INTESTINAL CELL GROWTH CONTROL--ROLE OF TYROSINE KINASE

描述（改编自研究者的摘要和/或目的）： 本提案的一个长期目标是确定Src家族的作用 酪氨酸激酶在恶性转化的调节过程中的作用。 私家侦探已经证明，Src的比活性升高， 结肠的大多数恶性和癌前病变。 此外，Src 活性随着肠隐窝细胞的分化而降低。 这些 结果表明Src的上调对于生长和 肠细胞的转化。 该提案的总体目标是 确定在正常肠道中下调Src的分子机制， 以及那些在结肠癌中上调Src的基因。 因此，假设 所测试的是与Src的特定结构域和与Src结合的蛋白质相关的蛋白质。 它们是激酶活性的重要调节剂。 因此电流 所有的努力都指向鉴定细胞蛋白质， 调节Src功能在肠成熟和恶性 转型 一个具体的目标是识别和表征蛋白质 与Src和人结肠的独特SH 3和/或SH 2结构域相互作用 癌细胞、正常肠和成纤维细胞。 三个战略将是 用于满足特定的目标。 这些主要是：1）lambda gt 11 表达文库筛选2）酵母双杂交系统，和3） 重组GST融合蛋白。 绑定到所需的站点和Src 一个相互作用的蛋白质将被确定，突变将被引入 进入结合位点，对Src活性的功能性影响将 进行评估。 PI已经证明，一种Src结合蛋白是 Syp酪氨酸磷酸酶。 Syp似乎在Tyr 527处使Src去磷酸化， 转化的F527 Src突变体使Syp在酪氨酸上磷酸化。 两 事件是上调酶活性的已知机制。 第二 具体的目的是扩展这些研究，并检查功能 Src-Syp相互作用的结果。 Src和Syp上的序列 而Syp上被F527 Src磷酸化的酪氨酸 一旦确定，突变将被引入这些位点， 将评估对酶活性的功能影响。 这些 研究可能会产生关于功能的重要信息， Src的一些方面，因为它们与人类结肠癌有关。

DESCRIPTION (adapted from investigator's abstract and/or aims): The long term objective of this proposal is to determine the role of the Src family of tyrosine kinases in the regulatory process of malignant transformation. The P.I. has demonstrated that the specific activity of Src is elevated in most malignant and pre-malignant lesions of the colon. Moreover, Src activity decreases as the intestinal crypt cells differentiate. These results suggest that upregulation of Src is important for growth and transformation of intestinal cells. The overall goal of the proposal is to define the molecular mechanisms that downregulate Src in normal intestine, and those that upregulate Src in colon cancer. Thus, the hypothesis to be tested is related to specific domains of Src and the proteins that bind to them, are important regulators of kinase activity. Thus, the current overall effort is directed toward identifying cellular proteins that modulate Src function during intestinal maturation and malignant transformation. One specific aim is to identify and characterize proteins that interact with the unique, SH3 and/or SH2 domains of Src and human colon carcinoma cells, normal intestine and fibroblasts. Three strategies will be utilized to answer the specific aims. These are mainly: 1) lambda gt11 expression library screening 2) the yeast two-hybrid system, and 3) recombinant GST fusion proteins. The site and Src required for binding to an interacting protein would be determined, mutations will be introduced into the binding site and the functional consequences on Src activity will be assessed. The PI has demonstrated that one Src-binding protein is the Syp tyrosine phosphatase. Syp appears to dephosphorylate Src at Tyr 527, and the transforming F527 Src mutant phosphorylates Syp on tyrosine. Both events are known mechanisms to upregulate enzymatic activity. The second specific aim is to extend these studies and examine the functional consequences of the Src-Syp interaction. The sequences on both Src and Syp required for binding and the tyrosine on Syp phosphorylated by F527 Src will be identified, mutations will be introduced into these sites and the functional consequences on enzymatic activity will be assessed. These studies are likely to yield important information regarding the functional aspects of Src as they relate to human colon cancer.