BIOCHEMICAL-ANALYSIS OF MEK ACTIVATION IN NIH3T3 FIBROBLASTS - IDENTIFICATION OF B-RAF AND OTHER ACTIVATORS

Numerous potential activators of MEK have been identified, including c -Raf-1, B Raf, c-Mos, and a family of MEK kinases. However, little inf ormation gives insight into the activators actually utilized in vivo. To address this, we have used column chromatography and a coupled MEK activation assay to identify in NTH3T3 cells, two major MEK activators , and a third insulin-specific activator. The first MEK activator has an apparent M(r) of 40,000-50,000, was immunologically distinct from A -Raf, B-Raf, c-Raf-1, c-MEKK, c-Mos, MEK1, and MEK2, and was rapidly a ctivated by serum, platelet-derived growth factor (PDGF), insulin, thr ombin, and phorbol ester. The second MEK activator was identified as B -Raf. Activation of 93-95 kDa B-Raf was observed in column fi actions and B-Raf immunoprecipitates from cytosolic and particulate fractions after stimulation with serum or PDGF, but not insulin. c-Raf-1 from cy tosol did not exhibit MEK activator activity; however, c-Raf-1 immunop recipitates from the particulate fraction revealed MEK activator activ ity that was enhanced after stimulation with PDGF or phorbol ester, bu t not serum or insulin. Both c-Mos and c-MEKK, were present in NTH3T3 fibroblasts but did not show MEK activator activity. These data provid e direct evidence that 93-95-kDa B-Rafisozymes and an unidentified 40- 50-kDa MEK activator are major agonist-specific MEK activators in NTH3 T3 fibroblasts.