Roles of the AMP-activated and cyclic-AMP-dependent protein kinases in the adrenaline-induced inactivation of acetyl-CoA carboxylase in rat adipocytes.
1. In isolated rat adipocytes, acetyl-CoA carboxylase is inactivated by treatment of the cells with adrenaline or the beta-agonist isoproterenol, but not by the alpha-agonist phenylephrine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30-40% increase in the labelling of enzyme isolated from 32P-labelled cells. 2. Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser-Ser77-Met-Ser79-Gly-Leu-His-Leu-Val-Lys, containing Ser-77 (phosphorylated by cyclic-AMP-dependent protein kinase) and Ser-79 (phosphorylated by the AMP-activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser-79 in both basal and hormone- or agonist-stimulated cells. Treatment of adipocytes with various agents which activate cyclic-AMP-dependent protein kinase by receptor-independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl-CoA carboxylase and increased phosphorylation at Ser-79. 3. The (Rp)-[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic-AMP-dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl-CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic-AMP-dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl-CoA carboxylase. 4. As shown by okadaic acid inhibition, greater than 95% of the acetyl-CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase-2A, with less than 5% attributable to protein phosphatase-1. Inhibition of protein phosphatase-1 via phosphorylation of inhibitor-1 is therefore unlikely to be the mechanism by which cyclic-AMP-dependent protein kinase indirectly increases phosphorylation of acetyl-CoA carboxylase. Various other potential mechanisms are discussed.
Biochemistry Department, The University, Dundee, Scotland.
T A
TA
Haystead
F
F
Moore
P
P
Cohen
D G
DG
Hardie
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors*; D000273:Adipose Tissue / Q000187:drug effects / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology*; D004837:Epinephrine / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012694:Serine
D005576:Colforsin; D012694:Serine; D000242:Cyclic AMP; D011494:Protein Kinases; D008025:Ligases; D000103:Acetyl-CoA Carboxylase; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine; D004837:Epinephrine