Gα and Gβγ Require Distinct Src-dependent Pathways to Activate Rap1 and Ras
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m204006200
ISSN1083-351X
AutoresJohn Schmitt, Philip A. Stork,
Tópico(s)Phosphodiesterase function and regulation
ResumoThe Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the β-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gβγ subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, β-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the β2-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Gαs to Rap1 and ERKs and the other through Gβγ to Ras and AKT. The Src tyrosine kinase is necessary for activation of extracellular signal-regulated kinases (ERKs) by the β-adrenergic receptor agonist, isoproterenol. In this study, we examined the role of Src in the stimulation of two small G proteins, Ras and Rap1, that have been implicated in isoproterenol's signaling to ERKs. We demonstrate that the activation of isoproterenol of both Rap1 and Ras requires Src. In HEK293 cells, isoproterenol activates Rap1, stimulates Rap1 association with B-Raf, and activates ERKs, all via PKA. In contrast, the activation by isoproterenol of Ras requires Gβγ subunits, is independent of PKA, and results in the phosphoinositol 3-kinase-dependent activation of AKT. Interestingly, β-adrenergic stimulation of both Rap1 and ERKs, but not Ras and AKT, can be blocked by a Src mutant (SrcS17A) that is incapable of being phosphorylated and activated by PKA. Furthermore, a Src mutant (SrcS17D), which mimics PKA phosphorylation at serine 17, stimulates Rap1 activation, Rap1/B-Raf association, and ERK activation but does not stimulate Ras or AKT. These data suggest that Rap1 activation, but not that of Ras, is mediated through the direct phosphorylation of Src by PKA. We propose that the β2-adrenergic receptor activates Src via two independent mechanisms to mediate distinct signaling pathways, one through Gαs to Rap1 and ERKs and the other through Gβγ to Ras and AKT. G protein-coupled receptors protein kinase A extracellular-regulated signal kinase hemagglutinin epidermal growth factor glutathione S-transferase Src family kinases β-adrenergic receptor kinase human embryonic kidney phosphoinositol 3-kinase Stimulation of G protein-coupled receptors (GPCRs)1 triggers a wide range of biochemical and physiological effects. GPCR activation of heterotrimeric G proteins signals to distinct effector molecules through both the G protein α and βγ subunits (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar, 2Birnbaumer L. Cell. 1992; 71: 10069-10072Abstract Full Text PDF Scopus (380) Google Scholar, 3Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). Gαs activation stimulates adenylyl cyclases to elevate intracellular cAMP and activation of PKA. PKA can regulate cell growth and differentiation through cross-talk with the mitogen-activated protein kinase or ERK (extracellular signal-regulated kinase) cascade (4Chen J. Iyengar R. Science. 1994; 263: 1278-1281Crossref PubMed Scopus (107) Google Scholar, 5Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 6Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar, 7Schmitt J.M. Stork P.J.S. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (127) Google Scholar, 8Stork P.J.S. Schmitt J.M. Trends Cell Biol. 2002; 12: 258-266Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar). In HEK293 cells, the β2-adrenergic receptor agonist isoproterenol stimulates endogenous receptors to activate ERKs through a PKA-dependent pathway (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 10Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1077) Google Scholar). The activation by isoproterenol of PKA has been shown to induce the activation of Ras via a Src-dependent mechanism that is mediated by Gβγ subunits (10Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1077) Google Scholar, 11Luttrell 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 (494) Google Scholar, 12Luttrell L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). However, isoproterenol and PKA can also activate Rap1 and ERKs in these cells (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In NIH3T3 fibroblast cells, PKA activation of Rap1 has been proposed to result from the direct phosphorylation by PKA on the Src tyrosine kinase (13Schmitt J.M. Stork P.J.S. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In NIH3T3 cells that do not express B-Raf, PKA and Rap1 antagonize Ras-dependent activation of ERKs (4Chen J. Iyengar R. Science. 1994; 263: 1278-1281Crossref PubMed Scopus (107) Google Scholar, 7Schmitt J.M. Stork P.J.S. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (127) Google Scholar). In addition to antagonizing Ras, Rap1 can activate ERKs in cells that express the mitogen-activated protein kinase kinase kinase B-Raf (14Ohtsuka T. Shimizu K. Yamamori B. Kuroda S. Takai Y. J. Biol. Chem. 1996; 271: 1258-1261Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). However, the contribution of Src in this action of Rap1 has not been examined. Because it has been shown that isoproterenol couples efficiently to both Ras and Rap1 in HEK293 cells, this model system provides an opportunity to examine the requirement of Src in each process. Surprisingly, we found that Src was required for the activation of both Ras and Rap1 by isoproterenol. However, it activated Rap1 and Ras through distinct mechanisms. Antibodies specific to phosphorylated-ERK (pERK) that recognize phosphorylated ERK1 (pERK1) and ERK2 (pERK2) at residues threonine 183 and tyrosine 185 were purchased from New England Biolabs (Beverly, MA). Antibodies specific to phosphorylated-AKT (pAKT) that recognize phosphorylated AKT at residue threonine 308 were purchased from Cell Signaling (Beverly, MA). Antibodies to Rap1, Raf-1, B-Raf, ERK2, c-Myc (9E10), Cbl, C3G, and agarose-conjugated antibodies to Myc and hemagglutinin (HA) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to HA (12CA5) were purchased from Roche Molecular Biochemicals (Indianapolis, IN). Anti-Ras antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). FLAG (M2) antibody, isoproterenol, and epidermal growth factor (EGF), were purchased from Sigma. Forskolin, PP2 (AG1879; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyazolo[3,4-d]pyrimidine), LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-bemzopyran-4-one), andN-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) were purchased from Calbiochem (Riverside, CA). Nickel-nitrilotriacetic acid-agarose was purchased from Qiagen Inc. (Chatswoth, CA). HEK293, SYF, and Src2+ cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum, penicillin/streptomycin, and l-glutamine at 37 °C in 5% CO2. Cells were maintained in serum-free Dulbecco's modified Eagle's medium for 16 h at 37 °C in 5% CO2 prior to treatment with various reagents for immunoprecipitation assay, and Western blotting. In all experiments, cells were treated with EGF (100 ng/ml), isoproterenol (10 μm), or forskolin (10 μm) for 5 min unless otherwise indicated. PP2 (10 μm), H89 (10 μm), and LY294002 (10 μm), were added to cells 20 min prior to treatment, unless otherwise indicated. Cell lysates and Western blotting were prepared as described (7Schmitt J.M. Stork P.J.S. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (127) Google Scholar). Briefly, protein concentrations were quantified using the Bradford protein assay. For detection of Raf-1, ERK2, Myc-ERK2, FLAG, Rap1, Ras, pERK1/2, Cbl, C3G, and pAKT, equivalent amounts of protein per treatment condition were resolved by SDS-PAGE, blotted onto polyvinylidene difluoride (Millipore Corp., Bedford, MA) membranes, and probed with the corresponding antibodies according to the manufacturer's guidelines. For immunoprecipitation of Myc-ERK2, Myc-Cbl, FLAG-Src, and HA-AKT equal amounts of cell lysate per condition were precipitated at 4 °C for 4–6 h in lysis buffer. Proteins were then resolved by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and probed with the indicated antibodies. In all cases, the results illustrated are from representative experiments, repeated at least three times. The Src, SrcS17A, and SrcS17D plasmids were all generated as previously described (13Schmitt J.M. Stork P.J.S. