STRUCTURE AND FUNCTION OF PAXILLIN

我最近发现了一种新的纽蛋白结合蛋白，桩蛋白。 该建议旨在进一步表征桩蛋白，其相互作用 和黏着斑蛋白的研究，以及鉴定其他paxillin结合蛋白的研究。 这些 数据有望有助于我们理解细胞骨架- 膜相互作用 桩蛋白的核苷酸序列将是 从桩蛋白cDNA获得。 得到的预测氨基酸序列 将分析与其他蛋白质的潜在同源性， 可能的酶或结构功能的建议。 适当 将对纯化的蛋白质进行生物化学测定， 证实/否定这些迹象。 将桩蛋白cDNA转染 全部或部分导入哺乳动物细胞，以确定 蛋白质是黏着斑定位所必需的。 平行的，部分的 或桩蛋白和黏着斑蛋白的全长融合蛋白将用于 体外测定以绘制它们的结合位点。 这两者的相互作用 还将在电子显微镜水平上检查蛋白质。 以下 通过劳斯肉瘤病毒转化，桩蛋白含有高水平的 磷酸酪氨酸 桩蛋白磷酸化在局灶性脑缺血中的作用 在病毒或化学诱导的转化过程中的粘附完整性将 追究 桩蛋白磷酸化对黏着斑蛋白的影响 结合将在体外测定。 为了确定小说 paxillin结合蛋白I将分离蛋白质复合物， 桩蛋白来自在培养物中生长的细胞，在其 与底层的界面。 细胞将被温和地透化， 在某些情况下，肌动蛋白细胞骨架被鬼笔环肽稳定。 蛋白 将从这些细胞中差异提取使用离子的变化， 强度、pH和二价阳离子浓度。 释放的蛋白质 与桩蛋白的结合将通过免疫沉淀共分离， 在非变性条件下的桩蛋白。 在适当的情况下， 将在化学交联剂的存在下进行，以稳定 桩蛋白及其相关蛋白之间的相互作用。 抗体将 针对任何新的桩蛋白结合蛋白产生，这些将是 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.