ONCOGENIC RAS ACTIVATION OF RAF MITOGEN-ACTIVATED PROTEIN KINASE-INDEPENDENT PATHWAYS IS SUFFICIENT TO CAUSE TUMORIGENIC TRANSFORMATION/

Substantial evidence supports a critical role for the activation of th e Raf-1/MEK/nitogen-activated protein kinase pathway in oncogenic Ras- mediated transformation. For example, dominant negative mutants of Raf -1, MEK, and mitogen-activated protein kinase all inhibit Ras transfor mation. Furthermore, the observation that plasma membrane-localized Ra f-1 exhibits the same transforming potency as oncogenic Ras suggests t hat Raf-1 activation alone is sufficient to mediate full Ras transform ing activity. However, the recent identification of other candidate Ra s effecters (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathwa ys may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), w hich are defective for Raf binding and activation, induced potent tumo rigenic transformation of some strains of NIH 3T3 fibroblasts. These R af-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA(19N) mutant, indicating that R as may cause transformation that occurs via coordinate activation of R af-dependent and -independent pathways that involves Rho family protei ns. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) res ulted in synergistic cooperation of their focus-forming activities, in dicating that Ras activates at least two Raf-independent, Ras effector -mediated signaling events.