Evidence that insulin and isoprenaline activate the cGMP-inhibited low-Km cAMP phosphodiesterase in rat fat cells by phosphorylation.
Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (less than 10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.
Degerman
E
E
Department of Medical and Psysiological Chemistry, University of Lund, Sweden.
Smith
C J
CJ
Tornqvist
H
H
Vasta
V
V
Belfrage
P
P
Manganiello
V C
VC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000273:Adipose Tissue / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D007545:Isoproterenol