title: Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells.
abstract: Activation of the mitogen-activated protein kinase cascade is a critical event in mitogenic growth factor signal transduction. Mitogen-activated protein kinase is directly activated by a dual specific kinase, MEK, which itself is activated by serine phosphorylation. The c-Raf kinase has been implicated in mediating the signal transduction from mitogenic growth factor receptors to MEK activation. Recently, the B-Raf kinase was shown to be capable of phosphorylating and activating MEK as a result of growth factor stimulation. In this report, we used the yeast two-hybrid screening to isolate MEK interacting proteins. All three members of the Raf family kinases were identified as positive clones when the mutant MEK1S218/222A, in which the two phosphorylation serine residues were substituted by alanines, was used as a bait, whereas no positive clones were isolated when the wild type MEK1 was used as a bait in a similar screening. These results suggest that elimination of the phosphorylation sites of a target protein (MEK1 in our study) may stabilize the interaction between the kinase (Raf) and its substrate (MEK1), possibly due the formation of a nonproductive complex. These observations seem to suggest a general strategy using mutants to identify the upstream kinase of a phosphoprotein or the downstream targets of a kinase. Although c-Raf and B-Raf have been implicated in growth factor-induced MEK activation, little is known about A-Raf. We observed that stimulation of Hela cells with epidermal growth factor resulted in a rapid and transient activation of A-Raf, which is then capable of phosphorylating and activating MEK1. Interestingly, A-Raf does not activate MEK2, although c-Raf can activate both MEK1 and MEK2. Our data demonstrated that A-Raf is, indeed, a MEK1 activator and may play a role in growth factor signaling.
authors.affiliation: Department of Biological Chemistry and the Institute of Gerontology, the University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: S J
authors.initials: SJ
authors.lastname: Noh
authors.forename: G
authors.initials: G
authors.lastname: Zhou
authors.forename: J E
authors.initials: JE
authors.lastname: Dixon
authors.forename: K L
authors.initials: KL
authors.lastname: Guan
mesh_terms: D003001:Cloning, Molecular; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity
chemical_list: D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D012694:Serine; D004815:Epidermal Growth Factor; C482128:MAP2K2 protein, human; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases