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.

帕西林是参与肌动蛋白膜的细胞骨架磷酸蛋白 在肌肉和非肌肉细胞中细胞粘附部位附着。我们的 帕克林蛋白cDNA的克隆表明该蛋白包含 一系列与蛋白质蛋白相互作用有关的不同领域。 该建议旨在确定分子的哪个区域 参与靶向Paxillin到细胞附着的专用部位 到被称为局灶性粘连的细胞外基质。蛋白质 将与Paxillin的各个领域相互作用。 并描述了。帕西林磷酸化在调节中的作用 这些关联也将进行研究。具体而言， 将蛋白质靶向局灶性粘连所需的帕西林是 通过稳定转染禽的定义区域确定 Paxillin cDNA进入NIH 3T3或CHO细胞。表达和亚细胞 禽帕西林的定位将由 分别使用免疫沉淀和免疫荧光显微镜使用使用 鸡肉特异性帕西林抗血清。或者，帕西林的片段 cDNA将由聚合酶链反应和相应的 在细菌中以谷胱甘肽S-转移酶表示的蛋白质片段 （GST）融合蛋白。纯化的融合蛋白将进行微注射 进入哺乳动物细胞，并用抗GST抗体定位。证据 局灶性粘附破坏，由特定的过表达产生 Paxillin构建体还将通过免疫荧光监测 显微镜。相同的GST - 帕西林融合蛋白将用作 降水结合测定中的亲和力矩阵以识别和分离 使用成纤维细胞和平滑肌的新型帕西林结合蛋白 裂解物。结合蛋白将使用 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，帕西林结合蛋白。任何检测到的新蛋白质都是 进一步纯化并在生化上表征。特别注意 将针对三个paxillin的进一步表征 结合蛋白；一个身份不明的100 kDa磷蛋白，一种丝氨酸激酶 和苏氨酸激酶。帕西林磷酸化是由 帕克林相关激酶的体外活性将通过 paxillin cDNA的缺失和定向的诱变，然后 磷酸肽和磷酸氨基酸分析以鉴定靶氨基 酸。这些数据将与帕西林的类似分析相关 由成纤维细胞体内诱导的磷酸化诱导 附着/脱离细胞外基质。重要性 焦点粘附组织中的磷酸化残基和 Paxillin靶向将通过转染Paxillin cDNA来解决 包含适当氨基酸的点突变。得出数据 从这些实验中，预计将有助于我们的理解 调节与细胞相关的细胞骨架组件的机制 粘附，从而为确定这种作用提供了基础 介导信号中的事件，来自细胞外环境， 这导致基因表达和细胞增殖。