title: PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway.
abstract: The serine/threonine kinase Raf-1 acts downstream of Ras in the MAPK pathway leading to ERK activation in response to mitogens. Raf-1 has oncogenic potential, but is normally controlled by a complex interplay of inhibitory and activating mechanisms. Although Raf-1 is phosphorylated in unstimulated cells, mitogens cause its membrane recruitment by Ras and subsequent phosphorylation on additional sites. Some of these events modulate Raf-1 kinase activity while others determine interactions with other proteins. These changes regulate the ability of Raf-1 to phosphorylate its downstream targets MEK1 and MEK2. Rho family small G proteins act synergistically with Raf-1 to stimulate the ERK pathway by a cross-cascade mechanism that enhances MEK phosphorylation by Raf-1. Here we show that both Raf-1 and MEK1 are phosphorylated by PAK1 and that mutations at PAK1 phosphorylation sites in either protein prevent cross-cascade activation. In contrast, MEK1 activation by constitutively-active Raf-1 is refractory to mutations at PAK1 phosphorylation sites. Phosphorylation of MEK1 on serine 298 does not appear to regulate the interaction between Raf-1 and MEK1, but rather the ability of Raf-1 to phosphorylate MEK1 with which it is complexed in vivo. Our findings indicate that PAK1 primes MEK1 for activation by Raf-1 and imply another level of regulation in the ERK cascade.
authors.affiliation: School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.
authors.forename: Lucy C
authors.initials: LC
authors.lastname: Coles
authors.forename: Peter E
authors.initials: PE
authors.lastname: Shaw
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D054462:p21-Activated Kinases
chemical_list: D013912:Threonine; D012694:Serine; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D054462:p21-Activated Kinases; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases