STRUCTURE AND FUNCTION OF PAXILLIN

巴西林是一种与肌动蛋白膜有关的细胞骨架磷蛋白 附着在肌肉和非肌肉细胞的细胞黏附部位。我们的 克隆的帕克西林基因表明，该蛋白含有一个 一系列与蛋白质相互作用有关的不同结构域。 这项提议旨在确定分子的哪些区域是 参与将帕西林定位于细胞附着的特定部位 细胞外基质，称为局灶性粘连。蛋白质 与巴西林的各个结构域相互作用将被识别 并以此为特征。巴西林磷酸化在调节中的作用 这些联系也将被研究。具体地说，S所在的地区 将蛋白质靶向焦点粘连所需的帕西林将是 通过稳定地转染禽类的特定区域而确定的 转化NIH3T3或CHO细胞。表达和亚细胞 禽巴西林的本地化将通过以下方式进行监测 免疫沉淀和免疫荧光显微镜分别使用 鸡肉特异性巴克西林抗血清。另一种选择是，分段的帕克西林 将通过聚合酶链式反应产生的cDNA和相应的 在细菌中表达的谷胱甘肽S转移酶蛋白片段 (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抗体定位。证据： 局灶性粘连破坏，由特定基因的过度表达引起 巴西林的构建，也将通过免疫荧光进行监测 显微镜。同样的GST-巴西林融合蛋白将被用于 用于鉴定和分离的沉淀结合分析中的亲和矩阵 利用成纤维细胞和平滑肌细胞构建新的帕西林结合蛋白 裂解物。结合蛋白将使用以下组合进行检测 代谢标记法、印迹法和体外激酶法。另外， 酵母相互作用陷阱系统将用于鉴定和克隆 直接，巴西林结合蛋白。任何检测到的新蛋白质都将是 进一步提纯和生化表征。特别关注 将针对三种帕克西林的进一步表征 结合蛋白；一个未知的100 kDa磷酸蛋白，一种丝氨酸激酶 和一种苏氨酸激酶。帕克西林磷酸化是由 帕西林相关激酶的体外活性将通过以下方法检测 巴西林基因的缺失和定点突变 用磷酸多肽和磷酸氨基酸分析鉴定目标氨基酸 酸。这些数据将与对帕西林的类似分析相关联 在体内诱导成纤维细胞的磷酸化 附着/脱离细胞外基质。它的重要性 局灶性黏附组织中的磷酸化残基和 通过转导帕西林基因来解决帕西林靶向问题 含有适当氨基酸的点突变。派生的数据 希望从这些实验中有助于我们理解 与细胞相关的调控细胞骨架组装的机制 粘附性，从而为确定这种 来自细胞外环境的中介信号中的事件， 这会导致基因表达和细胞增殖。

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.