DESCRIPTION (provided by applicant): We are interested in understanding how normal intestinal cells regulate their growth and how loss of that regulation results in malignant transformation. Our research focuses on molecular mechanisms by which the Src tyrosine kinases contributes to the regulation. We and others showed that downregulation of Src activity is important for differentiation, and upregulation is important for malignant transformation of intestinal cells. Thus, effort is directed towards identifying mechanisms that modulate Src activity during intestinal cell maturation and malignant transformation. We recently identified RACK1 as a novel Src substrate and binding partner and an inhibitor of Src activity and cell growth. We hypothesize that RACK1 is also an inhibitor of cell transformation by Src. We propose to test this as follows: Aim 1: We will assess RACK1's influence on cell transformation by Src. These studies involve analyzing NIH 3T3 and SV40LT-immortalized colonic epithelial cells that express v-Src or c-Src mutants together with wild-type or mutant RACK1 or inhibitory peptides, for anchorage-independent growth in soft agar, growth in low serum, focus formation on monolayers of normal cells, and tumor formation in nude mice; all capabilities that fully transformed v-Src cells have and normal cells lack. Aim 2: We will analyze mechanisms by which Src is activated in human colon carcinoma cells, and the functional consequences of the activation. These studies involve identifying mutations, posttranslational modifications, subcellular localization and protein-protein interactions of Src and RACK1, and assessing their influence on Src activity, cell transformation and tumorigenesis. These studies should generate significant new information regarding a novel inhibitor of Src and cell growth. Understanding how inhibitors of mitogenic signals work to regulate intestinal cell growth and how loss of that inhibition results in uncontrolled growth and malignant transformation, should impact our understanding of colonic carcinogenesis and lead to development of novel strategies for colon cancer therapy. Endogenous or peptide inhibitors of oncogenic kinases are potentially tumor suppressors; they represent exciting new targets for therapeutic intervention.