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The SrcY527F mutants were synthesized using PCR primers containing sequences corresponding to the 5′ end of the Src cDNA and sequences corresponding to the 3′ end of the Src cDNA, with the sequence corresponding to tyrosine 527 replaced with that for phenylalanine (Y527F). SrcWT, SrcS17A, and SrcS17D were amplified with these primers and subcloned into a FLAG-pcDNA3 to create SrcY527F, SrcS17A/Y527F, and SrcS17D/Y527F, respectively. Cbl-ct, encoding the carboxyl-terminal amino acids 541 to 906, was provided by Dr. Brian Druker (OHSU, Portland, OR). Hemagglutinin-tagged AKT (HA-AKT) was provided by Dr. Thomas Soderling (Vollum Institute, Portland, OR). The transducin (cone) cDNA was provided by the Guthrie cDNA Resource Center (www.guthrie.org/AboutGuthrie/Research/cDNA). Seventy to eighty percent confluent HEK293, SYF, or Src2+ cells were co-transfected with the indicated cDNAs using a LipofectAMINE 2000 kit (Invitrogen) according to the manufacturer's instructions. The control vector, pcDNA3 (Invitrogen Corp.), was included in each set of transfections to assure that each plate received the same amount of DNA. Following transfection, cells were allowed to recover in serum-containing media for 24 h. Cells were then starved overnight in serum-free Dulbecco's modified Eagle's medium before treatment and lysis. A GST fusion protein of the Rap1-binding domain of RalGDS (GST-RalGDS) was expressed inEscherichia coli following induction by isopropyl-1-thio-β-d-galactopyranoside (GST-RalGDS was a gift from Dr. Johannes Bos, Utrecht University, The Netherlands). Cells were grown as described and were stimulated at 37 °C for the indicated times and lysed in ice-cold lysis buffer (50 mmTris-Cl (pH 8.0), 10% glycerol, 1% Nonidet P-40, 200 mmNaCl, 2.5 mm MgCl2, 1 mmphenylmethylsulfonyl fluoride, 1 μm leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 mm NaF, 0.1 μmaprotinin, and 1 mm NaVO4). Active Rap1 was isolated as previously described by Franke et al. (15Franke B. Akkerman J.-W. Bos J.L. EMBO J. 1997; 16: 252-259Crossref PubMed Scopus (367) Google Scholar). Equivalent amounts of supernatants (500 μg) were incubated with the GST-RalGDS-Rap1 binding domain coupled to glutathione beads. Following a 1-h incubation at 4 °C, beads were pelleted and rinsed three times with ice-cold lysis buffer, proteins were eluted from the beads using 2× Laemmli buffer and applied to a 12% SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride membrane, blocked in 5% milk for 1 h, and probed with either α-Rap1/Krev-1 or FLAG antibody overnight at 4 °C, followed by incubation with an horseradish peroxidase-conjugated anti-rabbit IgG antibody (or an anti-mouse IgG for anti-FLAG Western blots). Proteins were detected using enhanced chemiluminescence. All experiments were repeated at least three times and representative gels are shown. HEK293 cells were grown as described, stimulated, and lysed in ice-cold lysis buffer. Activated Ras was assayed as previously described (7Schmitt J.M. Stork P.J.S. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (127) Google Scholar). Briefly, equivalent amounts of lysates from stimulated cells were incubated with GST-Raf1-RBD (Ras-binding domain) as specified by the manufacturer (Upstate Biotechnology, Lake Placid, NY). Proteins were eluted with 2× Laemmli buffer and applied to a 12% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane, blocked at room temperature for 1 h in 5% milk, and probed with either Ras or FLAG antibody overnight at 4 °C, followed by horseradish peroxidase-conjugated anti-mouse secondary antibodies. Proteins were detected using enhanced chemiluminescence. All experiments were repeated at least three times and representative gels shown. HEK293 cells were transfected using LipofectAMINE reagent with polyhistidine-tagged Rap1 (His-Rap1) as previously described (7Schmitt J.M. Stork P.J.S. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (127) Google Scholar, 9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Briefly, cells were lysed in ice-cold buffer containing 1% Nonidet P-40, 10 mm Tris, pH 8.0, 20 mm NaCl, 30 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, and 0.5 mg/ml aprotinin and supernatants were prepared by low speed centrifugation. Transfected His-tagged proteins were precipitated from supernatants containing equal amounts of protein using nickel-nitrilotriacetic acid-agarose and washed with 20 mm imidazole in lysis buffer and eluted with 500 mm imidazole and 5 mm EDTA in phosphate-buffered saline. The eluates containing His-tagged proteins were separated on SDS-PAGE and Raf-1 proteins were detected by Western blotting (7Schmitt J.M. Stork P.J.S. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (127) Google Scholar, 9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In HEK293 cells, isoproterenol activated endogenous Rap1 in a PKA-dependent manner, as previously shown (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Interestingly, this activation required Src family kinases (SFKs) as it was blocked by the inhibitor PP2 (16Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar) (Fig.1 A). Isoproterenol also activated endogenous Ras via SFKs, but this proceeded through a PKA-independent pathway (Fig. 1 B). To determine which SFK was mediating these effects, we utilized a pair of cell lines derived from mouse embryo fibroblasts that lack SFKs. One cell line, SYF, was developed from mice deficient in the genes encoding Yes, Fyn, and Src, and has been shown to lack SFK activity (17Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (647) Google Scholar). The second cell line, Src2+, originated from mice deficient in only Yes and Fyn and maintained the wild type Src gene and normal Src protein levels (17Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (647) Google Scholar). The ability of isoproterenol to activate Rap1 was absent in SYF cells (Fig. 2 A), but was retained in Src2+ cells (Fig. 2B). Similarly, the ability of isoproterenol to activate Ras was also absent in SYF cells (Fig. 2 C), but was retained in Src2+ cells (Fig.2 D). Additionally, both actions of isoproterenol could be reconstituted by transfecting wild type Src (SrcWT) into SYF cells (Fig. 2, A and C). Taken together, these data demonstrate that Src is required for activation by isoproterenol of both Ras and Rap1 in these cells. The ability of cAMP to activate Rap1 in selected cell types has recently been shown to require Src and PKA (13Schmitt J.M. Stork P.J.S. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), through the PKA-dependent phosphorylation of Src at serine 17 (Ser17). However, the requirement of Ser17 phosphorylation in hormonal signaling to Ras, Rap1, and ERKs has not been examined. To address the requirement of Ser17 phosphorylation in Ras and Rap1 signaling, we examined their activation by isoproterenol in cells expressing one of two SrcS17 mutants. Unlike SrcWT, the expression of a mutant Src, where Ser17 was replaced with an alanine (SrcS17A), was unable to reconstitute the activation by isoproterenol of Rap1 (Fig.2 A). Moreover, expression of a second Src mutant in which Ser17 was replaced with an aspartate (SrcS17D) resulted in constitutive activation of Rap1 (Fig. 2 A). This is similar to previous results from cells treated with forskolin, an activator of adenylyl cyclases (13Schmitt J.M. Stork P.J.S. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Importantly, this activation of Rap1 by SrcS17D was not further stimulated by isoproterenol, suggesting that SrcS17D was maximally activating Rap1. The expression of SrcS17A in Src2+ cells inhibited the activation by isoproterenol of Rap1 (Fig. 2 B), suggesting that SrcS17A was interfering with signals through endogenous Src in these cells. In contrast to the response in SYF cells, the activation of Rap1 by SrcS17D and isoproterenol in Src2+ cells was additive (Fig.2 B), presumably reflecting the contribution of the activation by isoproterenol of endogenous Src. Conversely, the activation by isoproterenol of Ras was not blocked by these mutants. In SYF cells, expression of SrcS17A reconstituted Ras activation by isoproterenol to a level similar to that seen using SrcWT (Fig. 2 C). Interestingly, in Src2+ cells, SrcS17A did not interfere with Ras activation by isoproterenol but appeared to enhance Ras activation in these cells (Fig. 2 D). On the other hand, SrcS17D was unable to activate Ras in either cell line (Fig. 2 C and D). These data suggest that SrcS17A was capable of mediating a Src-dependent pathway to Ras, demonstrating that the interference of Rap1 activation by SrcS17A in Src2+ cells was selective. Moreover, SrcS17D did not potentiate the stimulation by isoproterenol of Ras in Src2+cells (Fig. 2 D), suggesting that, unlike SrcS17A, SrcS17D alone could not participate efficiently in pathways to activate Ras. Surprisingly, SrcS17D could activate Ras to a moderate degree in SYF cells, but only in conjunction with isoproterenol (Fig. 2 C). This appears inconsistent with the selectivity of SrcS17D toward Rap1. One mechanism by which SrcS17D might function as an activator of Rap1 is to bind endogenous proteins that target Src toward Rap1. In this model, it might be expected that the selectivity of SrcS17D toward Rap1 would not be apparent if it was overexpressed. To