Specificity of Effector Activation by Rho Family GTPases

Rho 家族 GTP 酶激活效应器的特异性

• 批准号: 7935555
• 负责人: JEFFREY R PETERSON
• 金额: $ 22.93万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7935555
• 关键词: Affinity; Binding; Biochemical; Biological; Biological Models; Cell physiology; Cells; Clinical Pathways; Computer Simulation; Cultured Cells; Data; Disease; Drug Delivery Systems; Enzymes; Family; Future; GTP-Binding Proteins; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Immune System Diseases; In Vitro; Individual; Kinetics; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Mental Retardation; Molecular; Monitor; Monomeric GTP-Binding Proteins; Mus; Neoplasm Metastasis; Pathway interactions; Phosphorylation; Process; Proteins; Regulation; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Specificity; System; Testing; Validation; Wiskott-Aldrich Syndrome; Work; Xenopus; Xenopus laevis; base; egg; in vitro Model; inhibitor/antagonist; novel; novel therapeutic intervention; overexpression; p21 activated kinase; public health relevance; ras-Related G-Proteins; research study; response; rho; rho GTP-Binding Proteins; scaffold; small molecule; tumor; tumorigenesis

DESCRIPTION (provided by applicant): The Ras-related GTPases Rac and Cdc42 contribute to tumorigenesis and metastasis through the activation of diverse downstream effector proteins but how cells regulate activation of distinct subsets of effectors in different contexts is unknown. We have found that the number of Rac and Cdc42 molecules in cells is greatly exceeded by the total number of effector molecules and propose that competition between effectors must limit effector activation. This hypothesis is critically important because competition between effectors implies that loss or overexpression of an individual effector, such as can occur in cancer or Wiskott-Aldrich syndrome, would alter signaling by other pathways. This concept of an interconnected effector network, as opposed to distinct pathways that operate independently, has profound implications for the molecular basis of these diseases. Thus, understanding the interrelations of effector signaling pathways is crucial for predicting biological responses to pharmacological strategies targeting individual effector pathways. The goal of this proposal is to explicate the regulatory mechanisms controlling the specificity of effector activation by Rac and Cdc42. We have developed an experimental system using cytoplasmic extracts of Xenopus laevis eggs in which endogenous Rac and Cdc42 can be activated in a tunable manner and the activation of endogenous downstream effectors N-WASP and the p21-activated kinases (Paks) can be measured quantitatively. Our central hypothesis, based on preliminary studies, is that superimposed on a background of simple competition between effectors, two regulatory mechanisms bias effector activation; scaffolding proteins and pathway crosstalk. In our first aim we will determine the importance of competition on Rac/Cdc42 effector activation by modestly modulating protein levels of individual effectors and measuring the activation of others. In the second aim we will elucidate the role of a scaffolding protein, Toca-1, in biasing Rac/Cdc42 effector selection and will determine if guanine nucleotide exchange factors can serve as scaffolding factors to bias effector pathway activation. In the third aim we will illuminate the role of crosstalk in regulating Rac/Cdc42 effector selection by determining if phosphorylation of N- WASP by Paks regulates N-WASP activation by Rac/Cdc42. These studies will reveal fundamental mechanisms of signaling specificity that likely apply to other small GTPases, such as Ras, that activate multiple downstream pathways of clinical importance in cancer. PUBLIC HEALTH RELEVANCE: The Rho family GTPases Rac and Cdc42 regulate many other proteins that can contribute to the disease process in cancer, mental retardation, and immune disorders. Although many of the partners of Rac and Cdc42 are known, we do not know how the regulation of the various partners is coordinated. This proposal will illuminate how this coordination could be corrupted in disease and will provide crucial information for the selection of optimal drug targets for new therapeutic approaches.

描述（由申请人提供）：与RAS相关的GTPases RAC和CDC42通过激活不同下游效应子蛋白的激活而导致肿瘤发生和转移，但是细胞如何调节不同情况下效应子的不同子集的激活是未知的。我们发现，细胞中RAC和CDC42分子的数量大大超过了效应子分子的总数，并提出效应子之间的竞争必须限制效应子的激活。该假设至关重要，因为效应子之间的竞争意味着单个效应子的损失或过表达（例如癌症或Wiskott-Aldrich综合征可能发生）会改变其他途径的信号传导。与独立运作的不同途径相互联系的效应网络的概念对这些疾病的分子基础具有深远的影响。因此，了解效应子信号通路的相互关系对于预测针对单个效应途径的药理学策略的生物学反应至关重要。该提案的目的是解释控制RAC和CDC42效应子激活特异性的调节机制。我们已经使用爪蟾卵的细胞质提取物开发了一个实验系统，其中内源性RAC和CDC42可以以可调节的方式激活，并且可以用数量测量内源性下游效应物N-WASP和P21激活的激酶（PAK）的激活。我们基于初步研究的中心假设是，叠加在效应子之间简单竞争的背景下，两种调节机制偏见效应子激活。脚手架蛋白质和途径串扰。在我们的第一个目标中，我们将通过适度调节单个效应子的蛋白质水平并测量其他人的激活来确定竞争对RAC/CDC42效应子激活的重要性。在第二个目标中，我们将阐明脚手架蛋白，TOCA-1在偏置RAC/CDC42效应子选择中的作用，并将确定鸟嘌呤核苷酸交换因子是否可以用作造效器途径激活的脚手架因子。在第三个目的中，我们将通过确定paks是否通过RAC/CDC42来调节N-WASP激活N-WASP激活N-WASP的磷酸化是否是否磷酸化来阐明串扰在调节RAC/CDC42效应子选择中的作用。这些研究将揭示信号特异性的基本机制，该机制可能适用于其他小型GTPase（例如RAS），这些GTPase激活了癌症中临床重要性的多个下游途径。公共卫生相关性：Rho家族GTPases RAC和CDC42调节许多其他蛋白质，这些蛋白质可能有助于癌症，智力低下和免疫疾病的疾病过程。尽管RAC和CDC42的许多合作伙伴都是众所周知的，但我们不知道如何协调各个合作伙伴的监管。该提案将阐明如何在疾病中腐败这种协调，并为选择新的治疗方法的最佳药物目标提供关键信息。