Phosphorylation of phospholamban at threonine-17 in the absence and presence of β-adrenergic stimulation in neonatal rat cardiomyocytes

The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca2+ signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca2+ channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca2+ stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 muM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 muM S(-)-Bay K8644. Interestingly, S(-)-Bay K8644 alone was also able to induce Thr17 phosphorylation in a dose- and time-dependent fashion. Ryanodine (1.0 muM) reduced both the isoproterenol (0.1 muM) and S(-)-Bay K8644-(1 muM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 muM) diminished the S(-)-Bay K8644 and isoproterenol-associated Thr17 phosphorylation by 53.5 +/- 6.3% and 92.5 +/- 11.1%, respectively. Ser16 phosphorylation was not affected under these conditions. KN-93 reduced the Thr17 phosphorylation by S(-)-Bay K8644 and isoproterenol to levels of 1.1 +/- 0.3% and 8.6 +/- 2.1%, respectively, However, the effect of KN-93 was attenuated (47.8 +/- 3.6%) in isoproterenol prestimulated cells, Protein phosphatase inhibition by okadaic acid increased exclusively the Ser16 phosphorylation. rn summary bur results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca2+ at the level of CaMKII-mediated phospholamban phosphorylation in neonatal rat cardiomyocytes. We report conditions which exclusively produce Thr17 or Ser16 phosphorylation. We postulate that Ca2+ transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban. (C) 2000 Academic Press.