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 mi togenic signal transduction. Raf-l activation requires 14-3-3 binding to Ra f-1 as an essential step. This binding is regulated through phosporylation of Ser259 and Ser621 of Raf-1, each constituting part of the consensus moti f for the binding of Raf-1 to 14-3-3. However, Raf-l kinase kinase(s) that phosphorylates these sites remains unknown. In this report, we detected Raf -l kinase kinase activity using recombinant glutathione-S-transferase-Raf-1 fusion proteins as substrate of in situ gel kinase assay. Ser259 was phosp horylated by a kinase with a molecular weight of 90 kDa, which was suggeste d 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 m olecular weights of 85, 60, 50 and 48 kDa but not bg the kinase that phosph orylates Ser259. These results suggest that although Ser259 and Ser621 lie in the same amino acid sequence motif for 14-3-3 binding, these two regulat ory sites for this binding are phosphorylated by different protein kinases.