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

项目摘要 该提案旨在发现对高度依赖性细胞如何进行信号转导的机械见解。 保守的Ras-Raf-MEK-ERK途径通过调节蛋白质降解来调节。在人类中，突变 BRAF基因编码三种人类Rafs之一，是恶性肿瘤发展的驱动因素。 黑素瘤事实上，突变BRAF（V600 E）赋予组成型激酶活化，并且是最重要的激酶之一。 在这种癌症中发现的频繁突变。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复发频繁发生，这表明真正的耐药性是不可避免的。 有效的治疗将需要额外的药物策略。我们的工作，利用秀丽隐杆线虫作为一个 简单的模型来研究通过这种保守途径的信号转导，表明C.美丽的拉夫 信号传导通过抑制机制来控制，所述抑制机制包括磷酸化和蛋白质降解（DE la Cova和Greenwald，2012年）。对于这项研究，我们使用C。elegans模型来设计和开始 RNAi筛选以鉴定调节Raf的保守激酶和磷酸酶。我们发现CDK 2是 Raf蛋白降解所必需的，并且CDK 2的缺失增强了由突变体引起的表型， 活化的Raf被工程化以携带与BRAF相同的突变（V600 E）。我们的长期目标是确定新的 用于开发能够抑制Raf信号传导的药物的分子靶点。为了实现这一目标，我们 本申请的总体目标是（i）鉴定影响Raf的另外的激酶和磷酸酶 蛋白质降解和它们调节人细胞中BRAF的需要，和（ii）确定 激酶CDK 2调节Raf的机制和后果。我们的核心假设是， 保守的机制，如Raf蛋白的磷酸化，起到防止异常Raf活化的作用。 这个项目的基本原理是，我们发现的抑制性控制机制控制信号 通过Ras-Raf-MEK-ERK途径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.