title: 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.
authors.affiliation: Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, D-44026 Dortmund, Germany.
authors.forename: B K
authors.initials: BK
authors.lastname: Jaitner
authors.forename: J
authors.initials: J
authors.lastname: Becker
authors.forename: T
authors.initials: T
authors.lastname: Linnemann
authors.forename: C
authors.initials: C
authors.lastname: Herrmann
authors.forename: A
authors.initials: A
authors.lastname: Wittinghofer
authors.forename: C
authors.initials: C
authors.lastname: Block
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D018698:Glutamic Acid / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012441:Saccharomyces cerevisiae; D012694:Serine / Q000378:metabolism; D015533:Transcriptional Activation; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018698:Glutamic Acid; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins