The objective of this proposal is to investigate the mechanisms by which the Ca2+ and phospholipid-dependent protein kinase C is regulated, and to apply the understanding thus developed to the evaluation of a number of environmental toxins and hazardous compounds. Current assay methods for protein kinase C do not allow molecular analysis of enzyme regulation and activation. To overcome this problem, a novel assay system for enzyme activity utilizing mixed micelles will be developed and characterized. By varying the mole fractions of phospholipids and diacylglycerols present, the mixed micellar assay will be used to analyze the stoichiometry and specificity of protein kinase C activation. It also allows the evaluation of the specificity of antagonist action. This work will constitute the research effort during Phase I. A major portion of Phase I will be devoted to didactic course work through enrollment in the Duke University Program in Cell and Molecular Biology. Subsequent work will be directed towards the evaluation of a number of diacylglycerol analogues using the mixed micellar assay.
This aims at elucidation of structure-function relationships in protein kinase C regulation as well as developing specific antagonists to enzyme activation by diacylglycerols. These agonists and antagonists will also be evaluated using the A431 cultured cell line. Specific application of this work to health-related problems aims at extending the hypothesis that compounds with tumor promoting action modulate protein kinase C activity. The mixed micellar assay is ideally suited for in vitro testing of this hypothesis since it overcomes problems in agent delivery that arise in standard assays. Thus, certain carcinogens, tumor promoters and environmental agents will be evaluated. It is expected that the mixed micellar assay will emerge as a rapid screening method for such agents and compounds and be widely applicable like the Ames test for mutagens. The general application of this work to human health derives from the multiple biological functions of protein kinase C. This enzyme is pivotal in cell growth and differentiation, and the transduction of insulin, cholinergic, Alpha1 adrenergic, and other hormonal signals and growth factor effects. Therefore, the understanding of its regulation is relevant to cancer biology, endocrinology, pharmacology, and other disciplines. The development of specific antagonists will find immediate application in further studies in these fields.
