ILK-Actopaxin Interactions in Cell Signaling

ILK-Actopaxin 在细胞信号转导中的相互作用

• 批准号: 7568280
• 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
• 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/7568280
• 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; In Vitro; Integrin Signaling Pathway; Integrins; MEKs; Maintenance; Migration Assay; Molecular; Monitor; Musculoskeletal; Myosin Light Chains; Neoplasm Metastasis; Nocodazole; Phospho-Specific Antibodies; Phosphorylation; Physiological; Play; Potassium; Precipitation; Process; Protein Binding Domain; Proteins; RNA Interference; Regulation; Role; Scaffolding Protein; Signal Transduction; Site; Small Interfering RNA; Testing; Time; Tissues; Transfection; Video Microscopy; Western Blotting; actopaxin; cell behavior; cell motility; cofilin; insight; intersectin 1; mimetics; mutant; p21 activated kinase; paxillin; protein protein interaction; repaired; rho; rho GTP-Binding Proteins; scaffold; wound

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）：Actopaxin 是一种 42kDa 的粘着斑蛋白，可促进整合素信号传导以及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片状伪足的形成并刺激细胞迁移。 Actopaxin 还充当细胞骨架动力学关键调节因子（包括 ILK、paxillin 和 TESK1）的分子支架。该提案将重点关注最近发现的 Actopaxin 与 Cdc42/Rac GEF PIX、CdGAP 和内吞蛋白 b2-Adaptin 之间的相互作用在调节 Rho 家族 GTPase 信号传导和细胞运动中的重要性。目标 1 将利用瞬时转染肌动蛋白、PIX 和 PAK 突变体并结合荧光显微镜、刮伤、延时和博伊登室迁移，检查肌动蛋白的磷酸化如何通过 PIX-p21 激活激酶、PAK 轴调节片状伪足形成和细胞迁移化验。 Rho 家族 GTPase 活性将使用“Raichu”FRET 探针在细胞中进行评估。将产生磷酸表位特异性抗体来探测肌动蛋白磷酸化的时空调节。在目标 2 中，将像目标 1 一样检查 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。使用 CdGAP 和肌动蛋白突变体的 GST 下拉和共沉淀测定将描绘各自的结合域。这些结构域对于 CdGAP 亚细胞定位和活性的重要性将分别通过表观荧光显微镜和全内反射 (TIR-) 荧光显微镜以及体外 GAP 测定进行评估。目标 3 将通过刺激细胞极性和粘着斑分解（可能通过内吞事件）来研究 Actopaxin-b2-Adaptin 相互作用在细胞迁移中的作用。 Actopaxin 上的结合位点将使用 GST 融合蛋白和共沉淀来定义。将评估对亚细胞定位的影响。将结合突变体引入成纤维细胞中，分别利用钾消耗和诺考达唑洗脱测定来评估对细胞极性和粘着斑分解的影响。这些研究将进一步深入了解细胞与细胞外基质的相互作用如何向细胞骨架发出信号以促进细胞迁移，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转化的动态过程具有重要意义。 。