Structure and Function of Paxillin

描述（由申请人提供）：细胞迁移对于正常胚胎发育、组织修复和免疫监视至关重要，但也是智力迟钝、发育缺陷、肿瘤细胞侵袭和组织纤维化的促成因素。这是一个高度动态的过程，需要精确的空间和时间控制细胞粘附到细胞外基质（ECM）与肌动蛋白细胞骨架的重塑协调。Rho家族GTP酶在这种调节中起着核心作用，但控制其关键调节因子鸟嘌呤核苷酸交换因子（GEF）和GTP酶激活蛋白（GAP）活性的机制仍然知之甚少。桩蛋白（Paxillin）是一种多结构域的支架/衔接蛋白，它能将大量的结构和信号分子聚集到细胞粘附位点，从而在调节细胞迁移中起中枢作用。本提案的目的1将通过建立包括ARF GAP PKL/GIT 2、鸟嘌呤核苷酸交换因子Vav 2和酪氨酸磷酸酶PTP-PEST的局部信号网络来检验桩蛋白协调Rho家族GT3信号传导和粘着斑动力学的时空调节的假设。Hic-5是桩蛋白的近亲，在上皮-间充质转化期间上调，以通过Rho-ROCK信号传导促进细胞迁移，并且是细胞运动性以及肌成纤维细胞中病理生理基质重塑的重要调节剂。在目标2中，使用2D和3D矩阵模型系统，我们将剖析Hic-5控制细胞迁移和收缩的机制，并测试Hic-5独立和与桩蛋白一起调节这些过程的假设。为了实现这些目标，我们将通过RNA干扰抑制内源性蛋白表达或在成纤维细胞中表达突变蛋白，并利用共聚焦荧光延时显微镜，结合光漂白后荧光恢复（FRAP）和荧光共振能量转移（FRET）分析来评估细胞形态，极性和迁移以及粘着斑动力学和蛋白质-蛋白质相互作用的时空变化以及Rho家族GTdR活性。这将与细胞内信号传导变化的生化分析相结合，包括GEF活性测定、蛋白磷酸化分析和蛋白-蛋白相互作用。这些目标的完成将阐明桩蛋白和Hic-5的作用以及它们通过调节Rho GT3系统在调节细胞迁移中的潜在相互作用。公共卫生相关性：LL运动对于正常过程如胚胎发育和组织修复是必不可少的，但它也是癌症进展、组织纤维化和几种心血管和神经退行性疾病的关键因素。从拟议的研究中获得的信息将有助于我们了解细胞迁移机制是如何调节的，从而有可能确定迁移相关疾病矫正疗法的新靶点。

DESCRIPTION (provided by applicant): Cell migration is essential for normal embryonic development, tissue repair and immune surveillance, but is also a contributing factor in mental retardation, developmental defects, tumor cell invasion and tissue fibrosis. It is a highly dynamic process requiring exquisite spatial and temporal control of cell adhesion to the extracellular matrix (ECM) in coordination with remodeling of the actin cytoskeleton. The Rho family GTPases play a central role in this regulation but the mechanisms controlling the activity of their key regulators, the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) remain poorly understood. Paxillin is a multi-domain scaffold/adapter protein, which recruits numerous structural and signaling molecules to cell adhesion sites and thereby functions as a central hub in the regulation cell migration. Aim 1 of this proposal will test the hypothesis that paxillin coordinates the spatial-temporal regulation of Rho family GTPase signaling and focal adhesion dynamics by establishing a local signaling network comprising the ARF GAP PKL/GIT2, the guanine nucleotide exchange factor Vav2 and the tyrosine phosphatase PTP-PEST. Hic-5, a close relative of paxillin is upregulated during epithelial-mesenchymal transition to promote cell migration via Rho-ROCK signaling and is an important regulator of cell motility, as well as patho-physiologic matrix remodeling in myofibroblasts. In Aim 2, using 2D- and 3D-matrix model systems, we will dissect the mechanism through which Hic-5 controls cell migration and contractility and test the hypothesis that Hic-5 functions both independently and in conjunction with paxillin to regulate these processes. To accomplish these goals, we will suppress endogenous protein expression by RNA interference or express mutant proteins in fibroblasts and utilize confocal fluorescence time-lapse microscopy, combined with Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Resonance Energy Transfer (FRET) analysis to evaluate cell morphology, polarity and migration as well focal adhesion dynamics and spatial- temporal changes in protein- protein interactions and Rho family GTPase activity. This will be combined with biochemical analysis of changes in intracellular signaling to include GEF activity assays, protein phosphorylation profiling and protein- protein interactions. Completion of these Aims will elucidate the roles of paxillin and Hic-5 and their potential interactions in regulating cell migration vi modulation of the Rho GTPase system. PUBLIC HEALTH RELEVANCE: ll movement is essential for normal processes such as embryonic development and tissue repair but it is also a key factor in cancer progression, tissue fibrosis and several cardiovascular and neurodegenerative disorders. Information gained from the proposed study will contribute to our understanding of how the cell migration machinery is regulated and thereby will potentially identify novel targets for corrective therapies for migration-associated disorders.