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Induction of Rac-Guanine Nucleotide Exchange Activity of Ras-GRF1/CDC25Mm following Phosphorylation by the Nonreceptor Tyrosine Kinase Src

2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês

10.1074/jbc.275.8.5441

ISSN

1083-351X

Autores

Mari Kiyono, Yoshito Kaziro, Takaya Satoh,

Tópico(s)

Nitric Oxide and Endothelin Effects

Resumo

Ras-GRF1/CDC25Mm has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca2+ influx and G protein βγ subunit (Gβγ) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gβγ-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gβγ. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gβγ to regulate Ras-GRF1. Ras-GRF1/CDC25Mm has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca2+ influx and G protein βγ subunit (Gβγ) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gβγ-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gβγ. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gβγ to regulate Ras-GRF1. guanine nucleotide exchange factor Dbl homology dithiothreitol G protein βγ subunit(s) glutathioneS-transferase p21-activated kinase platelet-derived growth factor pleckstrin homology polyacrylamide gel electrophoresis Cdc42/Rac interactive binding The GDP/GTP state of low molecular weight GTP-binding proteins is regulated by guanine nucleotide exchange factors (GEFs)1 and GTPase-activating proteins. GEFs, when activated by upstream signals, stimulate GDP/GTP exchange reaction of a distinct set of GTP-binding proteins, leading to the accumulation of the active GTP-bound form within the cell. Activation of GEFs occurs by diverse mechanisms including phosphorylation, direct interaction with other proteins, and in some cases, subsequent translocation to the plasma membrane. The catalytic domain conserved among GEFs that target Ras is designated the CDC25 homology domain, which was originally identified as a region in the Saccharomyces cerevisiae Cdc25 protein essential for the function as an upstream regulator of Ras (1.Powers S. Semin. Cancer Biol. 1992; 3: 209-218PubMed Google Scholar). To date, mSos1/2 (2.Bowtell D. Fu P. Simon M. Senior P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6511-6515Crossref PubMed Scopus (239) Google Scholar,3.Chardin P. Camonis J.H. Gale N.W. Van Aelst L. Schlessinger J. Wigler M.H. Bar-Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (658) Google Scholar), Ras-GRF1/2 (4.Cen H. Papageorge A.G. Zippel R. Lowy D.R. Zhang K. EMBO J. 1992; 11: 4007-4015Crossref PubMed Scopus (97) Google Scholar, 5.Martegani E. Vanoni M. Zippel R. Coccetti P. Brambilla R. Ferrari C. Sturani E. Alberghina L. EMBO J. 1992; 11: 2151-2157Crossref PubMed Scopus (189) Google Scholar, 6.Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar, 7.Wei W. Mosteller R.D. Sanyal P. Gonzales E. McKinney D. Dasgupta C. Li P. Liu B. Broek D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7100-7104Crossref PubMed Scopus (74) Google Scholar, 8.Fam N.P. Fan W. Wang Z. Zhang L. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar), and Ras-GRP (9.Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (550) Google Scholar, 10.Tognon C.E. Kirk H.E. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (205) Google Scholar) have been characterized as a mammalian Ras-GEF containing the CDC25 homology domain. Particularly, the role of mSos in the regulation of Ras downstream of a variety of receptors including tyrosine kinase-type, cytokine, and G protein-coupled receptors has been clarified in detail. Virtually all types of receptors trigger the activation of specific tyrosine kinases, which in turn phosphorylate the receptor subunit itself and/or downstream molecules. Subsequently, a diverse array of proteins become associated with the tyrosine-phosphorylated receptor, thereby stimulating multiple signaling pathways. Recruitment of mSos complexed with the adaptor protein Grb2 to the tyrosine-phosphorylated receptor at the plasma membrane is considered to be a crucial step for the onset of Ras activation (11.Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (343) Google Scholar). Ras-GRFs contain various motifs in addition to the C-terminally located CDC25 homology domain, including the N-terminal pleckstrin homology (PH) domain, an IQ motif, a Dbl homology (DH) domain contiguous to the second PH domain (4.Cen H. Papageorge A.G. Zippel R. Lowy D.R. Zhang K. EMBO J. 1992; 11: 4007-4015Crossref PubMed Scopus (97) Google Scholar, 5.Martegani E. Vanoni M. Zippel R. Coccetti P. Brambilla R. Ferrari C. Sturani E. Alberghina L. EMBO J. 1992; 11: 2151-2157Crossref PubMed Scopus (189) Google Scholar, 6.Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar, 7.Wei W. Mosteller R.D. Sanyal P. Gonzales E. McKinney D. Dasgupta C. Li P. Liu B. Broek D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7100-7104Crossref PubMed Scopus (74) Google Scholar). Ras-GRF1 is reported to regulate Ras activity in response to Ca2+ influx, for which binding of calmodulin to the IQ motif is crucial (12.Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (393) Google Scholar). On the other hand, Ca2+-responsive, Ras-independent as well as constitutive, Ras-dependent pathways downstream of Ras-GRF1 were shown to direct Raf and extracellular signal-regulated kinase activities (13.Anborgh P.H. Qian X. Papageorge A.G. Vass W.C. DeClue J.E. Lowy D.R. Mol. Cell. Biol. 1999; 19: 4611-4622Crossref PubMed Scopus (70) Google Scholar). G protein βγ subunits (Gβγ) also stimulate Ras-GEF activity of Ras-GRF1 through serine phosphorylation (14.Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar, 15.Mattingly R.R. Saini V. Macara I.G. Cell Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar). Binding of Gβγ to the N-terminal PH domain of Ras-GRF1 was demonstrated (16.Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar), although it is unclear whether the binding is critical for Gβγ induction of Ras-GEF activity. Recently, oligomerization of Ras-GRFs through their DH domain was reported (13.Anborgh P.H. Qian X. Papageorge A.G. Vass W.C. DeClue J.E. Lowy D.R. Mol. Cell. Biol. 1999; 19: 4611-4622Crossref PubMed Scopus (70) Google Scholar). Oligomerization seems to be required for biological functions, because a mutation within the DH domain that abolishes oligomerization rendered Ras-GRFs incapable of inducing transformation of NIH 3T3 cells. Ras-GRF1 is expressed exclusively in brain, suggesting an important role in brain-specific signaling. Disruption of Ras-GRF1, in fact, resulted in defects in brain functions such as memory consolidation (17.Brambilla R. Gnesutta N. Minichiello L. White G. Roylance A.J. Herron C.E. Ramsey M. Wolfer D.P. Cestari V. Rossi-Arnaud C. Grant S.G.N. Chapman P.F. Lipp H. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (399) Google Scholar). In contrast, Ras-GRF2 is expressed not only in brain but also in several other tissues such as lung and spleen, suggesting a role distinct from that of Ras-GRF1 (8.Fam N.P. Fan W. Wang Z. Zhang L. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). Ras-GRP is expressed in nervous and hematopoietic cells (9.Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (550) Google Scholar, 10.Tognon C.E. Kirk H.E. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (205) Google Scholar). Besides the catalytic CDC25 homology domain, a pair of EF-hands, where Ca2+ binds, and the diacylglycerol-binding C1 domain were identified in Ras-GRP. Whereas deletion of the C1 domain eliminated the transforming activity of Ras-GRP, the EF-hands seemed to be dispensable (9.Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (550) Google Scholar, 10.Tognon C.E. Kirk H.E. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (205) Google Scholar). Consistent with this, Ras·GTP formation was enhanced by a diacylglycerol analog in the presence of Ras-GRP, suggesting an involvement of Ras-GRP in diacylglycerol-mediated activation of the Ras pathway (9.Ebinu J.O. Bottorff D.A. Chan E.Y.W. Stang S.L. Dunn R.J. Stone J.C. Science. 1998; 280: 1082-1086Crossref PubMed Scopus (550) Google Scholar). The Rho family of GTP-binding proteins consisting of Rho, Rac, and Cdc42 regulates various physiological responses through actin cytoskeleton rearrangements (18.Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2097) Google Scholar, 19.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar). Like Ras, Rho family proteins have been implicated as a molecular switch downstream of a wide variety of receptors. The activation of Rho family proteins in response to extracellular stimuli is thought to be directed by Rho family-specific GEFs. GEFs that act on Rho family proteins constitute the Dbl family, whose members possess DH and PH domains in tandem (20.Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar). The DH domain is responsible for catalysis, whereas the PH domain is believed to be implicated in membrane targeting and regulatory functions. Some Dbl family members are specific for an individual Rho family protein, whereas others act on all three subgroups. Although more than 20 Dbl family members have been identified, mechanisms underlying extracellular signal-dependent regulation of Dbl family GEFs remain largely unknown. Interestingly, mSos and Ras-GRFs possess the DH/PH domains as well, implying a role as a GEF for Rho family proteins. Indeed, mSos exhibits Rac-GEF activity when stimulated downstream of the Ras/phosphatidylinositol 3-kinase pathway, although Rac-GEF activity remains latent without upstream signals (21.Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (389) Google Scholar). Additionally, Ras-GRF1 functions as a Rac-GEF in response to signals mediated by Gβγ (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar), while Ras-GRF2 shows constitutive and Ca2+-stimulated Rac-GEF activity (23.Fan W. Koch C.A. de Hoog C.L. Fam N.P. Moran M.F. Curr. Biol. 1998; 8: 935-938Abstract Full Text Full Text PDF PubMed Google Scholar). Tyrosine phosphorylation of Ras-GRF1 has been implicated as a crucial step for the induction of Rac-GEF activity (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar). In this paper, we describe that Src elicits tyrosine phosphorylation of Ras-GRF1 in living cells and in a cell-free system, allowing Ras-GRF1 to act as a GEF for Rac. GEF activity toward other Rho family members, Rho and Cdc42, remained undetectable upon Src-dependent tyrosine phosphorylation. Ras-GEF activity was unaffected when phosphorylated by Src. Considering the observation that Gβγ induces Rac-specific GEF activity of Ras-GRF1 in a tyrosine phosphorylation-dependent manner, Src may be involved in G protein-coupled receptor stimulation of Ras-GRF1. The purified recombinant c-Src protein was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies including anti-Ras-GRF antibody (sc-226) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Rac1 antibody (R56220) (Transduction Laboratories), anti-phosphotyrosine antibody PY99 (sc-7020) (Santa Cruz Biotechnology), anti-Myc antibody 9E10 (BAbCO), anti-FLAG antibody M2 (Eastman Kodak Co.), anti-mouse Ig antibody (55480) (Cappel), and horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham Pharmacia Biotech) were purchased from commercial suppliers. PP2 and human platelet-derived growth factor (PDGF) BB were purchased from Calbiochem and R & D Systems, respectively. Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum. Transfection of 293 cells with expression plasmids was carried out as described previously (24.Satoh T. Kato J. Nishida K. Kaziro Y. FEBS Lett. 1996; 386: 230-234Crossref PubMed Scopus (49) Google Scholar). The rat Ras-GRF1 cDNA was a generous gift from Larry Feig (Tufts University School of Medicine, Boston, MA) (6.Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (290) Google Scholar). The cDNA encoding full-length Ras-GRF1 tagged with a c-Myc epitope at the N-terminal end was constructed by the polymerase chain reaction and subcloned into the mammalian expression vector pCMV5 (25.Andersson S. Davis D.L. Dahlbäck H. Jörnvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar), generating pCMV5-Myc-Ras-GRF1. pCMV5-Myc-Ras-GRF1(ΔN582) (which encodes c-Myc sequence and amino acids 583–1244 of Ras-GRF1) was generated in a similar way. pCMV5-FLAG-Ras-GRF1 was described previously (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar). pCMV5-FLAG-Ras-GRF1(N625) was constructed by subcloning an EcoRI–EcoRI fragment (which encodes FLAG sequence and amino acids 1–625 of Ras-GRF1) into pCMV5. Expression plasmids for Gβ1 and Gγ2 (pCMV5-β1 and pCMV5-γ2, respectively) were previously described (26.Ito A. Satoh T. Kaziro Y. Itoh H. FEBS Lett. 1995; 368: 183-187Crossref PubMed Scopus (54) Google Scholar). The cDNA encoding v-Src (kindly provided by Yasuo Fukami (Kobe University, Kobe, Japan)) was also subcloned into pCMV5. The expression plasmid for the wild-type PDGF receptor β was kindly provided by Lewis Williams (Chiron Corp.) (27.Satoh T. Fantl W.J. Escobedo J.A. Williams L.T. Kaziro Y. Mol. Cell. Biol. 1993; 13: 3706-3713Crossref PubMed Scopus (87) Google Scholar). For the expression of glutathione S-transferase (GST) fusion proteins in Escherichia coli, cDNAs encoding Ras-GRF1, Ras-GRF1(ΔN582), Rac1, RhoA, and Cdc42 were subcloned into pGEX-2T (Amersham Pharmacia Biotech). pGEX-PAK-CRIB (an expression plasmid for the c-Myc-tagged Cdc42/Rac interacting binding (CRIB) domain (amino acids 67–150) of rat α p21-activated kinase (PAK) fused to GST) (28.Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar) was kindly provided by Kenji Tago. All polymerase chain reaction products were sequenced to confirm the primary structure of encoded proteins. TheE. coli strain BL21 was transformed with the expression plasmid for each GST fusion protein. Protein expression was induced for 4 h with 0.5 mmisopropyl-1-thio-β-d-galactoside at 30 °C. Harvested cells were suspended and sonicated in lysis buffer A (8.1 mm Na2HPO4, 1.45 mmKH2PO4, 137 mm NaCl, 2.7 mm KCl, 5 mm EDTA, 150 mm(NH4)2SO4, 10% (v/v) glycerol, 0.1 mm phenylmethylsulfonyl fluoride, and 10 mmdithiothreitol (DTT)) and subjected to centrifugation at 23,000 ×g for 30 min. Supernatants were applied to a glutathione-Sepharose (Amersham Pharmacia Biotech) column, which was subsequently washed with GST wash buffer (20 mm Tris-HCl (pH 7.5), 2 mm MgCl2, 1 mm DTT). GST fusion proteins were eluted with glutathione buffer (50 mm Tris-HCl (pH 8.0), 10 mm glutathione), and dialyzed against dialysis buffer (25 mm Hepes-NaOH (pH 7.4), 20% (v/v) glycerol, 0.1 mm DTT). After adding 1 mm DTT, samples were stored at −80 °C. Recombinant Rac1, RhoA, and Cdc42 were overexpressed inE. coli as a GST fusion protein and purified by a glutathione-Sepharose column. GST was subsequently removed by digestion with thrombin, and GTP-binding proteins were further purified as described (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar). The recombinant Ha-Ras protein was described elsewhere (29.Satoh T. Nakamura S. Nakafuku M. Kaziro Y. Biochim. Biophys. Acta. 1988; 949: 97-109Crossref PubMed Scopus (38) Google Scholar). Cells were