Control of endogenous phosphorylation of the major cAMP-dependent protein kinase substrate in adipocytes by insulin and beta-adrenergic stimulation.
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.
Egan
J J
JJ
Membrane Regulation Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Greenberg
A S
AS
Chang
M K
MK
Londos
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D002478:Cells, Cultured; D007328:Insulin / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism
D007328:Insulin; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011943:Receptors, Adrenergic, beta; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D007545:Isoproterenol