Tumor formation in vivo and the in vitro transformation of cells cultured from primary tissues require multiples oncogenic events. These events result from the creation or introduction of dominant acting oncogenes and the inactivation of tumor suppressors. Susceptibility of a cell to the transforming properties of these genes may be modified by other cellular genes that, while not driving the oncogenic process, are necessary for the transduction of the transforming event. The central hypothesis of this proposal is that the protein product of the gene Kinase Suppressor of Ras (KSR) is a potent modifier of cell transformation by oncogenic Ras. The goal of this proposal is to explain the molecular mechanisms underlying the biological actions of this putative modifier. KSR was identified as a loss-of-function allele that suppresses the phenotype of activated Ras in Drosophila and C. elegans. These data indicated that normal KSR may be a positive regulator of Ras-regulated pathways in lower eukaryotes. Analysis of oncogenic Ras-mediated transformation in primary fibroblasts from KSR about mice indicate that KSR functions similarly in mammals. Additional analyses demonstrate that KSR is a specific effector of the Ras/Raf/MEK/ERK kinase cascade. KSR is phosphorylated, and mutation of a subset of these phosphorylation sites causes the redistribution of KSR from the cytoplasm to the nucleus. These observations have lead to the hypotheses that the phosphorylation of KSR determines its subcellular location and that the localization of KSR affects its ability to regulate the Ras/Raf/MEK/ERK kinase cascade and Ras-mediated cell transformation. These hypotheses will be tested by: 1) identifying the sequences in KSR that regulate its subcellular distribution; 2) determining the role of KSR phosphorylation in regulating the distribution of KSR between the cytoplasm and the nucleus; 3) characterizing the physical interaction of KSR with Raf, MEK and ERK and its biological consequences; and 4) determining how the contribution of KSR to cell transformation is affected by its subcellular distribution and phosphorylation.
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