dissolved in immunoprecipitation buffer (20 mm Tris-HCl (pH 7.5), 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, 0.5% (v/v) Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 0.1 mmNa3VO4, 3 mm β-glycerophosphate), and the supernatant of centrifugation (15,000 × g) for 10 min at 4 °C was used as a cell lysate. The anti-FLAG antibody M2 (2 μg) or the anti-Myc antibody 9E10 (2 μg) was mixed with a rabbit anti-mouse Ig antibody conjugated to protein A-Sepharose (Amersham Pharmacia Biotech). Lysates were mixed gently with the antibody·protein A-Sepharose complex for 2 h at 4 °C, and precipitates were washed four times with immunoprecipitation buffer. Precipitated proteins were subsequently separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was stained with the anti-phosphotyrosine antibody PY99 and a horseradish peroxidase-conjugated anti-mouse IgG antibody, followed by visualization by enhanced chemiluminescence detection reagents (NEN Life Science Products). GST-Ras-GRF1 and GST-Ras-GRF1(ΔN582) fusion proteins (1 μg) were incubated for 15 min at 25 °C in the presence or absence of 2 units of the recombinant c-Src protein. Assays were carried out in kinase reaction buffer (50 mm Hepes-NaOH (pH 7.3), 5 mmMgCl2, 5 mm MnCl2) containing 10% (v/v) of buffer A (20 mm Hepes-NaOH (pH 7.3), 5 mm MgCl2, 2 mm EGTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 2 mm DTT, 2 μg/ml aprotinin) and 10 μm[γ-32P]ATP (1,850 TBq/mol) or nonradiolabeled ATP. Phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography or immunoblotting using the anti-phosphotyrosine antibody PY99, respectively. GDP/GTP exchange assays were performed essentially as described (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar). 293 cells transfected with various combinations of plasmids were serum-starved for 24 h. Cells were rinsed with ice-cold phosphate-buffered saline (8.1 mm Na2HPO4, 1.45 mmKH2PO4, 137 mm NaCl, 2.7 mm KCl), dissolved in radioimmune precipitation buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 0.1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mm Na3VO4, 50 mm NaF, 50 mm β-glycerophosphate, 20 mm Na4P2O7), and centrifuged at 15,000 × g for 10 min. Subsequently, the anti-Myc antibody 9E10 (2 μg) and a rabbit anti-mouse Ig antibody conjugated to protein A-Sepharose were added to cell lysates, followed by incubation for 1 h at 4 °C with gentle mixing. Immunoprecipitates were washed twice with radioimmune precipitation buffer and twice with exchange buffer (10 mm Hepes-NaOH (pH 7.4), 5 mm MgCl2, 5 mm KCl, 1 mm EGTA) and subjected to GDP/GTP exchange assays. For [3H]GDP binding assays, immunoprecipitated proteins were incubated with Rac1 (200 ng) or Ha-Ras (40 ng) at 30 °C in exchange buffer supplemented with 2 mm DTT, 0.2 mg/ml bovine serum albumin, 1 mm ATP, and 1 μm[3H]GDP (1,265 TBq/mol). After incubation for specified periods, ice-cold wash buffer (10 mm Tris-HCl (pH 7.5), 10 mm MgCl2) was added, and samples were filtered through a nitrocellulose membrane, which was subjected to extensive washing with wash buffer. Radioactivity remaining on the filter was quantitated by the liquid scintillation counter. For [3H]GDP release assays, the small GTP-binding protein·[3H]GDP complex was prepared by incubation of each small GTP-binding protein in exchange buffer supplemented with 2 mm DTT, 5 mm EDTA, 0.2 mg/ml bovine serum albumin, 1 mm ATP, and 1 μm[3H]GDP (1,265 TBq/mol) for 90 min at 30 °C. 5 mm MgCl2 was added to terminate GDP/GTP exchange reaction. Immunoprecipitated proteins were incubated with the small GTP-binding protein·[3H]GDP complex at 30 °C in exchange buffer supplemented with 2 mm DTT, 0.2 mg/ml bovine serum albumin, 1 mm ATP, and 10 mm GDP. After incubation for specified periods, radioactivity remaining in the complex was quantitated by filter binding assays as described above. Phosphorylated or unphosphorylated GST-Ras-GRF1 was obtained by preincubation of GST-Ras-GRF1 with or without the recombinant c-Src protein in the presence of 10 μm nonradiolabeled ATP under conditions described above and subjected to GDP/GTP exchange assays. Affinity precipitation of Rac1 using GST-PAK-CRIB was performed essentially as described elsewhere (28.Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). E. coli HB101 cells transformed with pGEX-PAK-CRIB were grown at 25 °C to early logarithmic phase. Expression of GST-PAK-CRIB was induced by 1 mmisopropyl-1-thio-β-d-galactoside for 20 h at 25 °C. Harvested cells were suspended in lysis buffer B (50 mm Tris-HCl (pH 7.5), 20 mm MgCl2, 150 mm NaCl, 0.5% (v/v) Nonidet P-40, 20 μg/ml aprotinin, 1 mm Na3VO4) and disrupted by sonication. Disrupted cells were centrifuged at 25,000 × g for 20 min at 4 °C, and the supernatant was stored at −80 °C. Glutathione-Sepharose beads (Amersham Pharmacia Biotech) were mixed with the lysate containing 10 μg of GST-PAK-CRIB for 30 min at 4 °C and washed three times with buffer containing 50 mm Tris-HCl (pH 7.5), 20 mmMgCl2, and 150 mm NaCl. 293 cells were lysed in lysis buffer C (50 mm Tris-HCl (pH 7.5), 10 mmMgCl2, 200 mm NaCl, 2% (v/v) Nonidet P-40, 10% (v/v) glycerol, 2 mm phenylmethylsulfonyl fluoride, 1 mm DTT, 2 μg/ml each leupeptin and aprotinin, and 2 mm Na3VO4), and centrifuged at 15,000 × g for 10 min at 4 °C. Supernatants were incubated for 1 h at 4 °C with GST-PAK-CRIB conjugated to glutathione-Sepharose beads. Subsequently, Sepharose beads were washed three times with buffer containing 25 mm Tris-HCl (pH 7.5), 30 mm MgCl2, 40 mm NaCl, 1% (v/v) Nonidet P-40, and 1 mm DTT and twice with the same buffer without Nonidet P-40. Precipitated Rac1 was detected by immunoblotting using a monoclonal antibody against Rac1. As a first step to manifest a possible involvement of Src in the regulation of Ras-GRF1, Src-dependent tyrosine phosphorylation of Ras-GRF1 was examined by the use of a transient expression system in 293 cells. FLAG epitope-tagged full-length Ras-GRF1 was highly tyrosine-phosphorylated when co-expressed with v-Src as shown in Fig.1 A. The level of Ras-GRF1 tyrosine phosphorylation in response to v-Src as evaluated by immunoblotting using an anti-phosphotyrosine antibody was considerably higher than that induced by Gβγ co-expression (data not shown). A similar construct consisting of the N-terminal 625 amino acids (N625), which contains all known regulatory motifs located within the N-terminal portion, was also tyrosine-phosphorylated in a v-Src-dependent manner (Fig. 1 A). In addition, the Myc epitope-tagged N-terminally truncated mutant ΔN582, which contains only the C-terminally located CDC25 homology domain, and full-length Ras-GRF1 were tyrosine-phosphorylated by v-Src as illustrated in Fig. 1 B. Overall, multiple tyrosine residues seemed to become tyrosine-phosphorylated upon co-expression of v-Src. To show Src-dependent tyrosine phosphorylation of Ras-GRF1 under more physiological conditions, we examined the effect of PDGF because it is well known that Src acts downstream of the PDGF receptor (30.Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell. 1990; 62: 481-492Abstract Full Text PDF PubMed Scopus (480) Google Scholar, 31.Claesson-Welsh L. J. Biol. Chem. 1994; 269: 32023-32026Abstract Full Text PDF PubMed Google Scholar). The β-type PDGF receptor was transiently expressed with FLAG-tagged Ras-GRF1 in 293 cells, which were subsequently stimulated with PDGF. As illustrated in Fig. 1 C, PDGF stimulation induced tyrosine phosphorylation of Ras-GRF1, which was sensitive to the Src-specific inhibitor PP2. In contrast, tyrosine phosphorylation of the PDGF receptor remained unaffected upon PP2 treatment. Therefore, Src may be implicated as a kinase responsible for tyrosine phosphorylation of Ras-GRF1. On the basis of our previous observations that Gβγ induce Rac-GEF activity of Ras-GRF1 in a tyrosine phosphorylation-dependent manner, we next investigated the effect of Src-induced tyrosine phosphorylation on Rac-GEF activity. Myc epitope-tagged full-length Ras-GRF1 was immunoprecipitated from cells transfected with various combinations of expression plasmids, followed by Rac-GEF assays carried out essentially as described previously (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar). Binding of GDP to recombinant Rac1 and release of GDP from the Rac1·GDP complex under various conditions are shown in Fig. 2, A andB, respectively. Expression levels of Ras-GRF1 in individual transfectants were virtually the same as determined by immunoblotting (data not shown). As described previously, Ras-GRF1 recovered from Gβγ-co-expressing cells significantly enhances GDP binding to Rac1, whereas Ras-GRF1 exhibits virtually no Rac-GEF activity when immunoprecipitated from cells ectopically expressing Ras-GRF1 alone (Fig. 2 A). Ras-GRF1 from v-Src-co-expressing cells stimulated GDP binding to Rac1 like Gβγ-stimulated Ras-GRF1 (Fig.2 A). Consistent with this, the release rate of Rac1-bound GDP became enhanced following co-expression of v-Src as in the case of Gβγ (Fig. 2 B). Co-expression of Gβγ and v-Src did not result in additive increase in Rac-GEF activity (data not shown). To further investigate the activation of Rac1 within the cell, we employed a pull-down assay by the use of GST-PAK-CRIB as a probe (28.Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). As shown in Fig. 2 C, co-expression of Ras-GRF1 with v-Src resulted in synergistically enhanced formation of Rac·GTP. Fig. 3 shows the effect of Src-dependent tyrosine phosphorylation on GEF activity toward other Rho family members, RhoA and Cdc42. Relative amounts of GDP remaining complexed with respective GTP-binding proteins after incubation with v-Src-stimulated Ras-GRF1 for 60 min are shown. RhoA and Cdc42 were insensitive to Ras-GRF1 stimulated by co-expression of v-Src in contrast with Rac1. These results are analogous to previous observations that co-expression of Gβγ conferred GEF activity toward Rac1, but neither RhoA nor Cdc42, to Ras-GRF1 (22.Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (77) Google Scholar). Furthermore, the effect of v-Src on GEF activity toward Ha-Ras was examined (Fig. 4). Myc epitope-tagged Ras-GRF1 was immunoprecipitated and subjected to GDP binding assays for Ha-Ras. Ras-GRF1 exhibited constitutive Ras-GEF activity, which was not promoted by co-expression of v-Src in contrast to Gβγ (14.Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar, 15.Mattingly R.R. Saini V. Macara I.G. Cell Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar). Full-length Ras-GRF1 and its N-terminally deleted version ΔN582 were yielded inE. coli as GST fusion proteins, which were partially purified by using standard glutathione-Sepharose column chromatography. These recombinant Ras-GRF1 proteins were subjected to in vitro kinase assays using the recombinant c-Src protein. As illustrated in Fig. 5 A, both full-length and N-terminally deleted Ras-GRF1 were phosphorylated in a Src-dependent manner. Incorporation of 32P into the GST protein was not detected under similar conditions (data not shown). Lower molecular weight proteins that were phosphorylated by the recombinant c-Src protein are degradation products. To further confirm tyrosine phosphorylation in vitro, reaction mixtures without radioactive ATP were subjected to SDS-PAGE and immunoblotting using an

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