Discrimination of Amino Acids Mediating Ras Binding from Noninteracting Residues Affecting Raf Activation by Double Mutant Analysis

Abstract

The contribution of residues outside the Ras binding domain of Raf (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg59 and Raf-Arg67 from RafRBD are interacting residues complementary to Ras-Glu37 located in the Ras effector region. Raf-Arg59 and Raf-Arg67 also mediate interaction with Ras-Glu37 in Ras-dependent Raf activation. The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser257 in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation does not restore Raf binding to Ras-E37G. Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/E37G is still impaired as compared with activation by Ras-G12V. This strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase. The contribution of residues outside the Ras binding domain of Raf (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg59 and Raf-Arg67 from RafRBD are interacting residues complementary to Ras-Glu37 located in the Ras effector region. Raf-Arg59 and Raf-Arg67 also mediate interaction with Ras-Glu37 in Ras-dependent Raf activation. The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser257 in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation does not restore Raf binding to Ras-E37G. Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/E37G is still impaired as compared with activation by Ras-G12V. This strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase. c-Raf-1 (herein referred to as Raf) is a member of a serine/threonine protein kinase family implicated in the transduction of signals from the cell surface to the nucleus (1Daum G. Eisenmann-Tappe I. Fries H.W. Troppmair J. Rapp U. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (488) Google Scholar). Raf provides an immediate downstream target for Ras and is a pivotal regulator of cell proliferation and differentiation (2Koide H. Satoh T. Nakafuku M. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8683-8686Crossref PubMed Scopus (160) Google Scholar, 3Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (782) Google Scholar, 4Van Aelst L. Barr M. Marcus S. Polverino A. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6213-6217Crossref PubMed Scopus (505) Google Scholar, 5Voijtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar, 6Warne P.H. Rodriguez Viciana P. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar, 7Zhang 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). Ras recruits Raf to the plasma membrane, and the requirement for Ras in Raf activation can be overcome by fusing a membrane targeting signal to the Raf C terminus (8Leevers S.J. Paterson H.F. Marshall C.J. Nature. 1994; 369: 411-414Crossref PubMed Scopus (886) Google Scholar, 9Stokoe D. Macdonald S.G. Cadwallader K. Symons M. Hancock J.F. Science. 1994; 264: 1463-1467Crossref PubMed Scopus (847) Google Scholar). Efficient oncogenic transformation of cells by Ras requires cooperation of different Ras-dependent signaling pathways of which the activation of the Raf/MEK/ERK pathway is essential (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 11Joneson T. White M.A. Wigler M.H. Bar-Sagi D. Science. 1996; 271: 810-812Crossref PubMed Scopus (358) Google Scholar, 12Qiu R.-G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (813) Google Scholar). The activation of Raf appears to be a complex multistep process (13Morrison D.K. Cutler Jr., R.E. Curr. Opin. Cell Biol. 1997; 9: 174-179Crossref PubMed Scopus (538) Google Scholar). The regulatory N terminus of Raf contains two regions that are highly conserved between different members of the Raf family. A part of the first conserved region (CR1) encompassing amino acids 51–131 is responsible for Ras binding and has therefore been termed Ras binding domain (RafRBD). 1The abbreviations used are: RBD, Ras binding domain; mant-Gpp(NH)p, fluorescent analogue of guanylyl-5′-yl imidodiphosphate labeled with the mant group, 2′,3′-(N-methylanthroniloyl); CR, conserved region; CRD, cysteine rich domain; wt, wild-type; PCR, polymerase chain reaction; RSV, Rous sarcoma virus; MEK, mitogen-activated protein or extracellular signal-related kinase kinase; ERK, extracellular signal-related kinase. RafRBD constitutes an autonomous structural domain sufficient for GTP-dependent binding of Ras (14Chuang E. Barnard D. Hettich L. Zhang X.-f. Avruch J. Marshall M. Mol. Cell. Biol. 1994; 14: 5318-5325Crossref PubMed Scopus (158) Google Scholar, 15Emerson S.D. Waugh D.S. Scheffler J.E. Tsao K.L. Prinzo K.M. Fry D.C. Biochemistry. 