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一个因素。这是一个高度动态的过程，需要精细的空间和时间控制细胞粘附到细胞外基质（ECM），协调肌动蛋白细胞骨架的重塑。Rho家族GTPase在这一调控中发挥着核心作用，但控制其关键调控因子，鸟嘌呤核苷酸交换因子（gef）和GTPase激活蛋白（gap）活性的机制仍然知之甚少。Paxillin是一种多结构域支架/适配器蛋白，它将许多结构和信号分子招募到细胞粘附位点，从而在调节细胞迁移中发挥中心枢纽的作用。本研究的目的1将通过建立一个由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，来验证paxillin协调Rho家族GTPase信号通路和局灶黏附动力学的时空调节的假设。Hic-5是paxillin的近亲，在上皮-间质转化过程中上调，通过Rho-ROCK信号促进细胞迁移，是肌成纤维细胞细胞运动和病理生理性基质重塑的重要调节因子。在目标2中，使用2D和3d矩阵模型系统，我们将分析Hic-5控制细胞迁移和收缩的机制，并测试Hic-5独立或与paxillin一起调节这些过程的假设。为了实现这些目标，我们将在成纤维细胞中通过RNA干扰或表达突变蛋白来抑制内源性蛋白的表达，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、焦点粘附动力学、蛋白质-蛋白质相互作用和Rho家族GTPase活性的时空变化。这将与细胞内信号变化的生化分析相结合，包括GEF活性分析，蛋白质磷酸化分析和蛋白质-蛋白质相互作用。这些目标的完成将阐明paxillin和Hic-5的作用以及它们在调节Rho GTPase系统的细胞迁移中的潜在相互作用。公共卫生相关性：所有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理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纠正治疗的新靶点。