ILK-Actopaxin Interactions in Cell Signaling

描述（由申请人提供）：actitopaxin是一种42kDa的局灶黏附蛋白，促进整合素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板足形成并刺激细胞迁移。actipaxin还作为细胞骨架动力学关键调节因子的分子支架，包括ILK、paxillin和TESK1。本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。目的1将通过瞬时转染actopaxin、PIX和PAK突变体，结合荧光显微镜、刮伤、延时和Boyden室迁移试验，研究actopaxin的磷酸化如何通过PIX-p21活化的激酶、PAK轴调节板足形成和细胞迁移。Rho家族GTPase活性将使用“Raichu”FRET探针在细胞中进行评估。将产生磷酸化表位特异性抗体，以探测actitopaxin磷酸化的时空调节。在Aim 2中，CdGAP在整合素信号传导中的作用以及actopaxin结合在调节细胞迁移中的重要性将与Aim 1中一样被研究。使用CdGAP和actopaxin突变体的gst下拉和共沉淀分析将描绘各自的结合域。这些结构域对CdGAP亚细胞定位和活性的重要性将分别通过外延荧光显微镜和全内反射（TIR）荧光显微镜和体外GAP测定来评估。目的3将研究胞动蛋白b2-适应蛋白相互作用在细胞迁移中的作用，通过刺激细胞极性和局点粘附分解，可能通过内吞事件。将使用gst融合蛋白和共沉淀来确定actopaxin上的结合位点。对亚细胞定位的影响将被评估。结合突变体将被引入成纤维细胞，分别利用钾消耗和诺可达唑冲洗试验来评估对细胞极性和局灶黏着破坏的影响。这些研究将进一步深入了解细胞如何与细胞外基质相互作用，向细胞骨架发出信号，促进细胞迁移，从而对理解胚胎发生、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。

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.