ILK-Actopaxin Interactions in Cell Signaling

描述（由申请人提供）：Actopaxin是一种42 kDa的粘着斑蛋白，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。actopaxin氨基末端的磷酸化促进片状伪足形成并刺激细胞迁移。Actopaxin还充当细胞骨架动力学的关键调节因子（包括ILK、桩蛋白和TESK 1）的分子支架。本提案将集中于最近发现的actopaxin和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GT3信号传导和细胞运动中的重要性。目的1将研究actopaxin的磷酸化如何通过PIX-p21激活的激酶，PAK轴调节板状伪足的形成和细胞迁移使用actopaxin，PIX和PAK突变体的瞬时转染结合荧光显微镜，刮伤，延时和Boyden室迁移测定。将使用"Raichu" FRET探针在细胞中评价Rho家族GT3活性。将产生磷酸表位特异性抗体以探测actopaxin磷酸化的时空调节。在目标2中，CdGAP在整联蛋白信号传导中的作用和actopaxin结合在调节细胞迁移中的重要性将如目标1中那样检查。GST-下拉和共沉淀试验使用突变体的CdGAP和actopaxin将描绘各自的结合域。这些域的亚细胞定位的CdGAP和活性的重要性将分别通过epi和全内反射（TIR）荧光显微镜和体外GAP测定进行评估。目的3将检查actopaxin-b2-Adaptin相互作用在细胞迁移中的作用，通过刺激细胞极性和粘着斑解体，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。将评价对亚细胞定位的影响。将结合突变体引入成纤维细胞中，以分别使用钾耗竭和诺考达唑洗脱试验评估对细胞极性和粘着斑分解的影响。这些研究将进一步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。

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.