A series of wild-type and mutant rafgenes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine-and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and CR2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.The raffamily of proto-oncogenes consists of three active members: A-raf-1, B-raf, and c-raf-1 (2,4,5,20,22,23). The three proteins show the greatest stretch of homology in the carboxy-terminal half, which, in the case of c-raf-1, has been shown to have serine-and threonine-specific protein kinase activity (33, 48). Physiologically, Raf protein kinases function as information shuttles that communicate between the cell surface and nucleus. From Ki-ras revertant and anti-ras antibody microinjection studies, it is known that c-raf-1 acts independently of and perhaps downstream from Ras in signal transduction (2,22,36,41,42,52). In addition, we recently showed that Raf is hyperphosphorylated and enzymatically activated in cells which are transformed by src, fms, or ras, and in cells which have been treated with platelet-derived growth factor or 12-0-tetradecanoylphorbol-13-acetate (TPA) (35). In response to platelet-derived growth factor and TPA, Raf protein is also translocated from the cytoplasm to the perinuclear space, as is evident from immune fluorescence and cell fractionation studies (44). Ultimately, rafis thought to exert its effect by modulation of transcription factor activity via phosphorylation (42). Evidence for this comes from experiments in which cells transfected with activated raf genes showed increased transcriptional activity from an APl-dependent promoter in transient transfection assays, suggesting that Raf is involved in the regulation of transcription factors of the jun-AP1 gene family (56). Raf function is essential for normal rates of cell proliferation, as demonstrated by the lethal effect of mutations in the Drosophila melanogaster D-raf-1 locus (37).c-raf-1, the most-studied member of the family, encodes a * Corresponding auth...