SUBSTRATES OF P60SRC PROTEIN TYROSINE KINASE

活化的受体和非受体蛋白酪氨酸激酶（PTK）是 据推测，介导其在信号转导和细胞凋亡中的不同作用。 通过磷酸化酪氨酸上的细胞蛋白质进行转化， 改变它们的生化活动。 因为许多细胞基质 的PTK尚未进行分子表征，其功能仍然存在 未知的，我们对传递PTK的生化途径的理解- 诱导信号仍然不完整。 这项建议的中心主题是 表征新型PTK底物并确定其推定的 在信号转导和细胞转化中的作用。 使用单克隆 针对由src磷酸化的不同蛋白质制备的抗体 酪氨酸激酶，我最近克隆的cDNA编码八个细胞 底物，其中七个显示与在 国际蛋白质数据库。 我打算把重点放在 代表性的120千道尔顿（kDa）蛋白（p120），其 活化的p60 c-src转化的细胞中的磷酸化， T抗原，并响应表皮生长刺激细胞 血小板衍生生长因子（PDGF）和集落刺激因子（EGF） 因子1（CSF-1）暗示它可能在配体诱导的细胞凋亡中发挥重要作用。 信号传导和细胞转化。 我的目标是完成 p120 cDNA的表征，以确定其亚细胞拓扑结构， 不同细胞类型之间的分布，并对主要的 分子内的酪氨酸磷酸化位点。 使用此 信息，我希望开发模型生物系统，以定义为 p120的未知功能和确定酪氨酸的作用 磷酸化修饰其活性。 原则上，方法 我认为应该适用于描述另一个 src底物由我的抗体定义，我将包括实验 涉及这些蛋白质的情况下，他们的比较与 P120可能会提供特别的信息。

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.