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: 7524479
• 负责人: JEFFREY R PETERSON
• 金额: $ 31.26万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7524479
• 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 Diphosphate; 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; Numbers; Pathway interactions; Phosphorylation; Process; Protein Overexpression; Proteins; Public Health; Regulation; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Specificity; System; Testing; Validation; Wiskott-Aldrich Syndrome; Work; Xenopus; Xenopus laevis; base; concept; egg; in vitro Model; inhibitor/antagonist; mouse Gdi2 protein; novel; novel therapeutics; p21 activated kinase; 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）：Ras相关GTP酶Rac和Cdc 42通过激活不同的下游效应蛋白促进肿瘤发生和转移，但细胞如何在不同情况下调节效应子的不同子集的激活尚不清楚。我们发现细胞中Rac和Cdc 42分子的数量大大超过效应分子的总数，并提出效应分子之间的竞争必须限制效应分子的激活。这一假设是至关重要的，因为效应子之间的竞争意味着单个效应子的丢失或过表达，例如可能发生在癌症或Wiskott-Aldrich综合征中，将改变其他途径的信号传导。这种相互关联的效应网络的概念，而不是独立运作的不同途径，对这些疾病的分子基础有着深远的影响。因此，了解效应信号通路的相互关系是至关重要的预测生物反应的药理学策略，针对个别效应途径。本提案的目标是阐明调控机制控制的特异性效应激活的Rac和Cdc 42。我们已经开发了一个实验系统，使用非洲爪蟾卵的细胞质提取物，其中内源性Rac和Cdc 42可以被激活以可调的方式和内源性下游效应物N-WASP和p21激活的激酶（Paks）的激活可以定量测量。我们的中心假设，基于初步研究，是叠加在效应器之间的简单竞争的背景下，两个调节机制偏置效应器激活;支架蛋白和通路串扰。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效应子的激活来确定竞争对Rac/Cdc 42效应子激活的重要性。在第二个目标中，我们将阐明支架蛋白Toca-1在偏向Rac/Cdc 42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作为支架因子来偏向效应子途径激活。在第三个目标中，我们将通过确定Paks对N-WASP的磷酸化是否调节Rac/Cdc 42对N-WASP的激活来阐明串扰在调节Rac/Cdc 42效应物选择中的作用。这些研究将揭示信号特异性的基本机制，可能适用于其他小GTP酶，如Ras，激活癌症中具有临床重要性的多个下游途径。公共卫生关系：Rho家族GTP酶Rac和Cdc 42调节许多其他蛋白质，这些蛋白质可能有助于癌症、智力迟钝和免疫紊乱中的疾病过程。虽然Rac和Cdc 42的许多合作伙伴是已知的，但我们不知道如何协调各种合作伙伴的监管。该提案将阐明这种协调如何在疾病中被破坏，并将为新治疗方法的最佳药物靶点的选择提供关键信息。