Structure and Function of Paxillin

描述(由申请人提供)：Paxlin是一种多结构域，68 KDa的磷酸蛋白，定位于培养的哺乳动物细胞和活体中专门的肌动蛋白-膜-细胞外基质附着位置。我们将检验这个假设，即Paxlin在质膜上作为支架/适配蛋白来协调黏附和生长因子衍生信号之间的相互作用，从而导致肌动蛋白细胞骨架的重组和控制细胞迁移。目的1研究巴西林与PKL-PIX-PAK(p21-激活蛋白激酶)-NCK复合体相互作用的重要性，重点研究其在影响PAK功能中的作用。黏附和生长因子调节的PKL酪氨酸磷酸化的关键作用也将被确定。目的2利用PXILLIN缺失型和PTP-PEST缺失型成纤维细胞，研究PXLIN-PTP-PEST相互作用在控制PTP-PEST介导的细胞扩散、运动和p21-GTP酶信号转导中的重要作用。目的3将确定两个巴西林相关蛋白Hic-5和巴西林Delta的差异表达如何调节巴西林及其相关蛋白的整合素信号，从而影响上皮-间充质转化过程中细胞的迁移和形态。为了实现这些目标，细胞骨架组织将通过免疫荧光显微镜进行评估。延时视频显微镜、Boyden和Dunn小室分析将允许评估动态细胞形状和运动性变化。磷酸化特异性抗体将被用来识别局部黏附和细胞骨架蛋白以及MAP激酶和其他信号中间体的活性和位置的变化。Rho家族GTP酶活性将使用GST-GTP酶效应蛋白结合域进行评估。预计从拟议的研究中获得的信息将提供对细胞与环境相互作用和沟通以调节正常和肿瘤细胞迁移的分子机制的洞察，并可能确定未来可能作为治疗干预目标的蛋白质-蛋白质相互作用/途径。

DESCRIPTION (provided by applicant): Paxillin is a multi-domain, 68 KDa phosphoprotein that localizes to specialized actin-membrane-extracellular matrix attachment sites in cultured mammalian cells and in vivo. We will test the hypothesis that paxillin functions as a scaffold/adapter protein at the plasma membrane to coordinate the interplay between adhesion and growth factor-derived signals that result in reorganization of the actin cytoskeleton and control cell migration. Aim 1 will examine the importance of the interaction between paxillin and the PKL-PIX-PAK (p21-activated kinase)-Nck complex, focusing on its role in effecting PAK function. The critical role of adhesion and growth factor regulated PKL tyrosine phosphorylation will also be determined. Aim 2 will utilize paxillin null and PTP-PEST null fibroblasts to examine the essential role for paxillin-PTP-PEST interactions in controlling PTP-PEST-mediated effects on cell spreading, motility and p21-GTPase signaling. Aim 3 will determine how the differential expression of two paxillin related proteins, Hic-5 and paxillin delta modulate integrin signaling to paxillin and associated proteins to effect cell migration and morphology during epithelial-mesenchymal transition. To accomplish these Aims cytoskeletal organization will be evaluated by immunofluorescence microscopy. Time-lapse video microscopy, Boyden and Dunn chamber assays will permit evaluation of dynamic cell shape and motility changes. Phosphorylation specific antibodies will be used to identify changes in the activity and location of focal adhesion and cytoskeletal proteins as well MAP kinase and other signaling intermediates. Rho-family GTPases activities will be assessed using GST-GTPase effector protein binding domains. It is anticipated that information gained from the proposed study will provide insight into the molecular mechanisms by which cells interact and communicate with their environment to regulate normal and neoplastic cell migration and will potentially identify protein-protein interactions/pathways that might in the future serve as targets for therapeutic intervention.