CYTOSKEKETAL SUBSTRATES OF THE TYROSINE KINASE PP60 VSRC

酪氨酸激酶 PP60 VSRC 的细胞骨架底物

• 批准号: 3193375
• 负责人: JOHN R GLENNEY
• 金额: $ 12.73万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1994-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193375
• 关键词: affinity chromatography; cell membrane; chick embryo; cytoskeletal proteins; cytoskeleton; enzyme linked immunosorbent assay; enzyme substrate; fluorescence microscopy; gene expression; high performance liquid chromatography; immunological substance; laboratory mouse; laboratory rabbit; molecular site; monoclonal antibody; neoplastic transformation; oncogenes; phosphorylation; protein sequence; protein tyrosine kinase; radionuclides; tissue /cell culture

Many oncogenes are known to encode tyrosine-specific protein kinases presumably exerting their transforming effects by phosphorylating key substrates present in normal cells. Of the known tyrosine kinases and substrates, many are tightly linked to the plasma membrane-associated cytoskeleton. The proposed research is designed to identify new substrates of the transforming tyrosine kinase pp60v-src and to analyze the relevance of the phosphorylation to transformation. For this, cells expressing transforming wild type or non-transforming non-myristylated mutants of SRC will be examined. Proteins phosphorylated on tyrosine residues in transformed cells will be isolated by affinity chromatography using a monoclonal anti-phosphotyrosine antibody previously made in this laboratory. We show this antibody is far superior to others available. The proteins specifically eluted from this antibody column with the hapten phosphotyrosine will then be used as antigen to produce monoclonal antibodies to individual components. We have already produced antibodies to several new proteins in this way. We will also examine phosphotyrosine-containing peptides derived from transformed and non-transformed cells. Peptides will be sequenced and anti-peptide antibodies will be made to select phosphorylation sites. We will focus on those substrates for which there is a correlation with transformation. We have identified a 22K substrate which displays such a correlation. Antibodies to individual substrates will be used to determine the subcellular localization of the proteins by immunofluorescence microscopy and subcellular fractionation of normal and transformed chick embryo fibroblasts. We will isolate the 22K substrate from normal chick tissues and examine the association with other cytoskeletal proteins. Peptides from the 22K protein will be sequenced and used to search the protein sequence database for possible homology to other known proteins. These studies will advance our knowledge of both the membrane skeleton of fibroblasts and alterations in the cytoskeleton occurring upon malignant transformation.

已知许多癌基因编码酪氨酸特异性蛋白激酶 推测它们通过磷酸化关键酶来发挥转化作用 存在于正常细胞中的底物。已知的酪氨酸激酶和 底物，许多与质膜相关的紧密连接 细胞骨架拟议的研究旨在确定新的基板 转化酪氨酸激酶pp60v-src的表达，并分析其相关性。 磷酸化转化的过程。为此，表达 SRC的转化野生型或非转化非豆蔻酰化突变体 将被审查。酪氨酸残基磷酸化蛋白质 转化的细胞将通过亲和层析分离， 单克隆抗磷酸酪氨酸抗体先前在此 实验室我们表明这种抗体是远远优于其他可用的上级。的 用半抗原从该抗体柱特异性洗脱的蛋白质 然后将磷酸酪氨酸用作抗原以产生单克隆抗体。 抗体对单个组分的影响。我们已经制造出了 几种新的蛋白质我们亦会研究 含有磷酸酪氨酸的肽， 非转化细胞。肽将被测序， 将制备抗体以选择磷酸化位点。我们将专注于 与转化相关的底物。我们 已经确定了显示这种相关性的22K衬底。 将使用针对单个底物的抗体来确定 免疫荧光显微镜下蛋白质的亚细胞定位 正常鸡胚和转化鸡胚的亚细胞分级 成纤维细胞我们将从正常鸡组织中分离22K底物 并检查与其他细胞骨架蛋白的关联。肽 22K蛋白将被测序并用于搜索蛋白质序列 与其他已知蛋白质的可能同源性。这些研究将 推进我们对成纤维细胞膜骨架的认识， 恶性转化时细胞骨架的改变。