Agonist-dependent Phosphorylation of the G Protein-coupled Receptor Kinase 2 (GRK2) by Src Tyrosine Kinase
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.34411
ISSN1083-351X
AutoresSusana Sarnago, Ana Elorza, Federico Mayor,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoGRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparentK m for GRK2 of 12 nm, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of β2-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of ∼5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling. GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparentK m for GRK2 of 12 nm, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of β2-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of ∼5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling. G protein-coupled receptors Dulbecco's modified Eagle's medium G protein-coupled receptor kinase protein kinase C mitogen-activated protein kinase polyacrylamide gel electrophoresis radioimmune precipitation buffer.> Activation of G protein-coupled receptors (GPCRs)1 triggers a process termed desensitization, initiated by agonist-dependent phosphorylation of the receptor by specific G protein-coupled receptor kinases (GRKs) (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1053) Google Scholar). This phosphorylation event leads to the recruitment of cytosolic proteins known as β-arrestins to the receptor-signaling complex, with the subsequent uncoupling from heterotrimeric G proteins and loss of receptor responsiveness (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1053) Google Scholar, 2Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 238-319Crossref Scopus (853) Google Scholar, 3Lohse M. Krasel C. Winstel R. Mayor Jr., F. Kidney Int. 1996; 49: 1047-1052Abstract Full Text PDF PubMed Scopus (56) Google Scholar). GRK2 is a ubiquitous member of the GRK family that has been shown to phosphorylate different GPCRs (4Chuang T.T. Iacovelli L. Sallese M. DeBlasi A. Trends Pharmacol. Sci. 1996; 17: 416-421Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 5Aragay A.M. Ruiz-Gómez A. Penela P. Sarnago S. Elorza A. Jimenez-Sainz M.C. Mayor Jr., F. FEBS Lett. 1998; 430: 37-40Crossref PubMed Scopus (63) Google Scholar). Recent evidence indicates that GRK2 and β-arrestin not only promote receptor uncoupling but also directly participate in GPCR sequestration, thus triggering receptor dephosphorylation and recycling to the plasma membrane (reviewed in Refs. 6Mayor F. Penela P. Ruiz-Gómez A. Trends Cardiovasc. Med. 1998; 8: 235-241Crossref Scopus (28) Google Scholar, 7Lefkowitz R.J. J. Biol. 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Consistent with an important physiological role, GRK2 activity and subcellular localization appear to be subject to complex regulatory processes that involve the interaction of diverse domains of the kinase with G protein βγ subunits, several lipids, anchoring proteins, and the agonist-bound form of receptors. The interaction of GRK2 with βγ subunits through its C-terminal pleckstrin homology domain helps to target the kinase to the membrane and enhances its activity toward the receptor (11Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar, 12Daaka Y. Pitcher J.A. Richardson M. Stoffel R.H. Robishaw J.D. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2180-2185Crossref PubMed Scopus (153) Google Scholar). The interaction of GRK2 with phosphatidylinositol 4,5-bisphosphate by means of the N-terminal portion of its pleckstrin homology domain seems also to be necessary for full kinase activation (13Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 14DebBurman S.K. Ptasienski J. Benovic J.L. Hosey M.M. J. Biol. Chem. 1996; 271: 22552-22562Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). GRK2 activity toward receptors is also modulated by phosphorylation by PKC on a yet unidentified residue located in the C terminus of GRK2. Phosphorylation by PKC leads to an increased GRK2 activity, probably due to an enhanced kinase association to the plasma membrane (15Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (141) Google