The long term goal of the research is to contribute to an understanding of neoplastic transformation, growth regulation and lipid second messengers in signal transduction systems through the study of protein kinase C. Protein kinase C functions to transduce sn-1,2-diacylglycerol second messengers produced in response to growth factors, neurotransmitters, hormones and some oncogene products. Protein kinase C is the major receptor of phorbol esters and other tumor promoters. The goals are to understand the structure, function and regulation of protein kinase C, whether mutant forms of protein kinase C will cause cellular transformation, and to elucidate the mechanism by which certain anti-tumor agents inhibit protein kinase C.
The specific aims are: 1) to express the rat brain c-DNAs encoding protein kinase C in E. coli, to purify these and compare their properties with the brain enzyme; 2) to define the smallest polypeptide capable of high affinity phorbol ester binding; 3) to develop colony autoradiographic methods for expressed protein kinase C activity and phorbol binding in E. coli and to select mutants defective in activity and phorbol binding; 4) to determine whether wild type or mutant forms of protein kinase C will cause cellular transformation directly or by collaboration with other oncogenes; 5) to further define the mechanism of regulation by determining the structural features within phospholipids and diacylglycerol required for activation, and by performing direct binding studies of phospholipid, diacylglycerol and Ca2+ to protein kinase C and 6) to elucidate the mechanism of protein kinase C inhibition by anti-tumor agents and other inhibitors and to discover and/or design protein kinase inhibitors. These studies will employ mixed micellar methods of analysis developed by our lab and rat brain c- DNAs cloned and sequenced by our lab. These studies will contribute to understanding the function of protein kinase C in signal transduction and the molecular mechanisms of regulation of cell growth. The studies will critically assess the role of protein kinase C in oncogenesis and as a target of anti-tumor agents.
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