Insulin has profound effects on a number of diverse cellular processes. It produces these """"""""immediate and delayed"""""""" effects subsequent to its interaction with a specific receptor on the cell membrane. The coupling mechanism(s) between the insulin-receptor interaction and the metabolic effects produced by insulin remains unclear. Recently, it has been found that ATP-citrate lyase is phosphorylated in response to the action of insulin, glucagon, and Beta-agonist. In addition it is known that ATP-citrate lyase is phosphorylated at a minimum of three amino acid residues within two different peptide sequences designated as peptide a and b. Our studies show that insulin decreases the phosphorylation of peptide b and increases the phosphorylation of peptide a. The decrease in peptide b phosphorylation is due to the dephosphorylation of threonine and the increase in peptide a phosphorylation is due to an increase in serine phosphorylation. The specific protein kinases and phosphatases that control these phosphorylations are unknown. This proposal outlines steps to unravel the relationship of the insulin directed peptide b phosphothreonine dephosphorylation and the specific phosphatase involved and the insulin directed phosphorylation of peptide a. Specifically we will (i) determine the amino acid sequence of peptide b and of peptide B the analogous sequence phosphorylated in vitro. (ii) fully describe the effects of insulin and isoproterenol on peptide b phosphorylation and dephosphorylation and (iii) define the role of and control for the protein kinase and phosphatases specific for these phosphorylations. Detailed investigation of the relationship of ATP-citrate lyase kinase to the control of phosphatase activity will undertaken. Isolation and purification of a specific phosphothreonyl phosphatase will be rigorously pursued. Our recent demonstration of a new insulin directed increase in the phosphorylation of a low molecular weight protein, phosphoprotein """"""""m"""""""" with properties of a modulator protein will be integrated into our scheme of the mechanism of insulin action. As these studies on the insulin-directed control of ATP-citrate lyase phosphorylation and dephosphorylation take place analogous studies on the """"""""multi-site phosphorylations"""""""", """"""""second site regulation"""""""" and hormone control of acetyl CoA carboxylase phosphorylation-dephosphorylation will be undertaken.
