Cancer often arises when the control of normal cell function goes awry due to defects in critical signal transduction pathways. The signaling pathway regulated by the RasGTPase is one such pathway and it functions to modulate vital cellular processes, including proliferation, differentiation, survival, and senescence. Members of the Raf serine/threonine kinase family are key intermediates in the Ras pathway, serving to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. There are three mammalian Raf proteins, A-Raf, B-Raf, and C-Raf (also known as Raf-1). As might be expected for proteins so centrally involved in cell signaling, the Raf kinases can directly contribute to oncogenic transformation and other human disease states. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently results in constitutive signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. In addition, mutations in the Raf proteins themselves can function as disease drivers. Germline-mutations in C-Raf are causative for Noonan and LEOPARD syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) syndromes, with B-Raf mutations occurring in 75% of CFC patients. Moreover, somatic mutations in B-Raf are observed in 70% of malignant melanomas as well as in many colorectal, ovarian, lung and papillary thyroid carcinomas. Our research has elucidated several important mechanisms contributing to the regulation of both normal and mutant 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, our work has provided insight regarding how the KSR1 scaffold is recruited from the cytosol to the plasma membrane in response to growth factor signals. Through structure/function analysis, our studies showed that the conserved CA1 region of KSR1 forms an extended sterile-alpha-motif domain, which upon growth factor signaling, becomes exposed and binds phospholipids found in the plasma membrane. As a result, KSR1-bound MEK is localized to the cell surface where it can be phosphorylated by activated Raf kinases. Finally, our studies have revealed that the expression levels of KSR1 can alter the effects of ATP-competitive Raf inhibitors on oncogenic Ras to 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 shown that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events (e. g., phosphorylation on inhibitory feedback sites and the phosphorylation of residues that mediate 14-3-3 binding) and protein interactions (e.g., 14-3-3 binding and Raf dimerization). Moreover, we have found that Raf dimerization is critical for upregulated signaling induced by human disease-associated Raf mutants with moderate, low or impaired kinase activity, or in cases where the pathway is induced by activated RTK or Ras proteins. Our work has further revealed that somatic mutations which modulate Raf dimerization have the potential to alter the progression and treatment of human disease states with elevated Ras pathway signaling. Finally, our work has provided the first 'proof of principle'that inhibiting Raf dimerization can suppress Raf signaling under conditions where dimerization is required. Taken together, these findings have important implications for the treatment of human disease states with elevated Ras pathway signaling and identify the Raf dimer interface as a therapeutic target.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Freeman, Alyson K; Ritt, Daniel A; Morrison, Deborah K (2013) The importance of Raf dimerization in cell signaling. Small GTPases 4:
Freeman, Alyson K; Ritt, Daniel A; Morrison, Deborah K (2013) Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. Mol Cell 49:751-8
Morrison, Deborah K (2009) The 14-3-3 proteins: integrators of diverse signaling cues that impact cell fate and cancer development. Trends Cell Biol 19:16-23
McKay, Melissa M; Ritt, Daniel A; Morrison, Deborah K (2009) Signaling dynamics of the KSR1 scaffold complex. Proc Natl Acad Sci U S A 106:11022-7