Mechanisms of protein degradation that control signal transduction by Ras-Raf-MEK-ERK

项目摘要 这项提议旨在发现对信号转导是如何由高度 保守的Ras-Raf-MEK-ERK通路受蛋白质降解调控。在人类中，突变 基因BRAF编码三个人类RAF之一，是恶性肿瘤发展过程中的驱动事件 黑色素瘤。事实上，突变BRAF(V600E)赋予构成蛋白激活，是最重要的 在这种癌症中发现了频繁的突变。抑制Raf激酶可以抑制黑色素瘤的生长 活动。然而，耐药性的发展和疾病的复发频繁发生，表明真正的 有效的治疗需要额外的药物策略。我们的工作，利用秀丽线虫作为 通过这一保守途径研究信号转导的简单模型表明，线虫Raf 信号通过包括磷酸化和蛋白质降解(De)在内的抑制机制进行控制 拉科瓦和格林沃尔德，2012年)。对于这项拟议的研究，我们使用线虫模型来设计和开始 RNAi筛选以确定调节Raf的保守的激酶和磷酸酶。我们发现CDK2是 Raf蛋白降解所必需，且CDK2的丢失增强了突变体引起的表型， 激活的Raf被设计为携带与BRAF(V600E)相同的突变。我们的长期目标是发现新的 开发能够抑制Raf信号的药物的分子靶点。为了这个目标，我们的 本申请的总体目标是：(I)确定影响Raf的其他激酶和磷酸酶 蛋白质在人体细胞中的降解及其调节BRAF的需要，以及(Ii)确定 激酶CDK2调节Raf的机制和后果。我们的中心假设是 保守的机制，如Raf蛋白的磷酸化，可以防止Raf的异常激活。 这个项目的基本原理是我们发现了控制信号的抑制控制机制 Ras-Raf-MEK-ERK通路的转导将为黑色素瘤的药物治疗提供新的策略。

Project Summary This proposal aims to discover mechanistic insights into how signal transduction by the highly conserved Ras-Raf-MEK-ERK pathway is modulated by regulated protein degradation. In humans, mutation of the gene BRAF, encoding one of three human Rafs, is a driver event in the development of malignant melanoma. Indeed, the mutation BRAF(V600E) confers constitutive kinase activation and is one of the most frequent mutations found in this cancer. Melanoma growth can be suppressed by inhibition of Raf kinase activity. However, development of drug resistance and disease relapse occurs frequently, indicating that truly effective treatments will require additional drug strategies. Our work, utilizing Caenorhabditis elegans as a simple model to investigate signal transduction by this conserved pathway, demonstrates that C. elegans Raf signaling is kept in check by inhibitory mechanisms that include phosphorylation and protein degradation (de la Cova and Greenwald, 2012). For this proposed research, we use the C. elegans model to design and begin an RNAi screen to identify conserved kinases and phosphatases that regulate Raf. We find that CDK2 is necessary for Raf protein degradation, and that loss of CDK2 enhances phenotypes caused by a mutant, activated Raf engineered to carry the same mutation as BRAF(V600E). Our long-term goal is to identify new molecular targets for the development of drugs capable of inhibiting Raf signaling. Toward this goal, our overall objectives in this application are to (i) identify additional kinases and phosphatases that impact Raf protein degradation and their requirement for regulating BRAF in human cells, and (ii) determine the mechanism and consequences of Raf regulation by the kinase CDK2. Our central hypothesis is that conserved mechanisms, such as phosphorylation of the Raf protein, act to prevent aberrant Raf activation. The rationale for this project is that our discovery of inhibitory control mechanisms governing signal transduction by the Ras-Raf-MEK-ERK pathway will inform new strategies of drug therapy for melanoma.