Studies on the Regulation of Rac signaling

Rac信号调控的研究

• 批准号: 7025094
• 负责人: RACHEL J BUCHSBAUM
• 金额: $ 24.92万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025094
• 关键词: RNA interference; apoptosis; biological signal transduction; calcium flux; cell line; cell migration; extracellular matrix; gene expression; gene targeting; genetic manipulation; guanine nucleotide binding protein; immunofluorescence technique; immunoprecipitation; nerve /myelin protein; phosphorylation; protein localization; protein protein interaction; transfection; transport proteins; yeast two hybrid system

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to explore the interactions of the Rac exchange factor Tiam1 with three different scaffold protein complexes. Both Tiam1 and Rac have multiple roles in cell signaling, and both have been implicated in malignant cell behaviors, including altered cell motility, invasion, apoptosis and survival. The factors regulating Tiam1 and Rac signaling specificity and the resulting effects on cellular behavior have not yet been well characterized. This proposal tests the hypothesis that by interacting with a specific scaffold protein, Tiam1 brings activated Rac into a complex with a particular downstream effector pathway, thereby contributing to Rac signaling specificity and Rac-mediated effects on cellular function. The first part of the project investigates in detail the factors that may contribute to regulating Tiam1 interaction with each scaffold protein complex, including calcium signals, phosphorylation, Ras-mediated signals, ligand stimulation, and extra-cellular matrix. Methods used here include transfection, immunoprecipitation, immunofluorescence, cell lines with inducible protein expression, and RNA interference. The results of these experiments will form the basis for future study of the specific upstream pathways promoting these associations. The second part of the project investigates the effect of directed Tiam1/Rac signaling through each scaffold protein complex on cellular migration/invasion and survival/apoptosis. This will entail additional methods including mutational analysis with reverse two hybrid and differential interaction trap techniques in yeast and engineering of stable DNA-based RNAi knock-out lines using a retroviral vector, along with transwell migration and invasion assays and apoptosis assays. This work will develop a powerful set of reagents for future study of how Tiam1 interactions with particular scaffold protein complexes affect signaling pathways downstream of Rac. Taken together, the results of this work should provide better understanding of how scaffold protein complexes direct Tiam1 and Rac signaling specificity. Determining how specific Tiam1-scaffold interactions can influence Rac-mediated cellular behavior will provide important information as to the regulation of signals governing normal and malignant cell behavior. Ultimately this may allow development of therapeutic reagents targeted specifically against Rac-mediated cancer cell pathophysiology.

描述（由申请人提供）：该提案的广泛长期目标是探索RAC交换因子TIAM1与三种不同的脚手架蛋白复合物的相互作用。 TIAM1和RAC在细胞信号传导中均具有多个作用，并且都与恶性细胞行为有关，包括改变细胞运动，侵袭，凋亡和生存。调节TIAM1和RAC信号特异性以及对细胞行为的影响的因素尚未得到很好的特征。该建议检验了以下假设：通过与特定的支架蛋白相互作用，TIAM1将激活的RAC带入具有特定下游效应途径的复合物，从而有助于RAC信号特异性和RAC介导的对细胞功能的影响。该项目的第一部分详细研究了可能导致TIAM1相互作用与每个支架蛋白复合物的相互作用的因素，包括钙信号，磷酸化，RAS介导的信号，配体刺激和细胞外基质。此处使用的方法包括转染，免疫沉淀，免疫荧光，具有诱导蛋白表达的细胞系和RNA干扰。这些实验的结果将成为对促进这些关联的特定上游途径的未来研究的基础。该项目的第二部分研究了通过每个支架蛋白复合物对细胞迁移/侵袭和生存/凋亡的定向TIAM1/RAC信号传导的影响。这将需要进行其他方法，包括使用逆转录病毒载体的酵母中的两种杂种和差异相互作用陷阱技术以及稳定的基于DNA的RNAi敲除线的突变分析，以及Transwell迁移和入侵分析和凋亡测定法。这项工作将开发一组强大的试剂，以将来研究TIAM1与特定脚手架蛋白复合物如何影响RAC下游的信号通路。综上所述，这项工作的结果应更好地了解脚手架蛋白复合物如何直接tiam1和rac信号传导特异性。确定特定的TIAM-SCADGOLD相互作用如何影响RAC介导的细胞行为，将提供有关调节正常细胞行为和恶性细胞行为的信号的重要信息。最终，这可能允许开发针对RAC介导的癌细胞病理生理学的治疗试剂。