Protein Kinase A-mediated Phosphorylation of the Gα13 Switch I Region Alters the Gαβγ13-G Protein-coupled Receptor Complex and Inhibits Rho Activation
2002; Elsevier BV; Volume: 278; Issue: 1 Linguagem: Inglês
10.1074/jbc.m209219200
ISSN1083-351X
AutoresJeanne M. Manganello, Jinsheng Huang, Tohru Kozasa, T. Voyno-Yasenetskaya, Guy C. Le Breton,
Tópico(s)Neuropeptides and Animal Physiology
ResumoThe present studies mapped the protein kinase A (PKA) phosphorylation site of Gα13 and studied the consequences of its phosphorylation. Initial experiments using purified human Gα13 and the PKA catalytic subunit established that PKA directly phosphorylates Gα13. The location of this phosphorylation site was next investigated with a new synthetic peptide (G13SRIpep) containing the PKA consensus sequence (Arg-Arg-Pro-Thr203) within the switch I region of Gα13. G13SRIpep produced a dose-dependent inhibition of PKA-mediated Gα13 phosphorylation. On the other hand, the Thr-phosphorylated derivative of G13SRIpeppossessed no inhibitory activity, suggesting that Gα13Thr203 may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Gα13-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Gα13 phosphorylation stabilized coupling of Gα13 with platelet thromboxane A2 receptors but destabilized coupling of Gα13 to its βγ subunits. In order to determine the functional consequences of this phosphorylation on Gα13 signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Gα13 wild type (Gα13-WT) or Gα13-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%;p < 0.002) of Rho activation in Gα13wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Gα13 Thr203. The present studies mapped the protein kinase A (PKA) phosphorylation site of Gα13 and studied the consequences of its phosphorylation. Initial experiments using purified human Gα13 and the PKA catalytic subunit established that PKA directly phosphorylates Gα13. The location of this phosphorylation site was next investigated with a new synthetic peptide (G13SRIpep) containing the PKA consensus sequence (Arg-Arg-Pro-Thr203) within the switch I region of Gα13. G13SRIpep produced a dose-dependent inhibition of PKA-mediated Gα13 phosphorylation. On the other hand, the Thr-phosphorylated derivative of G13SRIpeppossessed no inhibitory activity, suggesting that Gα13Thr203 may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Gα13-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Gα13 phosphorylation stabilized coupling of Gα13 with platelet thromboxane A2 receptors but destabilized coupling of Gα13 to its βγ subunits. In order to determine the functional consequences of this phosphorylation on Gα13 signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells overexpressing human Gα13 wild type (Gα13-WT) or Gα13-T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%;p < 0.002) of Rho activation in Gα13wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Gα13 Thr203. protein kinase C thromboxane A2 protein kinase A 8-bromo-cyclic AMP 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 4-morpholineethanesulfonic acid Chinese hamster ovary wild type Protein phosphorylation is a well established and ubiquitous mechanism for regulating protein function. Such regulation can impact cellular signaling at multiple levels including enzymatic activity, protein structure, protein translocation, and protein-protein interactions, among others. Regarding seven-transmembrane receptors, evidence has been provided that phosphorylation of specific sites can serve to alter receptor-G protein coupling, initiate receptor internalization, and ultimately modulate cross-membrane receptor signaling (1Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Whereas such effects of receptor phosphorylation have been well documented, considerably less is known regarding the prevalence or the possible signaling consequences of G protein phosphorylation (2Sagi-Eisenberg R. Trends Biochem. Sci. 1989; 14: 355-357Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 3Fields T.A. Casey P.J. J. Biol. Chem. 1995; 270: 23119-23125Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 4Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 12562-12567Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 5Aragay A.M. Quick M.W. J. Biol. Chem. 1999; 274: 4807-4815Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 6Murthy K.S. Makhlouf G.M. J. Biol. Chem. 2000; 275: 30211-30219Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Nevertheless, several reports have provided evidence that G protein phosphorylation can alter heterotrimer complex formation and downstream