The insulin- and isoproterenol-activated, cGMP-inhibited, low Km cAMP phosphodiesterase (PDE) has been isolated and purified from rat and bovine adipose tissues and utilized for the production of specific polyclonal rabbit antibodies (anti-cAMP PDE). Incubation of intact rat fat cells with maximally effective concentrations of insulin (0.1 nM) or isoproterenol (100 nM) increased particulate (100,000 g) cAMP PDE activity by approximately 50 and 100%, respectively. Under these conditions, in 32P-labeled rat adipocytes both hormones induced [32P]phosphoserine phosphorylation of a predominant 135 kDa and a minor 44 kDa particulate protein immunoprecipitated by anti-cAMP PDE. Little or no phosphorylation was detected in the absence of hormones. The two phosphoproteins were identified as or closely related to cAMP PDE (with the 44 kDa likely a proteolytic fragment) by the following findings: 1) Immunoprecipitation of the 135 kDa phosphoprotein paralleled loss of enzyme activity in the super- natant, and preincubation of the anti-cAMP PDE with pure rat or bovine PDE selectively blocked the immunoprecipitation of the phospho-proteins. 2) These proteins copurified with cAMP PDE activity through DEAE-Sephacel chromatography and were obtained by highly selective affinity chromatography on CIT-agarose, the agarose-immobilized isothiocyante derivative of cilostamide, a specific and potent inhibitor of the particulate enzyme. These results indicate that catecholamines and insulin induce phosphorylation of cAMP PDE in fat cells through activation of cAMP-dependent protein kinase and, presumably, an insulin-sensitive serine protein kinase, respectively, and suggest that these phosphorylations are related to activation of the enzyme.
