Bartel S, Vetter D, Schlegel W P, Wallukat G, Krause E G, Karczewski P
Max Delbrück Center for Molecular Medicine, Berlin, 13125, Germany.
J Mol Cell Cardiol. 2000 Dec;32(12):2173-85. doi: 10.1006/jmcc.2000.1243.
The site-specific phospholamban phosphorylation was studied with respect to the interplay of cAMP- and Ca(2+)signaling in neonatal rat cardiomyocytes. To elucidate the signal pathway(s) for the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII) we studied Thr17 phosphorylation of phospholamban in dependence of Ca(2+)channel activation by S(-)-Bay K8644 and in dependence of the depletion of the sarcoplasmic reticulum Ca(2+)stores by ryanodine or thapsigargin in the absence or presence of beta -adrenergic stimulation. The isoproterenol (0.1 microM)-induced Thr17 phosphorylation was potentiated 2.5-fold in presence of 1 microM 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 microM) reduced both the isoproterenol (0.1 microM) and S(-)-Bay K8644-(1 microM) mediated Thr17 phosphorylation by about 90%. Thapsigargin (1 microM) 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. In summary, our results reflect a cross-talk between beta -adrenoceptor stimulation and intracellular Ca(2+)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 Ca(2+)transport systems of the sarcoplasmic reticulum are critical determinants for the activation of CaMKII that catalyzes phosphorylation of phospholamban.
针对新生大鼠心肌细胞中cAMP和Ca(2+)信号的相互作用，研究了位点特异性的受磷蛋白磷酸化。为了阐明激活Ca(2+)/钙调蛋白依赖性蛋白激酶（CaMKII）的信号通路，我们研究了在有无β-肾上腺素能刺激的情况下，S(-)-Bay K8644激活Ca(2+)通道以及雷诺丁或毒胡萝卜素耗尽肌浆网Ca(2+)储存时受磷蛋白的苏氨酸17磷酸化情况。在存在1μM S(-)-Bay K8644的情况下，异丙肾上腺素（0.1μM）诱导的苏氨酸17磷酸化增强了2.5倍。有趣的是，单独的S(-)-Bay K8644也能够以剂量和时间依赖性方式诱导苏氨酸17磷酸化。雷诺丁（1.0μM）使异丙肾上腺素（0.1μM）和S(-)-Bay K8644（1μM）介导的苏氨酸17磷酸化均降低了约90%。毒胡萝卜素（1μM）使S(-)-Bay K8644和异丙肾上腺素相关的苏氨酸17磷酸化分别降低了53.5±6.3%和92.5±11.1%。在这些条件下，丝氨酸16磷酸化不受影响。KN-93将S(-)-Bay K8644和异丙肾上腺素诱导的苏氨酸17磷酸化分别降低到1.1±0.3%和8.6±2.1%的水平。然而，在异丙肾上腺素预刺激的细胞中，KN-93的作用减弱（47.8±3.6%）。冈田酸抑制蛋白磷酸酶仅增加了丝氨酸16磷酸化。总之，我们的结果反映了新生大鼠心肌细胞中β-肾上腺素能受体刺激与细胞内Ca(2+)在CaMKII介导的受磷蛋白磷酸化水平上的相互作用。我们报告了专门产生苏氨酸17或丝氨酸16磷酸化的条件。我们推测肌浆网的Ca(2+)转运系统是催化受磷蛋白磷酸化的CaMKII激活的关键决定因素。
