ILK-Actopaxin Interactions in Cell Signaling

ILK-Actopaxin 在细胞信号转导中的相互作用

基本信息

批准号：
7356055
负责人：
Christopher E Turner
金额：
$ 39.25万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-02-15 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7356055
关键词：
Actins Adhesions Antibodies Binding Binding Sites Biochemical Biological Assay Cardiovascular system Cell Communication Cell Polarity Cell Proliferation Cells Chimeric Proteins Co-Immunoprecipitations Complex Cytoskeletal Modeling Cytoskeleton Defect Deletion Mutagenesis Embryonic Development Endocytosis Epitopes Event Extracellular Matrix Family Fibroblasts Fluorescence Microscopy Fluorescence Resonance Energy Transfer Focal Adhesions Funding Growth Factor Guanosine Triphosphate Phosphohydrolases ILK gene In Vitro Integrin Signaling Pathway Integrins Localized MEKs Maintenance Migration Assay Molecular Monitor Musculoskeletal Myosin Light Chains Neoplasm Metastasis Nocodazole Phospho-Specific Antibodies Phosphorylation Physiological Potassium Precipitation Process Protein Binding Domain Proteins RNA Interference Regulation Role Role playing therapy Scaffolding Protein Signal Transduction Site Small Interfering RNA Testing Time Tissues Transfection Video Microscopy Western Blotting actopaxin cell behavior cell motility cofilin human wyatt protein insight integrin-linked kinase intersectin 1 mimetics mouse wyatt protein mutant p21 activated kinase paxillin protein protein interaction repaired rho rho GTP-Binding Proteins scaffold wound

项目摘要

DESCRIPTION (provided by applicant): Actopaxin is a 42kDa focal adhesion protein that facilitates integrin signaling and interactions with the actin cytoskeleton. Phosphorylation of the actopaxin amino-terminus promotes lamellipodia formation and stimulates cell migration. Actopaxin also serves as a molecular scaffold for key regulators of cytoskeletal dynamics including ILK, paxillin, and TESK1. This proposal will focus on the importance of recently identified interactions between actopaxin and the Cdc42/Rac GEF PIX, CdGAP and the endocytic protein b2-Adaptin in the regulation of Rho family GTPase signaling and cell motility. Aim 1 will examine how phosphorylation of actopaxin regulates lamellipodia formation and cell migration via the PIX-p21-activated kinase, PAK axis using transient transfection of actopaxin, PIX and PAK mutants combined with fluorescence microscopy, scrape wound, time-lapse and Boyden chamber migration assays. Rho family GTPase activity will be evaluated in cells using "Raichu" FRET probes. Phospho-epitope specific antibodies will be generated to probe the spatio-temporal regulation of actopaxin phosphorylation. In Aim 2 the role of CdGAP in integrin signaling and the importance of actopaxin binding in regulating cell migration will be examined as in Aim 1. GST-pulldown and co precipitation assays using mutants of CdGAP and actopaxin will delineate the respective binding domains. The importance of these domains for subcellular localization of CdGAP and activity will be assessed by epi- and Total Internal Reflection- (TIR-) fluorescence microscopy and in vitro GAP assays respectively. Aim 3 will examine a role for the actopaxin-b2-Adaptin interaction in cell migration through the stimulation of cell polarity and focal adhesion disassembly, potentially through endocytic events. The binding site on actopaxin will be defined using GST-fusion proteins and co-precipitation. Effects on subcellular localization will be evaluated. Binding mutants will be introduced into fibroblasts to assess effect on cell polarity and focal adhesion disassembly utilizing potassium depletion and nocodazole washout assays respectively. These studies will provide further insight into how cell interactions with the extracellular matrix signals to the cytoskeleton to promote cell migration, and thus is of importance to understanding the dynamic processes of embryogenesis, tissue maintenance and repair as well as cardiovascular and musculoskeletal defects and metastatic transformation.

描述（由申请人提供）：肌动蛋白是一种42KDA焦点粘附蛋白，可促进整联蛋白信号传导和与肌动蛋白细胞骨架的相互作用。肌动蛋白氨基末端的磷酸化促进了薄膜脂蛋白的形成并刺激细胞迁移。肌动蛋白也是细胞骨架动力学关键调节剂在内的分子支架，包括ILK，Paxillin和Tesk1。该提案将重点介绍肌动蛋白与CDC42/RAC GEF PIX，CDGAP和内吞蛋白B2-ADAPTIN在RHO家族GTPase信号传导和细胞运动性调节中的重要性。 AIM 1将研究肌动蛋白的磷酸化如何通过PIX-P21激活的激酶，使用肌动蛋白肌动蛋白，PIX和PAK突变体的瞬时转染与荧光显微镜，Scrape伤口，时间失气和Boyden Chamber迁移的磷酸化，并通过PIX-P21激活的激酶，PAK轴迁移。 RHO家族GTPase活性将在细胞中使用“ Raichu” FRET探针进行评估。将生成磷酸 - 异常特异性抗体，以探测肌动蛋白肌动蛋白磷酸化的时空调节。在AIM 2中，CDGAP在整联蛋白信号传导中的作用以及肌动蛋白结合在调节细胞迁移中的重要性将被检查，如AIM 1。使用CDGAP和肌动蛋白的突变体GST-Pulldown和Co降水分析，将划定各自的结合域。这些结构域对CDGAP和活性亚细胞定位的重要性将分别通过表演内部反射和总内反射 - （TIR-）荧光显微镜以及体外差距测定。 AIM 3将通过刺激细胞极性和局灶性粘附拆卸，可能通过内吞事件来研究肌动蛋白-B2-磷脂的相互作用在细胞迁移中的作用。肌动蛋白上的结合位点将使用GST融合蛋白和共沉淀定义。将评估对亚细胞定位的影响。结合突变体将被引入成纤维细胞中，以评估分别利用钾耗竭和诺科杜唑冲洗分析的细胞极性和局灶性粘附拆卸作用。这些研究将进一步了解细胞与细胞外基质信号与细胞骨架的相互作用如何促进细胞迁移，因此对于理解胚胎发生，组织维持和修复以及心血管和肌肉骨骼缺陷和转移的动态过程至关重要。