Ser(16) prevails over Thr(17) 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 b etween Ca2+ and cAMP pathways, we have investigated the beta-adrenergic-ind uced phosphorylation of Ser(16) and Thr(17) Of phospholamban in perfused ra t hearts using antibodies recognizing phospholamban phosphorylated at eithe r 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(1 6) induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine corr elated to increased cardiac relaxation (r = 0.96), whereas phosphorylation of Thr(17) did not. Elevation of extracellular Ca2+ did not induce phosphor ylation at Thr(17); only in the presence of a submaximal dose of isoprotere nol, phosphorylation at Thr(17) increased eightfold without additional effe cts on relaxation rate. Thr(17) phosphorylation was partially affected by r yanodine and was completely abolished in the presence of 1 mu mol/l verapam il or nifedipine. The data indicate that 1) phosphorylation of phospholamba n at Ser(16) by cAMP-dependent protein kinase is the main regulator of beta -adrenergic-induced cardiac relaxation definitely preceding Thr(17) phospho rylation and 2) the beta-adrenergic-mediated phosphorylation of Thr(17) by Ca2+- calmodulin-dependent protein kinase required influx of Ca2+ through t he L-type Ca2+ channel.