The Ras pathway is a critical signal transduction cascade involved in regulating cellular proliferation, differentiation, and survival. Members of the Raf serine/threonine kinase family are key intermediates in this pathway, functioning to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. Three Raf proteins are found in mammalian cells, Raf-1, A-Raf, and B-Raf. As might be expected for proteins so centrally involved in cell signaling, the Raf kinases also contribute to oncogenic transformation and cancer. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently induces deregulated signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. Moreover, constitutively active Raf proteins can themselves cause cell transformation. In particular, mutation of the B-Raf family member is observed in 67% of malignant melanomas as well as in many colorectal, ovarian, and papillary thyroid carcinomas. During this past fiscal year, our research has elucidated several important mechanisms contributing to the regulation of both normal and oncogenic Raf signaling. Our studies have revealed that the KSR1 scaffold plays a critical role in modulating the intensity and duration of Raf signaling emanating from the plasma membrane in response to growth factor treatment. In addition, we have found that the KSR expression levels can alter the effects of Raf inhibitors on oncogenic Ras/ERK signaling. Specifically, KSR1 competes with C-Raf for inhibitor-induced binding to B-Raf and in doing so attenuates the paradoxical activating effect of these drugs on ERK signaling. Our studies have also revealed that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events (e. g., phosphorylation on inhibitory feedback sites) and protein interactions (e.g., 14-3-3 binding and heterodimerization with C-Raf). Taken together, these findings identify potential targets for therapeutic intervention in tumors with constitutive Ras- or Raf-dependent signaling.
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