Regulated Proteolysis in RAS Signal Transduction

描述(申请人提供)：RAS信号通路控制所有多细胞生物体中细胞的生长、存活和死亡。在大约30%的人类癌症中发现RAS原癌基因的去调控。因此，癌症生物学的一个重要目标是了解正常和异常RAS信号背后的分子机制。果蝇R7光感受器细胞命运规范是研究RAS信号转导的一种遗传模型。在这个系统中，我们已经证明RAS的激活引导了一个由SINA(SINA)介导的泛素介导的蛋白分解途径。SINA编码高度保守的E3泛素连接酶家族成员，与脊椎动物的神经元分化、细胞凋亡、应激反应、肿瘤抑制、β-连环蛋白、APC和P53信号转导有关。泛素介导的蛋白质降解的底物特异性主要由E3连接酶决定，我们的研究重点是Sina E3连接酶，它是果蝇RAS信号通路的一个重要下游成分。本研究的目的是：(1)以果蝇为模式生物，研究Sina E3家族依赖性蛋白降解在RAS信号转导中的作用和调控；(2)鉴定新的Sina途径成分，并确定其在RAS信号转导调控蛋白降解中的功能；(3)研究哺乳动物Sina同源基因(SIAHs)在RAS介导的肿瘤发生中的作用，阐明SINA依赖的蛋白分解在动物发育和人类肿瘤过程中如何促进细胞生长、分化和凋亡。我们的中心假设是，Sina依赖的蛋白分解在发育过程中发挥着重要作用。我们将通过检测Sina功能缺失表型，通过遗传筛选和生化纯化识别新的Sina途径成分，并通过遗传学、分子和生化分析相结合的方法确定它们的功能，来检验这一假设。我们还将验证SIHA在肿瘤发生过程中RAS信号转导中所需的假设。我们检测到SINA在正常哺乳动物发育过程中的快速分裂细胞和人类肿瘤中的表达增强，这表明SINA可能是RAS介导的细胞增殖所必需的。我们打算鉴定SINA蛋白在果蝇中的功能和调控，并将我们所学到的应用于人胰腺癌细胞。由于许多已知的新浪底物在肿瘤发生中具有已证明的作用，拟议中的研究很可能将确定抗癌治疗的小说靶点，并有助于我们对癌症生物学的理解。

DESCRIPTION (provided by applicant): The RAS signaling pathway controls cell growth, survival and death in all multicellular organisms. Deregulation of RAS proto-oncogenes is found in approximately 30% of human cancer. Thus an important goal in cancer biology is to understand the molecular mechanisms underlying normal and aberrant RAS signals. Drosophila R7 photoreceptor cell fate specification is a genetically tractable model in which to study RAS signal transduction. In this system, we have shown that RAS activation directs a ubiquitin-mediated proteolysis pathway mediated by SEVEN-IN-ABSENTIA (SINA). SINA encodes a member of a highly conserved family of E3 ubiquitin ligases and has been implicated in neuronal differentiation, apoptosis, stress response, tumor suppression, beta-catenin, APC and p53 signaling in vertebrates. The substrate specificity of ubiquitin-mediated proteolysis is primarily determined by the E3 ligases, and the focus of our proposal is the SINA E3 ligase, an essential downstream component of RAS signaling pathway in Drosophila. The objectives of this research proposal are (1) to determine the developmental roles and regulation of SINA E3 family-dependent proteolysis in RAS signaling using Drosophila as a model organism, (2) to identify new SINA pathway components and determine their function in regulated proteolysis in RAS signal transduction, and (3) to investigate the roles of the mammalian SINA homologues (SIAHs) in RAS-mediated oncogenesis and to elucidate how the SIAH-dependent proteolysis promotes cell growth, differentiation and apoptosis during animal development and human cancers. Our central hypothesis is that the SINA-dependent proteolysis plays an important role in development. We will test this hypothesis by examining sina loss-of function phenotypes, by identifying new SINA pathway components by genetic screens and biochemical purification, and by determining their functions by a combination of genetic, molecular and biochemical analyses. We will also test the hypothesis that SIHA is required for RAS signal transduction during oncogenesis. We have detected enhanced expression of SINA in rapidly dividing cells during normal mammalian development and in human carcinomas, suggesting that SINA may be required for RAS-mediated cell proliferation in carcinogenesis. We propose to identify the function and regulation of SINA proteins in Drosophila, and apply what we have learned to human pancreatic cancer cells. Since many known SINA substrates have a demonstrated role in oncogenesis, it is likely that the proposed research will identify novels targets for anticancer therapies and contribute to our understanding of cancer biology.