CHARACTERIZATION OF THE RELATIONSHIP BETWEEN RAF AND GROWTH REGULATORS

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Cytoplasmic serine/threonine-specific kinases, such as raf and protein kinase C, play an integrative role in the transduction of signals from the cell membrane to the nucleus. To dissect raf-specific signaling pathways, we designed raf inhibition experiments employing monoclonal antibodies as well as expression of antisense RNA. The isolation of cells resistant to transformation by v-raf constituted a second strategy to identify factors which regulate raf-dependent signaling pathways. These cells, termed CHP25, express a functional v-raf oncogene. However, they are non-tumorigenic, do not form colonies in soft agar, and possess a flat morphology. CHP25 cells are resistant to transformation by sis, ras, and tyrosine- and serine/threonine-kinase encoding oncogenes suggesting that Raf functions downstream of most membrane-associated and cytoplasmic signal inducers. In contrast to v-raf-transformed cells in which the endogenous Raf-I protein kinase is constitutively activated, v-Raf in CHP25 cells does not activate endogenous Raf-1 kinase. Since mitogen regulation of Raf-I kinase in CHP25 cells is intact, we conclude that CHP25 cells are blocked at the level of Raf-I substrate phosphorylation. Consistent with this interpretation, CHP25 cells show specific alterations of early gene induction. The serum induction of c-fos and junD, as well as the serum and 12-0-tetradecanoylphorbol-13-acetate (TPA) induction of junB and egr-1, are almost completely abolished. There is a downstream bypass to the Raf-I block since v-fos can re-transform CHP25. However, v-myc and v-jun, c-jun, and junB are ineffective. We conclude that Raf-I signaling is essential for transformation of NIH/3T3 cells by peripheral oncogenes and for regulation of a subset of early response genes by TPA and serum growth factors. The Raf-I inhibition experiment points to jun as a substrate critical for raf oncogene transformation. Other substrates include a ppl2O phosphoprotein which is currently being purified.