Ser16 prevails over Thr17 phospholamban phosphorylation in the β-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 Ser(16) and Thr(17) Of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser(16) and Thr(17) with strikingly different half-maximal values (EC50 = 4.5 +/- 1.6 and 28.2 +/- 1.4 nmol/l, respectively). The phosphorylation of Ser(16) induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr(17) did not. Elevation of extracellular Ca2+ did not induce phosphorylation at Thr(17); only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr(17) increased eightfold without additional effects on relaxation rate. Thr(17) phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 mu mol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser(16) by cAMP-dependent protein kinase is the main regulator of beta-adrenergic-induced cardiac relaxation definitely preceding Thr(17) phosphorylation and 2) the beta-adrenergic-mediated phosphorylation of Thr(17) by Ca2+- calmodulin-dependent protein kinase required influx of Ca2+ through the L-type Ca2+ channel.