The objectives of the research outlined in this proposal are to define the role of transforming gene products (such as pp60-v-src and p21-v-Ha-ras), serum growth factors or tumor promoting phorbol ester in growth control and cell transformation. The interactions of the transforming proteins with cellular membranes will be characterized and attempts made to determine if any cellular membrane components are required for membrane association by using purified protein components and synthetic lipid vesicles in reconstitution experiments. We shall study cellular enzymes that may be instrumental in causing the phenotypic changes displayed by cells in response to these transforming proteins or tumor promoters. Examples of such cellular enzymes include the serine-specific ribosomal protein S6 kinase, enzymes involved in the regulation of cyclic AMP levels and enzymes involved in the generation of the second messengers diacylglycerol and inositol trisphosphate. When feasible, these cellular enzymes will be used to generate polyclonal or monoclonal antibody as an additional aid in functional characterization and for use in obtaining cDNA or genomic molecular clones for these proteins. We shall compare quiescent cells to cells stimulated to proliferate with regard to synthesis of specific proteins, protein phosphorylation patterns and expression of cellular genes. These studies should provide new insights concerning the biochemical pathways that lead to cellular proliferation and malignant transformation. We shall utilize 1) multiple techniques for protein purification; 2) molecular cloning of cDNA libraries; 3) polyclonal and monoclonal antibodies; 4) viral transformation mutants.
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