Hagemann D, Kuschel M, Kuramochi T, Zhu W, Cheng H, Xiao R P
Laboratory of Cardiovascular Science, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
J Biol Chem. 2000 Jul 21;275(29):22532-6. doi: 10.1074/jbc.C000253200.
Both Ser(16) and Thr(17) of phospholamban (PLB) are phosphorylated, respectively, by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). PLB phosphorylation relieves cardiac sarcoplasmic reticulum Ca(2+) pump from inhibition by PLB. Previous studies have suggested that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) phosphorylation by CaMKII and is essential to the relaxant effect of beta-adrenergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electrical field stimulation (0. 1-3 Hz) or both. A beta(1)-adrenergic agonist, norepinephrine (10(-9)-10(-6) m), in the presence of an alpha(1)-adrenergic antagonist, prazosin (10(-6) m), selectively increases the PKA-dependent phosphorylation of PLB at Ser(16) in quiescent myocytes. In contrast, electrical pacing induces an opposite phosphorylation pattern, selectively enhancing the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic effect on phosphorylation of Thr(17) but not Ser(16). Frequency-dependent Thr(17) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened contractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr(17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change of heart rate.
受磷蛋白（PLB）的丝氨酸（Ser）16和苏氨酸（Thr）17分别被环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）和钙（Ca2+）/钙调蛋白依赖性蛋白激酶II（CaMKII）磷酸化。PLB磷酸化可解除心肌肌浆网Ca2+泵受PLB的抑制。以往研究表明，PKA对Ser16的磷酸化是CaMKII对Thr17磷酸化的前提条件，且对β-肾上腺素能刺激的舒张作用至关重要。为确定Thr17的PLB磷酸化的作用，我们研究了分离的成年大鼠心肌细胞中PLB的双位点磷酸化情况，以响应β1-肾上腺素能刺激或电场刺激（0.1 - 3Hz）或两者同时刺激。在α1-肾上腺素能拮抗剂哌唑嗪（10-6m）存在的情况下，β1-肾上腺素能激动剂去甲肾上腺素（10-9 - 10-6m）可选择性增加静息心肌细胞中PKA依赖性的PLB在Ser16位点的磷酸化。相反，电起搏诱导出相反的磷酸化模式，以频率依赖性方式选择性增强CaMKII介导的Thr17的PLB磷酸化。当电刺激（2Hz）和β1-肾上腺素能刺激联合时，可导致PLB的双磷酸化，并对Thr17而非Ser16的磷酸化产生协同作用。频率依赖性的Thr17磷酸化与细胞收缩的50%舒张时间（t50）的缩短密切相关，这独立于Ser16磷酸化的舒张作用，但与之相加，导致在高刺激频率下收缩舒张加快。因此，我们得出结论，在完整的心肌细胞中，Thr17的PLB磷酸化在没有先前Ser16磷酸化的情况下发生，且频率依赖性的Thr17的PLB磷酸化可能为心肌细胞适应心率突然变化提供一种内在机制。
