Enhanced activation of cAMP-dependent protein kinase by rapid synthesis and degradation of cAMP.
Activation of cAMP-dependent protein kinase II by static and dynamic steady-state cAMP levels was studied by reconstituting an in vitro model system composed of hormone-sensitive adenylate cyclase, cyclic nucleotide phosphodiesterase, and cAMP-dependent protein kinase II. The rates of cAMP synthesis were regulated by incubating isolated membranes from AtT20 cells with various concentrations of forskolin. In the presence of 3-methylisobutylxanthine, the rate of protein kinase activation was proportional to the rate at which cAMP was synthesized, and there was a direct relationship between the degree of activation and the level of cAMP produced. The activation profiles of protein kinase generated in the presence of exogenous cAMP or cAMP produced by activation of adenylate cyclase in the absence of cAMP degradation were indistinguishable. Dynamic steady-state levels of cAMP were achieved by incubating the membranes with forskolin in the presence of purified cyclic nucleotide phosphodiesterase. Under these conditions, the apparent activation constant of protein kinase II for cAMP was reduced by 65-75%. This increased sensitivity to activation by cAMP was seen when phosphotransferase activity was measured directly in reaction mixtures containing membranes, protein kinase, and histone H2B or when regulatory and catalytic subunits were first separated by immunoprecipitation of holoenzyme and regulatory subunits with specific anti-serum. Our results are consistent with the hypothesis that rapid cAMP turnover may function as a mechanism for amplifying hormonal signals which use the cAMP-dependent protein kinase system.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D011494:Protein Kinases / Q000378:metabolism*
D005576:Colforsin; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D015056:1-Methyl-3-isobutylxanthine