ILK-Actopaxin Interactions in Cell Signaling

描述(申请人提供)：Actopaxin是一种42 kDa的焦点黏附蛋白，促进整合素信号转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脂体的形成，刺激细胞迁移。Actopaxin也是细胞骨架动力学关键调节因子的分子支架，包括ILK、PXLIN和TESK1。这项建议将集中在最近发现的Actopaxin与CDC42/RAC全环蛋白PIX、CDGAP和内吞蛋白b2-Adaptin之间相互作用在调节Rho家族GTPase信号和细胞运动中的重要性。目的1通过瞬时转染Actopaxin、PIX和PAK突变体，结合荧光显微镜、擦伤、时间推移和Boyden小室迁移实验，研究Actopaxin的磷酸化如何通过PIX-p21激活的激酶、PAK轴调节片状脂体的形成和细胞迁移。Rho家族GTP酶活性将使用“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索actopaxin磷酸化的时空调节。在目标2中，将像目标1中那样研究CDGAP在整合素信号转导中的作用以及actopaxin结合在调节细胞迁移中的重要性。使用cdGAP和actopaxin突变体的GST-Pull和共沉淀分析将描绘各自的结合域。这些结构域对亚细胞定位和活性的重要性将分别通过表观和全内反射(TIR)荧光显微镜和体外GAP分析来评估。目标3将研究actopaxin-b2-Adaptin相互作用在细胞迁移中的作用，可能是通过刺激细胞极性和局部黏附分解，可能通过内吞事件。Actopaxin上的结合部位将使用GST融合蛋白和共沉淀来确定。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，以分别利用钾耗竭和诺可达唑洗脱试验评估对细胞极性和局部黏附解离的影响。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，从而对理解胚胎发生、组织维护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.