Dephosphorylation of Ser-259 regulates Raf-1 membrane association.
Mitogenic stimulation of Raf-1 is a complex yet incompletely understood process involving membrane relocalization and phosphorylation of activating residues. We recently reported that Raf-1-associated protein phosphatase 2A contributes to kinase activation, an effect mediated via Ser-259 of Raf-1. Here, we show that mitogens stimulate Ser-259 dephosphorylation and Raf-1/protein phosphatase 2A association concomitantly with membrane accumulation and activation of Raf-1. Blocking Ser-259 dephosphorylation inhibits the two latter events, but it does not prevent activation of a S259A Raf-1 mutant, which is preferentially localized at the membrane independently of mitogenic stimulation. Inhibition of Ser-259 dephosphorylation has no effect on the activation of membrane-tethered Raf-1 (Raf-1CAAX). These data show that Ser-259 dephosphorylation contributes to Raf-1 activation by supporting its membrane accumulation rather than by increasing the specific activity of the kinase and provide a mechanistic basis for the support of kinase activation by Raf-1-associated protein phosphatase 2A.
Kubicek
Markus
M
Department of Cell Biology and Microbiology, Institute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohr Gasse 9, 1030 Vienna, Austria.
Pacher
Margit
M
Abraham
Dietmar
D
Podar
Klaus
K
Eulitz
Manfred
M
Baccarini
Manuela
M
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008954:Models, Biological; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D054648:Protein Phosphatase 2; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D019319:Okadaic Acid; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2