Project Summary This proposal aims to discover mechanistic insights into how signal transduction by the highly conserved Ras-Raf-MEK-ERK pathway is modulated by regulated protein degradation. In humans, mutation of the gene BRAF, encoding one of three human Rafs, is a driver event in the development of malignant melanoma. Indeed, the mutation BRAF(V600E) confers constitutive kinase activation and is one of the most frequent mutations found in this cancer. Melanoma growth can be suppressed by inhibition of Raf kinase activity. However, development of drug resistance and disease relapse occurs frequently, indicating that truly effective treatments will require additional drug strategies. Our work, utilizing Caenorhabditis elegans as a simple model to investigate signal transduction by this conserved pathway, demonstrates that C. elegans Raf signaling is kept in check by inhibitory mechanisms that include phosphorylation and protein degradation (de la Cova and Greenwald, 2012). For this proposed research, we use the C. elegans model to design and begin an RNAi screen to identify conserved kinases and phosphatases that regulate Raf. We find that CDK2 is necessary for Raf protein degradation, and that loss of CDK2 enhances phenotypes caused by a mutant, activated Raf engineered to carry the same mutation as BRAF(V600E). Our long-term goal is to identify new molecular targets for the development of drugs capable of inhibiting Raf signaling. Toward this goal, our overall objectives in this application are to (i) identify additional kinases and phosphatases that impact Raf protein degradation and their requirement for regulating BRAF in human cells, and (ii) determine the mechanism and consequences of Raf regulation by the kinase CDK2. Our central hypothesis is that conserved mechanisms, such as phosphorylation of the Raf protein, act to prevent aberrant Raf activation. The rationale for this project is that our discovery of inhibitory control mechanisms governing signal transduction by the Ras-Raf-MEK-ERK pathway will inform new strategies of drug therapy for melanoma.