Activation and phosphorylation of the 'dense-vesicle' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.
Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.
Kilgour
E
E
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Anderson
N G
NG
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation; D008099:Liver / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases