Molecular mechanisms of hormone action can be studied by determining their effects on specific phosphorylations. Our studies of insulin's action on the phosphorylation and de novo synthesis of fat cell proteins suggest new biochemical mechanisms to explain insulin's physiological actions. New techniques of SDS-slab acrylamide gel electrophoresis and autoradiography permit study of the effects of insulin, catecholamines, cyclic AMP and other agents on fat cell protein metabolism with increased sensitivity and specificity. We found that in the absence of adenylate cyclase stimulation, insulin stimulated the phosphorylation of soluble fraction phosphoprotein-2 (130,000 daltons) and of membrane associated phosphoprotein-6b (62,000 daltons). Epinephrine action enhances the phosphorylation of phosphoprotein-5 (65-69,000 daltons) and markedly increases the dephosphorylation of phosphoprotein-6b. The present proposal will: (a) identify and purify the protein kinase involved in insulin stimulated phosphoprotein phosphorylations; (b) determine the stimulation mechanism for the cyclic AMP independent protein kinase (direct or indirect via "second messengers"); (c) study the enzymology of the insulin stimulated protein phosphorylations; (d) study the reciprocal relationships between the effects of insulin and catecholamines on insulin and epinephrine protein phosphorylations; (e) will correlate these changes in protein phosphorylations with normal hormonal changes in physiology (lipolysis, glucose transport); (f) study the mechanisms for the epinephrine enhanced dephosphorylation of phosphoprotein-6b and of the insulin enhanced dephosphorylation of phosphoprotein-5 and (g) identify the function of the major phosphorylated proteins in fat cells. An understanding of the mechanism of insulin stimulated protein phosphorylations will describe unique intracellular circuits which may be critical in the propagation of insulin's action and may help explain some of the physiological changes which occur in human diseases such as diabetes mellitus, atheroscelerosis.