1994; 33: 7745-7752Crossref PubMed Scopus (33) Google Scholar, 16Fridman M. Tikoo A. Varga M. Murphy A. Nur-E-Kamal M.S.A. Maruta H. J. Biol. Chem. 1994; 269: 30105-30108Abstract Full Text PDF PubMed Google Scholar, 17Scheffler J.E. Waugh D.S. Bekesi E. Kiefer S.E. LoSardo J.E. Neri A. Prinzo K.M. Tsao K.-L. Wegrzynski B. Emerson S.D. Fry D.C. Biochemistry. 1994; 35: 22340-22346Google Scholar, 18Herrmann C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). The crystal structure of RafRBD complexed to the Ras homologue Rap1A has been solved (19Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (561) Google Scholar, 20Nassar N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar). Functional analysis of the interaction between Ras and RafRBD demonstrated that the mutation of Raf-Arg89 is sufficient to abrogate Ras-dependent Raf activation completely and that the activation of Raf correlates quantitatively with the binding affinity between Ras and RafRBD (21Fabian J.R. Vojtek A.B. Cooper J.A. Morrison D.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5982-5986Crossref PubMed Scopus (157) Google Scholar, 22Block C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). the role of RafRBD in Ras binding is in the contribution of other in the Raf regulatory domain to Ras-Raf interaction and signaling is from A of that the domain and the RafCR2 domain also be involved in Ras-Raf interaction and Ras-dependent Raf activation (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar, S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, S. S. C.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.A. C.J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. K. K. M. S. T. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, B. Marshall M.S. Avruch J. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). to the role of the RafCR2 that the interaction of the Ras effector mutant with Raf by the Raf-S257L mutation that is the RafCR2 the two-hybrid system (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). transformation by Ras-G12V/E37G be by of Ras-G12V/E37G with that the Raf-S257L mutation is to restore the binding of Raf to and as a Ras-dependent transformation to be to the activation of Raf-S257L by Ras-G12V/E37G (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). This further that the RafCR2 domain be involved in Ras-dependent Raf binding and activation. the RafCR2 region to mediate interaction of Raf with that of Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the mutation of to to constitutive activation of Raf that the RafCR2 region an role in the control of Raf activation J.R. Morrison D.K. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). structural are analysis of the RafCR2 domain provides the tool to investigate role in Ras-Raf protein-protein interaction and Raf activation. analysis has in protein-protein interaction J.A. PubMed Scopus Google Scholar, T. J.A. Science. 1995; PubMed Scopus Google as the Ras effector domain M.S. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). mutations of residues a protein-protein to on binding affinity J.A. Biochemistry. PubMed Scopus Google effect has been to interacting protein by the double mutant approach to identify complementary interacting amino acids G. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). we used the double mutant approach to identify amino acids in Raf that in interaction with Ras and to the role of Raf-Ser257 in Ras-Raf to biochemical analysis of mutations the RafRBD with to binding affinity C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google analysis of the interaction of of the Raf regulatory N terminus has been by a to expressed in E. affinity C. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Herrmann and C. we by of protein-protein interaction the two-hybrid which has been to between and interactions J. R. E. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). We developed a quantitative two-hybrid system to the effect of RafCR2 mutations on Ras-Raf binding we the effect of and RafCR2 mutations on Ras-dependent Raf activation a we the role of Raf-Ser257 and the RafCR2 domain by Ras-Raf binding and activation. The of the for and and of and mutant RafRBD been C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). protein as J. M. Wittinghofer A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). RafRBD as a for Ras C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). The affinity of RafRBD and RafRBD mutants for Ras therefore by Ras with fluorescent with RafRBD proteins. The of from Ras used to the in with of the complex with a of and different of and the as the of The by the to of been C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). and as C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). The mutant by two D. M. R. 1994; PubMed Scopus Google Scholar). used to the in the first a the and a the The the which by of the to of the the RafCR2 domain the a and a and of the RafCR2 the the mutant domain the Raf The two-hybrid Ras by of Ras residues of the or the Ras-G12V/E37G The Ras C terminus in to with the plasma Ras the binding domain D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The Raf regulatory domain and mutations and the activation domain by The of the and R. A. J. 1993; PubMed Scopus Google Scholar). cells to on and with a of of by the of or of a of C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google of an protein R. Mol. Cell. Biol. 1996; PubMed Scopus Google and of or a Raf used for of the or the of cells and and and as R. A. J. 1993; PubMed Scopus Google Scholar). activity by to The two-hybrid system in developed by and D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and here by the binding with Raf and cells to PubMed Scopus Google with of of the two-hybrid and on and and to interaction between the proteins. used as the activity by cells on and the by in for on and in and The an A of The activity to in with the cells in of by of of and of by the cell for and for in and cells the The with of The for in a to the cell The activity and the activity to the We the contribution of amino acids to Ras-Raf binding in by the effect of mutations RafRBD on the Ras-Raf binding affinity C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). the effect of a mutation in a protein-protein surface to the effect of the first complementary interacting amino acids can be by the of G. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). identify interacting residues in RafRBD that are complementary to Ras-Glu37 we binding affinity between and RafRBD mutants in The assay to binding the that RafRBD as a for Ras C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). to RafRBD the of the binding of RafRBD to and to Ras The mutation of the affinity to and to and of and Raf-Arg89 on the binding of as in the mutant and the mutation and the binding to the interaction to remained The of and binding to by and to a in binding to Ras-E37G. that Raf-Arg59 and Raf-Arg67 to as and effect on the interaction with Ras-E37G. that the double mutant approach is to identify amino acids in Ras effector that to residues the Ras effector for the between RafRBD or RafRBD mutants and and mutant between Ras and mutant RafRBD are from between Ras and RafRBD the which of of from Ras as a of the RafRBD as C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). The of the is between Ras and mutant RafRBD are from 22Block C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google in a between Ras and RafRBD the which of of from Ras as a of the RafRBD as C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). The of the is the mutation of the between Ras and RafRBD to in we the effect of mutation on Raf activation in we used a assay in This assay a by binding R. A. J. 1993; PubMed Scopus Google which are high affinity for to mediate activation of the signaling pathway R. A. 1993; PubMed Scopus Google Scholar). of Ras-G12V/E37G with Raf the activity to of the activity observed activation of Raf by This of activity to the of mutations RafRBD mutations in RafRBD that Ras-Raf binding affinity to a in activity to not that to the effect of mutations RafRBD on of Raf by we the of Ras-G12V/E37G The or of Ras-G12V/E37G to an in activity in the of Raf the of Raf of activity observed with This demonstrated that the by Ras-G12V/E37G by the activation of Raf and that Ras-G12V/E37G not Raf to the The signal by the of Ras-G12V/E37G that by activation of Raf by Ras-G12V. This to the effect of RafRBD mutants on activation by The double mutant approach complementary interacting residues the interaction of Ras with RafRBD in the activation of Raf by we the effect of RafRBD mutations the Raf protein on the activation of the pathway by We that correlates quantitatively with the binding affinity between Ras and RafRBD C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). which in a of Raf activation by Ras-G12V/E37G we RafRBD mutants for activation by of and which in to in affinity of RafRBD to also to an of Raf activity in the of the Ras-G12V/E37G the affinity of the mutant strongly still a activation of of as compared with the the which be by to of the with The effect of the and the mutation on the activation by Ras-G12V/E37G is in to the effect of mutations on the activation by which is by C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). the of mutations RafRBD on Ras-dependent we the of activation of Raf mutants by Ras-G12V/E37G activation by The demonstrates that residues Raf-Arg59 and Raf-Arg67 in RafRBD contribute strongly to activation of Raf by not a role in activation by identify Raf-Arg59 and Raf-Arg67 as interacting residues complementary to the Ras-Glu37 effector in the Ras-dependent activation of Ras-G12V/E37G to be to with Raf in the two-hybrid system (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). mutation of Raf-S257L the RafCR2 domain been to restore binding of Raf to the Ras-G12V/E37G that the RafCR2 domain and in Raf-Ser257 a role in Ras-Raf interaction (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). bacterially expressed Raf the regulatory domain a to C. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google not to the contribution of the RafCR2 domain to the Ras-Raf interaction in binding affinity by we to investigate the two-hybrid the two-hybrid system D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google we the with a to the binding domain in with the regulatory domain of Raf in the activation domain of with to and in a signal for a interaction between and the RafCR2 mutants or to on and an signal with Ras-G12V. to the binding between and RafCR2 mutants observed interaction between Ras-G12V/E37G and or Raf-S257L be we to that Raf-S257L can restore binding of Ras-G12V/E37G and the two-hybrid system as This the of the chain of Raf-Ser257 to Ras-Raf binding affinity the quantitative contribution of amino acids to Ras-Raf binding we a quantitative two-hybrid We the two-hybrid assay the RafRBD mutants for which the contribution to Ras-Raf binding affinity been C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). RafRBD mutants in the of the Raf regulatory we observed a quantitative between binding in with RafRBD and the activity with the two-hybrid assay in of Raf mutants has of of in the two-hybrid system not This demonstrates that the two-hybrid system can be used as a quantitative tool to the effect of amino of Raf that are not for affinity in We the activity by the The activity by the mutant to that of the regulatory domain This shows that mutation of does not Ras-Raf binding affinity and strongly suggests that the chain of Raf-Ser257 does not contribute to the interaction between Ras and The of contribution of Raf-Ser257 of the RafCR2 domain to the Ras-Raf interaction the of the mutation the activation of Raf in the by with or Ras-G12V/E37G characteristics to the activation of Raf by Ras mutants the activation of to be as compared with the activation of a of Raf-Ser257 in the activation of This to the activation of Raf-S257L by The Raf-S257L mutation in which with the activity by that been to be constitutively J.R. Morrison D.K. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). the activity of Raf-S257L the of the assay the we the of Raf-S257L The activity the activity that is by Ras-dependent Raf activation This to investigate the effect of Ras on Raf-S257L with the activity of Raf-S257L that mutation of Raf-S257L can be further by Ras-G12V. with the activation of Raf-S257L by the activation of Raf-S257L by Ras-G12V/E37G still demonstrates that the Raf-S257L mutant does not restore the of Raf to be by to a of Raf of the Raf-S257L mutation on Raf activity and by the of of Raf-S257L or the pathway as in of the of Raf-S257L to a of or of by Raf and the Raf-S257L mutant by of of or The of or used here as is still amino acids that are not part of the Ras-Raf surface in Ras-Raf interaction and activation of we by first complementary interacting residues the Ras-Raf surface double mutant The of approach as a to the contribution of residues that are located outside RafRBD to the Ras-Raf binding been that the of Raf binding by the mutation in the Ras effector region be by mutation of Raf-S257L in the RafCR2 domain (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google we on analysis of the role of amino acids Raf-Ser257 in Ras-Raf interaction and activation. we residues in Raf that contribute to Ras-Raf binding affinity by interacting with and which therefore complementary interacting residues of Biochemical analysis that Raf-Arg59 and Raf-Arg67 of RafRBD are interacting residues complementary to analysis that Raf-Arg67 is to in the complex (19Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (561) Google and that Raf-Arg59 is to in the complex of with RafRBD N. Horn G. Herrmann C. Block C. Janknecht R. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 723-729Crossref PubMed Scopus (177) Google Scholar). with we by analysis that Raf-Arg59 and Raf-Arg67 contribute to the interaction with The mutation been to signaling and transformation (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). We the effect of the mutation on signaling and Raf mutants a assay that Ras-Raf binding affinity C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). of the Raf to activation by Ras-G12V/E37G as compared with activation by Ras-G12V. This is in with the activity by Ras-G12V/E37G that (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google Scholar). to the Raf/MEK/ERK be that the effect of the mutation on Ras-Raf interaction and activation is the effect of the mutation that signaling completely (21Fabian J.R. Vojtek A.B. Cooper J.A. Morrison D.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5982-5986Crossref PubMed Scopus (157) Google Scholar, 22Block C. Janknecht R. Herrmann C. Nassar N. Wittinghofer A. Nat. Struct. Biol. 1996; 3: 244-250Crossref PubMed Scopus Google Scholar). the of Ras-dependent Raf signaling and to the effect of Raf the of Ras-G12V/E37G the activation of RafRBD mutants by Ras-G12V/E37G that the effect of RafRBD mutations on binding affinity in is completely in with the effect on Ras-dependent Raf signaling in residues Raf-Arg59 and which we to be complementary to Ras-Glu37 by in affinity also complementary to Ras-Glu37 in Ras-dependent Raf activation. This activation of Raf mutants by activation by The of by or Ras-G12V/E37G with other by Raf mutants with mutations that not the binding to mutation of and activation by activation by Ras-G12V/E37G the of Raf mutants in the assay can be used as a to the contribution of amino to and to Ras-dependent Raf activation in The criteria for complementary also to be for mutations of Ras interacting residues in Raf outside of been that the Raf-S257L mutation binding to and transformation (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google we the effect of Raf-Ser257 mutations on Ras-Raf binding and Ras-dependent Raf activation. and the and the Raf-S257L mutant interaction with we to a of the interaction with by the Raf-S257L mutant the two-hybrid The for to the of (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google be to the different of the two-hybrid assay we been to two-hybrid system for between and the affinity of to Raf-S257L be appears to that the Raf-S257L mutation not restore the interaction to We the two-hybrid system as a quantitative to the binding of the Ras-Raf The of quantitative two-hybrid system with RafRBD the of the regulatory the activity by RafRBD mutants in a with the of mutants we that the two-hybrid system can be used to the effect of point mutations on the affinity of Ras-Raf interaction and of other protein-protein interactions as in the mutant activity to we demonstrated that is to between in shows that the does not contribute to the Ras-Raf This also that the chain of Raf-Ser257 to the Ras-Raf the two-hybrid system are in with the we with which the Ras-dependent Raf activation. in the quantitative two-hybrid the activation characteristics as to we observed a constitutive activity of the Raf-S257L mutant which also in the mutation in the RafCR2 strongly constitutively the activity of Raf-S257L further by Ras-G12V. of Raf activity Ras-G12V/E37G used of the that Raf-S257L the interaction with and to a interaction between the observed activity of Ras-G12V/E37G Raf-S257L (10White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 1-20Abstract Full Text PDF PubMed Scopus (628) Google which be for the of Ras-Raf interaction by the Raf-S257L we can that Raf-S257L Ras-Raf we that Ras-G12V/E37G and the constitutively Raf-S257L different signaling The activation of pathways in to not be on Ras-Raf the RafCR2 domain appears to be an independent regulatory domain that can further signals the Raf signaling pathway in to Ras signal be the of RafCR2 by kinase B. Zhang Y. S. S. R. Nature. 1995; PubMed Scopus Google Scholar, Y. B. S. S. S. M. H. R. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). a of Raf be to the activation of the and of the of which the been solved F. I. J. Nature. 1997; PubMed Scopus Google Scholar, M.J. Nature. 1997; PubMed Scopus Google Scholar). the regulatory domain on the of the kinase the kinase domain The of by different the kinase. We to be a that also to in which different the regulatory region of the protein kinase may signals kinase activation. is that constitutively activating point mutations in the regulatory region of Raf-S257L or a common mechanism by with the inactive ground state of kinase. We D. for protein and R. for two-hybrid