Scholar, 17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This would provide a cross-talk or feedback mechanism by which activation of PKC by different transduction systems may affect GPCR modulation and signaling. In this regard, emerging evidence indicates that stimulation of Gq- or Gi-coupled GPCR also leads to the activation of the mitogen-activated protein kinase (MAPK) cascade in a Ras-dependent way (see Refs. 7Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar and 18Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar for recent reviews). It has been shown that activation of lysophosphatidic acid and β2-adrenergic and other GPCR receptors stimulate phosphorylation on tyrosine residues of Shc and Gab1 adapter proteins and other plasma membrane-associated proteins (Refs. 18Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 19Van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar, 20Chen Y. Grall D. Salcini A.E. Pelicci P.G. Pouyssegur J. Van Obberghen-Schiling E. EMBO J. 1996; 15: 1037-1044Crossref PubMed Scopus (104) Google Scholar, 21Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar and references therein). Recruitment and activation of cytosolic tyrosine kinases of the Src family play a critical role in the GPCR-mediated regulation of the MAPK cascade (Refs. 18Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar and 21Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 22Wan Y. Kurosaki T. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (256) Google Scholar, 23Luttrell L.M. Ferguson S.S.G. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1234) Google Scholar and references therein). Interestingly, it has been recently suggested that GRK2 and β-arrestin directly participate in the process of MAPK cascade activation, either by facilitating receptor internalization and/or the interaction with additional proteins (7Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 24Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 25Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). On the other hand, overexpression of the c-Src kinase enhances β-adrenergic-induced cAMP accumulation in murine fibroblasts (26Bushman W.A. Wilson L.K. Luttrell D.K. Moyers S.J. Parsons S.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7462-7466Crossref PubMed Scopus (35) Google Scholar, 27Moyers J.S. Bouton A.H. Parsons S.J. Mol. Cell. Biol. 1993; 13: 2391-2400Crossref PubMed Google Scholar) and promotes a loss of responsiveness of luteinizing hormone receptor in MA10 cells (28Taylor C.C. Limback D. Terranova P.F. Endocrinology. 1996; 137: 5735-5738Crossref PubMed Scopus (17) Google Scholar). These results suggest a role for Src-like tyrosine kinases in the modulation of G protein-coupled receptor signaling and desensitization. In this context, we have investigated the possibility that GRK2 could be regulated by Src-mediated tyrosine phosphorylation. We find that GRK2 is a new substrate for c-Src and, what is more important, that agonist stimulation of β2-adrenergic receptors leads to rapid phosphorylation of GRK2 on tyrosine residues, which results in an enhancement of GRK2 intrinsic activity. Our results indicate that tyrosine phosphorylation of GRK2 is inherent to GPCR activation, thus suggesting an important role in the modulation of GRK2 cellular functions. Bovine GRK2 was overexpressed and purified from baculovirus-infected Sf9 cells as described (29Murga C. Ruiz-Gómez A. Garcı́a-Higuera I. Kim C.M. Benovic J.L. Mayor Jr., F. J. Biol. Chem. 1996; 271: 985-994Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Purity of the GRK2 preparation as determined by SDS-polyacrylamide gel electrophoresis was >95%. Recombinant baculovirus for GRK2 was kindly provided by Dr. J. L. Benovic at the Thomas Jefferson Cancer Institute of Philadelphia. The cDNAs encoding human pp60c-src and the constitutively active Y527F pp60c-src were provided by Dr. S. Gutkind (NIH, Bethesda, MD). COS-7 cells, HEK-293 cells, and human Jurkat T lymphocytes were from the American Type Culture Collection (Manassas, VA). Culture media and LipofectAMINE were from Life Technologies, Inc. Protein A-Sepharose, isoproterenol, and heparin (M r 6,000) were obtained from Sigma. [γ-32P]ATP was purchased from Amersham Pharmacia Biotech, and [35S]methionine- and [35S]cysteine-labeling mixture was from NEN Life Science Products. Purified pp60c-src was obtained from Upstate Biotechnology. All other reagents were of the highest grade commercially available. COS-7 and HEK-293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum at 37 °C in a humidified 7% CO2 atmosphere. Transfections were performed on 70% confluent monolayers in 100-mm dishes for immunoprecipitation assays or 60-mm dishes for 35S-labeling experiments. For transient transfection, cells were incubated at 37 °C in serum-free OPTIMEM (5 ml containing 5 μg of DNA/100-mm dish or 3 μg/60-mm dish) plus 8 μl of LipofectAMINE reagent/μg of DNA. Empty vector was added to transfections as needed to keep the total amount of DNA added/dish constant. After 5 h of exposure to the transfection medium, monolayers were refed with culture medium and incubated overnight. Assays were performed 48 h after transfection. Transient expression was confirmed by immunoblot analysis of whole-cell lysates using specific antisera (mAb-1 from Upstate Biotechnology for c-Src and Ab9 (30Ruiz-Gómez A. Mayor Jr., F. J. Biol. Chem. 1997; 272: 9601-9604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) for GRK2). Stimulation of COS-7 cells was performed 48 h after transfection and was carried out at 37 °C in serum-free DMEM supplemented with 20 mm HEPES, pH 7.5, and 1 mm ascorbic acid (Sigma). Isoproterenol was used at the concentration depicted in the figure legends. In some experiments, cells were serum-starved for 90 min before the experiment as reported (31Lin F.-T. Daaka Y. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 31640-31643Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Treatments with the Src inhibitor PP2 (5 μm, Calbiochem) were initiated 15 min before isoproterenol stimulation. For metabolic labeling, transiently transfected HEK-293 cells were kept for 2 h in methionine and cysteine-free DMEM and then incubated for 15 min in DMEM supplemented with 250 μCi/ml [35S]methionine- and [35S]cysteine-labeling mixture as reported (32Penela P. Ruiz-Gómez A. Castaño J.G. Mayor Jr., F. J. Biol. Chem. 1998; 273: 35238-35244Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Labeled cells were used for antiphosphotyrosine immunoprecipitation experiments as described below. After SDS-polyacrylamide gel electrophoresis (PAGE), the presence of GRK2 was subsequently analyzed by35S detection or immunoblot. For immunoprecipitation, the cells were washed twice with ice-cold phosphate-buffered saline supplemented with 1 mm sodium orthovanadate, solubilized in 700 μl/100-mm dish or 400 μl/60-mm dish of RIPA buffer (50 mm Tris, pH 7.5, 0.3 mNaCl, 1% (w/v) sodium deoxycholate, 1% (v/v) TX-100, 0.1% SDS, 1 mm sodium orthovanadate, plus a mixture of protease inhibitors). After gentle rocking for 90 min at 4 °C, the lysates were clarified by centrifugation, and an aliquot (30 μl) was used to assess protein overexpression. Before GRK2 immunoprecipitation, the supernatants were precleared by overnight incubation with a preimmune serum plus 0.5 mg/ml bovine serum albumin. After incubation at 4 ° with Protein A-Sepharose for 1 h and centrifugation, the beads (preimmune controls) were washed with ice-cold RIPA buffer and resuspended in sample buffer. GRK2 was then immunoprecipitated from the supernatants with the specific AbFP1 polyclonal antibody as described (29Murga C. Ruiz-Gómez A. Garcı́a-Higuera I. Kim C.M. Benovic J.L. Mayor Jr., F. J. Biol. Chem. 1996; 271: 985-994Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 32Penela P. Ruiz-Gómez A. Castaño J.G. Mayor Jr., F. J. Biol. Chem. 1998; 273: 35238-35244Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Alternatively, tyrosine-phosphorylated proteins were immunoprecipitated by incubating with 50 μl of a 50% suspension of an agarose-conjugated phosphotyrosine antibody