Mutational activation of c-raf-1 and definition of the minimal transforming sequence.

Abstract

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...

Strict regulation of c-Raf kinase levels is required for early organogenesis of the vertebrate inner ear

“…In order to clone c-raf and Raf-C4 cDNAs into RCAS, c-raf and Raf-C4 were subcloned into the PAJ10 adapter plasmid to obtain ClaI ends. PAJ10 is a Bluescript plasmid containing a multicloning adapter anked by ClaI sites that was a kind gift of Dr D Martin Zanca (CSIC, Salamanca, Spain), c-raf was cleaved from Elneo c-raf (Heidecker et al, 1990) by EcoRI-ApaI digestion and inserted into PAJ10 (PAJ10/RAF). Then craf was removed from PAJ10 by digestion with ClaI and inserted into RCAS at the ClaI site (RCAS/RAF).…”

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

“…A 14.3.3 dimer may bridge CR2 to a separate binding site in CR3, maintaining an inactive conformation (Luo et al, 1995). Removal of CR1 and CR2 or removal of the 14.3.3 binding site in CR2 (serine 259) results in the oncogenic activation of Raf-1 (Heidecker et al, 1990;Morrison, 1996).…”

Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis

“…MEK then phosphorylates MAPK on threonine and tyrosine, and the activated MAPK which is a serine/threonine kinase is able to phosphorylate several nuclear transcription factors inluding Myc, Elk and Rsk (Siegfried and Zi , 1990;Kolch et al, 1991;Alexandropoulos et al, 1992;Blenis, 1993;Thomas, 1992;Crews et al, 1992). It is now known that not only can the activated Ras oncogene cause cellular transformation (Barbacid, 1987;Trahey et al, 1987), but that activated v-Raf or a constitutively activated MAPKK can also result in transformation (Heidecker et al, 1990;Cleveland et al, 1994;Samuels et al, 1993;Mansour et al, 1994).…”