AN INHIBITORY RAF-1 MUTANT SUPPRESSES EXPRESSION OF A SUBSET OF V-RAF-ACTIVATED GENES

The Proto-oncogene Raf-1 is a cytoplasmic serine/threonine kinase impl icated in the signaling process in cell proliferation. To determine if Raf-1 is sufficient and necessary to transmit mitogenic signals to gr owth-responsive genes, we examined the effect of constitutively activa ted (v-raf) or inhibitory (Raf-C4) Raf-1 proteins on reporter gene act ivation in transient expression assays of NIH 3T3 cells. In serum-star ved cells, v-raf strongly activated transcription from the promoters o f the immediate-early genes c-fos and egr-2, as well as the proximal o r B promoter of the late growth response gene rep-3 (rep-3b). Two othe r late response gene promoters, cad and dhfr, were only modestly activ ated by v-raf, however. An individual serum response element from the c-fos or egr-2 promoter conferred both serum-inducibility and v-raf-re sponsiveness to a heterologous promoter. Consistent with the degree to which antisense c-raf-1 RNA and dominant-negative Raf-1 proteins inte rfere with NIH 3T3 cell proliferation, Raf-C4 reduced serum-induced tr anscription from the egr-2 and rep-3b promoters in a dose-dependent ma nner by 50%. In contrast, Raf-C4 did not significantly reduce transcri ption from the c-fos or cad promoters or the serum response element-dr iven heterologous promoters. We conclude that Raf-1 is both sufficient and necessary to activate a subset of early and late growth response genes.