Abstract

In isolated, 32Pi-loaded, rat adipocytes, we have examined phosphorylation of the major cAMP-dependent protein kinase (A-kinase) substrate, a protein that appears to be associated with the lipid storage droplet and migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 65-67-kDa doublet. In control cells, a strong phosphorylation signal is detected as the (+/- cAMP) A-kinase activity ratio ranges from approximately 0.1 to approximately 0.3-0.4 with increasing isoproterenol concentrations. By contrast, insulin-treated cells exhibiting A-kinase activity ratios over the range of 0.1-0.25 contain less 32P in the 65-67-kDa protein than control cells exhibiting identical A-kinase activity ratios. At higher activity ratios (greater than 0.3), this reduction in phosphorylation of the 65-67-kDa protein by insulin disappears. It is concluded that insulin stimulates a phosphatase activity that acts on the 65-67-kDa protein. Insulin actions aside, these studies reveal two interesting phenomena. 1) Whereas elevated, steady-state A-kinase activities are established rapidly (1-2 min) upon isoproterenol stimulation, phosphorylation of the 65-67-kDa substrate proceeds through a burst, followed by a decline to a steady-state level by 10-12 min. An "adaptation" mechanism, providing for a constant response to a constant stimulus, may underlie this lack of parallelism between the time course of phosphorylation and A-kinase activity. 2) Removal of [32Pi] orthophosphate immediately before isoproterenol stimulation leads to a rapid (t approximately 10 min) loss in labeling of the 65-67-kDa protein, whereas the phosphorylation state of other phosphoproteins are not changed. These data suggest that elevation of A-kinase activity leads to a rapid exchange of external Pi with an ATP pool that is used by A-kinase.