We have examined the phosphorylation and the serine/threonine-specific kinase activity of the protooncogene product Raf-1 (formerly c-raf) in response to oncogenic transformation or growth-factor treatment of mouse 3T3 cells. Expression of the membrane-bound oncogene products encoded by v-fms, v-src, v-sis, polyoma virus middle-sized tumor antigen, and Ha-ras increased the apparent molecular weight and phosphorylation of the Raf-1 protein, while expression of the nuclear oncogene and protooncogene products encoded by v-fos and c-myc did not. Changes in electrophoretic mobility and phosphorylation occurred rapidly in response to treatment of cells with platelet-derived growth factor, acidic fibroblast growth factor, epidermal growth factor, and the protein kinase C activator phorbol 12-myristate 13-acetate, but not insulin. The phosphorylation of the Raf-1 protein occurred primarily on serine and threonine residues. However, a subpopulation of Raf-1 molecules was phosphorylated on tyrosine residues in cells transformed by v-src or stimulated with platelet-derived growth factor. Transformation by v-src, or treatment with platelet-derived growth factor or phorbol 12-myristate 13-acetate, activated the Raf-1-associated serine/kinase activity as measured in immune-complex kinase assays. These findings suggest that proliferative signals generated at the membrane result in the phosphorylation of the Raf-1 protein and the activation of its serine/threonine kinase activity. Raf-1 activation may thus serve to transduce signals from the membrane to the cytoplasm and perhaps on to the nucleus.

Our recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation, we have employed the following strategy. First, we have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60c-src, the CSF-1 receptor, or the platelet-derived growth factor (PDGF) receptor (Piwnica-Worms et al. 1987; Morrison et al. 1988). Second, we have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues (Kaplan et al. 1986, 1987; Piwnica-Worms et al. 1987; Morrison et al. 1988 and in prep.). In this endeavor, we are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the...