Role of HOS in cell transformation and apoptosis

描述（由申请人提供）：SCF(HOS/ β - trcp) E3泛素连接酶以磷酸化依赖的方式控制细胞生长、转化和存活的一些重要调节因子的蛋白水解。我们和其他人证明了HOS/ β - trcp蛋白的水平在肿瘤细胞中增加，并且这些水平对于这些底物的降解至关重要，这限制了细胞的生长和存活。Hos水平是通过丝裂原活化蛋白激酶（MARK）途径诱导的。在继续研究HOS在癌症发病机制中的作用时，我们将重点放在干扰素α (IFNa)受体IFNAR1亚基的降解上，该亚基最近被确定为HOS的一种新型底物。hos控制的IFNAR1的泛素化和降解依赖于一种尚未确定的激酶的特异性磷酸化。这一通路的调控在接受IFNa治疗的恶性黑色素瘤患者中尤为重要。我们假设，在黑色素瘤细胞中，HOS识别基序内IFNAR1的磷酸化以及组成性有丝分裂信号诱导HOS导致IFNAR1的加速降解和I型IFN受体的下调，这反过来限制了黑色素瘤细胞对IFNa的抗增殖和促凋亡作用的敏感性。在Aim 1中，我们将确定在携带野生型或突变BRAF癌基因的黑色素瘤细胞中，HOS表达和IFNAR1降解率是如何被调节的。我们将进一步利用药理学和遗传学手段来确定MARK通路在调控HOS水平和活性中的作用。在第二个目标中，我们将研究MARK通路、HOS活性和IFNAR1稳定性在控制人类黑色素瘤细胞对IFNa的细胞反应程度中的作用。这些研究将包括分析IFNa信号传导的范围和ifn刺激基因的表达，这些基因赋予IFNa的抗增殖和促凋亡作用，以及黑色素瘤细胞对IFNa诱导的生长抑制和凋亡的敏感性，在体外和体内。最后，在Specific Aim 3中，我们将使用生化、药理学和遗传学方法来鉴定和表征IFNAR1激酶及其在体外和细胞中IFNAR1泛素化和降解中的作用。

DESCRIPTION (provided by applicant): 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.