from Transduction Laboratories. All immunoprecipitated samples were boiled for 5 min before resolution by 8% SDS-PAGE and transference to nitrocellulose membranes. Tyrosine phosphorylation was detected by immunoblot using horseradish peroxidase-conjugated antiphosphotyrosine monoclonal antibodies (HPRO-PY20 or PY99-HRP from Transduction Laboratories or Santa Cruz Laboratories, respectively). Immunoprecipitated GRK2 protein was detected in the same blots by incubating stripped filters with Ab9 antibody. Blots were developed using a chemiluminescent method (ECL, Amersham Pharmacia Biotech). For detection of GRK2 activity in the immunoprecipitates, 40-μl aliquots of the latter were washed with phosphorylation buffer to remove the detergent, and casein or rhodopsin were added to a final concentration of 30 μm or 1 μm, respectively. The phosphorylation reaction was initiated by adding 20 μl of kinase reaction buffer (30 mm Tris-HCl, pH 7.2, 8 mm MgCl2, 1.4 mm EDTA, 1 mm EGTA, 4.5 mm NaF, 0.25 mm Na3VO4, 160 μm ATP, and 5,000 cpm/pmol [γ-32P]ATP). After incubation for 30–60 min at 30 °C, the reaction was stopped with SDS sample buffer, and the phosphorylated proteins were resolved by SDS-PAGE, revealed by autoradiography, and quantitated as reported (29Murga C. Ruiz-Gómez A. Garcı́a-Higuera I. Kim C.M. Benovic J.L. Mayor Jr., F. J. Biol. Chem. 1996; 271: 985-994Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 33Freeman J.L.R. de la Cruz E.M. Pollard T.D. Lefkowitz R.J. Pitcher J.A. J. Biol. Chem. 1998; 273: 20653-20657Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). For the in vitro phosphorylation studies, recombinant GRK2 protein at the concentrations depicted in the figure legends was incubated with purified c-Src kinase (9.2 nm, Upstate Biotechnology) in a final volume of 15–30 μl of kinase reaction buffer (100 mm Tris-HCl, pH 7.2, 25 mmMnCl2, 2 mm EGTA, 0.25 mmNa3VO4, 125 mm magnesium acetate, 50 μm ATP, and 2,000 cpm/pmol [γ-32P]ATP) in the presence or absence of 0.25 ng/μl heparin to inhibit GRK2 autophosphorylation (34Benovic J.L. Stone C.W. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 6707-6710Abstract Full Text PDF PubMed Google Scholar). After the indicated time at 30 °C, the reaction was stopped by the addition of 2× SDS-PAGE sample buffer. Phosphorylated proteins were resolved by electrophoresis in 8% polyacrylamide gels and visualized by autoradiography. Enolase phosphorylation assays were performed as described (35Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell Biol. 1990; 62: 481-492Scopus (478) Google Scholar). To explore the possibility that GRK2 could be a target for Src-like tyrosine kinases, recombinant GRK2 was incubated with purified c-Src under phosphorylating conditions. The presence of Src promoted a marked increase in GRK2 phosphorylation (Fig.1 A). The phosphorylation of GRK2 is likely due to the activity of Src, since these initial experiments were performed in the presence of heparin, a well known inhibitor of GRK2 activity and autophosphorylation (34Benovic J.L. Stone C.W. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 6707-6710Abstract Full Text PDF PubMed Google Scholar). Control experiments indicated that heparin had no effect on Src intrinsic activity toward other substrates such as enolase (Fig. 1 B). To further confirm that GRK2 was directly phosphorylated by Src on tyrosine residues, we performed similar phosphorylation experiments followed by immunoblot analysis using antiphosphotyrosine antibodies. Fig. 1 C shows that these antibodies only recognized GRK2 after previous incubation with Src. These results indicate that GRK2 is a substrate for Src tyrosine kinase in vitro. To explore if phosphorylation of GRK2 by Src could have a physiological meaning, we sought to determine the kinetic parameters of the phosphorylation reaction. Since results similar to those shown in Fig.1, A and C, were obtained in the absence of heparin (data not shown), we routinely performed the experiments in the latter conditions using basal GRK2 autophosphorylation controls to assess Src-mediated