Ras- and Raf-induced down-modulation of non-muscle tropomyosin are MEK- independent

Transformation is accompanied by the down-regulation of the high molecular weight isoforms of non-muscle tropomyosin. Several lines of evidence suggest that tropomyosin down-regulation may be essential for ras- induced tumorigenicity. It is unclear which of the many signaling pathways downstream of Ras are involved in tropomyosin down-regulation. Here we demonstrate that Raf activation induces tropomyosin down-regulation comparable to that induced by Ras. Expression of the effector-domain mutant Ras-G12V,Y40C, which is unable to bind Raf, induced only modest down- modulation of tropomyosin. Treatment with the MEK-specific inhibitor PD98059 had little effect on tropomyosin levels in ras- or raf-transformed cells. In contrast, a mutant form of MEK-1, MEK-1-S218A,S222A, restored tropomyosin levels in ras-transformed NIH3T3 cells almost to the levels observed in non- transformed cells. MEK-1-S218A,S222A does not inhibit MEK phosphorylation and is a poor inhibitor of ERK phosphorylation. These data suggest that this mutant form of MEK-1 interferes with a yet uncharacterized pathway controlled by Raf. We conclude that the ras-induced down-modulation of tropomyosin is predominantly Raf-mediated, but MEK-independent, and that a novel pathway exists downstream of Raf which may play an important role in regulation of the cytoskeleton.