INTESTINAL CELL GROWTH CONTROL--ROLE OF TYROSINE KINASE

描述(改编自调查员的摘要和/或目标)：长篇 本提案的任期目标是确定Src家族的作用 酪氨酸激酶在恶性转化过程中的调控作用。 P.I.已经证明了Src的比活性在 大多数恶性和癌前病变的结肠。此外，源 随着肠道隐窝细胞的分化，活跃度降低。这些 结果表明，src的上调对生长和 肠道细胞的转化。该提案的总体目标是 明确正常肠道中下调Src的分子机制， 以及那些在结肠癌中上调Src的基因。因此，假设是 检测与Src的特定结构域以及与其结合的蛋白质有关 它们是激酶活性的重要调节因子。因此，当前的 所有的努力都是为了识别细胞蛋白质， 肠道成熟和癌变过程中对Src功能的调节 转型。一个特别的目标是鉴定和表征蛋白质。 与Src和人结肠的独特的SH3和/或SH2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 用来回答特定的目标。主要有：1)lambda gt11 表达文库筛选2)酵母双杂交系统，3) 重组GST融合蛋白。绑定到所需的站点和源 相互作用的蛋白质将被确定，突变将被引入 进入结合部位以及对Src活性的功能影响将 被评估。PI已经证明了一种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F527Src突变体使SYP在酪氨酸上磷酸化。两者都有 事件是已知的上调酶活性的机制。第二 具体目标是扩展这些研究并检查功能 Src-SYP相互作用的后果。Src和SYP上的序列 需要结合和F527Src磷酸化的SYP上的酪氨酸将 ，突变将被引入这些位置，并且 将对酶活性的功能后果进行评估。这些 研究可能会得出关于功能基因的重要信息 Src与人类结肠癌的关系。

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.