GRK2 phosphorylation in each experimental determination. Fig. 2 Aindicates that GRK2 is readily phosphorylated by c-Src reaching a plateau at 15–20 min of incubation. The stoichiometry of phosphorylation of 10 nm GRK2 induced by equimolar Src in the absence of heparin was 0.86 ± 0.08 mol of phosphate incorporated/mol of GRK2 (mean ± S.E. of 4 independent experiments). This value is similar to that reported for PKC phosphorylation of GRK2 (0.5–0.9 mol Pi/mol of GRK2; see Refs. 15Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar and 16Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (141) Google Scholar) and suggests that GRK2 is phosphorylated by c-Src on 1 tyrosine residue. Double-reciprocal plot analysis (Fig. 2 B) reveals that c-Src displays an apparent K m for GRK2 of ∼12 nm, with a V max of 1.3 nmol/min/mg of protein. This K m is lower than that reported (36Akiyama T. Kadowaki T. Nishida E. Kadooka T. Ogawara H. Fukami Y. Sakai H. Takaku F. Kasuga M. J. Biol. Chem. 1986; 261: 14797-14803Abstract Full Text PDF PubMed Google Scholar, 37Sato K-I. Yamamoto H. Otsuki T. Aoto M. Tokmakov A.A. Hayashi F. Fukami Y. FEBS Lett. 1997; 410: 136-140Crossref PubMed Scopus (21) Google Scholar, 38De Corte V. Gettemans J. Vandekerckhove J. FEBS Lett. 1997; 401: 191-196Crossref PubMed Scopus (73) Google Scholar) for well known Src substrates such as fodrin (0.3 μm), tubulin (2 μm), gelsolin (6 μm), or Shc (90 nm). Therefore, our data indicate that GRK2 is a high affinity substrate for c-Src in vitro and suggest that tyrosine phosphorylation of GRK2 would also take place in the cellular environment. To determine whether GRK2 could be phosphorylated by Src in vivo, we coexpressed this kinase with wild type Src or the constitutively active Y527F Src mutant (39Kmiecik T.E. Shalloway D. Cell. 1987; 49: 65-73Abstract Full Text PDF PubMed Scopus (407) Google Scholar) in COS-7 cells. The pattern of phosphorylated proteins in cell lysates was investigated using antiphosphotyrosine antibodies. As shown in the left panelof Fig. 3 A, a protein that comigrates with GRK2 is markedly tyrosine-phosphorylated only in cells transfected with constitutively active Src. Similar levels of GRK2 (upper right panel) and Src (lower right panel) overexpression were obtained in the different conditions, as assessed by reprobing the blots with specific antibodies. In addition, antiphosphotyrosine immunoblot analysis of GRK2 immunoprecipitated from cells transiently co-transfected with the kinase and Y527F Src demonstrate that GRK2 is indeed phosphorylated on tyrosine residues under these conditions (Fig. 3 B). The titration experiment shown in Fig. 3 B indicates a correlation between the phosphotyrosine content of immunoprecipitated GRK2 and an increased expression of the Src mutant, suggesting a direct relationship between the levels of Src activity and tyrosine phosphorylation of the GRK2 cellular pool. We also performed similar experiments in HEK-293 cells. Our data show that GRK2 could be immunoprecipitated with an agarose-conjugated phosphotyrosine antibody only after coexpression with the Src mutant (Fig. 3 C and data not shown). GRK2 protein present in the phosphotyrosine immunoprecipitates was detected either by Western blot using a GRK2 antibody or by35S-radiolabeling and autoradiography. It is worth noting that a tyrosine-phosphorylated band with a slightly slower migration than GRK2 was detected by the kinase antibody (Fig. 3 C,upper panel) but not by 35S-labeling (Fig.3 C, lower panel). Such a band is also present in some experiments in GRK2 immunoprecipitates analyzed with phosphotyrosine antibodies (Fig.4 A and data not shown). It is tempting to suggest that such a band represents a minor cellular pool of another form of tyrosine-phosphorylated GRK2, which deserves to be characterized in future experiments.Figure 4Agonist-induced tyrosine phosphorylation of GRK2. A, time course of isoproterenol-stimulated GRK2 tyrosine phosphorylation. COS-7 cells transiently transfected with GRK2, c-Src, and β2-adrenergic receptor (1 μg of cDNA of each/plate) were