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

Ras 和生长因子对 c-Raf-1 的调节

• 批准号: 6561581
• 负责人: GURI TZIVION
• 金额: $ 26.19万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6561581
• 关键词: SDS polyacrylamide gel electrophoresis; animal extract; antineoplastics; biological signal transduction; cell cycle proteins; cell transformation; chimeric proteins; gene deletion mutation; growth factor; growth inhibitors; guanine nucleotide binding protein; immunoprecipitation; mitogen activated protein kinase; phosphorylation; protein binding; protein protein interaction; protein structure function; protein transport; protooncogene; transfection; western blottings

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.

描述（由申请人提供）：Ras-Raf-MAPK 通路通过将信号从膜结合受体传递到细胞核和细胞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（如生长、应激、生存和炎症）的反应。该途径的异常导致异常生长和增殖，并且在许多情况下，导致恶性转化。在各种人类癌症中发现的高频率激活 Ras 突变，加上 Raf 在众多生理过程中的作用得到充分记录，使得 Ras-Raf-MAPK 通路成为各种癌症、炎症和其他食物药物开发的主要目标。 c-Raf-1 是一种也在转化病毒中发现的细胞原癌基因，是 MAPK 激活的主要 Ras 效应子，并且是 Ras-MAPK 级联的主要且不可或缺的部分。尽管许多实验室一直在研究 Raf 调控，但这种高度复杂机制的许多方面仍然未知。申请人发表的工作和本提案中提出的初步结果有助于阐明其中几个方面，并提供一种抑制 Raf 活性的新方法。本申请的目的是进一步理解Raf调节，重点关注Ras和磷酸化在Raf调节中的具体作用，并基于本申请中描述的有效的体外Raf抑制剂开发有用的体内Raf抑制剂。这些目标将通过追求以下三项具体援助来实现： 1. 描述 Ras 在 Raf-1 激活过程中的作用。将关注区分 Ras 在将 Raf 募集至膜和置换 14-3-3 方面的作用，以及 Ras-Ras 和 Raf-Raf 二聚化在激活过程中的作用。 2.确定Raf-1 S471和T481位点在大鼠调节和功能中的作用。这一目标将通过检查 S471 和 T481 磷酸化的调节并评估其取代和磷酸化的功能意义来实现。 3. 表征 Raf-1 抑制肽抑制 Raf 的机制，并确定能够在体内抑制细胞 Raf-1 的肽递送方法。拟议的研究将通过提供 Ras 作用的分子细节和新发现的 Raf 磷酸化位点在 Raf 激活中的作用，增强我们对 Raf 调控的理解。此外，拟议的研究为开发体内 Raf 抑制剂提供了前景，该抑制剂除了是一种非常有用的研究工具外，还将为开发涉及细胞过度增殖的疾病（如癌症和炎症）的治疗药物提供基础。