Raf family protein kinases function as second messengers in mitogenic signal transduction events. So far, most of the work has been done with the c-Raf-1 isozyme. We have now performed biochemical characterization of the endogenous A-Raf kinase. Like c-Raf-1, the protein of the A-Raf proto-oncogene is a serine/ threonine kinase that is activated after stimulation of cells with platelet derived growth factor and other mitogens. Incubation with these mitogens leads to a shift in electrophoretic mobility, which is caused by an increased phosphate incorporation into the A-Raf protein. To investigate the Ras-dependent regulation of the A-Raf kinase, we used stable transfectants of PC12 pheochromocytoma cells with constitutive expression of a dominant negative Ras mutant (M17) and a dexamethasone inducible mutant in NIH/3T3 cells. The expression of the Ras mutant blocks A-Raf phosphorylation and kinase activity of stimulated cells in the same way as c-Raf-1 phosphorylation and activation is blocked. In transient transfection assays, it is shown that the N-terminal half, the regulatory portion of the A-Raf kinase, is blocking Ras induction of c-Raf-1-dependent reporter gene expression to the same extent as the regulatory part of the c-Raf-1 kinase itself. This indicates regulation of these isozymes by a common mechanism.
