The objective of this study is to increase our understanding of the mechanism by which hormones which act via the adenylate cyclase-cAMP-dependent protein kinase system regulate cellular processes such as growth, differentiation, and hormone secretion. We propose to study the mechanism of cAMP-dependent protein kinase activation in intact cells stimulated with hormones and other agents which raise intracellular levels of cAMP. We have developed a novel method to measure the state of activation of each cAMP-dependent protein kinase isoenzyme. This technique measures the activity associated with immunoprecipitated regulatory subunits. Immunoprecipitation will be used to determine the activation profiles of cAMP-dependent protein kinases in cells stimulated with hormones which raise intracellular cAMP and in cells treated with hormones which inhibit a rise in cAMP such as somatostatin. Studies designed to characterize the mechanism of activation of protein kinases are also proposed. A reconstituted system composed of partially purified membranes which have a hormone-sensitive adenylate cyclase, purified cAMP-dependent protein kinase and cyclic nucleotide phosphodiesterase will be used to determine whether the rate of cAMP turnover affects the rate of protein phosphorylation. These studies will also enhance our understanding of the kinetics of protein kinase activation since the rate limiting step will be the generation of cAMP. Studies are also proposed to characterize the cAMP-dependent protein kinase isoenzymes in AtT20 mouse pituitary tumor cells. These cells contain protein kinase I, free RI subunits and the neural-specific protein kinase II. The studies are designed to: a) determine the reason for the presence of free RI subunits, b) measure the rate of synthesis and turnover of protein kinase isoenzymes and, c) establish mutant cells which are defective in cAMP-dependent protein kinase isoenzyme expression. We will attempt to correlate functional properties of the cAMP-dependent protein kinase isoenzymes with physiological response of the cell to hormonal stimulation (adrenocorticotropin release). These studies should enhance our understanding of the pathophysiology of certain metabolic diseases and of malignant cell growth and differentiation.
