Raf-1 Is Involved in the Regulation of the Interaction between Guanine Nucleotide Exchange Factor and Ha-Ras

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1–149) and Raf (51–131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras·Raf complex, showed that the binding of Raf and GEF to Ha-Ras·GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras·GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras·GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133–314) of phosphatidylinositol 3-kinase p110α. Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell. The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1–149) and Raf (51–131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras·Raf complex, showed that the binding of Raf and GEF to Ha-Ras·GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras·GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras·GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133–314) of phosphatidylinositol 3-kinase p110α. Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell. The Ha-Ras p21 protein is a central element for the transmission of extracellular signals within the cell, interacting with a growing number of regulators and effectors (1Macara I.G. Lounsbury K.M. Richards S.A. Mckiernan C. Bar-Sagi D. FASEB J. 1996; 10: 625-630Crossref PubMed Scopus (211) Google Scholar, 2Wittinghofer A. Nassar N. Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar). These are proteins containing modular domains, whose interactions with other ligands are only partially known (3Marshall C. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar, 4Ponting C. Benjamin D. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 5Pawson T. Nature. 1995; 373: 573-579Crossref PubMed Scopus (2234) Google Scholar). The regulators enhance the two basic intrinsic activities of Ras: 1) the hydrolysis of GTP, and 2) the GDP/GTP exchange, two reactions stimulated by GTPase-activating proteins (GAPs) 1The abbreviations used are: GAP, GTPase-activating protein; p120-GAP, human rasGAP; GEF, guanine nucleotide exchange factor; C-Sdc25p, catalytic domain ofSaccharomyces cerevisiae of 550 amino acid residues; CDC25Mm, mouse rasGEF (p140-GRF); C-CDC25Mm-285, catalytic domain of CDC25Mm comprising 285 amino acid residues; RBD, Ras-binding domain; PI3K, phosphatidylinositol 3-kinase; GST, glutathioneS-transferase; GTPγS, guanosine 5′-O-(thio-triphosphate); SPA, scintillation proximity assay; ME, 2-mercaptoethanol. and the guanine nucleotide exchange factors (GEFs), respectively (6Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). The effectors transmit the downstream signal to pathways controlling growth, differentiation, and other fundamental processes of the cell. Despite a multitude of in vitro andin vivo studies, the coordination mechanisms by which the activity of Ha-Ras is regulated are only now beginning to be defined. It is important to mention here that the interaction of protein ligands with Ha-Ras bears a specific difference in that GAPs and effectors show a much higher affinity for Ha-Ras·GTP than for Ha-Ras·GDP, whereas GEFs act on Ras·GTP and Ras·GDP with nearly the same efficiency (3Marshall C. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar,7Schaber M.D. Garsky V.M. Boylan D. Hill W.S. Scolnick E.M. Marshall M.S. Sigal L.S. Gibbs J.B. Proteins. 1989; 6: 306-315Crossref PubMed Scopus (57) Google Scholar, 8Créchet J.B. Poullet P. Mistou M.-Y. Parmeggiani A. Camonis J. Boy-Marcotte E. Damak F. Jacquet M. Science. 1990; 248: 866-868Crossref PubMed Scopus (91) Google Scholar, 9Haney S.A. Broach J.R. J. Biol. Chem. 1994; 269: 16541-16548Abstract Full Text PDF PubMed Google Scholar, 10Jacquet E. Baouz S. Parmeggiani A. Biochemistry. 1995; 34: 12347-12354Crossref PubMed Scopus (29) Google Scholar, 11Poullet P. Créchet J.B. Bernardi A. Parmeggiani A. Eur. J. Biochem. 1995; 227: 537-544Crossref PubMed Scopus (17) Google Scholar). The common property of GAPs and Ras effectors to show a pronounced specificity for activated Ras is in line with the proposed role of p120-GAP not only as GTPase-activating protein but also as an effector of Ras (12Pronk G.I. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar). In line with this, the best investigated effector of Ha-Ras, c-Raf-1, competes with p120-GAP and neurofibromin for binding to Ras·GTP (2Wittinghofer A. Nassar N. Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar, 13Zhang X.F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (689) Google Scholar, 14Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar, 15Herrmann C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Another effector of Ha-Ras is RalGDS, a GDP/GTP exchange factor of RalA p24 and RalB p24. RalGDS interacts with Ras in a GTP-dependent manner and inhibits the binding of both Raf-1 and GAP to Ha-Ras (16Kikuchi A. Demo S.D. Ye Z.H. Chen Y.W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar). In this context, we have recently described that in vitrop120-GAP protects the active state of p21 from unproductive exchanges of bound GTPγS the of the GEF-stimulated GDP/GTP exchange on Ras C. Baouz S. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is that other protein ligands of Ras, as act to GAP to with GEF for binding to Ha-Ras·GTP and to be not only downstream of p21 but also upstream on the activation mechanism to the GDP/GTP exchange on Ha-Ras In agreement with this is the that in a of a factor the to to phosphatidylinositol 3-kinase another effector of Ras is to the GDP/GTP exchange on Ras a active of the of bound to Ras p21 T. Williams L.T. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar, A. Williams L.T. Science. 1995; PubMed Scopus Google Scholar). in vitro an interference between effectors as Raf and GEFs in this we have the influence of Raf on the action of GEF to more on the mechanism controlling the activation of Ha-Ras. have that the binding of Raf and GEF to Ha-Ras·GTP is mutually exclusive. we have the of to the GEF-stimulated exchange on the active state of Ha-Ras. The between GEF and Raf is proposed to a mechanism to promote the GDP/GTP This a role to the effector as a of the downstream signal of Ras and as an upstream of Ras Raf residues was by M. S. Marshall of of and in containing Raf residues were by P. N. The were in of containing of both proteins was by a of The which was to was for and the was in of and containing and the of the was for and the which was with of was for The was with and the protein with was to with and ME, and The protein were for more than was with a containing the of the cerevisiae the 550 residues and was in containing The for the and of C-Sdc25p, as described by Poullet P. Créchet J.B. Bernardi A. Parmeggiani A. Eur. J. Biochem. 1995; 227: 537-544Crossref PubMed Scopus (17) Google was used with the The of was a of for The which were by for were with The was in of ME, and the was for and the was for with of with ME, and The was with The protein bound to the was with human in of for The was and the was The of were with ME, and and the RBD (amino acid residues 133–314) of were by J. Downward The were in of containing The of RBD protein was by a of The which was to was for and the was in of containing and the of the was for and the which was with of was for The was with and the protein with was to with ME, and and of the was by the binding The and were by Ha-Ras with a of and for in and was to the The was by the of a of nucleotide with GEF, and the various were and on of and the was in a scintillation The were as described E. Baouz S. Parmeggiani A. Biochemistry. 1995; 34: 12347-12354Crossref PubMed Scopus (29) Google Scholar, J.B. Poullet P. Bernardi A. Parmeggiani A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). The interaction between Raf and Ras was by the of GEF to with for binding to the scintillation proximity The described by M. J. Biochem. 1994; PubMed Scopus Google and C. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google was used with the The were by Ha-Ras p21 with a of for in C. was to the The was in as a containing of C-Sdc25p, full-length CDC25Mm, in was was to by of a of and protein in and in which GEF was were also was in the to a of The were for and for and for the was in a scintillation of the the signal was as a of the binding to the in the of GEF CDC25Mm catalytic domain C-CDC25Mm-285, and Ha-Ras were and as described S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was a Nature. PubMed Scopus Google Scholar). was by the protein as the in the of was also by binding as by the both GEF and Raf act on the active state of Ha-Ras, we have for between the activities of two a of the effect of Raf on the GEF activity on Ras, we the influence of Raf on the intrinsic exchange activity of Ha-Ras, a that investigated by C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google a and The two Raf RBDs Raf and Raf were as and on the from the were as nucleotide the of GTPγS from Ha-Ras the of Raf the of the as a of of inhibited the of from Ha-Ras in agreement with the of C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google a of for the affinity between and Ras. the in of a was the same conditions used for an effect was also in the of In this the was from to which to a of the intrinsic exchange on Ha-Ras p21 The between and (1–149) was higher than that for and in agreement with the affinity of (1–149) for Ras p21 S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, S. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google of (1–149) on the intrinsic exchange on Ha-Ras. In the intrinsic of were from in the of of (1–149) In the of of intrinsic exchange on Ha-Ras were the intrinsic exchange on Ha-Ras in the of (1–149) that was as The the of The exchange on Ha-Ras can be stimulated by guanine nucleotide exchange the catalytic domain of S. cerevisiae Sdc25p a GEF that on Ha-Ras p21 and J.B. Poullet P. Mistou M.-Y. Parmeggiani A. Camonis J. Boy-Marcotte E. Damak F. Jacquet M. Science. 1990; 248: 866-868Crossref PubMed Scopus (91) Google Scholar, M.-Y. Jacquet E. Poullet P. P. Wittinghofer A. Parmeggiani A. J. PubMed Scopus Google Scholar). in and enhances the exchange on Ha-Ras, but this effect can be by the of of (1–149) of and C-Sdc25p, of the from to in a concentration-dependent manner results, can that the to the GEF-stimulated exchange on Ha-Ras to In the of the of the in a concentration-dependent manner from to which to an of the exchange on Ha-Ras the to Raf RBD on exchange on Ha-Ras in the and of GEF, our results show that Raf RBDs not only the intrinsic exchange on the active of Ras but also the of GEF to this of GTPγS with the same results not also the Raf effect was on the active state of Ha-Ras, the of Raf. Raf not with the state of Ha-Ras, effect of Raf RBDs on the exchange on p21 could be not The of Raf to with GEF to a with to the effect of Raf to act upstream and downstream on Ras could also be to other ligands of Ras, as in the of by in vivo results T. Williams L.T. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar, A. Williams L.T. Science. 1995; PubMed Scopus Google Scholar). this we have used the RBD (amino acid residues 133–314) of P.R. P.H. M. Downward J. J. 1996; PubMed Scopus Google and effect on the activity of the exchange the action of of RBD on of the in the and of a of C-Sdc25p, the of from to in a of with GTPγS the same results, a of the exchange on Ha-Ras could that the of RBD on activity was In to Raf-1 the RBD not to influence the intrinsic exchange activity of Ha-Ras and The of GEF activity was the of a between Raf proteins and the exchange factor for binding to the active of Ha-Ras, the the effect of Raf not the interaction of Raf RBD with the active of Ras the binding of GEF to Ras, we in which Ras was from the Ras·Raf complex, and the of the was by This can be used to the binding of binding a under conditions of protein with was used to the binding of to Ras with M. J. Biochem. 1994; PubMed Scopus Google Scholar, C. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This to the effect of GEF on the interaction between and in of in a in the signal by the binding to These results are with the that is a between and for binding to The of of binding was to the of our results was the of from the In this we have not in studies J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that CDC25Mm Ha-Ras in we have the interactions between Raf and Ras in a by the of full-length CDC25Mm to with for binding to in this of full-length CDC25Mm the signal by the Raf binding to Ras the of CDC25Mm in higher than the is to to the of the protein S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we could only a of GEF that a of binding in the This to that with the same of higher to of catalytic domain E. Baouz S. Parmeggiani A. Biochemistry. 1995; 34: 12347-12354Crossref PubMed Scopus (29) Google we could more than of the binding This is the that an effector can the interaction between the active of Ras and GEF, that two proteins be bound to the active of Ras. the of the effect of Raf RBDs on GEF in the of the active of Ha-Ras to the of in vitro with the physiological conditions in the cell, we the influence of Raf RBD in a in which the of p21 were in the and the of the and GTPγS to the to in the cell. the action of full-length CDC25Mm on Ha-Ras in the and of and a and GTPγS of a of CDC25Mm, the of enhances the of the exchange on Ha-Ras, as by the of from the Ha-Ras the under conditions by on the on Ha-Ras, was not we have this a in which Ha-Ras p21 was with and the GTPγS was with In this we the exchange as of stimulated by CDC25Mm in the and of In both the of the active of Ras by GEF proteins was by the of the results in A. These data show that the Raf of the GEF activity by sequestration of the active of Ha-Ras the interaction between the exchange factor and Ha-Ras·GDP, physiological In the GDP/GTP of Ras the of of the for the hydrolysis of GTP, could the of as GTP/GTP show a higher binding affinity for the state of Ha-Ras, this property is and is not by to a for the GDP/GTP exchange on Ras. is that mechanisms the interaction between the exchange factor and Ha-Ras·GDP, In our work C. Baouz S. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we have that by GEF of the Ha-Ras exchange was inhibited by vitro. This by GAP of the GEF sequestration of the active of Ha-Ras was to the Ha-Ras exchange, an of GAP that could to regulate the Ha-Ras activity in the cell. Consistent with this is the observation that under specific conditions in the effect of p120-GAP be inhibited (12Pronk G.I. Bos J.L. Biochim. Biophys. Acta. 1994; 1198: 131-147PubMed Google Scholar, J. P.H. Nature. 1990; PubMed Scopus Google Scholar, M. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. McCormick F. T. C. Mol. Cell. Biol. PubMed Scopus Google Scholar, J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google that the of p120-GAP with the active of p21 is with proposed as of downstream signals of Ha-Ras A. P. McCormick F. Cell. 1990; Full Text PDF PubMed Scopus Google Scholar, F. F. F. Cell PubMed Scopus Google Scholar). the active of Ha-Ras to with an number of ligands (1Macara I.G. Lounsbury K.M. Richards S.A. Mckiernan C. Bar-Sagi D. FASEB J. 1996; 10: 625-630Crossref PubMed Scopus (211) Google Scholar, 2Wittinghofer A. Nassar N. Trends Biochem. Sci. 1996; 21: 488-491Abstract Full Text PDF PubMed Scopus (137) Google Scholar, C. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar, 4Ponting C. Benjamin D. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 5Pawson T. Nature. 1995; 373: 573-579Crossref PubMed Scopus (2234) Google other effectors be in a mechanism of of Ha-Ras bound to that is to that described for This a and to the multiple interactions of Ha-Ras and the physiological of the GDP/GTP exchange in the cell. that the the of Ras are in the cell. Raf-1 activity to be regulated by and Mol. 1995; PubMed Scopus Google Scholar). It that Raf can be by protein and that of Raf-1 affinity for Ras p21 A. Williams L.T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). a Raf-1 was to be of the mechanisms by which Ha-Ras between In and have to be in controlling the activity of rasGEF CDC25Mm, the of and are in to be S. Jacquet E. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Nature. 1995; PubMed Scopus Google Scholar, I.G. Nature. 1996; PubMed Scopus Google Scholar). more on the mechanisms for a activation of Ras as a of with regulators and we have the influence of c-Raf-1 on the nucleotide exchange of Ha-Ras in this a the Ras binding domains of Raf have to be to not only the intrinsic GDP/GTP exchange on Ras, as by another C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google but also the GEF-stimulated The specific of this effect is by on the active state of Ha-Ras, the of by Raf-1 RBDs of the exchange on p21 was The observation that Raf RBDs as nucleotide GTPγS in the Ras·Raf C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google the of this effect is the of a between the exchange factor and Raf for binding to the active of Ras is only a of the of the intrinsic GDP/GTP exchange on this Ha-Ras·GTP was by GEF proteins from the Ras·Raf complex, and the of was by the scintillation proximity this we could that the of the GEF activity was to a between Raf and GEF for binding to the active of Ha-Ras. for the that the binding of Raf-1 to the active of Ras the interaction of the with nucleotide exchange that the binding of Raf and GEF to Ha-Ras is mutually exclusive. by to the of Ras as a for interaction with the various effectors (3Marshall C. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (475) Google Scholar). that residues this as of the are for and the specificity of the interaction (1Macara I.G. Lounsbury K.M. Richards S.A. Mckiernan C. Bar-Sagi D. FASEB J. 1996; 10: 625-630Crossref PubMed Scopus (211) Google Scholar, S.A. M. E. P. A. 1995; Google Scholar, N. C. C. Wittinghofer A. Biol. 1996; 8: Scopus Google Scholar, M. F. F. A. M. M. S. T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). to the interaction between Ras and GEF, a role to which is of the P. Créchet J.B. Bernardi A. Parmeggiani A. Eur. J. Biochem. 1995; 227: 537-544Crossref PubMed Scopus (17) Google Scholar, A. Créchet J.B. F. C. E. E. Parmeggiani A. J. PubMed Scopus Google Scholar, Marshall 1993; 8: Google Scholar, F. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J. D. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar, K.M. S. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar, S. A. J. D. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J.B. Bernardi A. Parmeggiani A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and C. Baouz S. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of our the of Ha-Ras in with the GEF of the of protein Bar-Sagi D. J. Nature. PubMed Scopus Google Scholar). This a interaction of not only the but also of the with GEF, a that could not be from M.-Y. Jacquet E. Poullet P. P. Wittinghofer A. Parmeggiani A. J. PubMed Scopus Google Scholar). the of the interaction of Ras with Raf and GEF that the and that are in the binding of both GEF and The between ligands in our work on the of binding on in and also in in vitro that protein the of Ha-Ras p21 binding to RalGDS and that the binding of RalGDS to the active of Ras p21 inhibits the interaction of Ras p21 with Raf-1 and GAP (16Kikuchi A. Demo S.D. Ye Z.H. Chen Y.W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar, A. Williams L.T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The human protein also as a protein with a pronounced affinity for the state of Ras in with Raf-1 for binding to Ha-Ras in vitro J. Mol. Cell. Biol. 1995; PubMed Google Scholar). This the results of this work the existence of mechanisms controlling the interactions of Ras with multiple These mechanisms are on for binding to p21 that are regulated in the by the of the active state of the various ligands of Ras. The of the interactions in the of Ras can be by the between p21 and P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar, T. Williams L.T. Mol. Cell. Biol. 1993; PubMed Scopus Google Scholar, A. Williams L.T. Science. 1995; PubMed Scopus Google Scholar, P.R. P.H. M. Downward J. J. 1996; PubMed Scopus Google Scholar). In the cell, that Ras can the of Ras to to activation to an important of downstream P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar, P.R. P.H. M. Downward J. J. 1996; PubMed Scopus Google Scholar). an of Ras was in to active in A. Williams L.T. Science. 1995; PubMed Scopus Google that not only act downstream of p21 but also act upstream to Ha-Ras on the activation mechanism to the GDP/GTP In this context, to more on the of the upstream downstream role of PI3K, we have to this with our results on Raf by the influence of RBD on the GEF-stimulated exchange on the active of Ha-Ras. to we have that the of RBD the GTP/GTP exchange on Ras stimulated by in with Raf and other effector of Ras as RalGDS, by RBD on the intrinsic exchange on Ras was our results show that not only Raf-1 but also to exchange on the active of Ha-Ras The between GEF and Raf PI3K, as the for p120-GAP and GEF C. Baouz S. Bernardi A. Parmeggiani A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google could a regulatory under more physiological as by the observation that by mimicking the cellular ratio, the of Raf enhances the GEF-stimulated GDP/GTP GTPγS exchange on Ha-Ras. This of interactions of p21 and effectors on the of the active state the role of protein ligands of Ras, as Raf and in to p120-GAP, and the existence of coordination mechanisms controlling the of a with our results, the In the of Ras proteins are in the bound an signal is by activation of Ras·GDP is by the binding of GEF proteins and the of the Ras comparable affinity for and GTP, is to GTP, with the of in the a the This to the of a of Ras proteins by Ras·GTP and Ras·GDP the role of GEF is to as of Ras as the interaction with activated Ras proteins be a GDP/GTP interactions of Ras could be by binding to effectors that the active of Ras. This the interaction of GEF with the Ras·GDP The suggests a mechanism the activity of a binding between effectors and GEF regulators that the physiological of the GDP/GTP of Ras and the activity of the Ras in the cell. are to M. S. P. N. and J. Downward for the c-Raf-1 c-Raf-1 and RBD, are to P. N. for and on this our J. E. and for and and S. Baouz and E. Jacquet for CDC25Mm and