signaling events. Specifically, Kozasa and Gilman (4Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 12562-12567Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) demonstrated that in vitro phosphorylation of Gα12 and Gαz by PKC1 results in conformational changes that inhibit interaction of the Gα and Gβγ subunits. A similar PKC-mediated phosphorylation effect on Gα-Gβγ interaction was observed by Fields and Casey (3Fields T.A. Casey P.J. J. Biol. Chem. 1995; 270: 23119-23125Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) for Gαzand by Murthy et al. (6Murthy K.S. Makhlouf G.M. J. Biol. Chem. 2000; 275: 30211-30219Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) for Gαi1/2. Offermanns et al. demonstrated that a PKC-dependent phosphorylation of Gα12 and Gα13 occurred when human platelets were activated by thrombin, the TXA2 mimetic U46619, or the phorbol ester phorbol 12-myristate 13-acetate (PMA) (7Offermanns S., Hu, Y.H. Simon M.I. J. Biol. Chem. 1996; 271: 26044-26048Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Collectively, the above results therefore suggest that different Gα subunits can indeed serve as substrates for PKC. Whereas the functional consequences of such phosphorylation remain largely unknown, the strategic position of G proteins in the signaling cascade suggests that phosphorylation-mediated changes in G protein conformation or activity could serve as a significant regulatory point in the signaling process. In this connection, we recently provided evidence that TXA2receptor-coupled Gα13 is phosphorylated through a PKA-mediated process (8Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These results represented the first demonstration that a platelet Gα subunit is phosphorylated by cAMP-dependent kinase and provided a possible explanation for the high sensitivity of TXA2 receptor signaling to increased cAMP levels. The present studies extend these findings by characterizing the specific PKA phosphorylation site in Gα13 and identifying the molecular and functional consequences of this phosphorylation. Our results demonstrate that PKA phosphorylates Gα13 at Thr203, which is situated within the functionally important switch I region of the Gα subunit (9Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar, 10Milburn M.V. Tong L. deVos A.M. Brunger A. Yamaizumi Z. Nishimura S. Kim S.H. Science. 1990; 247: 939-945Crossref PubMed Scopus (849) Google Scholar, 11Tong L.A. de Vos A.M. Milburn M.V. Kim S.H. J. Mol. Biol. 1991; 217: 503-516Crossref PubMed Scopus (217) Google Scholar, 12Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (538) Google Scholar, 13Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (531) Google Scholar, 14Coleman D.E. Sprang S.R. Biochemistry. 1998; 37: 14376-14385Crossref PubMed Scopus (83) Google Scholar). This phosphorylation results in stabilization of Gα13 coupling to TXA2 receptors and destabilization of Gα13 coupling to its βγ subunits. Separate experiments also provided evidence that a functional consequence of this phosphorylation is decreased basal Rho activation levels. Thus, PKA-mediated Gα13 Thr203phosphorylation appears to represent a novel mechanism for the regulation of signaling through the Gα13 pathway. G13SRIpep(CLLARRPTKGIHEY) and G13SRIpepP (CLLARRPpTKGIHEY (where pT represents phosphothreonine)) peptides were synthesized by Multiple Peptide Systems (San Diego, CA). An N-terminal cysteine was added to each peptide to aid in coupling to carrier protein for future antibody production. Outdated human platelet units were obtained from Heartland Blood Centers (Aurora, IL). G418 was obtained from Calbiochem; PKAcat (protein kinase A, catalytic subunit, bovine heart), protein A-Sepharose, aprotinin, leupeptin, coumeric acid, luminol, 8-Br-cAMP, NaF, and CHAPS were obtained from Sigma; [γ-32P]ATP (4,500 Ci/mmol) was purchased from ICN Biochemicals, Inc.; G13-N, Myc, Gα12, and Gβcommon IgG and Rho A polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rabbit polyclonal anti-peptide antibodies to TXA2 receptor (P2 Ab) and Gαq (QL Ab) were prepared as previously described (15Borg C. Lam S.C. Dieter J.P. Lim C.T. Komiotis D. Venton D.L. Le Breton G.C. Biochem. Pharmacol. 1993; 45: 2071-2078Crossref PubMed Scopus (18) Google Scholar); the Wizard Plus Midiprep DNA Purification kit was from Promega (Madison, WI); LipofectAMINE Plus and LipofectAMINE 2000 were purchased from Invitrogen; the QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA); horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) was from Bio-Rad; the BCA protein assay kit was from Pierce; and the Rho activation assay kit was from Upstate Biotechnology, Inc. (Lake Placid, NY). Primers (T203A-DS, 5′-GGA TGC CTT TGG CGG GTC TTC TGG CAA GCA GAA TAT CTT G-3′; T203A-US, 5′-GCC AGA AGA CCC GCC AAA GGC ATC CAT GAA TAC GAC TTT G-3′) were from Integrated DNA Technologies, Inc. (Coralville, IA). Gα13 (16Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (741) Google Scholar) was purified from Sf9 cells, as previously described (17Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). For phosphorylation of purified Gα subunits, 5 pmol of Gα subunit was added to 20 mm [γ-32P]ATP (4,500 Ci/mmol), PKAcat (protein kinase A, catalytic subunit (50 ng; 60 units) or PKA vehicle (100 mm NaCl, 20 mm MES, pH 6.5, 30 mm β-mercaptoethanol, 100 mm EDTA, 50% ethylene glycol) in phosphorylation buffer (25 mmTris-HCl, pH 7.5, 5 mm MgCl2, 125 mm CaCl2, 1 mm dithiothreitol) in a total reaction volume of 115 μl, and the mixture was incubated for 30 min at 30 °C. The reaction was terminated by adding Laemmli (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) sample buffer (62.5 mm Tris-HCl, pH 6.5, 3% SDS, 10% glycerol). Proteins were separated by SDS-PAGE and were visualized by silver staining (19Morrissey J.H. Anal. Biochem. 1981; 117: 307-310Crossref PubMed Scopus (2942) Google Scholar) followed by autoradiography. COS-7 cells and CHO cells were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose, 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B. COS-7 cells were transiently transfected when the cells were 80–90% confluent in 100-mm culture dishes. The medium was replaced with serum-free Dulbecco's modified Eagle's medium, and the cells were transfected using the LipofectAMINE Plus method (Invitrogen) according to the manufacturer's protocol, using 10 μg of DNA and 20 μl of LipofectAMINE per transfection. After 3 h at 37 °C, the medium was supplemented with 1 volume of 20% fetal bovine serum, Dulbecco's modified Eagle's medium. Experiments were performed 48 h after transfection. Transient transfection of COS-7 cells with cDNAs for Gα13, Gβ1, and Myc-tagged Gγ2subunits was performed using LipofectAMINE 2000 reagent, according to the manufacturer's instructions. Transfections in CHO cells with human Gα13 wild type (Gα13-WT) or Gα13-T203A mutant plasmids were performed as described above. After 3 days of transfection, G418 (500 μg/ml) was added for selection. After 3–5 weeks, the stable cell lines were established and confirmed by Western blot. The transfected cells were washed three times with cold PBS and were harvested with lysis buffer (50 mmHepes, pH 7.4, 10 mm MgCl2, 1 mmEDTA, 100 mm NaCl, 1% Triton X-100, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 20 μg/ml leupeptin). The lysates were centrifuged to remove insoluble material, and the protein concentration of the supernatant was measured using the BCA protein assay method. The protein concentration of each lysate was adjusted to be equal within an experiment and was typically 0.50–2.0 mg/ml. Aliquots of 0.6 ml were incubated overnight at 4 °C with 10 μg/ml Gα13antibody and were subsequently incubated with protein A-Sepharose beads (55 μl of a 10% (w/v) suspension) for 4 h at 4 °C. The immune complexed beads were washed once with lysis buffer and were supplemented with resuspension buffer (50 mm Hepes, pH 7.5, 10 mm MgCl2, 1 mm EGTA, 0.014% Tween 20, 1 mm dithiothreitol). 85 μl of protein-bound beads were then incubated with 50 μm[γ-32P]ATP, 150 ng of PKA, 1 mm cAMP, and the indicated concentration of blocking peptide in a total volume of 125 μl. The mixture was incubated for 30 min at 30 °C, after which time the beads were pelleted by centrifugation and washed three times with resuspension buffer. The beads were then suspended in Laemmli sample buffer plus resuspension buffer, and immune complexes were eluted by boiling. The eluted proteins were subjected to SDS-PAGE, silver staining, and autoradiography. Phosphorylated proteins on autoradiograms were quantified using a densitometer (Protein Databases, Inc., Huntington Station, NY), and values were normalized for the amount of silver-stained protein on the gel. Gα13 was cloned from a human adenocarcinoma LoVo cell line and was subcloned into a pcDNA3 vector. The Gα13 mutation with the substitution of Ala for Thr203 was performed using the Stratagene QuikChange site-directed mutagenesis kit according to the manufacturer's protocol. The final mutant was verified by DNA sequencing. 