Ser16 prevails over Thr17 phospholamban phosphorylation in the beta-adrenergic regulation of cardiac relaxation.
Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the beta-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 +/- 1.6 and 28. 2 +/- 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+ did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 micromol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of beta-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the beta-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+ through the L-type Ca2+ channel.
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007476:Ionophores / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology; D009924:Organ Culture Techniques; D010766:Phosphorylation; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism* / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D000099211:Phospholamban