Structure and Function of Paxillin

Paxillin 的结构和功能

• 批准号: 7933357
• 负责人: Christopher E Turner
• 金额: $ 12.39万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7933357
• 关键词: Actins; Adhesions; Antibodies; Binding; Biochemical; Biological Assay; Biological Models; Cardiovascular system; Cell Adhesion; Cell physiology; Cells; Cellular Morphology; Cicatrix; Complex; Confocal Microscopy; Coupled; Cytoskeleton; Defect; Development; Disease; Embryonic Development; Environment; Exhibits; Extracellular Matrix; Family; Family member; Fibroblasts; Fibrosis; Fluorescence; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Focal Adhesions; GTPase-Activating Proteins; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Image; Immunologic Surveillance; Mediating; Mental Retardation; Microscopy; Modeling; Molecular; Movement; Myofibroblast; Neoplasm Metastasis; Neurodegenerative Disorders; PTPN12 gene; Pathway interactions; Phosphorylation; Physiological; Play; Process; Protein Tyrosine Phosphatase; Proteins; RNA Interference; Recruitment Activity; Regulation; Relative (related person); Role; Signal Transduction; Signaling Molecule; Site; Specificity; Structure; System; Testing; Time; Tissues; Tumor Cell Invasion; Tyrosine Phosphorylation; Video Microscopy; Western Blotting; Work; Wound Healing; adapter protein; angiogenesis; cell motility; epithelial to mesenchymal transition; genetic regulatory protein; in vivo; migration; mutant; novel; paxillin; protein expression; protein protein interaction; public health relevance; rho; rho GTP-Binding Proteins; scaffold; time use; tumor progression

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.

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，但也是智力低下、发育缺陷、肿瘤细胞侵袭和组织纤维化的一个促成因素。这是一个高度动态的过程，需要对细胞与细胞外基质（ECM）的粘附进行精确的空间和时间控制，并与肌动蛋白细胞骨架的重塑相协调。 Rho 家族 GTP 酶在此调节中发挥核心作用，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) 活性的机制仍知之甚少。桩蛋白是一种多结构域支架/接头蛋白，它将大量结构和信号分子募集到细胞粘附位点，从而充当调节细胞迁移的中枢。该提案的目标 1 将通过建立由 ARF GAP PKL/GIT2、鸟嘌呤核苷酸交换因子 Vav2 和酪氨酸磷酸酶 PTP-PEST 组成的局部信号网络来检验桩蛋白协调 Rho 家族 GTPase 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-间质转化过程中上调，通过 Rho-ROCK 信号传导促进细胞迁移，并且是细胞运动以及肌成纤维细胞病理生理基质重塑的重要调节因子。在目标 2 中，我们将使用 2D 和 3D 矩阵模型系统剖析 Hic-5 控制细胞迁移和收缩性的机制，并测试 Hic-5 独立发挥作用以及与桩蛋白结合调节这些过程的假设。为了实现这些目标，我们将通过RNA干扰抑制内源蛋白表达或在成纤维细胞中表达突变蛋白，并利用共焦荧光延时显微镜，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-蛋白质的时空变化 相互作用和 Rho 家族 GTP 酶活性。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、蛋白质磷酸化分析和蛋白质-蛋白质相互作用。完成这些目标将阐明桩蛋白和 Hic-5 的作用以及它们在通过调节 Rho GTPase 系统来调节细胞迁移中的潜在相互作用。公共健康相关性：运动对于胚胎发育和组织修复等正常过程至关重要，但它也是癌症进展、组织纤维化以及多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，从而有可能确定针对迁移相关疾病的纠正疗法的新靶点。