Phosphorylation of Serine 916 of Ras-GRF1 Contributes to the Activation of Exchange Factor Activity by Muscarinic Receptors
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.37379
ISSN1083-351X
Autores Tópico(s)Ion channel regulation and function
ResumoThe Ras-GRF1 exchange factor is strongly implicated in the control of neuronal Ras. The activity of Ras-GRF1 is regulated by increases in intracellular calcium and the release of Gβγ subunits from heterotrimeric G-proteins. Increases in Ras-GRF1 activity toward Ras that are stimulated by receptors coupled to G-proteins are associated with enhanced phosphorylation of Ras-GRF1 on one or more serine residues. Co-expression of Ras-GRF1 with subtype 1 human muscarinic receptors in COS-7 cells allowed mapping of a carbachol-stimulated phosphorylation site to a region composed of residues 916–976. Site-directed mutagenesis replaced each of the serine residues within this region with alanine and demonstrated that serine 916 is a major site of in vivo phosphorylation of Ras-GRF1 in both COS-7 cells and NIH-3T3 fibroblasts. Serine 916 was a substrate for protein kinase A both in vivo and in vitro, suggesting a novel link between the cAMP and Ras signaling systems. Carbachol-dependent phosphorylation of serine 916 occurred through a protein kinase A-independent pathway, however. Full-length Ras-GRF1 that contains an alanine 916 mutation was only partially activated by carbachol, suggesting that phosphorylation at residue 916 is necessary for full activation. Phosphorylation of serine 916 in response to forskolin treatment did not, however, increase the activity of Ras-GRF1, indicating that it is not sufficient for activation. The Ras-GRF1 exchange factor is strongly implicated in the control of neuronal Ras. The activity of Ras-GRF1 is regulated by increases in intracellular calcium and the release of Gβγ subunits from heterotrimeric G-proteins. Increases in Ras-GRF1 activity toward Ras that are stimulated by receptors coupled to G-proteins are associated with enhanced phosphorylation of Ras-GRF1 on one or more serine residues. Co-expression of Ras-GRF1 with subtype 1 human muscarinic receptors in COS-7 cells allowed mapping of a carbachol-stimulated phosphorylation site to a region composed of residues 916–976. Site-directed mutagenesis replaced each of the serine residues within this region with alanine and demonstrated that serine 916 is a major site of in vivo phosphorylation of Ras-GRF1 in both COS-7 cells and NIH-3T3 fibroblasts. Serine 916 was a substrate for protein kinase A both in vivo and in vitro, suggesting a novel link between the cAMP and Ras signaling systems. Carbachol-dependent phosphorylation of serine 916 occurred through a protein kinase A-independent pathway, however. Full-length Ras-GRF1 that contains an alanine 916 mutation was only partially activated by carbachol, suggesting that phosphorylation at residue 916 is necessary for full activation. Phosphorylation of serine 916 in response to forskolin treatment did not, however, increase the activity of Ras-GRF1, indicating that it is not sufficient for activation. guanine nucleotide exchange factor subtype 1 human muscarinic receptors cAMP-dependent protein kinase polyacrylamide gel electrophoresis glutathione S-transferase isobutylmethyl xanthine protein kinase inhibitor N-[Tris-(hydroxymethyl)methyl]glycine The Ras GTPases are known to play central roles in pathways of cellular growth and differentiation (1Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3758) Google Scholar). Their function in terminally differentiated cells such as neurons is less clear, but they have been suggested to participate in learning and memory (2Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar). As timed, molecular switches they cycle between active (GTP-bound) and inactive (GDP-bound) conformations under the control of guanine nucleotide exchange factors (GEFs)1 and GTPase-activating proteins (3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1751) Google Scholar). The balance of the effective GEF and GTPase-activating protein activities determines the activation state of Ras proteins. Regulation of the activity or subcellular localization of a specific GEF is now recognized as the major control for Ras activity in many instances (4Downward J. Nature. 1998; 396: 416-417Crossref PubMed Scopus (36) Google Scholar).The Ras-GRF1 exchange factor (5Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar), which was also previously known as CDC25Mm (6Cen H. Papageorge A.G. Vass W.C. Zhang K. Lowy D.R. Mol. Cell. Biol. 1993; 13: 7718-7724Crossref PubMed Scopus (46) Google Scholar, 7Martegani E. Vanoni M. Zippel R. Coccetti P. Brambilla R. Ferrari C. Sturani E. Alberghina L. EMBO J. 1992; 11: 2151-2157Crossref PubMed Scopus (188) Google Scholar), is implicated in neurotransmission. Ras-GRF1 is highly expressed in neurons of the central nervous system (8Wei W. Schreiber S.S. Baudry M. Tocco G. Broek D. Mol. Brain Res. 1993; 19: 339-344Crossref PubMed Scopus (27) Google Scholar, 9Ferrari C. Zippel R. Martegani E. Gnesutta N. Carrera V. Sturani E. Exp. Cell Res. 1994; 210: 353-357Crossref PubMed Scopus (30) Google Scholar, 10Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar) and is present in postsynaptic densities (11Sturani E. Abbondio A. Branduardi P. Ferrari C. Zippel R. Martegani E. Vanoni M. Denis-Donini S. Exp. Cell Res. 1997; 235: 117-123Crossref PubMed Scopus (55) Google Scholar). Mice that lack the expression of Ras-GRF1 have defects in the consolidation of memory (12Brambilla 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.-P. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (397) Google Scholar) and in postnatal growth (13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar). The latter may be due to a decrease in circulating insulin-like growth factor-1 levels that is secondary to changes in the hypothalamus (13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar).In contrast to the Sos exchange factors, which couple tyrosine kinase-derived signals to the activation of Ras (14Buday L. Downward J. Mol. Cell. Biol. 1993; 13: 1903-1910Crossref PubMed Scopus (106) Google Scholar, 15Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1003) Google Scholar), Ras-GRF1 links heterotrimeric G-proteins (16Shou C. Wurmser A. Ling K. Barbacid M. Feig L.A. Oncogene. 1995; 10: 1887-1893PubMed Google Scholar, 17Zippel R. Orecchia S. Sturani E. Martegani E. Oncogene. 1996; 12: 2697-2703PubMed Google Scholar, 18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar, 19Mattingly R.R. Saini V. Macara I.G. Cell. Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar) and calcium signals (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (390) Google Scholar) to Ras. Further, regulation through Sos occurs by translocation of the GEF from the cytosol to its substrate, Ras, at the plasma membrane (21Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar), whereas Ras-GRF1 has not been found to undergo obvious subcellular redistribution (22Buchsbaum R. Telliez J.-B. Goonesekera S. Feig L.A. Mol. Cell. Biol. 1996; 16: 4888-4896Crossref PubMed Scopus (91) Google Scholar). Muscarinic acetylcholine receptors, for example, stimulate an increase in the phosphorylation state of Ras-GRF1 that is closely associated with an increase in its exchange activity toward Ras (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar). In this study, serine 916 of Ras-GRF1 is shown to be phosphorylated in response to the activation of co-expressed muscarinic receptors. This residue is an in vivo and in vitro substrate for PKA, but another kinase is likely to be responsible for its phosphorylation in response to carbachol stimulation. Phosphorylation of serine 916 is necessary but not sufficient for maximal activation of Ras-GRF1.DISCUSSIONThis study identifies serine 916 of the Ras-GRF1 exchange factor as a target for phosphorylation in vivo in COS-7 cells and NIH-3T3 fibroblasts in response to both the activation of co-expressed muscarinic receptors and the activation of PKA. Mutation of serine 916 to alanine prevents full activation of the exchange activity of Ras-GRF1 toward Ras. Clearly, however, phosphorylation at serine 916 does not provide a complete explanation of the activation of Ras-GRF1 by G-protein-coupled receptors because it is not, by itself, sufficient to replicate this activation. It is likely that there are additional sites at which phosphorylation occurs in response to agonist stimulation and these events are also required for activation. The absence of these further phosphorylation events would explain the absence of activation in response to forskolin, which may be only able to induce phosphorylation at serine 916. Evidence that there are indeed increases in phosphorylation in response to carbachol at sites that are not so stimulated by forskolin is shown in Fig. 6. Whether phosphorylation at serine 916 in response to a cAMP/PKA signal may contribute to the overall regulation of Ras-GRF1 activity in the cell is unknown, but it is possible that this could provide an additional point of cross-talk between the cAMP and Ras signaling systems (36Bourne H.R. Nature. 1995; 376: 727-729Crossref PubMed Scopus (62) Google Scholar).Ras-GRF1 is expressed exclusively in the neurons of the postnatal central nervous system in rodents (10Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar), where, based on the phenotypes of knockout mice that apparently lack expression of Ras-GRF1 (12Brambilla 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.-P. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (397) Google Scholar, 13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar), it plays a significant physiological role (37Finkbeiner S. Dalva M.B. BioEssays. 1998; 20: 691-695Crossref PubMed Scopus (8) Google Scholar, 38Orban P.C. Chapman P.F. Brambilla R. Trends Neurosci. 