CYTOSKEKETAL SUBSTRATES OF THE TYROSINE KINASE PP60 VSRC

已知许多癌基因编码酪氨酸特异性蛋白激酶。 推测通过磷酸化关键字来发挥其转化作用 正常细胞中存在底物。已知的酪氨酸激酶和 底物中，很多都与质膜紧密相连 细胞骨架。这项拟议的研究旨在确定新的底物 转化酪氨酸激酶pp60v-src的表达及其相关性分析 从磷酸化到转化。为此，细胞表达 转化SRC野生型或非转化非肉豆蔻化突变体 将会被检查。酪氨酸残基上的磷酸化蛋白质 转化的细胞将通过亲和层析分离，使用 本实验室制备的抗磷酸酪氨酸的单抗 实验室。我们表明，这种抗体远远优于现有的其他抗体。这个 用半抗原从该抗体柱中特异性洗脱的蛋白质 然后，将磷酸酪氨酸作为抗原来产生单克隆 针对个别成分的抗体。我们已经产生了抗体来对抗 几种新的蛋白质以这种方式出现。我们还将检查 含磷酸酪氨酸的多肽来自转化和 未转化的细胞。多肽将被测序并抗肽 将制作抗体来选择磷酸化位点。我们将重点关注 那些与转化相关的底物。我们 已经确定了显示这种相关性的22K底物。 针对个别底物的抗体将被用来确定 免疫荧光显微镜下蛋白质的亚细胞定位 正常鸡胚胎和转化鸡胚胎的亚细胞分离 成纤维细胞。我们将从正常的鸡组织中分离出22K底物 并研究与其他细胞骨架蛋白的关系。多肽来自 将对22K蛋白进行测序，并用于搜索蛋白质序列 可能与其他已知蛋白质同源的数据库。这些研究将 加深我们对成纤维细胞膜骨架和 癌变时细胞骨架的改变。

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.