MECHANISMS OF RAF ACTIVATION

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Raf-1 protein serine/threonine kinase (PSK) functions as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C (PKC) at the membrane with activation of early growth response genes in the nucleus. Recently we have shown that another member of the Raf family, B-Raf, is activated in PC12 cells after nerve growth factor stimulation. Receptor coupling of Raf-1 and B-Raf activation is controlled by p21Ras. Growth factor stimulation of cells results in the increased phosphorylation and concomitant kinase activation of Raf-1 and B-Raf. In addition to regulatory phosphorylations, a role for phospho- lipid-derived products in the activation of Raf-1 has been suggested by the presence of a cysteine finger motif in the N-terminal half of the Raf- 1 protein, which is conserved in other Raf PSK family members and related to a similar structure in PKC. We have now extended our studies on Raf-1 and B-Raf to A-Raf, and were able to show that (i) kinase activation of A- Raf occurs under identical conditions and with similar kinetics as previously seen with Raf-1, (ii) PKC-alpha phosphorylates and activates Raf-1, (iii) products derived from the hydrolysis of phosphatidylcholine activate Raf-1, and (iv) we have identified the PSK oncogenes mos and cot as potential Raf kinase kinases.