1999; 22: 38-44Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). It is reasonable to question, therefore, whether the results from the COS-7 and NIH-3T3 model systems reflect a mechanism that is relevant to physiological events in neuronal signaling. We have previously shown that Ras-GRF1 in neonatal rat brains is a phosphoprotein, the phosphorylation state of which is increased in response to the muscarinic agonist carbachol, and that carbachol increases the exchange activity toward Ras that is present in lysates of rat brains (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar). Whether serine 916 of Ras-GRF1 is also a site for phosphorylation in neurons remains, however, to be determined. It should also be noted that Ras-GRF1 may be expressed more widely in human tissues than in rodents (39Guerrero C. Rojas J.M. Chedid M. Esteban L.M. Zimonjic D.B. Popescu N.C. Font de Mora J. Santos E. Oncogene. 1996; 12: 1097-1107PubMed Google Scholar).The Ras-GRF2 exchange factor (40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar) is highly homologous to Ras-GRF1, with much of the difference being short insertion sequences that are present only in Ras-GRF1. These insertions produce the larger size of 140 kDa for Ras-GRF1 rather than 135 kDa for Ras-GRF2. Both Ras-GRF1 and Ras-GRF2 bind calmodulin through their ilimaquinone domains and can couple ionomycin-induced increases in cytosolic calcium into increased activation of mitogen-activated protein kinase (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (390) Google Scholar, 40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). It is striking, therefore, that serine 916 of Ras-GRF1 occurs within one of the insert regions and so has no homologous residue in Ras-GRF2. Serine 916 and the adjacent residues are, however, fully conserved between Ras-GRF1 from rodents and humans (5Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar, 6Cen H. Papageorge A.G. Vass W.C. Zhang K. Lowy D.R. Mol. Cell. Biol. 1993; 13: 7718-7724Crossref PubMed Scopus (46) Google Scholar, 41Wei W. Das B. Park W. Broek D. Gene. 1994; 151: 279-284Crossref PubMed Scopus (17) Google Scholar). Because I have found that phosphorylation of serine 916 plays a functional role in the activation of Ras-GRF1 by muscarinic receptors, it will be interesting to test whether Ras-GRF2 is regulated in a similar manner.Serine 916 lies N-terminal to the CDC25 domain in Ras-GRF1 that has the exchange factor activity for Ras (42Coccetti P. Mauri I. Alberghina L. Martegani E. Parmeggiani A. Biochem. Biophys. Res. Comm. 1995; 206: 253-259Crossref PubMed Scopus (34) Google Scholar) and is just C-terminal to PEST sequences that confer sensitivity, at least in vitro, to proteolysis (43Baouz S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. 1997; 272: 6671-6676Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This region of Ras-GRF2 contains a cyclin destruction box (40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). Because phosphorylation may be a signal for protein turnover (44Wulczyn F.G. Krappmann D. Scheidereit C. Nucleic Acids Res. 1998; 26: 1724-1730Crossref PubMed Scopus (16) Google Scholar), and because this region of Ras-GRF1 may be concerned with regulation of protein stability, it is possible that phosphorylation of serine 916 also plays a role in the termination of signaling through induced down-regulation of Ras-GRF1.Another distinction between Ras-GRF1 and Ras-GRF2 had been that I 2R. R. Mattingly, unpublished results. and others (45Freshney N.W. Goonesekera S.D. Feig L.A. FEBS Lett. 1997; 407: 111-115Crossref PubMed Scopus (44) Google Scholar, 46Anborgh 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) had not found any exchange activity toward GTPases of the Rho family in Ras-GRF1, whereas Ras-GRF2 has clear exchange activity for Rac (47Fan W.T. Koch C.A. deHoog C.L. Fam N.P. Moran M.F. Curr. Biol. 1998; 8: 935-938Abstract Full Text Full Text PDF PubMed Google Scholar). Recently, however, Kiyono et al. (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar) have demonstrated that Rac exchange factor activity can be induced in Ras-GRF1 by the co-expression of G-protein βγ subunits. The DBL and plekstrin homology domains that mediate Rac exchange factor activity (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar) are also required for the regulation of the Ras exchange activity of Ras-GRF1 in response to ionomycin (45Freshney N.W. Goonesekera S.D. Feig L.A. FEBS Lett. 1997; 407: 111-115Crossref PubMed Scopus (44) Google Scholar). This result suggests that control of these two activities may be closely linked. Further, the induction of Rac exchange activity by Gβγ is also associated with an increase in the phosphorylation state of Ras-GRF1, although in this case the event is likely to be phosphotyrosine (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar). We previously showed that Gβγ induced an increase in the Ras exchange activity of Ras-GRF1 (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar), and this led to the