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 i s 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.
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