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

EVIDENCES FOR A FUNCTION OF Raf-1 AND PHOSPHATIDYLINOSITOL 3-KINASE UPSTREAM TO Ras*
  • Carmela Giglione
    Affiliations
    From the Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France
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  • Andrea Parmeggiani
    Correspondence
    To whom correspondence should be addressed:Tel.: 33-1-6933-4180; Fax: 33-1-6933-4840;
    Affiliations
    From the Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the European Community contract BIOTECH BIO4-CT96-1110, the Ligue Nationale Française Contre le Cancer, the Association pour la Recherche sur le Cancer (Grant 6377), and the Fédération Nationale des Centres de Lutte Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 25, 1998DOI:https://doi.org/10.1074/jbc.273.52.34737
      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 (
      • Macara I.G.
      • Lounsbury K.M.
      • Richards S.A.
      • Mckiernan C.
      • Bar-Sagi D.
      ,
      • Wittinghofer A.
      • Nassar N.
      ). These are proteins containing modular domains, whose interactions with other ligands are only partially known (
      • Marshall C.
      ,
      • Ponting C.
      • Benjamin D.
      ,
      • Pawson T.
      ). 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)
      The 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.
      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 (
      • Boguski M.S.
      • McCormick F.
      ). 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 (
      • Marshall C.
      ,
      • Schaber M.D.
      • Garsky V.M.
      • Boylan D.
      • Hill W.S.
      • Scolnick E.M.
      • Marshall M.S.
      • Sigal L.S.
      • Gibbs J.B.
      ,
      • Créchet J.B.
      • Poullet P.
      • Mistou M.-Y.
      • Parmeggiani A.
      • Camonis J.
      • Boy-Marcotte E.
      • Damak F.
      • Jacquet M.
      ,
      • Haney S.A.
      • Broach J.R.
      ,
      • Jacquet E.
      • Baouz S.
      • Parmeggiani A.
      ,
      • Poullet P.
      • Créchet J.B.
      • Bernardi A.
      • Parmeggiani A.
      ). 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 (
      • Pronk G.I.
      • Bos J.L.
      ). 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 (
      • Wittinghofer A.
      • Nassar N.
      ,
      • Zhang X.F.
      • Settleman J.
      • Kyriakis J.M.
      • Takeuchi-Suzuki E.
      • Elledge S.J.
      • Marshall M.S.
      • Bruder J.T.
      • Rapp U.R.
      • Avruch J.
      ,
      • Warne P.H.
      • Viciana P.R.
      • Downward J.
      ,
      • Herrmann C.
      • Martin G.A.
      • Wittinghofer A.
      ). 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 (
      • Kikuchi A.
      • Demo S.D.
      • Ye Z.H.
      • Chen Y.W.
      • Williams L.T.
      ).
      In this context, we have recently described that in vitrop120-GAP protects the active state of p21 from unproductive exchanges of bound GTPγS after the accomplishment of the GEF-stimulated GDP/GTP exchange on Ras (
      • Giglione C.
      • Parrini M.C.
      • Baouz S.
      • Bernardi A.
      • Parmeggiani A.
      ). It is possible that other protein ligands of Ras, such as Raf, may act similar to GAP to compete with GEF for binding to Ha-Ras·GTP and consequently to be involved not only downstream of p21 but also upstream on the activation mechanism leading to the GDP/GTP exchange on Ha-Ras p21. In agreement with this is the finding that in fibroblast cell lines, a mutant of a platelet-derived growth factor receptor lacking the ability to bind to phosphatidylinositol 3-kinase (PI3K), another putative effector of Ras p21, is unable to stimulate the GDP/GTP exchange on Ras p21. Moreover, when expressed inXenopus oocytes, a constitutively active form of PI3K increased the amount of GTP bound to Ras p21 (
      • Satoh T.
      • Fantl W.J.
      • Escobedo J.A.
      • Williams L.T.
      • Kaziro Y.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ).
      Because no in vitro experiment demonstrating an interference between effectors such as Raf or PI3K and GEFs has yet been reported, in this work, we have analyzed the influence of Raf on the action of GEF to shed more light on the mechanism controlling the activation of Ha-Ras. We have found that the binding of Raf and GEF to Ha-Ras·GTP is mutually exclusive. Furthermore, we have provided evidence supporting the ability of PI3K to inhibit the GEF-stimulated exchange on the active state of Ha-Ras. The competition between GEF and Raf or PI3K is proposed to represent a mechanism to selectively promote the productive GDP/GTP exchange. This would confer a dual role to the effector as a transmitter of the downstream signal of Ras and as an upstream regulator of Ras activation.

      DISCUSSION

      In the GDP/GTP cycle of Ras proteins, the reversibility of all partial steps of the nucleotide·p21 interaction, except for the hydrolysis of GTP, could allow the occurrence of futile reactions, such as GTP/GTP exchange. Even if rasGEFs show a slightly higher binding affinity for the GDP-bound state of Ha-Ras, this differential property is comparatively small and is not sufficient by itself to support a marked preference for the physiologically productive GDP/GTP exchange on Ras. Therefore, it is likely that additional mechanisms exist favoring the interaction between the exchange factor and Ha-Ras·GDP, its biological substrate.
      In our previous work (
      • Giglione C.
      • Parrini M.C.
      • Baouz S.
      • Bernardi A.
      • Parmeggiani A.
      ), we have shown that stimulation by GEF of the Ha-Ras GTPγS/GTPγS exchange was specifically inhibited by p120-GAPin vitro. This inhibition by GAP of the GEF activityvia sequestration of the active form of Ha-Ras was found to favor the GEF-induced Ha-Ras GDP/GTPγS exchange, revealing an additional function of GAP that could contribute to regulate the Ha-Ras activity in the cell. Consistent with this is the observation that under specific conditions in vivo, the GTPase- activating effect of p120-GAP may be inhibited (
      • Pronk G.I.
      • Bos J.L.
      ,
      • Downward J.
      • Graves P.H.
      • Warne R.
      • Sydonia R.
      • Cantrell D.A.
      ,
      • Torti M.
      • Marti K.B.
      • Altschuler D.
      • Yamamoto K.
      • Lapetina E.G.
      ,
      • Moran M.F.
      • Polakis P.
      • McCormick F.
      • Pawson T.
      • Ellis C.
      ,
      • Sermon B.A.
      • Eccleston J.F.
      • Skinner R.H.
      • Lowe P.N.
      ), suggesting that the protective association of p120-GAP with the active form of p21 is correlated with its proposed function as transmitter of downstream signals of Ha-Ras (
      • Yatani A.
      • Okabe K.
      • Polakis P.
      • Halenbeck R.
      • McCormick F.
      • Brown A.M.
      ,
      • Tocque B.
      • Delumeau I.
      • Parker F.
      • Maurier F.
      • Multon M.C.
      • Schweighoffer F.
      ).
      Because the active form of Ha-Ras has been found to interact with an increasing number of ligands (
      • Macara I.G.
      • Lounsbury K.M.
      • Richards S.A.
      • Mckiernan C.
      • Bar-Sagi D.
      ,
      • Wittinghofer A.
      • Nassar N.
      ,
      • Marshall C.
      ,
      • Ponting C.
      • Benjamin D.
      ,
      • Pawson T.
      ), other effectors may be involved in a mechanism of protection of Ha-Ras bound to GTP that is similar to that described for p120-GAP. This would constitute a simple and efficient system to coordinate the multiple interactions of Ha-Ras and would help favor the physiological course of the GEF-dependent GDP/GTP exchange reaction in the cell. However, one should take into account that the competitive phenomena at the level of Ras are very likely further modulated in the cell. Raf-1 activity appears to be regulated by phosphorylation/dephosphorylation events, ligand proteins, and lipids (
      • Morrison D.K.
      ). It has been reported that Raf can be phosphorylated by protein kinase A, and that phosphorylation of Raf-1 reduces its affinity for Ras p21 (
      • Kikuchi A.
      • Williams L.T.
      ). As a consequence, Raf-1 phosphorylation was suggested to be one of the mechanisms by which Ha-Ras distinguishes between its ligands. In turn, phosphorylation, calmodulin, and calpain have been found to be involved in controlling the activity of neuronal rasGEF CDC25Mm, although the nature of these effects and how they are coordinated remain in large part to be clarified (
      • Baouz S.
      • Jacquet E.
      • Bernardi A.
      • Parmeggiani A.
      ,
      • Farnsworth C.L.
      • Feshney N.W.
      • Rosen L.B.
      • Ghosh A.
      • Greenberg M.E.
      • Feig L.A.
      ,
      • Mattingly R.R.
      • Macara I.G.
      ).
      To shed more light on the possible molecular mechanisms accounting for a transient activation of Ras as a result of coupling or uncoupling with its regulators and effectors, we have studied the influence of c-Raf-1 on the GEF-induced nucleotide exchange of Ha-Ras in this work, using purified components. As a main result, the Ras binding domains of Raf have been found to be able to inhibit not only the intrinsic GDP/GTP exchange on Ras, as reported by Herrmann et al.using another method (
      • Herrmann C.
      • Martin G.A.
      • Wittinghofer A.
      ), but also the GEF-stimulated GTPγS/GTPγS exchange. The specific nature of this effect is supported by its strict dependence on the active state of Ha-Ras, the target of Raf; no inhibition by Raf-1 RBDs of the GEF-dependent GDP/GDP exchange on p21 was detected. The observation that Raf RBDs behave as nucleotide dissociation inhibitors, trapping GTPγS or GTP in the Ras·Raf complex (
      • Herrmann C.
      • Martin G.A.
      • Wittinghofer A.
      ), raised the question of whether this effect is the result of a competition between the exchange factor and Raf for binding to the active form of Ras or is only a consequence of the Raf-dependent inhibition of the intrinsic GDP/GTP exchange on p21. To analyze this aspect, Ha-Ras·GTP was displaced by GEF proteins from the Ras·Raf complex, and the amount of residual complex was assessed by using the scintillation proximity assay. With this system, we could demonstrate that the inhibition of the GEF activity was due to a reversible competition between Raf and GEF for binding to the active form of Ha-Ras. Moreover, it has been shown for the first time that the binding of Raf-1 to the active form of Ras blocks the interaction of the latter with its nucleotide exchange factor, showing that the binding of Raf and GEF to Ha-Ras is mutually exclusive.
      Extensive analysis by site-directed mutagenesis has pointed to the switch 1 region of Ras as a crucial area for interaction with the various effectors (
      • Marshall C.
      ). Moreover, it has been shown that residues outside this region, such as those of the switch 2 region, are responsible for molecular recognition and the specificity of the interaction (
      • Macara I.G.
      • Lounsbury K.M.
      • Richards S.A.
      • Mckiernan C.
      • Bar-Sagi D.
      ,
      • Moodie S.A.
      • Paris M.
      • Villafranca E.
      • Kirshmeier P.
      • Willumsen B.M.
      • Wolfman A.
      ,
      • Nassar N.
      • Horn G.
      • Herrmann C.
      • Block C.
      • Janknecht R.
      • Wittinghofer A.
      ,
      • Akasaka K.
      • Tamada M.
      • Wang F.
      • Kariya K.
      • Shima F.
      • Kikuchi A.
      • Yamamoto M.
      • Shirouzu M.
      • Yokoyama S.
      • Kataoka T.
      ). With regard to the interaction between Ras and GEF, a direct role has been assigned to helix α2, which is part of the switch 2 region (
      • Poullet P.
      • Créchet J.B.
      • Bernardi A.
      • Parmeggiani A.
      ,
      • Verrotti A.
      • Créchet J.B.
      • Di Blasi F.
      • Seidita G.
      • Mirisola M.G.
      • Kavounis C.
      • Nastopoulos V.
      • Burderi E.
      • De Vendittis E.
      • Parmeggiani A.
      • Fasano O.
      ,
      • Howe L.R.
      • Marshall C.J.
      ,
      • Mirisola M.G.
      • Seidita G.
      • Verrotti A.C.
      • Di Blasi F.
      • Fasano O.
      ,
      • Mosteller R.D.
      • Han J.
      • Broek D.
      ,
      • Quilliam L.A.
      • Kato K.K.
      • Rabun K.M.
      • Hisaka M.M.
      • Huff S.Y.
      • Campbell-Burk S.
      • Der C.J.
      ,
      • Quilliam L.A.
      • Hisaka M.M.
      • Zhang S.
      • Lowry A.
      • Mosteller R.D.
      • Han J.
      • Drugan J.K.
      • Brock D.
      • Campbell S.L.
      • Der C.J.
      ,
      • Créchet J.B.
      • Bernardi A.
      • Parmeggiani A.
      ), and helix α3 (
      • Giglione C.
      • Parrini M.C.
      • Baouz S.
      • Bernardi A.
      • Parmeggiani A.
      ). During the submission of our manuscript, the three-dimensional model of Ha-Ras in complex with the GEF region of the son of sevenless protein has been reported (
      • Boriack-Sjodin P.A.
      • Margarit S.M.
      • Bar-Sagi D.
      • Kuriyan J.
      ). This model emphasizes a direct interaction of not only the switch 2 region but also of the switch 1 region with GEF, a contact that could not be deduced from site-directed mutagenesis (
      • Mistou M.-Y.
      • Jacquet E.
      • Poullet P.
      • Rensland H.
      • Gideon P.
      • Schlichting L.
      • Wittinghofer A.
      • Parmeggiani A.
      ). Therefore, the present knowledge of the interaction sites of Ras with Raf and GEF indicates that the switch 1 and 2 regions represent structural elements that are directly involved in the binding of both GEF and effectors. The interferences between these ligands reported in our work thus depend on the overlap of their binding sites on p21, inducing exclusion phenomena.
      Recently, it has been demonstrated in intact cells and also in experiments in vitro that protein kinase A regulates the selectivity of Ha-Ras p21 binding to either RalGDS or Raf-1, and that the binding of RalGDS to the GTP-bound active form of Ras p21 inhibits the interaction of Ras p21 with Raf-1 and GAP (
      • Kikuchi A.
      • Demo S.D.
      • Ye Z.H.
      • Chen Y.W.
      • Williams L.T.
      ,
      • Kikuchi A.
      • Williams L.T.
      ). The human protein Rin has also been identified as a protein with a pronounced affinity for the GTP-bound state of Ras in vivo, competing with Raf-1 for binding to Ha-Ras in vitro (
      • Han L.
      • Colicelli J.
      ). This further supports the results of this work indicating the existence of coordinated mechanisms controlling the interactions of Ras with its multiple ligands. These mechanisms are based on mutual competitions for binding to p21 that are very likely regulated in the cell by the level of the active state of the various ligands of Ras.
      The complexity of the interactions in the network of Ras can be further illustrated by the relationship between p21 and PI3K (
      • Warne P.H.
      • Viciana P.R.
      • Downward J.
      ,
      • Satoh T.
      • Fantl W.J.
      • Escobedo J.A.
      • Williams L.T.
      • Kaziro Y.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Viciana P.R.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.
      • Downward J.
      ). In the intact cell, evidence has been obtained that Ras can stimulate PI3K activity; the ability of Ras to contribute to PI3K activation seems to represent an important portion of its downstream signaling (
      • Warne P.H.
      • Viciana P.R.
      • Downward J.
      ,
      • Viciana P.R.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.
      • Downward J.
      ). Nonetheless, an elevated level of GTP-bound Ras was found in response to constitutively active PI3K expressed in Xenopusoocytes (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ), suggesting that PI3K would not only act downstream of p21 but also act upstream to Ha-Ras on the activation mechanism leading to the GDP/GTP exchange. In this context, to shed more light on the issue of the upstream versus downstream role of PI3K, we have attempted to correlate this aspect with our results on Raf by studying the influence of PI3K RBD on the GEF-stimulated exchange on the active form of Ha-Ras. Similar to Raf, we have found that the presence of PI3K RBD reduced the GTP/GTP exchange on Ras stimulated by GEF; in contrast with Raf and other effector molecules of Ras such as RalGDS, no apparent inhibition by PI3K RBD on the intrinsic exchange on Ras was detected.
      Taken together, our results show that not only Raf-1 but also PI3K contributes to prevent GEF-dependent exchange on the active form of Ha-Ras p21. The mutual exclusion between GEF and Raf or PI3K, as the one previously reported for p120-GAP and GEF (
      • Giglione C.
      • Parrini M.C.
      • Baouz S.
      • Bernardi A.
      • Parmeggiani A.
      ), could represent a regulatory process under more physiological conditions, as suggested by the observation that by mimicking the cellular GDP:GTP molar ratio, the presence of Raf enhances the GEF-stimulated GDP/GTP or GTPγS exchange on Ha-Ras. This “feedback influence” of interactions of p21 and effectors on the level of the active state emphasizes the dual role of several protein ligands of Ras, such as Raf and PI3K in addition to p120-GAP, and reveals the existence of coordination mechanisms controlling the level of p21·GTP.
      To derive a model consistent with our results, one should take into account the following aspects. In the absence of external stimulation, Ras proteins are normally found in the inactive GDP bound state. When an external signal is transmitted by tyrosine kinase- or G protein-coupled receptors, rapid activation of Ras·GDP is achieved by the binding of GEF proteins and subsequent GDP release. After the dissociation of GDP, the nucleotide-free Ras protein, despite its comparable affinity for GDP and GTP, is likely to bind GTP, with the excess of GTP over GDP (10:1) in the cell assuring a preferential GTP binding, at least at the beginning. This leads to the formation of a mixed pool of Ras proteins composed by Ras·GTP and Ras·GDP complexes. Because the role of GEF is to activate as many molecules of Ras as possible, the interaction with already activated Ras proteins would be physiologically useless, delaying a productive GDP/GTP cycle. Futile interactions of Ras could be prevented, or at least greatly reduced, by binding to effectors that sequestrate the active form of Ras. This would favor the interaction of GEF with its biologically relevant substrate, the Ras·GDP complex. The model suggests a general mechanism modulating the activity of RAS through a competitive binding between effectors and GEF regulators that may determine the physiological course of the GDP/GTP cycle of Ras and thus the activity of the Ras pathway in the cell.

      Acknowledgments

      We are indebted to Drs. M. S. Marshall, P. N. Lowe, and J. Downward for providing the vectors encoding c-Raf-1 (1–149), c-Raf-1 (51–131), and PI3K RBD, respectively. We are grateful to Dr. P. N. Lowe for sending SPA beads and advice on this method. We thank our laboratory colleagues Drs. J. B. Créchet, E. Jacquet, and I. Krab for discussion and advice and Drs. S. Baouz and E. Jacquet for providing purified CDC25Mm and C-CDC25Mm-285.

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