identification of the serine 916 phosphorylation site in this study. In view of the close parallels between the control of the Ras and Rac exchange activities of Ras-GRF1, it is possible that phosphorylation at serine 916 may also participate in the control of Rac exchange activity.An overall picture of the regulation of Ras-GRF1 is still, therefore, incomplete. Indeed, many GEFs for Ras superfamily GTPases are subject to complex regulation through phosphorylation (49Fleming I.N. Elliott C.M. Buchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 50Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), allosteric interactions (51Ebinu 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 (545) Google Scholar, 52Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar), and subcellular redistribution (21Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar). GEFs may also play a role in the selection of targets for their substrate GTPases (53Giglione C. Parmeggiani A. J. Biol. Chem. 1998; 273: 34737-34744Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In the case of Ras-GRF1 and its activation by ionomycin, there is now evidence that it can participate in the activation of Raf, the Ras effector, through a mechanism that may be independent of further activation of Ras (46Anborgh 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). It is likely that these complexities reflect the underlying function of Ras-GRF1 and other GEFs to serve as key integrators and controllers of signaling pathways with activities beyond the simple control of GTP binding to their substrates. The Ras GTPases are known to play central roles in pathways of cellular growth and differentiation (1Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3758) Google Scholar). Their function in terminally differentiated cells such as neurons is less clear, but they have been suggested to participate in learning and memory (2Rosen L.B. Ginty D.D. Weber M.J. Greenberg M.E. Neuron. 1994; 12: 1207-1221Abstract Full Text PDF PubMed Scopus (595) Google Scholar). As timed, molecular switches they cycle between active (GTP-bound) and inactive (GDP-bound) conformations under the control of guanine nucleotide exchange factors (GEFs)1 and GTPase-activating proteins (3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1751) Google Scholar). The balance of the effective GEF and GTPase-activating protein activities determines the activation state of Ras proteins. Regulation of the activity or subcellular localization of a specific GEF is now recognized as the major control for Ras activity in many instances (4Downward J. Nature. 1998; 396: 416-417Crossref PubMed Scopus (36) Google Scholar). The Ras-GRF1 exchange factor (5Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar), which was also previously known as CDC25Mm (6Cen H. Papageorge A.G. Vass W.C. Zhang K. Lowy D.R. Mol. Cell. Biol. 1993; 13: 7718-7724Crossref PubMed Scopus (46) Google Scholar, 7Martegani E. Vanoni M. Zippel R. Coccetti P. Brambilla R. Ferrari C. Sturani E. Alberghina L. EMBO J. 1992; 11: 2151-2157Crossref PubMed Scopus (188) Google Scholar), is implicated in neurotransmission. Ras-GRF1 is highly expressed in neurons of the central nervous system (8Wei W. Schreiber S.S. Baudry M. Tocco G. Broek D. Mol. Brain Res. 1993; 19: 339-344Crossref PubMed Scopus (27) Google Scholar, 9Ferrari C. Zippel R. Martegani E. Gnesutta N. Carrera V. Sturani E. Exp. Cell Res. 1994; 210: 353-357Crossref PubMed Scopus (30) Google Scholar, 10Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar) and is present in postsynaptic densities (11Sturani E. Abbondio A. Branduardi P. Ferrari C. Zippel R. Martegani E. Vanoni M. Denis-Donini S. Exp. Cell Res. 1997; 235: 117-123Crossref PubMed Scopus (55) Google Scholar). Mice that lack the expression of Ras-GRF1 have defects in the consolidation of memory (12Brambilla 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.-P. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (397) Google Scholar) and in postnatal growth (13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar). The latter may be due to a decrease in circulating insulin-like growth factor-1 levels that is secondary to changes in the hypothalamus (13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar). In contrast to the Sos exchange factors, which couple tyrosine kinase-derived signals to the activation of Ras (14Buday L. Downward J. Mol. Cell. Biol. 1993; 13: 1903-1910Crossref PubMed Scopus (106) Google Scholar, 15Egan S.E. Giddings B.W. Brooks M.W. Buday L. Sizeland A.M. Weinberg R.A. Nature. 1993; 363: 45-51Crossref PubMed Scopus (1003) Google Scholar), Ras-GRF1 links heterotrimeric G-proteins (16Shou C. Wurmser A. Ling K. Barbacid M. Feig L.A. Oncogene. 1995; 10: 1887-1893PubMed Google Scholar, 17Zippel R. Orecchia S. Sturani E. Martegani E. Oncogene. 1996; 12: 2697-2703PubMed Google Scholar, 18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar, 19Mattingly R.R. Saini V. Macara I.G. Cell. Signal. 1999; 11: 603-610Crossref PubMed Scopus (22) Google Scholar) and calcium signals (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (390) Google Scholar) to Ras. Further, regulation through Sos occurs by translocation of the GEF from the cytosol to its substrate, Ras, at the plasma membrane (21Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar), whereas Ras-GRF1 has not been found to undergo obvious subcellular redistribution (22Buchsbaum R. Telliez J.-B. Goonesekera S. Feig L.A. Mol. Cell. Biol. 1996; 16: 4888-4896Crossref PubMed Scopus (91) Google Scholar). Muscarinic acetylcholine receptors, for example, stimulate an increase in the phosphorylation state of Ras-GRF1 that is closely associated with an increase in its exchange activity toward Ras (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar). In this study, serine 916 of Ras-GRF1 is shown to be phosphorylated in response to the activation of co-expressed muscarinic receptors. This residue is an in vivo and in vitro substrate for PKA, but another kinase is likely to be responsible for its phosphorylation in response to carbachol stimulation. Phosphorylation of serine 916 is necessary but not sufficient for maximal activation of Ras-GRF1. DISCUSSIONThis study identifies serine 916 of the Ras-GRF1 exchange factor as a target for phosphorylation in vivo in COS-7 cells and NIH-3T3 fibroblasts in response to both the activation of co-expressed muscarinic receptors and the activation of PKA. Mutation of serine 916 to alanine prevents full activation of the exchange activity of Ras-GRF1 toward Ras. Clearly, however, phosphorylation at serine 916 does not provide a complete explanation of the activation of Ras-GRF1 by G-protein-coupled receptors because it is not, by itself, sufficient to replicate this activation. It is likely that there are additional sites at which phosphorylation occurs in response to agonist stimulation and these events are also required for activation. The absence of these further phosphorylation events would explain the absence of activation in response to forskolin, which may be only able to induce phosphorylation at serine 916. Evidence that there are indeed increases in phosphorylation in response to carbachol at sites that are not so stimulated by forskolin is shown in Fig. 6. Whether phosphorylation at serine 916 in response to a cAMP/PKA signal may contribute to the overall regulation of Ras-GRF1 activity in the cell is unknown, but it is possible that this could provide an additional point of cross-talk between the cAMP and Ras signaling systems (36Bourne H.R. Nature. 1995; 376: 727-729Crossref PubMed Scopus (62) Google Scholar).Ras-GRF1 is expressed exclusively in the neurons of the postnatal central nervous system in rodents (10Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar), where, based on the phenotypes of knockout mice that apparently lack expression of Ras-GRF1 (12Brambilla 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.-P. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (397) Google Scholar, 13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar), it plays a significant physiological role (37Finkbeiner S. Dalva M.B. BioEssays. 1998; 20: 691-695Crossref PubMed Scopus (8) Google Scholar, 38Orban P.C. Chapman P.F. Brambilla R. Trends Neurosci. 1999; 22: 38-44Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). It is reasonable to question, therefore, whether the results from the COS-7 and NIH-3T3 model systems reflect a mechanism that is relevant to physiological events in neuronal signaling. We have previously shown that Ras-GRF1 in neonatal rat brains is a phosphoprotein, the phosphorylation state of which is increased in response to the muscarinic agonist carbachol, and that carbachol increases the exchange activity toward Ras that is present in lysates of rat brains (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar). Whether serine 916 of Ras-GRF1 is also a site for phosphorylation in neurons remains, however, to be determined. It should also be noted that Ras-GRF1 may be expressed more widely in human tissues than in rodents (39Guerrero C. Rojas J.M. Chedid M. Esteban L.M. Zimonjic D.B. Popescu N.C. Font de Mora J. Santos E. Oncogene. 1996; 12: 1097-1107PubMed Google Scholar).The Ras-GRF2 exchange factor (40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar) is highly homologous to Ras-GRF1, with much of the difference being short insertion sequences that are present only in Ras-GRF1. These insertions produce the larger size of 140 kDa for Ras-GRF1 rather than 135 kDa for Ras-GRF2. Both Ras-GRF1 and Ras-GRF2 bind calmodulin through their ilimaquinone domains and can couple ionomycin-induced increases in cytosolic calcium into increased activation of mitogen-activated protein kinase (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (390) Google Scholar, 40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). It is striking, therefore, that serine 916 of Ras-GRF1 occurs