Raf Function Is Required for Proliferation of NIH|3T3 Cells and Transformation by Nonnuclear Oncogenes

Studies of oncogenes have established that their cellular homologs are part of a kinase cascade mechanism that regulates the translation of exogenous growth factor stimulation into a mitogenic gene response [reviewed in 1]. The cellular homolog of the v-raf oncogene [2], c-raf-1 [3], encodes a mitogen-regulated serine/threonine-specific protein kinase, whose specific activity is enhanced by a large number of growth factors [4-13]. Moreover, treatment of NIH/3T3 fibroblasts with platelet-derived growth factor (PDGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) causes translocation of a fraction of Raf-1 from the cytosol to the nucleus [12]. A similar fraction of v-Raf is found associated with the nucleus also in the absence of stimulation (W. B. Anderson and U. R. Rapp, unpublished results). These observations led to the speculation that Raf-1 serves as a shuttle enzyme which converts peripheral signals into a transcriptional response that results in mitogenesis [14]. Consequently, a key step in transformation can be envisioned to uncouple Raf-1 kinase from mitogen regulation, either through constitutive stimulation by upstream signal transducers or mutational activation of raf itself. This hypothesis was tested in NIH/3T3 fibroblasts using three complementary approaches to interfere with the function of normal and transforming versions of Raf-1. These strategies included expression of c-raf-1 antisense RNA or kinase inactive c-raf-1 mutants, as well as the analysis of a cellular mutant phenotype that can suppress v-raf transformation.