Regulated Proteolysis in RAS Signal Transduction

描述（由申请人提供）：RAS信号通路控制所有多细胞生物体中的细胞生长、存活和死亡。RAS原癌基因的失调在大约30%的人类癌症中发现。因此，癌症生物学的一个重要目标是了解正常和异常RAS信号的分子机制。果蝇R7光感受器细胞命运特化是研究RAS信号转导的一个遗传学上易处理的模型。在这个系统中，我们已经表明，RAS激活指导泛素介导的蛋白水解途径介导的七-在-缺（SINA）。SINA编码高度保守的E3泛素连接酶家族的成员，并且已经涉及脊椎动物中的神经元分化、凋亡、应激反应、肿瘤抑制、β-连环蛋白、APC和p53信号传导。泛素介导的蛋白质水解的底物特异性主要由E3连接酶决定，我们的研究重点是果蝇RAS信号通路的重要下游组分SINA E3连接酶。本研究计划的目的是：（1）以果蝇为模式生物，确定SINA E3家族依赖的蛋白水解在RAS信号转导中的发育作用和调控，（2）鉴定新的SINA通路组分，并确定它们在RAS信号转导中调节蛋白水解的功能，以及（3）研究哺乳动物SINA同源物（SIAH）在RAS介导的肿瘤发生中的作用，并阐明SIAH依赖的蛋白水解如何促进细胞生长，在动物发育和人类癌症中的分化和凋亡。我们的中心假设是，依赖于SINA的蛋白水解在发育中起着重要作用。我们将检验这一假设，通过检查sina的功能丧失的表型，通过识别新的SINA通路的基因筛选和生化纯化的组件，并确定其功能的组合，遗传，分子和生化分析。我们还将检验SIHA是肿瘤发生过程中RAS信号转导所必需的假设。我们已经检测到增强表达的SINA在快速分裂的细胞在正常哺乳动物的发展和人类癌症，表明SINA可能需要RAS介导的细胞增殖在致癌作用。我们建议确定果蝇中SINA蛋白的功能和调节，并将我们所了解的应用于人类胰腺癌细胞。由于许多已知的SINA底物在肿瘤发生中具有已证实的作用，因此拟议的研究可能会确定抗癌疗法的新靶点，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.