SUBSTRATES OF P60SRC PROTEIN TYROSINE KINASE

激活的受体和非受体蛋白酪氨酸激酶(PTKs)是 推测介导它们在信号转导和细胞中的不同作用 通过将细胞蛋白磷酸化到酪氨酸上进行转化，从而 改变它们的生化活动。因为许多细胞底物 许多PTKs尚未被分子表征，它们的功能仍然存在 未知的是，我们对传递PTK的生化途径的理解- 诱导信号仍然不完整。这项提议的中心主题是 表征新的PTK底物并确定其可能的性质 S在信号转导和细胞转化中的作用。使用单克隆 针对SRC磷酸化的不同蛋白制备抗体 酪氨酸激酶，我最近克隆了编码八个细胞的cDNA 底物，其中7个与以下序列没有总体同源性 国际蛋白质数据库。我计划把重点放在角色塑造上 具有代表性的120千道尔顿(KDa)蛋白质(P120) 活化p60c-src转化的细胞中的磷酸化 T抗原，以及对表皮生长刺激细胞的反应 因子(EGF)、血小板衍生生长因子(PDGF)和集落刺激 因子1(CSF-1)提示它可能在配体诱导中起中心作用。 信号和细胞转化。我的目标是完成分子 P120基因的鉴定，以确定其亚细胞拓扑结构和 在不同细胞类型中的分布，并以生化方法绘制主要 分子内酪氨酸磷酸化的位置。使用这个 信息，我希望开发定义为AS的模型生物学系统 目前尚不清楚p120的功能和确定酪氨酸的作用 磷酸化修饰其活性。原则上，这些方法 我的设想应该适用于对他者的刻画 由我的抗体定义的SRC底物，我将包括实验 在与这些蛋白质比较的情况下涉及这些蛋白质 P120可能被证明是特别有信息量的。

Activated receptor and nonreceptor protein tyrosine kinases (PTKs) are presumed to mediate their diverse effects in signal transduction and cell transformation by phosphorylating cellular proteins on tyrosine and thereby modifying their biochemical activities. Because many cellular substrates of PTKs have not been molecularly characterized and their functions remain unknown, our understanding of the biochemical pathways that relay PTK- induced signals remains incomplete. The central theme of this proposal is to characterize novel PTK substrates and to determine their putative role(s) in signal transduction and cell transformation. Using monoclonal antibodies prepared to different proteins phosphorylated by the src tyrosine kinase, I have recently cloned cDNAs encoding eight cellular substrates, seven of which show no overall homology to sequences in international protein databases. I plan to focus on the characterization of a representative 120 kilodalton (kDa) protein (p120) whose phosphorylation in cells transformed by activated p60c-src, polyoma middle T antigen, and in response to stimulation of cells by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and colony-stimulating factor 1 (CSF-1) implies that it may play a central role in ligand-induced signaling and cell transformation. My goals are to complete the molecular characterization of p120 cDNA, to determine its subcellular topology and distribution among different cell types, and to biochemically map the major sites of tyrosine phosphorylation within the molecule. Using this information, I hope to develop model biological systems for defining the as yet unknown function of p120 and for determining the role of tyrosine phosphorylation in modifying its activity. In principle, the approaches that I envision should be applicable to the characterization of the other src substrates defined by my antibodies, and I will include experiments involving these proteins under circumstances where their comparison with p120 might prove particularly informative.