Proteolysis of a number of important regulators of cell growth, transformation and survival is controlled by the SCF(HOS/beta-Trcp) E3 ubiquitin ligases in a phosphorylation-dependent manner. We and others demonstrated that levels of HOS/beta-Trcp proteins are increased in tumor cells and that these levels are pivotal for degradation of those substrates, which limit cell growth and survival. Hos levels are induced via mitogen-activated protein kinase (MARK) pathway. In continuing our studies of the role of HOS in pathogenesis of cancer we focus on degradation of the IFNAR1 subunit of interferon alpha (IFNa) receptor, which we recently identified as a novel substrate of HOS. HOS-controlled ubiquitination and degradation of IFNAR1 depends on its specific phosphorylation by a yet unidentified kinase. Regulation of this pathway is especially important in malignant melanoma patients who are treated with IFNa. We hypothesize that, in melanoma cells, phosphorylation of IFNAR1 within the HOS recognition motif as well as the induction of HOS by constitutive mitogenic signaling leads to accelerated degradation of IFNAR1 and down regulation of the Type I IFN receptor, which, in turn, limits the sensitivity of melanoma cells to anti-proliferative and proapoptotic effects of IFNa.
In Aim 1 we will determine how HOS expression and the rate of degradation of IFNAR1 are regulated in melanoma cells harboring either wild type or mutated BRAF oncogene. We will further use pharmacologic and genetic means to determine the role of MARK pathway in regulation of HOS levels and activities. In the 2nd aim we will investigate a contribution of MARK pathway, HOS activities and IFNAR1 stability in controlling the extent of cellular responses of human melanoma cells to IFNa. These studies will include analysis of the extent of IFNa signaling and expression of IFN-stimulated genes, which confer anti-proliferative and pro-apoptotic effects of IFNa, as well as of the sensitivity of melanoma cells to IFNa-induced growth suppression and apoptosis in vitro and in vivo. Finally, in Specific Aim 3 we will use biochemical, pharmacological and genetic approaches to identify and characterize IFNAR1 kinase(s) and its role in IFNAR1 ubiquitination and degradation in vitro and in cells. ? ?
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