Autophosphorylation of rat liver type II cAMP-dependent protein kinase.
A monoclonal antibody was prepared against the regulatory subunit (RII) of rat liver type II cAMP-dependent protein kinase. Autophosphorylated and nonphosphorylated RII in extracts from rat liver or hepatocytes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and quantified by immunoblot analysis with this antibody. Under basal conditions, 90% of hepatocyte RII was in the phosphorylated form. Incubating hepatocytes with 8-bromo-cAMP and a phosphodiesterase inhibitor resulted in activation of cAMP-dependent protein kinase and glycogenolysis but did not affect phospho RII levels. RII phosphorylation was also unaffected by the inclusion of sufficient insulin to cause a decrease in cAMP-dependent protein kinase activity and glycogenolysis. The results indicate that unlike other cell types, dissociation of rat hepatocyte type II cAMP-dependent protein kinase does not result in dephosphorylation of RII. The biochemical basis for the apparent lack of RII dephosphorylation in intact hepatocytes was examined by comparison with smooth muscle where RII is rapidly dephosphorylated. Rat liver extract contained 4-fold less RII and had an 80-fold lower rate of dephosphorylation of endogenous RII compared to bovine smooth muscle extract. The differences in the rates of RII dephosphorylation in tissue extracts were not observed using purified RII from either tissue. These data suggested that the slow rate of RII dephosphorylation in rat hepatocytes is due to a difference in the susceptibility of endogenous rat liver RII to dephosphorylation rather than a difference in phosphatase activity.
Scott
C W
CW
Department of Pharmacology, Southwestern Graduate School, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Mumby
M C
MC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D012368:4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; D000242:Cyclic AMP; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D005947:Glucose