within one of the insert regions and so has no homologous residue in Ras-GRF2. Serine 916 and the adjacent residues are, however, fully conserved between Ras-GRF1 from rodents and humans (5Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar, 6Cen H. Papageorge A.G. Vass W.C. Zhang K. Lowy D.R. Mol. Cell. Biol. 1993; 13: 7718-7724Crossref PubMed Scopus (46) Google Scholar, 41Wei W. Das B. Park W. Broek D. Gene. 1994; 151: 279-284Crossref PubMed Scopus (17) Google Scholar). Because I have found that phosphorylation of serine 916 plays a functional role in the activation of Ras-GRF1 by muscarinic receptors, it will be interesting to test whether Ras-GRF2 is regulated in a similar manner.Serine 916 lies N-terminal to the CDC25 domain in Ras-GRF1 that has the exchange factor activity for Ras (42Coccetti P. Mauri I. Alberghina L. Martegani E. Parmeggiani A. Biochem. Biophys. Res. Comm. 1995; 206: 253-259Crossref PubMed Scopus (34) Google Scholar) and is just C-terminal to PEST sequences that confer sensitivity, at least in vitro, to proteolysis (43Baouz S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. 1997; 272: 6671-6676Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This region of Ras-GRF2 contains a cyclin destruction box (40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). Because phosphorylation may be a signal for protein turnover (44Wulczyn F.G. Krappmann D. Scheidereit C. Nucleic Acids Res. 1998; 26: 1724-1730Crossref PubMed Scopus (16) Google Scholar), and because this region of Ras-GRF1 may be concerned with regulation of protein stability, it is possible that phosphorylation of serine 916 also plays a role in the termination of signaling through induced down-regulation of Ras-GRF1.Another distinction between Ras-GRF1 and Ras-GRF2 had been that I 2R. R. Mattingly, unpublished results. and others (45Freshney N.W. Goonesekera S.D. Feig L.A. FEBS Lett. 1997; 407: 111-115Crossref PubMed Scopus (44) Google Scholar, 46Anborgh 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) had not found any exchange activity toward GTPases of the Rho family in Ras-GRF1, whereas Ras-GRF2 has clear exchange activity for Rac (47Fan W.T. Koch C.A. deHoog C.L. Fam N.P. Moran M.F. Curr. Biol. 1998; 8: 935-938Abstract Full Text Full Text PDF PubMed Google Scholar). Recently, however, Kiyono et al. (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar) have demonstrated that Rac exchange factor activity can be induced in Ras-GRF1 by the co-expression of G-protein βγ subunits. The DBL and plekstrin homology domains that mediate Rac exchange factor activity (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar) are also required for the regulation of the Ras exchange activity of Ras-GRF1 in response to ionomycin (45Freshney N.W. Goonesekera S.D. Feig L.A. FEBS Lett. 1997; 407: 111-115Crossref PubMed Scopus (44) Google Scholar). This result suggests that control of these two activities may be closely linked. Further, the induction of Rac exchange activity by Gβγ is also associated with an increase in the phosphorylation state of Ras-GRF1, although in this case the event is likely to be phosphotyrosine (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar). We previously showed that Gβγ induced an increase in the Ras exchange activity of Ras-GRF1 (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar), and this led to the identification of the serine 916 phosphorylation site in this study. In view of the close parallels between the control of the Ras and Rac exchange activities of Ras-GRF1, it is possible that phosphorylation at serine 916 may also participate in the control of Rac exchange activity.An overall picture of the regulation of Ras-GRF1 is still, therefore, incomplete. Indeed, many GEFs for Ras superfamily GTPases are subject to complex regulation through phosphorylation (49Fleming I.N. Elliott C.M. Buchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 50Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), allosteric interactions (51Ebinu 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 (545) Google Scholar, 52Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar), and subcellular redistribution (21Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar). GEFs may also play a role in the selection of targets for their substrate GTPases (53Giglione C. Parmeggiani A. J. Biol. Chem. 1998; 273: 34737-34744Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In the case of Ras-GRF1 and its activation by ionomycin, there is now evidence that it can participate in the activation of Raf, the Ras effector, through a mechanism that may be independent of further activation of Ras (46Anborgh 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). It is likely that these complexities reflect the underlying function of Ras-GRF1 and other GEFs to serve as key integrators and controllers of signaling pathways with activities beyond the simple control of GTP binding to their substrates. This study identifies