction of G_z that 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 G_z to GAP activity (Fig. 6 and Table I). Sensitivity of non-phosphorylated S16A G_z to either RGSZ1 or RGS4 was equivalent to that of phospho-Ser¹⁶ G_z and was not further altered by phosphorylation of Ser²⁷. S27A G_z was more sensitive to GAP activity than was S16A G_z, but was still a far worse GAP substrate than wild-type G_z. 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 G on the sensitivity of mutated or phosphorylated G_z to GAPs was similarly reproducible.

The intrinsic rates at which S16A and S27A G_z 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 G_z provided a control to show that the effects of mutation or phosphorylation of Ser¹⁶ and Ser²⁷ are local. Limited tryptic proteolysis increased the k_hydrol for each of these proteins to ~0.022 min¹ (data not shown), the value characteristic of the proteolyzed wild-type, non-phosphorylated protein (35).

G Inhibits the Protein Kinase Activity of PAK1-- During the course of the experiments described above, we noticed that G reproducibly inhibited the protein kinase activity of PAK1-(232-544) (Fig. 7A). 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 G have been described (39). 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 G had no effect on PAK1 activity (Fig. 7A). G also inhibited the ability of PAK1 to phosphorylate either MBP (Fig. 7) or MEK1 (data not shown). G thus appears to inhibit PAK directly rather than simply binding the G_z substrate and blocking access to the kinase. The inhibitory activity of G 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).

View larger version (40K): [in this window] [in a new window]   Fig. 7.   G inhibits the protein kinase activity of PAK1. A, PAK1-(232-544) (250 nM) was incubated for 60 min under standard phosphorylation conditions with either 150 nM Gz or 200 nM MBP and decreasing concentrations of G as indicated by the wedges (2300, 760, 250, 83, 28, and 9 nM). Similar results were obtained in 12 experiments, and the identical result was obtained using GST-PAK1 instead of PAK1-(232-544). A constant amount of G storage buffer was added to all reactions. B, inhibition of the kinase activity of PAK1 by G was not blocked by formation of a heterotrimer with Gi1. PAK1-(232-544) (0.25 µM) was incubated with 0.2 µM MBP, 1.25 µM G (except lane 1), and different concentrations of GDP-bound myristoylated Gi1 for 60 min under standard conditions. The concentrations of Gi1 were 5, 2.5, 1.25, 0.625, 0.313, 0.151, 0.075, and 0 µM as indicated by the wedge. Under similar conditions, GDP-bound Gq (up to 0.31 µM; data not shown) and phosducin (up to 6.7 µM) also had no effect on MBP phosphorylation in the presence or absence of G. A and B show autoradiograms of polyacrylamide gels of phosphorylation reaction mixtures.

G slightly but reproducibly stimulated PAK1 autophosphorylation in the presence or absence of added substrate (example in Fig. 7A), indicating that G binds directly to PAK1 to alter its function. In yeast, G also binds (and stimulates) the PAK homolog Ste20p directly (1, 2), and its stimulatory activity is blocked by binding to G. Surprisingly, G was a potent inhibitor of PAK1 protein kinase activity both when free or when complexed as a heterotrimer with GDP-bound G_z or G_i (Fig. 7, A and B) or G_q (data not shown). Addition of the G-binding protein phosducin (up to 6.7 µM) also failed to reverse inhibition by G (Fig. 7B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 G. 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 G_z activation by inhibiting the GAP activity of RGS proteins, including the RGSZ subfamily of G_z-selective GAPs. Second, phosphorylation of G_z decreases its affinity for G subunits and thus attenuates the inhibitory effects of G on G_z activation. The net result is a two-pronged potentiation of G_z signaling by PAK. The reduced affinity of phospho-G_z for G will promote G release and might thereby potentiate G signaling, but G_z is expressed at such low levels that it may release too little G to have significant impact on intracellular signaling.

Several lines of evidence are consistent with the hypothesis that PAK phosphorylates G_z under physiological conditions in cells. In vitro, PAK1-phosphorylated purified G_z to a stoichiometry of 1 mol of phosphate/mol of G_z. The rate of phosphorylation was also reasonably fast in comparison with PKC, which phosphorylates G_z in platelets stimulated by either thrombin or phorbol ester (13, 19). Phosphorylation of G_z 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 G_z was of the same order as that catalyzed by PKC in response to 12-O-tetradecanoylphorbol-13-acetate despite the fact that PKC phosphorylates G_z on two sites rather than one. Finally, phosphorylation of G_z 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 G_z.

PAK1 displays marked selectivity for G_z relative to other G subunits and for Ser¹⁶ relative to other potential phosphorylation sites in G_z. Selectivity for Ser¹⁶ allowed us to delineate the individual contributions of phosphorylation of Ser¹⁶ and Ser²⁷ to regulation of G_z 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 G. PKC-catalyzed phosphorylation of Ser²⁷ in the S16A mutant also decreased affinity for G. On the other hand, phosphorylation of both residues in wild-type G_z had no more effect than phosphorylation of Ser¹⁶ alone. These data confirm the idea that the extreme N-terminal helix of G subunits is crucial for interaction with RGS proteins (29, 35), despite the fact that this interaction was not observed in the crystal structure of the G_i1-RGS4 complex (40).

The ability of G 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 G was nearly complete, was effective with multiple protein substrates, and occurred over a physiological range of G concentrations. Second, PAK inhibition by G extends the pattern of G protein regulation of the PAK family that was established in yeast. However, whereas Shk1p is activated, perhaps indirectly, by G in S. pombe and Ste20p is directly activated by G in S. cerevisiae, PAK1 is directly inhibited by mammalian G. The ability of G to regulate PAK1-(232-544) also indicates that the G-binding site on PAK is unrelated to the binding site for Rac and Cdc42, which lies near the PAK N terminus (41). A tantalizing possibility is that mammalian G proteins may regulate PAKs through multiple mechanisms.

Inhibition of PAK by G in vitro fulfilled most criteria for physiological validity, so it was initially puzzling that inhibition was not blocked either by GDP-bound G_i or by phosducin. However, yeast G binds Ste20p through the N-terminal helix of G (2, 42), which is not occluded in the phosducin-G complex (33). Whereas the N-terminal helix of G makes extensive contact with G (43, 44), the face of the helix that binds PAK may remain accessible in some conformations of the G-G heterotrimer. Contact sites for other G-regulated effectors cluster on the face of the G torus rather than near its N terminus (45) and are thus fully blocked by G. It should be possible to use such structural information to evaluate the biological relevance of PAK inhibition by G in cells where PAK activity can be monitored in response to receptor-regulated G release.

	ACKNOWLEDGEMENTS

We thank Jimmy Woodson and Steven Stippec for excellent technical assistance, David Manning for the plasmids that encode mutant forms of G_z, Kevin Berman for TAO1, Tohru Kozasa for PKC, and Rachel Gaudet and Paul Sigler for phosducin.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants GM30355, GM53032, and GM16926 and Robert A. Welch Foundation Grant I-0982.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Molecumetics, Inc., 2023 120th Ave. N. E., Bellevue, WA 98005-2199.

^§ To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-8717; Fax: 214-648-2994; E-mail: ross@utsw.swmed.edu.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; PAK, p21-activated protein kinase; GAP, GTPase-activating protein; GST, glutathione S-transferase; GTPS, guanosine 5'-O-thiotriphosphate; MBP, myelin basic protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES