title: Positive and negative regulation of Raf kinase activity and function by phosphorylation.
abstract: Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA.
authors.forename: H
authors.initials: H
authors.lastname: Chong
authors.forename: J
authors.initials: J
authors.lastname: Lee
authors.forename: K L
authors.initials: KL
authors.lastname: Guan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D019908:Proto-Oncogene Proteins c-raf