Uncoupling Raf1 from MEK1/2 impairs only a subset of cellular responses toRaf activation

The Raf family of serine/threonine protein kinases is intimately involved i n the transmission of cell regulatory signals controlling proliferation and differentiation. The best characterized Raf substrates are MEK1 and MEK2. The activation of MEK1/2 by Raf is required to mediate many of the cellular responses to Raf activation, suggesting that MEK1/2 are the dominant Raf e ffector proteins. However, accumulating evidence suggests that there are ad ditional Raf substrates and that subsets of Raf-induced regulatory events a re mediated independently of Raf activation of MEK1/2. To examine the possi bility that there is bifurcation at the level of Raf in activation of MEK1/ 2-dependent and MEK1/2-independent cell regulatory events, we engineered a kinase-active Raf1 variant (RafBXB(T481A)) with an amino acid substitution that disrupts MEK1 binding. We find that disruption of MEK1/2 association u ncouples Raf from activation of ERK1/2, induction of serum-response element -dependent gene expression, and induction of growth and morphological trans formation. However, activation of NF-kappaB-dependent gene expression and i nduction of neurite differentiation were unimpaired. In addition, Raf-depen dent activation of p90 ribosomal S6 kinase was only slightly impaired. Thes e results support the hypothesis that Raf kinases utilize multiple downstre am effectors to regulate distinct cellular activities.