serine 916 of the Ras-GRF1 exchange factor as a target for phosphorylation in vivo in COS-7 cells and NIH-3T3 fibroblasts in response to both the activation of co-expressed muscarinic receptors and the activation of PKA. Mutation of serine 916 to alanine prevents full activation of the exchange activity of Ras-GRF1 toward Ras. Clearly, however, phosphorylation at serine 916 does not provide a complete explanation of the activation of Ras-GRF1 by G-protein-coupled receptors because it is not, by itself, sufficient to replicate this activation. It is likely that there are additional sites at which phosphorylation occurs in response to agonist stimulation and these events are also required for activation. The absence of these further phosphorylation events would explain the absence of activation in response to forskolin, which may be only able to induce phosphorylation at serine 916. Evidence that there are indeed increases in phosphorylation in response to carbachol at sites that are not so stimulated by forskolin is shown in Fig. 6. Whether phosphorylation at serine 916 in response to a cAMP/PKA signal may contribute to the overall regulation of Ras-GRF1 activity in the cell is unknown, but it is possible that this could provide an additional point of cross-talk between the cAMP and Ras signaling systems (36Bourne H.R. Nature. 1995; 376: 727-729Crossref PubMed Scopus (62) Google Scholar). Ras-GRF1 is expressed exclusively in the neurons of the postnatal central nervous system in rodents (10Zippel R. Gnesutta N. Matus-Leibovitch N. Mancinelli E. Saya D. Vogel Z. Sturani E. Mol. Brain Res. 1997; 48: 140-144Crossref PubMed Scopus (62) Google Scholar), where, based on the phenotypes of knockout mice that apparently lack expression of Ras-GRF1 (12Brambilla 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.-P. Sturani E. Klein R. Nature. 1997; 390: 281-286Crossref PubMed Scopus (397) Google Scholar, 13Itier J.-M. Tremp G.L. Leonard J.-F. Multon M.-C. Ret G. Schweighoffer F. Tocque B. Bluet-Pajot M.-T. Cormier V. Dautry F. Nature. 1998; 393: 125-126Crossref PubMed Scopus (92) Google Scholar), it plays a significant physiological role (37Finkbeiner S. Dalva M.B. BioEssays. 1998; 20: 691-695Crossref PubMed Scopus (8) Google Scholar, 38Orban P.C. Chapman P.F. Brambilla R. Trends Neurosci. 1999; 22: 38-44Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). It is reasonable to question, therefore, whether the results from the COS-7 and NIH-3T3 model systems reflect a mechanism that is relevant to physiological events in neuronal signaling. We have previously shown that Ras-GRF1 in neonatal rat brains is a phosphoprotein, the phosphorylation state of which is increased in response to the muscarinic agonist carbachol, and that carbachol increases the exchange activity toward Ras that is present in lysates of rat brains (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar). Whether serine 916 of Ras-GRF1 is also a site for phosphorylation in neurons remains, however, to be determined. It should also be noted that Ras-GRF1 may be expressed more widely in human tissues than in rodents (39Guerrero C. Rojas J.M. Chedid M. Esteban L.M. Zimonjic D.B. Popescu N.C. Font de Mora J. Santos E. Oncogene. 1996; 12: 1097-1107PubMed Google Scholar). The Ras-GRF2 exchange factor (40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar) is highly homologous to Ras-GRF1, with much of the difference being short insertion sequences that are present only in Ras-GRF1. These insertions produce the larger size of 140 kDa for Ras-GRF1 rather than 135 kDa for Ras-GRF2. Both Ras-GRF1 and Ras-GRF2 bind calmodulin through their ilimaquinone domains and can couple ionomycin-induced increases in cytosolic calcium into increased activation of mitogen-activated protein kinase (20Farnsworth C.L. Freshney N.W. Rosen L.B. Ghosh A. Greenberg M.E. Feig L.A. Nature. 1995; 376: 524-527Crossref PubMed Scopus (390) Google Scholar, 40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). It is striking, therefore, that serine 916 of Ras-GRF1 occurs within one of the insert regions and so has no homologous residue in Ras-GRF2. Serine 916 and the adjacent residues are, however, fully conserved between Ras-GRF1 from rodents and humans (5Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (289) Google Scholar, 6Cen H. Papageorge A.G. Vass W.C. Zhang K. Lowy D.R. Mol. Cell. Biol. 1993; 13: 7718-7724Crossref PubMed Scopus (46) Google Scholar, 41Wei W. Das B. Park W. Broek D. Gene. 1994; 151: 279-284Crossref PubMed Scopus (17) Google Scholar). Because I have found that phosphorylation of serine 916 plays a functional role in the activation of Ras-GRF1 by muscarinic receptors, it will be interesting to test whether Ras-GRF2 is regulated in a similar manner. Serine 916 lies N-terminal to the CDC25 domain in Ras-GRF1 that has the exchange factor activity for Ras (42Coccetti P. Mauri I. Alberghina L. Martegani E. Parmeggiani A. Biochem. Biophys. Res. Comm. 1995; 206: 253-259Crossref PubMed Scopus (34) Google Scholar) and is just C-terminal to PEST sequences that confer sensitivity, at least in vitro, to proteolysis (43Baouz S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. 1997; 272: 6671-6676Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This region of Ras-GRF2 contains a cyclin destruction box (40Fam N.P. Fan W.T. Wang Z. Zhang L.J. Chen H. Moran M.F. Mol. Cell. Biol. 1997; 17: 1396-1406Crossref PubMed Scopus (133) Google Scholar). Because phosphorylation may be a signal for protein turnover (44Wulczyn F.G. Krappmann D. Scheidereit C. Nucleic Acids Res. 1998; 26: 1724-1730Crossref PubMed Scopus (16) Google Scholar), and because this region of Ras-GRF1 may be concerned with regulation of protein stability, it is possible that phosphorylation of serine 916 also plays a role in the termination of signaling through induced down-regulation of Ras-GRF1. Another distinction between Ras-GRF1 and Ras-GRF2 had been that I 2R. R. Mattingly, unpublished results. and others (45Freshney N.W. Goonesekera S.D. Feig L.A. FEBS Lett. 1997; 407: 111-115Crossref PubMed Scopus (44) Google Scholar, 46Anborgh 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) had not found any exchange activity toward GTPases of the Rho family in Ras-GRF1, whereas Ras-GRF2 has clear exchange activity for Rac (47Fan W.T. Koch C.A. deHoog C.L. Fam N.P. Moran M.F. Curr. Biol. 1998; 8: 935-938Abstract Full Text Full Text PDF PubMed Google Scholar). Recently, however, Kiyono et al. (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar) have demonstrated that Rac exchange factor activity can be induced in Ras-GRF1 by the co-expression of G-protein βγ subunits. The DBL and plekstrin homology domains that mediate Rac exchange factor activity (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar) are also required for the regulation of the Ras exchange activity of Ras-GRF1 in response to ionomycin (45Freshney N.W. Goonesekera S.D. Feig L.A. FEBS Lett. 1997; 407: 111-115Crossref PubMed Scopus (44) Google Scholar). This result suggests that control of these two activities may be closely linked. Further, the induction of Rac exchange activity by Gβγ is also associated with an increase in the phosphorylation state of Ras-GRF1, although in this case the event is likely to be phosphotyrosine (48Kiyono M. Satoh T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4826-4831Crossref PubMed Scopus (76) Google Scholar). We previously showed that Gβγ induced an increase in the Ras exchange activity of Ras-GRF1 (18Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (154) Google Scholar), and this led to the identification of the serine 916 phosphorylation site in this study. In view of the close parallels between the control of the Ras and Rac exchange activities of Ras-GRF1, it is possible that phosphorylation at serine 916 may also participate in the control of Rac exchange activity. An overall picture of the regulation of Ras-GRF1 is still, therefore, incomplete. Indeed, many GEFs for Ras superfamily GTPases are subject to complex regulation through phosphorylation (49Fleming I.N. Elliott C.M. Buchanan F.G. Downes C.P. Exton J.H. J. Biol. Chem. 1999; 274: 12753-12758Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 50Ichiba T. Hashimoto Y. Nakaya M. Kuraishi Y. Tanaka S. Kurata T. Mochizuki N. Matsuda M. J. Biol. Chem. 1999; 274: 14376-14381Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), allosteric interactions (51Ebinu 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 (545) Google Scholar, 52Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (671) Google Scholar), and subcellular redistribution (21Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (924) Google Scholar). GEFs may also play a role in the selection of targets for their substrate GTPases (53Giglione C. Parmeggiani A. J. Biol. Chem. 1998; 273: 34737-34744Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In the case of Ras-GRF1 and its activation by ionomycin, there is now evidence that it can participate in the activation of Raf, the Ras effector, through a mechanism that may be independent of further activation of Ras (46Anborgh 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). It is likely that these complexities reflect the underlying function of Ras-GRF1 and other GEFs to serve as key integrators and controllers of signaling pathways with activities beyond the simple control of GTP binding to their substrates. I thank Dr. D. L. Brautigan for the use of the Mono-Q chromatography system; Dr. D. Lowy for his original gift of the plasmid encoding CDC25Mm; Drs. P. B. Joel, C. E. Smith, and O. Tatsis for technical advice; Dr. A. Wolfman for his gift of recombinant c-Ha-Ras; and C. L. Smith for preparation of GST fusion proteins. I am indebted to Dr. I. G. Macara, in whose laboratory at the Markey Center for Cell Signaling of the University of Virginia these studies were initiated.
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