Regulation of c-Raf-1 by Ras and Growth Factors

描述(申请人提供)：Ras-Raf-MAPK通路通过将信号从膜结合的受体传递到核质靶标来调节许多生理过程，这些靶标协调细胞对各种因素的反应，如生长、应激、生存和炎症。这一途径的异常会导致异常的生长和增殖，在许多情况下，会导致恶性转化。在多种人类癌症中发现的高频率激活RAS突变，以及Raf在许多生理过程中的作用，使得Ras-Raf-MAPK通路成为治疗各种癌症、炎症和其他营养物质药物开发的主要靶点。c-Raf-1是一种在转化病毒中也发现的细胞原癌基因，是激活MAPK的主要RAS效应因子，是Ras-MAPK级联反应中重要且不可或缺的部分。尽管许多实验室一直在研究Raf的调控，但这一高度复杂的机制的许多方面仍不清楚。申请人发表的工作和本提案中提出的初步结果有助于揭示其中的几个方面，并为抑制Raf活性提供了一种新的方法。本申请的目的是加深对Raf调节的了解，重点是Ras和磷酸化在Raf调节中的具体作用，并开发基于本申请中描述的有效的体外Raf抑制剂的体内有用的Raf抑制剂。这些目标将通过追求以下三个具体的ALM来实现：1.确定RAS在RAF-1激活过程中的作用。将注意区分RAS在将Raf招募到膜上和取代14-3-3方面的作用，以及RAS-RAS和Raf-Raf二聚在活化过程中的作用。2.确定Raf-1 S471和T481位点在大鼠调节和功能中的作用。这一目标将通过检测S471和T481磷酸化的调节以及评估它们的替代和磷酸化的功能意义来实现。3.鉴定Raf-1抑制肽抑制Raf的机制，并确定体内抑制细胞Raf-1的多肽递送方法。这项拟议的研究将通过提供RAS在Raf激活中的作用和新发现的Raf磷酸化位点的分子细节来加深我们对Raf调控的理解。此外，这项拟议的研究为开发体内Raf抑制剂提供了前景，除了是一个非常有用的研究工具外，还将为开发治疗癌症和炎症等涉及细胞过度增殖的疾病的药物提供基础。

DESCRIPTION (provided by applicant): The Ras-Raf-MAPK pathway regulates many physiological processes by transmitting signals from membrane-bound receptors to nuclear and cytoplasmic targets that coordinate cellular response to a variety of factors, such as growth, stress, survival and inflammation. Aberrations in this pathway result in abnormal growth and proliferation, and in many cases, cause malignant transformation. The high frequency of activating Ras mutations found in various human cancers, together with the well-documented role of Raf in numerous physiological processes, make the Ras-Raf-MAPK pathway a prime target for drug development for various cancers, inflammation and other aliments, c-Raf-1, a cellular protooncogene also found in transforming viruses, is the primary Ras effector for MAPK activation and is a major and indispensable part of the Ras-MAPK cascade. Although many laboratories have been studying Raf regulation, many aspects of this highly complex mechanism remain unknown. Published work by the applicant and preliminary results presented in this proposal help to unravel several of these aspects and provide a novel approach for inhibiting Raf activity. The objectives of the present application are to further the understanding of Raf regulation, focusing on specific roles of Ras and phosphorylation in Raf regulation, and to develop a useful in vivo Raf inhibitor based on a potent in vitro Raf inhibitor, described in this application. These objectives will be accomplished by pursuing the following three specific alms: 1. Characterize the role of Ras in the Raf-1 activation process. Attention will be given to distinguishing between the roles of Ras in recruiting Raf to the membrane and displacing 14-3-3, and to the role of Ras-Ras and Raf-Raf dimerization in the activation process. 2. Determine the role of Raf-1 S471 and T481 sites in Rat regulation and function. This aim will be accomplished by examining the regulation of S471 and T481 phosphorylation and by evaluating the functional significance of their substitution and phosphorylation. 3. Characterize the mechanism underlying Raf inhibition by the Raf-1 inhibitor peptide, and identify peptide-delivery methods allowing inhibition of cellular Raf-1 in vivo. The proposed study will enhance our understanding of Raf regulation by providing molecular details on the role of Ras and newly identified Raf phosphorylation sites in Raf activation. In addition, the proposed study offers the prospect for developing an in vivo Raf inhibitor, which in addition to being a highly useful research tool, would provide a basis for developing therapeutic agents for diseases involving excessive cell proliferation such as cancer and inflammation.