stimulated or not (C lane) for the indicated times with isoproterenol (ISO, 10 μm) as described under "Experimental Procedures." Immunoprecipitates (IPP) of GRK2 from RIPA buffer lysates were resolved by 8% SDS-PAGE, blotted, and analyzed with antiphosphotyrosine (anti-pTyr) antibodies (upper panel); after stripping, the presence of GRK2 was analyzed in the same gel using a specific GRK2 antibody (lower panel). Thearrows indicate the migration of GRK2 as assessed by the position of recombinant GRK2 resolved in the same gel (GRK2 Std). A gel representative of three independent experiments is shown. B, quantitation of isoproterenol-stimulated tyrosine phosphorylation of GRK2. Tyrosine phosphorylation of GRK2 was measured by scanner laser densitometry, and the data were normalized to the amount of GRK2 protein present in the immunoprecipitates, as assessed with the GRK2 antibodies. Nonstimulated controls were taken as the basal condition. Each data point represent the mean ± S.E. from at least three independent experiments. C, isoproterenol-induced tyrosine phosphorylation of GRK2 is blocked by a specific Src inhibitor. COS-7 cells as in panel A were treated with 5 μm PP2 or vehicle as detailed under "Experimental Procedures" before stimulation for the indicated times with isoproterenol (10 μm) as described above. Tyrosine phosphorylation of GRK2 was assessed as in panel A. The arrows indicate the migration of GRK2. A gel representative of two independent experiments is shown.View Large Image Figure ViewerDownload (PPT) Taken together, our results indicate that GRK2 is phosphorylated by activated Src tyrosine kinase in living cells. We next tested the hypothesis that GPCR activation could be the physiological trigger of GRK2 phosphorylation on tyrosine residues. Several recent reports have established that stimulation of β-adrenergic and other GPCR leads to c-Src-mediated phosphorylation of several cellular proteins. Phosphorylation of adapter proteins such as Shc and Gab1 is followed by Ras-dependent modulation of the MAPK cascade (reviewed in Refs. 7Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar and 18Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Activation of β2-adrenergic receptors also leads to Src-mediated phosphorylation of dynamin, which appears to be related to increased receptor internalization and signaling through the MAPK cascade (40Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Therefore, we investigated whether β2-adrenergic receptor stimulation would lead to GRK2 tyrosine phosphorylation. COS-7 cells were transiently transfected with β2-adrenergic receptor, GRK2, and wild type c-Src and treated with the β-agonist isoproterenol for different periods of time. GRK2 was then immunoprecipitated, and tyrosine phosphorylation was analyzed using antiphosphotyrosine antibodies. Fig. 4 shows that isoproterenol stimulation results in a marked increase in tyrosine phosphorylation of GRK2, which is maximal (4–5-fold) within 5 min of agonist exposure. These results demonstrate that β2-adrenergic receptor activation triggers the rapid tyrosine phosphorylation of GRK2. Interestingly, when similar experiments were performed in the presence of a specific Src inhibitor, the agonist-stimulated GRK2 tyrosine phosphorylation was markedly reduced (Fig. 4 C), thus suggesting that this process is in fact due to the activity of Src. The use of transient transfection experimental systems raises the question of whether GRK2 phosphorylation on tyrosine residues could also be observed in more physiological conditions. In this regard, preliminary data in our laboratory 2S. Sarnago, A. Elorza, and F. Mayor, Jr., manuscript in preparation. indicate that tyrosine phosphorylation of endogenous GRK2 can be detected in Jurkat cells and that its tyrosine phosphorylation levels are modulated upon stimulation with agents that activate tyrosine kinases of the Src family or with CXCR4 receptor agonists (data not shown). To determine whether tyrosine phosphorylation of GRK2 affected its function, we compared the activity of GRK2 in cells expressing this kinase in the absence or presence of the constitutively active Y527F Src mutant. Y527F Src expression promotes an increased level of GRK2 tyrosine phosphorylation that can be detected both in cell lysates (Fig. 5 A) or after immunoprecipitation with specific GRK2 antibodies (Fig. 5 B). When immunoprecipitates containing similar quantities of GRK2 (Fig.5 B, lower panel) were tested for the ability of GRK2 to phosphorylate soluble GRK2 substrates, a clear increase in GRK2 activity was detected in the tyrosine-phosphorylated samples (between a 1.4- and 2.5-fold increase in activity toward casein, Fig.5 C). Similar results are obtained when a GPCR such as rhodopsin is used as a substrate (1.7 ± 0.29-fold increase in phosphorylation, Fig. 5 D). The range of this stimulatory effect is similar to that reported for other GRK2 modulatory mechanisms such as phosphorylation by PKC or interaction with lipids (1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1053) Google Scholar, 15Chuang T.T. LeVine III, H. De Blasi A. J. Biol. Chem. 1995; 270: 18660-18665Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Winstel R. Freund S. Krasel C. Hoppe E. Lohse M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2105-2109Crossref PubMed Scopus (141) Google Scholar, 17Pronin A.N. Benovic J.L. J. Biol. Chem. 1997; 272: 3806-3812Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). It is worth noting that the observed effect is likely an underestimation, since only a fraction of the immunoprecipitated GRK2 protein would be phosphorylated on tyrosine residues. The fact that the activity of GRK2 is increased toward both soluble and membrane- bound substrates suggests a direct effect of Src phosphorylation on its catalytic activity. Taken together, our data indicate that GRK2 is a substrate for c-Srcin vitro and in living cells and that stimulation of the β2-adrenergic receptor leads to rapid tyrosine phosphorylation of GRK2 and modulation of GRK2 function. Since activation of β2-adrenergic receptor has been shown to increase Src-mediated protein phosphorylation (18Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 19Van Biesen T. Hawes B.E. Luttrell D.K. Krueger K.M. Touhara K. Porfiri E. Sakaue M. Luttrell L.M. Lefkowitz R.J. Nature. 1995; 376: 781-784Crossref PubMed Scopus (525) Google Scholar, 20Chen Y. Grall D. Salcini A.E. Pelicci P.G. Pouyssegur J. Van Obberghen-Schiling E. EMBO J. 1996; 15: 1037-1044Crossref PubMed Scopus (104) Google Scholar, 21Luttrell L.M. 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This suggested function of GRK2 is in line with the recently proposed role for GRKs and arrestins in "signal switching" (7Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar) and may also have physiological implications. In this regard, GRK2 appears to be important in the function and disfunction of the cardiovascular system (reviewed in Refs. 1Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1053) Google Scholar, 5Aragay A.M. Ruiz-Gómez A. Penela P. Sarnago S. Elorza A. Jimenez-Sainz M.C. Mayor Jr., F. FEBS Lett. 1998; 430: 37-40Crossref PubMed Scopus (63) Google Scholar, and 6Mayor F. Penela P. Ruiz-Gómez A. Trends Cardiovasc. Med. 1998; 8: 235-241Crossref Scopus (28) Google Scholar). Recent data indicate that c-Src activation plays a critical role in hypertrophic growth regulation of cardiac myocites (Refs. 46Fuller S.J. Gillespie-Brown J. Sugden P.H. J. Biol. 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The increasing role of GRKs and arrestins in GPCR signal transduction would require further research to better understand their mechanisms of regulation and their interaction with other cellular proteins. We thank Drs. J. L. Benovic and J. S. Gutkind for experimental tools and Dr. Ana Ruiz-Gómez for generously providing purified recombinant GRK2. Drs. P. Penela, A. Ruiz-Gómez, C. Ribas, and A. Aragay are acknowledged for critical reading of the manuscript. We also thank A. Morales for secretarial assistance and Professor F. Mayor for continuous encouragement.
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