STRUCTURE AND FUNCTION OF PAXILLIN

我最近发现了一种新的纽蛋白结合蛋白--巴西林。 这项提议旨在进一步表征巴西林及其相互作用 与纽蛋白结合，并鉴定其他巴西林结合蛋白。这些 数据有望有助于我们对细胞骨架的理解- 膜相互作用。巴西林的核苷酸序列将是 从巴西林基因中获得。由此预测的氨基酸序列 将分析与其他蛋白质的潜在同源性，并为 提示可能的酶或结构功能。恰如其分 将对纯化的蛋白质进行生化分析以 确认/反驳这些迹象。将帕克西林的cdna基因导入 全部或部分进入哺乳动物细胞，以确定 蛋白质是定位局部粘连所必需的。并行、部分 或者帕克西林和纽蛋白的全长融合蛋白将用于 体外实验绘制它们的结合部位图。这两者的相互作用 蛋白质也将在电子显微镜水平上进行检查。跟随 由劳斯肉瘤病毒转化，巴西林含有高水平的 磷酸酪氨酸。帕克西林磷酸化在病灶丢失中的作用 病毒或化学诱导的转化过程中的黏附完整性将 被调查。帕西林磷酸化对纽蛋白的影响 结合作用将在体外进行测定。在试图辨认小说 巴西林结合蛋白I将分离含有以下成分的蛋白复合体 在培养的细胞中生长的具有局灶性粘连的巴西林 与底物对接。细胞将被轻轻渗透，并在 在某些情况下，用鬼臼毒素稳定了肌动蛋白细胞骨架。蛋白质 将从这些细胞中利用离子变化进行差异化提取 强度、pH和二价阳离子浓度。蛋白质在体内释放 与巴西林将通过免疫沉淀共同分离 非变性条件下的巴西林。在适当的情况下，实验 将在化学交联剂存在的情况下进行稳定 巴西林与其相关蛋白之间的相互作用。抗体会 针对任何新的巴西林结合蛋白而产生，这些蛋白将是 用于进一步研究蛋白质在细胞骨架中的作用 膜组织。

I have recently identified a novel vinculin-binding protein, paxillin. This proposal is aimed at further characterizing paxillin, its interaction with vinculin and at identifying other paxillin-binding proteins. These data are expected to contribute to our understanding of cytoskeletal- membrane interactions. The nucleotide sequence of paxillin will be obtained from paxillin cDNA. The resulting predicted amino acid sequence will be analyzed for potential homologies with other proteins and for suggestions of possible enzymatic or structural function. Appropriate biochemical assays will be performed on the purified protein to confirm/disprove such indications. The paxillin cDNA will be transfected in whole or in part into mammalian cells to determine which regions of the protein are required for focal adhesion localization. In parallel, partial or full length fusion proteins of paxillin and vinculin will be used in in vitro assays to map their binding sites. The interaction of these two proteins will also be examined at the electron microscope level. Following transformation by Rous sarcoma virus, paxillin contains high levels of phosphotyrosine. The role of paxillin phosphorylation in the loss of focal adhesion integrity during viral or chemically induced transformation will be investigated. The effects of paxillin phosphorylation on vinculin binding will be determined in vitro. In an attempt to identify novel paxillin-binding proteins I will isolate protein complexes containing paxillin from cells grown in culture possessing focal adhesions at their interface with the substratum. Cells will be gently permeabilized and, in some cases, the actin cytoskeleton stabilized with phalloidin. Proteins will be differentially extracted from these cells using changes in ionic strength, pH and divalent cation concentrations. Proteins released in conjunction with paxillin will be co-isolated by immunoprecipitation of paxillin under non-denaturing conditions. Where appropriate, experiments will be performed in the presence of chemical cross-linkers to stabilize interactions between paxillin and its associated proteins. Antibodies will be generated against any novel paxillin-binding proteins and these will be used to study further the proteins regarding their role in cytoskeletal membrane organization.