MITOGEN-ACTIVATED PROTEIN-KINASE EXTRACELLULAR SIGNAL-REGULATED PROTEIN-KINASE ACTIVATION BY ONCOGENES, SERUM, AND 12-O-TETRADECANOYLPHORBOL-13-ACETATE REQUIRES RAF AND IS NECESSARY FOR TRANSFORMATION

The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyr osine kinase receptors in the membrane with control of transcription f actor activity in the nucleus. We have examined whether Raf is obligat ory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope -tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as represe ntatives of three classes of oncogenes (protein tyrosine kinases abl/s rc, Ras, and protein serine/ threonine kinases mos/cot) were all Raf-d ependent for stimulation of MAPK. All of the MAPK stimulating oncogene s were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the abilit y to stimulate MAPK/ERK appears to be critical for transformation by o ncogenic Raf-1 as ERK-1 and -2 synergized with v-raf in a focus induct ion assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibi tory. Raf/ERK synergism was also observed in transcriptional transacti vation of the oncogene-response element in the polyoma enhancer. We co nclude that this Raf signaling pathway, which connects to many upstrea m activators and downstream effecters, is essential for transformation by most oncogenes.