STRUCTURE AND FUNCTION OF PAXILLIN

PAXILLIN 的结构和功能

• 批准号: 2459447
• 负责人: Christopher E Turner
• 金额: $ 21.99万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-05 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459447
• 关键词: 3T3 cells; CHO cells; actin binding protein; cell adhesion; cell cell interaction; chick embryo; chimeric proteins; electron microscopy; fluorescence microscopy; immunoprecipitation; laboratory mouse; laboratory rabbit; membrane activity; microinjections; molecular cloning; phosphorylation; polymerase chain reaction; protein kinase; protein signal sequence; protein structure function; protein transport; site directed mutagenesis; transfection

Paxillin is a cytoskeletal phosphoprotein involved in actin-membrane attachment at sites of cell adhesion in muscle and non-muscle cells. Our cloning of the paxillin cDNA indicates that this protein contains a series of distinct domains implicated in protein-protein interactions. This proposal is designed to determine which regions of the molecule are involved in targeting paxillin to specialized sites of cell attachment to the extracellular matrix known as focal adhesions. Proteins interacting with the individual domains of paxillin will be identified and characterized. The role of paxillin phosphorylation in regulating these associations will also be studied. Specifically, the region(s) of paxillin necessary for targeting the protein to focal adhesions will be determined through stable transfection of defined regions of avian paxillin cDNA into NIH 3T3 or CHO cells. Expression and subcellular localization of the avian paxillin will be monitored by immunoprecipitation and immunofluorescence microscopy respectively, using chicken-specific paxillin antisera. Alternatively, segments of paxillin cDNA will be generated by polymerase chain reaction and the corresponding protein fragments expressed in bacteria as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins will be microinjected into mammalian cells and localized with anti-GST antibodies. Evidence for focal adhesion disruption, resulting from over-expression of particular paxillin constructs, will also be monitored by immunofluorescence microscopy. The same GST-paxillin fusion proteins will be used as affinity matrices in precipitation binding assays to identify and isolate novel paxillin binding proteins using fibroblast and smooth muscle lysates. Binding proteins will be detected using a combination of metabolic labeling, blotting and in vitro kinase assays. Additionally, the yeast Interaction Trap system will be used to identify and clone directly, paxillin binding proteins. Any novel proteins detected will be further purified and characterized biochemically. Particular attention will be directed towards further characterization of three paxillin binding proteins; an unidentified 100 kDa phosphoprotein, a serine kinase and a threonine kinase. Paxillin phosphorylation resulting from the activity in vitro of the paxillin-associated kinases will be examined by deletion and site-directed mutagenesis of the paxillin cDNA followed by phosphopeptide and phosphoamino acid analysis to identify target amino acids. These data will be correlated with a similar analysis of paxillin phosphorylation induced in vivo in fibroblasts stimulated by attachment/detachment to/from the extracellular matrix. The importance of the phosphorylated residues in focal adhesion organization and paxillin targeting will be addressed by transfection of paxillin cDNA containing point mutations of the appropriate amino acids. Data derived from these experiments are expected to contribute to our understanding of the mechanisms regulating cytoskeletal assembly associated with cell adhesion, thereby providing a foundation for determining the role of such events in mediating signals, derived from the extracellular environment, that lead to gene expression and cell proliferation.

Paxillin 是一种参与肌动蛋白膜的细胞骨架磷蛋白 附着在肌肉和非肌肉细胞的细胞粘附位点。我们的 桩蛋白 cDNA 的克隆表明该蛋白含有 涉及蛋白质-蛋白质相互作用的一系列不同结构域。 该提案旨在确定分子的哪些区域 参与将桩蛋白靶向细胞附着的特定位点 细胞外基质称为粘着斑。蛋白质 与桩蛋白的各个结构域的相互作用将被识别 并进行了表征。桩蛋白磷酸化的调节作用 还将对这些协会进行研究。具体来说，以下地区 将蛋白质靶向粘着斑所必需的桩蛋白将是 通过稳定转染禽类特定区域来确定 将桩蛋白 cDNA 导入 NIH 3T3 或 CHO 细胞。表达和亚细胞 禽桩蛋白的定位将由以下人员监测 分别使用免疫沉淀和免疫荧光显微镜 鸡特异性桩蛋白抗血清。或者，桩蛋白片段 通过聚合酶链式反应生成cDNA，并生成相应的 在细菌中表达为谷胱甘肽 S-转移酶的蛋白质片段 (GST) 融合蛋白。纯化的融合蛋白将被显微注射 进入哺乳动物细胞并用抗 GST 抗体进行定位。证据 粘着斑破坏，由特定的过度表达引起 桩蛋白构建体，也将通过免疫荧光进行监测 显微镜。相同的 GST-桩蛋白融合蛋白将用作 沉淀结合测定中的亲和矩阵以识别和分离 使用成纤维细胞和平滑肌的新型桩蛋白结合蛋白 裂解物。将使用以下组合来检测结合蛋白 代谢标记、印迹和体外激酶测定。此外， 酵母相互作用陷阱系统将用于识别和克隆 直接，桩蛋白结合蛋白。任何检测到的新蛋白质都将被 进一步纯化和生化表征。特别注意 将针对三种桩蛋白的进一步表征 结合蛋白；一种未鉴定的 100 kDa 磷蛋白，一种丝氨酸激酶 和苏氨酸激酶。桩蛋白磷酸化是由 桩蛋白相关激酶的体外活性将通过以下方法进行检查 桩蛋白 cDNA 的缺失和定点诱变，然后 磷酸肽和磷酸氨基酸分析以识别目标氨基酸 酸。这些数据将与桩蛋白的类似分析相关联 体内诱导的成纤维细胞磷酸化 与细胞外基质的附着/分离。重要性 粘着斑组织中的磷酸化残基和 桩蛋白靶向将通过转染桩蛋白 cDNA 来解决 含有适当氨基酸的点突变。数据推导 这些实验预计将有助于我们的理解 调节与细胞相关的细胞骨架组装的机制 粘附力，从而为确定这种作用提供了基础 来自细胞外环境的介导信号事件， 导致基因表达和细胞增殖。