examine further the finding that SrcS17D could participate in the activation by isoproterenol of Ras in SYF cells, we compared the effect of increasing concentrations of both transfected SrcWT and SrcS17D in these cells (Fig. 3). Increasing amounts of transfected SrcWT potentiated the activation by isoproterenol of Ras at all concentrations examined, especially at low to moderate doses (Fig.3). In contrast, the ability of SrcS17D to carry a signal from isoproterenol to Ras was only apparent at the highest level of expression examined. Therefore, whereas activation by SrcS17D of Rap1 was not further enhanced by isoproterenol, isoproterenol-dependent activation of Ras by SrcS17D was contingent on overexpression. These data are consistent with a model that SrcS17D interacts with an endogenous protein that channels Src toward a Rap1 pathway, but that when overexpressed, SrcS17D can act independently of this pathway. Next, we examined the mechanism of ERK activation by isoproterenol in HEK293 cells (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The ability of isoproterenol to activate Rap1 was modestly enhanced following transfection of SrcWT, but was completely blocked following transfection of SrcS17A (Fig.4 A). These data suggest that SrcS17A can interfere with the ability of endogenous Src to mediate the activation by isoproterenol of Rap1 in HEK293 cells. Expression of SrcS17D activated Rap1 constitutively in these cells, and this was not significantly enhanced by isoproterenol (Fig. 4 A). Upon its activation, Rap1 binds the effector B-Raf to activate ERKs (5Vossler M. Yao H. York R. Rim C. Pan M.-G. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar,9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). This recruitment of B-Raf has been used as an index of Rap1 activation in a variety of cell types (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 18Guo F. Kumahara E. Saffen D. J. Biol. Chem. 2001; 276: 25568-25581Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 19Liao Y. Satoh T. Gao X. Jin T.G., Hu, C.D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Klinger M. Kudlacek O. Seidel M. Freissmuth M. Sexl V. J. Biol. Chem. 2002; 277: 32490-32497Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The ability of isoproterenol to stimulate the association of B-Raf with Rap1 was blocked by SrcS17A. SrcS17D stimulated the association of B-Raf with Rap1, and this was modestly enhanced by isoproterenol (Fig.4 B). Taken together, these data demonstrate that phosphorylation of Ser17 is required for both Rap1 activation and function. It has previously been suggested that the activation by cAMP of Rap1 by forskolin requires Cbl and the Rap1 exchanger C3G, in NIH3T3 cells (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar,13Schmitt J.M. Stork P.J.S. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). To examine whether a similar mechanism might underlie signaling from GPCRs, we examined isoproterenol signaling in HEK293 cells expressing two interfering mutants: Cbl-ct, a carboxyl-terminal fragment that blocks Cbl function (13Schmitt J.M. Stork P.J.S. Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and CBR, a truncated protein containing the Crk-binding region of C3G that blocks C3G binding to Crk (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 21York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J.S. Nature. 1998; 392: 622-625Crossref PubMed Scopus (762) Google Scholar). The ability of isoproterenol to induce the recruitment by Rap1 of B-Raf was blocked by both Cbl-ct and CBR (Fig. 4 B). Isoproterenol also induced the association of Cbl with SrcWT but not SrcS17A (Fig. 4 C). SrcS17D induced this association in the absence of isoproterenol (Fig.4 C). Similarly, isoproterenol induced an association between C3G and transfected Cbl that was mimicked by SrcS17D, but not SrcS17A (Fig. 4 D). In addition, we detected an isoproterenol-dependent association between wild type Src and C3G that was also mimicked by SrcS17D (Fig. 4 E). This suggests that the association of Cbl/C3G with Src following isoproterenol stimulation was dependent on phosphorylation of Ser17. Mutation of tyrosine 527 of Src to phenylalanine (Y527F) produces a constitutively active (oncogenic) Src by eliminating the inhibitory phosphorylation at Tyr527 (22Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar, 23Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1086) Google Scholar). This mutant constitutively activated both Ras and Rap1 in HEK293 cells (Fig.5, A and B). However, introduction of S17A in SrcY527F created a new mutant (SrcS17A/Y527F) that was unable to activate Rap1, whereas SrcS17D/Y527F activated Rap1 constitutively (Fig. 5 A). Both SrcS17A/Y527F and SrcS17D/Y527F could activate Ras, suggesting that S17A selectively interfered with oncogenic activation by Src of Rap1. In addition, both Cbl-ct and CBR interfered with Rap1 activation, but had no effect on Ras activation, suggesting that the action of endogenous Cbl and C3G were specific for Rap1. The inability of SrcS17A to interfere with Ras signaling was seen by examining hormonally activated Src in HEK293 cells. Both SrcWT and SrcS17A, but not by SrcS17D, modestly enhanced the activation by isoproterenol of Ras (Fig.6 A). Ras activation by isoproterenol is thought to be mediated via Gβγ (10Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1077) Google Scholar, 24Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). This was confirmed by experiments in HEK293 cells that showed that the activation by isoproterenol of Ras was blocked by expression of a truncated β-adrenergic receptor kinase (βARK-ct) (Fig.6 A), and by expression of transducin, a retinal-specific Gαs subunit (Fig. 6 B). Both βARK-ct and transducin block signals generated from βγ subunits by binding to endogenous βγ (2Birnbaumer L. Cell. 1992; 71: 10069-10072Abstract Full Text PDF Scopus (380) Google Scholar, 25Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar). As a control, we show that EGF activation of Ras was not blocked by expression of transducin (Fig. 6 B). In contrast, transducin did not block Rap1 activation by isoproterenol (Fig. 6 C). Taken together, these data suggest that while Ras and Rap1 are both activated by Src-dependent mechanisms downstream of the β-adrenergic receptor, only Ras activation involves Gβγ. To examine the downstream consequences of Src-dependent signaling in HEK293 cells, we measured ERK activation, using pERK antibodies (pERK1/2). Activation by isoproterenol of ERKs required both PKA and SFKs, as phosphorylation of ERK was prevented by both H89 and PP2 (Fig. 7 A). Similar results were seen using forskolin (Fig. 7 B). In contrast, the phosphoinositol 3-kinase (PI3K) inhibitor, LY294002, did not block ERK phosphorylation (Fig. 7 A). Expression of SrcS17A blocked the activation by isoproterenol of ERKs (Fig.8 A), and SrcS17D constitutively activated ERKs (Fig. 8 B), consistent with a model that the activation by Src of Rap1 was necessary and sufficient for the activation by isoproterenol of ERKs in these cells. Moreover, isoproterenol was unable to further activate ERKs in SrcS17D-expressing cells, suggesting that the phosphorylation of SrcS17 was the predominant mode of ERK activation by isoproterenol.Figure 8Activation by isoproterenol of ERKs requires serine 17 phosphorylation of Src. A, PKA phosphorylation of Src on serine 17 is necessary for ERK activation by cAMP. HEK293 cells were transfected with Myc-ERK2 in the presence or absence of SrcS17A, and stimulated with isoproterenol, as indicated. Myc-ERK2 was immunoprecipitated from cell lysates using an agarose-coupled Myc antibody followed by Western blotting for phospho-ERK (pMycERK2, upper panel) or total Myc-ERK2 with a Myc antibody, as a control for protein loading (lower panel). B, expression of SrcS17D mimics the activation by isoproterenol of ERKs. HEK293 cells were transfected with Myc-ERK2 in the presence or absence of SrcS17D, and stimulated with isoproterenol, as indicated. Cell lysates were examined as inA for phosphorylated pMyc-ERK2 and expression of pMyc-ERK2.View Large Image Figure ViewerDownload (PPT) Previously, we and others have suggested that Ras was not required for ERK activation by isoproterenol in HEK293 cells (9Schmitt J.M. Stork P.J.S. J. Biol. Chem. 2000; 275: 25342-25350Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 20Klinger M. Kudlacek O. Seidel M. Freissmuth M. Sexl V. J. Biol. Chem. 2002; 277: 32490-32497Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This is despite the fact that Ras is activated by isoproterenol (Fig. 6,A and B). To examine the physiological role of Ras signaling in these cells, we examined a well known Ras effector, PI3K and its target, AKT (26Downward J. Curr.
Referência(s)