The objective of this study is to increase our understanding of the mechanisms by which hormones that 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 and inactivation 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. Experiments measuring the reassociation of R and C subunits subsequent to removal of the hormone will also be performed so that we can determine the rates of protein kinase inactivation in intact cells. Studies to determine the effects of phosphorylation on the binding of calmodulin to brain fodrin will also be carried out. These studies will use a solid phase binding assay on nitrocellulose filters to measure the binding of 125I-labeled calmodulin to purified fodrin. Isolated fodrin subunits will be separated by SDS-polyacrylamide gel electrophoresis and transfected to nitrocellulose filters by the Western blot procedure. After blocking of non-specific site the filters will then be incubated in buffer containing 125I-labeled calmodulin and the amount of calmodulin bound will be determine by liquid scintillation spectrometry. Binding of calmodulin to native brain fodrin will also be investigated using a solid phase binding assay. The procedures are the same as for separated subunits except that the samples are spotted directly on the filters. The effects of phosphorylation of fodrin by cAMP-dependent protein kinase and calmodulin-dependent protein kinase on the binding of 125I-labeled calmodulin will be evaluated. These studies should enhance our knowledge of the physiological role of protein kinases in mediating hormonal responses and give us a better understanding of the pathophysiology of certain metabolic diseases.
