INHIBITION OF V-RAF-DEPENDENT C-FOS EXPRESSION AND TRANSFORMATION BY A KINASE DEFECTIVE MUTANT OF THE MITOGEN-ACTIVATED PROTEIN-KINASE ERK2

Receptor-bound growth factors elicit intracellular signals that lead t o the phosphorylation and activation of numerous intracellular kinases and transcription factors with consequent changes in patterns of gene expression. Several oncogene products are able to mimic these signals , resulting in cell transformation and proliferation. For example, the introduction of oncogenic forms bf Raf-l kinase into fibroblasts indu ces transformation and leads to the constitutive expression of, among others, the c-fos proto-oncogene. Here it is shown that the elevation of c-fos promoter activity brought about by v-raf is mediated by TCF/E lk-1, which forms a ternary complex with SRF at the serum response ele ment and is a substrate for mitogen-activating protein kinases in vitr o. In NIH 3T3 fibroblasts, v-raf activates Erk2, acid overexpression o f an interfering mutant of Erk2 both blocks the ability of v-raf to ac tivate the c-fos promoter and suppresses transformation. Mutation of i ndividual mitogen-activating protein kinase phosphoacceptor sites in T CF/Elk-1 also compromises v-raf-activated expression of a Gal-Elk/Gal- chloramphenicol acetyltransferase reporter system. However, in at leas t one instance the introduction of glutamate, but not aspartate, at a phosphoacceptor. site is compatible with activation. These results pro vide compelling evidence that phosphorylation of TCF/Elk-1 by Erk2 is a major link in the Raf-1 kinase dependent signal transduction pathway that activates c-fos expression.