Detection of kinases that phosphorylate 14-3-3 binding sites of Raf-1 using in situ gel kinase assay.
Raf-1 is a serine/threonine protein kinase that plays a critical role in mitogenic signal transduction. Raf-1 activation requires 14-3-3 binding to Raf-1 as an essential step. This binding is regulated through phosphorylation of Ser259 and Ser621 of Raf-1, each constituting part of the consensus motif for the binding of Raf-1 to 14-3-3. However, Raf-1 kinase kinase(s) that phosphorylates these sites remains unknown. In this report, we detected Raf-1 kinase kinase activity using recombinant glutathione-S-transferase-Raf-1 fusion proteins as substrate of in situ gel kinase assay. Ser259 was phosphorylated by a kinase with a molecular weight of 90 kDa, which was suggested to be Rsk judging from the molecular size, the time course of activation after EGF stimulation and the elution pattern from an anion-exchange column. The Raf-1 fragment containing Ser621 was phosphorylated by kinases with molecular weights of 85, 60, 50 and 48 kDa but not by the kinase that phosphorylates Ser259. These results suggest that although Ser259 and Ser621 lie in the same amino acid sequence motif for 14-3-3 binding, these two regulatory sites for this binding are phosphorylated by different protein kinases.
Kinuya
M
M
Department of Biochemisty I, National Defense Medical College, Tokorozawa, Saitanma, Japan.
Takishima
K
K
Mamiya
G
G
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005782:Gels; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation / Q000502:physiology; D010764:Phosphorylase Kinase / Q000032:analysis*; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D012694:Serine / Q000378:metabolism
D005782:Gels; D011994:Recombinant Proteins; D012694:Serine; D004815:Epidermal Growth Factor; D005982:Glutathione Transferase; D010764:Phosphorylase Kinase; D019908:Proto-Oncogene Proteins c-raf