12 units of outdated human platelets were pooled and incubated with 3 mm aspirin for 45 min. Solubilized platelet membranes were then prepared as previously described (8Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), yielding a typical protein concentration of 4.5 mg/ml. Solubilized membranes were phosphorylated and purified by ligand affinity chromatography, as described (8Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, solubilized proteins were incubated with 1 mm 8-Br-cAMP or vehicle (H2O), 100 μl of ATP, 60 mmCaCl2, and incubation medium (45 mm histidine HCl, 50 mm KH2PO4, 20 mm NaF, 120 mm KCl, pH 7.4) for 30 min at 30 °C. The phosphorylated samples were supplemented with 500 mm KCl, 0.5 mg/ml asolectin, 20% glycerol, 0.2 mm EGTA, 10 mm CHAPS and were incubated on the ligand affinity columns overnight at 4 °C. Unbound proteins were washed with buffer D (20 mm Tris base, 10 mmCHAPS, 20% glycerol, 500 mm KCl, 0.2 mm EGTA, 0.5 mg/ml asolectin, pH 7.4) supplemented with 20 mm NaF and 2 mm orthovanadate. TXA2 receptor-G protein complexes were eluted from the column with the TXA2antagonist BM13.177 in buffer D at a flow rate of 0.125 ml/min. A 3-ml elution fraction was collected, dialyzed in 10 mmNH4HCO3, and then lyophilized. Proteins were reconstituted into 150 μl of H2O and were subjected to SDS-PAGE. Rho activation was determined as previously described (20Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1369) Google Scholar). CHO-Gα13-WT and CHO-Gα13-T203A mutant cells were seeded on six-well plates, grown to 80% confluence, and serum-starved for 24 h. Following treatment with 8-Br-cAMP (1 mm) at 37 °C for 15 min, the cells were washed once with PBS and harvested with 300 μl of lysis buffer (50 mm Tris-HCl, pH 7.5 containing 100 mm NaCl, 1 mm EDTA, 5 mmMgCl2, 10% glycerol, 50 mm NaF, 1 mm Na3VO4, 1 mmdithiothreitol, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml each of leupeptin and pepstatin, and 0.2% Nonidet P-40). The cell lysates were clarified by centrifugation at 15,000 × gfor 1 min and incubated with 20 μg of glutathioneS-transferase-Rho binding domain fusion protein conjugated with glutathione beads at 4 °C for 1 h. The beads were washed three times with lysis buffer and subjected to SDS-PAGE on a 12% gel. Bound RhoA was detected by Western blot using a polyclonal antibody against RhoA. Data were analyzed according to the analysis of variance using Dunnett's multiple comparison post-test or Student's t test, as indicated, using GraphPad PRISM statistical software (San Diego, CA). Statistical significance is defined as p < 0.05, p < 0.01, orp < 0.002, as indicated. Whereas we previously demonstrated that cAMP induces phosphorylation of TXA2 receptor-coupled Gα13 in human platelets (8Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), these results did not determine whether the observed phosphorylation is directly mediated by PKA or whether additional kinases are involved. In order to address this question, additional studies were performed using human Gα13 purified from Sf9 cells (16Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (741) Google Scholar) with [γ-32P]ATP in the presence or absence of the PKA catalytic subunit (PKAcat). Electrophoretic separation and silver staining of these samples revealed the presence of a single protein at 44 kDa corresponding to Gα13 (Fig. 1 a, lanes 1 and 2) but no such band in the presence of PKA alone (Fig. 1 a, lane 3). Analysis of this gel by autoradiography revealed substantial phosphorylation of Gα13 (at 44 kDa) when PKA is added (Fig. 1 b,lane 1) but no phosphorylation (Fig. 1 b, lane 2) in the absence of PKA. These experiments therefore establish that PKA alone is sufficient to phosphorylate Gα13. The specificity of this Gα13 phosphorylation was examined in parallel experiments by assaying a separate platelet G protein, Gαi (purified from Sf9 cells as previously described) (17Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Fig. 1 aillustrates a single protein band (lanes 4 and5) corresponding to Gαi. However, it can also be seen (Fig. 1 b, lanes 4 and5) that no phosphorylation of Gαi was observed in either the presence or the absence of PKA. Collectively, these results demonstrate that PKA directly phosphorylates Gα13and that this reaction does not require intermediate kinases or other co-factors. A 14-amino acid synthetic peptide (G13SRIpep) corresponding to the specific Gα13 amino acid sequence Leu197–Tyr209 was initially used to locate the PKA phosphorylation site of Gα13. This amino acid region was selected because analysis of the primary structure of the Gα13 subunit revealed only one PKA consensus sequence represented by Arg-Arg-Pro-Thr203. On this basis, G13SRIpep, which encompasses this region, was used to probe the Leu197-Tyr209 region for potential phosphorylation. Thus, it was reasoned that PKA-mediated phosphorylation of Gα13 would be blocked or reduced by a competing peptide substrate (G13SRIpep) containing an amino acid sequence that is identical to that contained within Gα13. In these experiments, human wild type Gα13 was immunoprecipitated from transiently transfected COS-7 cell using protein A-Sepharose beads coupled to an anti-peptide antibody directed against the N terminus of Gα13(G13-N IgG). The α subunit-antibody-bead complex was then incubated with PKAcat and [γ-32P]ATP in the presence or absence of G13SRIpep. Following incubation, the proteins were eluted from the beads and were subjected to gel electrophoresis and autoradiography. It can be seen that the efficiency of immunoprecipitation was comparable for each Gα13-transfected COS-7 cell sample (Fig. 2 a, lanes 1–6). It can also be seen that PKA induced a substantial Gα13 phosphorylation (Fig. 2 b, lane 1). Furthermore, upon the addition of G13SRIpep, there was a dramatic dose-dependent inhibition of this PKA-mediated effect (Fig. 2 b, lanes 2–5). Quantitation of this peptide inhibition is illustrated in Fig. 2 c, where it can be seen that Gα13 phosphorylation was significantly blocked at a peptide concentration of as little as 50 μm, and was completely inhibited at a peptide concentration of 500 μm. Fig. 2 a, lanes 7 and8, demonstrate that the vector-transfected COS-7 cells do not reveal detectable amounts of Gα13 under these conditions, and consistent with this finding, there was no observed PKA-induced phosphorylation in the 44-kDa region of the immunoprecipitated protein samples (Fig. 2 b,lanes 7 and 8). These findings using transfected COS-7 cells therefore confirm our previous results in platelets showing PKA-mediated phosphorylation of Gα13and suggest that the PKA phosphorylation site of Gα13 is contained within the Leu197–Tyr209 sequence. However, since it is possible that the ability of G13SRIpep to block phosphorylation was due to a nonspecific peptide effect, a control peptide was evaluated in subsequent experiments. Specifically, it was reasoned that the most appropriate control would employ a peptide with an amino acid sequence identical to that of G13SRIpep, the only difference being substitution of a phosphorylated Thr in the peptide sequence. Thus, the phosphorylated peptide should serve as a poor substrate for PKA. On this basis, G13SRIpepP was synthesized and tested for its ability to affect PKA-mediated phosphorylation under the same experimental conditions as those used for G13SRIpep. As can be seen in Fig3 a, lanes 1–4, equal amounts of Gα13 protein were immunoprecipitated under each experimental condition. It can also be seen (Fig. 3 b, lane 1) that PKA addition again resulted in substantial Gα13phosphorylation. However, in contrast to the effects of G13SRIpep, the addition of G13SRIpepP was completely ineffective in blocking this PKA-mediated phosphorylation (Fig. 3 b,lanes 2 and 3), even at a concentration (500 μm) that resulted in complete inhibition by G13SRIpep. The quantitative analysis of these phosphorylation profiles is illustrated in Fig. 3 c. Since Gα13 contains only a single PKA consensus sequence within Leu197–Tyr209, these findings therefore suggest 1) that G13SRIpepspecifically acts to block PKA-induced phosphorylation of Gα13; 2) that the location of the PKA phosphorylation site is contained within the Leu197–Tyr209sequence of Gα13; and 3) that Thr203 serves as the phosphorylation site within this sequence.Figure 3No Inhibition of PKA-induced phosphorylation of Gα13 by G13SRI-P peptide. a, silver stain of immunoprecipitated Gα13 protein. b, autoradiogram of phosphorylated Gα13; c, quantitative analysis of PKA-induced phosphorylation; n = 4. Statistical analysis was performed by analysis of variance. *, p < 0.05; **, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to provide independent confirmation that Thr203 is in fact the residue phosphorylated by PKA, a mutant Gα13 (Gα13-T203A) was produced in which Thr203 was changed to Ala by site-directed mutagenesis. In these experiments, the Gα13-T203A mutant (or wild type Gα13) was immunoprecipitated from transiently transfected COS-7 cells using the G13-N antibody, and the α subunit-antibody-bead complex was subjected to phosphorylation, as described earlier. It can be seen (Fig. 4 a, lanes 1–4) that the efficiency of immunoprecipitation was comparable for both wild type and mutant protein. However, a comparison of the phosphorylation profiles reveals that substitution of Thr203 with Ala resulted in a complete loss of PKA-mediated Gα13 phosphorylation (Fig. 4 b). In this regard, some experiments revealed a minor phosphorylation in the mutant with or without PKA. However, this apparent phosphorylation was not found to be significantly different from vector control, and no detectable phosphorylation was observed in the mutant autoradiogram (Fig. 4 b, lanes 3 and 4). The complete absence of PKA-induced phosphorylation in the T203A mutant, in combination with the previous results, therefore provides evidence that PKA phosphorylates a single site at Thr203contained within switch I region of Gα13. The next series of experiments investigated the molecular consequences of this PKA-mediated Thr203 phosphorylation. Because phosphorylation is known to alter protein conformation and function, it is possible that Thr203 phosphorylation may change the conformational or chemical characteristics of Gα13 and thereby alter its normal function in the receptor-coupled signaling process. Indeed, this possibility might be anticipated for two reasons: 1) Thr203 resides within switch I region of the Gα13 subunit, and 2) the switch I region is considered important for modulating activation of the α subunit and for regulating its affinity for the βγ subunits (16Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (741) Google Scholar). Based on these considerations, we examined whether PKA phosphorylation of the switch I region Thr203 residue affects the stability of the Gαβγ13 heterotrimeric complex. In these studies, COS-7 cells were transfected with either wild type or mutant Gα13 (Gα13-T203A) as well as with β and Myc-tagged γ2. The βγ subunits were then immunoprecipitated with anti-Myc antibody. It can be seen that co-transfection of β and Myc-tagged γ2 cDNAs alone (without Gα13) showed no Gα13 protein in either the lysate or the immunoprecipitate (Fig. 5, row a,lane 3, and row b,lane 3). As expected, Gα13 was immunoprecipitated (Fig. 5 row a, lane 2) and present in the lysate (Fig. 5, row b, lane 2) when the cells were co-transfected with β, Myc-tagged γ2 cDNAs, and wild type Gα13. On the other hand, no such Gα13 immunoprecipitation was observed (Fig. 5,row a, lane 1) when the co-transfection was done with Gα13-T203A mutant. Furthermore, Western blotting of total cell lysates demonstrated that both constructs of Gα13 were expressed at the same levels (Fig. 5, row b, lanes 1 and2), suggesting that the inability of mutated Gα13 to complex with β1γ2 was not due to insufficient expression. These findings therefore suggest that mutation of Gα13 at Thr203 disrupts heterotrimer stability. The next series of experiments extended the above finding and investigated possible consequences of Gα13 Thr203 phosphorylation on stability of the receptor-Gα13 complex. To this end, affinity purification of the receptor-G protein complex was employed. This technique has previously been used to co-purify native TXA2receptors with their associated Gα proteins (21Knezevic I. Borg C. Le Breton G.C. J. Biol. Chem. 1993; 268: 26011-26017Abstract Full Text PDF PubMed Google Scholar, 22Djellas Y. Manganello J.M. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 14325-14330Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and was used to demonstrate PKA phosphorylation of TXA2 receptor-coupled Gα13 (8Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In the present experiments, solubilized platelet membranes were prepared and incubated in the presence or absence of 1 mm 8-Br-cAMP plus [γ-32P]ATP. The TXA2 receptor-G protein complexes were then purified by affinity chromatography (8Manganello J.M. Djellas Y. Borg C. Antonakis K. Le Breton G.C. J. Biol. Chem. 1999; 274: 28003-28010Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and the column eluate was assayed for TXA2 receptor, Gα13, Gαq, and β subunits by immunoblotting. It can be seen that in the absence of 8-Br-cAMP (Fig. 6 a,lane 1) TXA2 receptors co-purified with their associated G protein heterotrimeric complexes. Thus, the TXA2 receptor elute fractions yielded positive immunoreactivity for the Gα13, Gαq, and Gβ subunits. It can also be seen, however, that 8-Br-cAMP treatment produced a significant shift in this elution profile (i.e. a substantial increase in co-eluted Gα13 and a substantial decrease in co-eluted Gβ) (Fig. 6 a, lane 2). On the other hand, 8-Br-cAMP treatment produced no change in the amounts of either Gαq or TXA2receptor (Fig. 6 a, lanes 1 and2). Quantitation of these results is illustrated in Fig. 6 b. The ability of 8-Br-cAMP to cause such a sel
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