Luo W, Chu G, Sato Y, Zhou Z, Kadambi V J, Kranias E G
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, USA.
J Biol Chem. 1998 Feb 20;273(8):4734-9. doi: 10.1074/jbc.273.8.4734.
Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 --> Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the beta-agonist stimulatory responses in the mammalian heart.
受磷蛋白是肌浆网Ca2+-ATP酶活性和心肌收缩性的关键调节因子。在异丙肾上腺素刺激过程中，受磷蛋白的丝氨酸16和苏氨酸17位点均发生磷酸化。为了确定受磷蛋白双位点磷酸化的生理意义，我们构建了在受磷蛋白基因敲除小鼠的心脏区域表达野生型或丝氨酸16突变为丙氨酸的突变型受磷蛋白的转基因模型。对突变型或野生型受磷蛋白水平相似的转基因品系进行了平行研究。Langendorff灌注表明，敲除小鼠的基础心脏高动力功能通过重新导入野生型或突变型受磷蛋白得到了同等程度的逆转。然而，与野生型心脏相比，异丙肾上腺素刺激与突变型受磷蛋白收缩参数的反应低得多有关。这些减弱的反应是由于在32P标记灌注实验中评估的突变型受磷蛋白缺乏磷酸化。体内受磷蛋白缺乏磷酸化并非由于丝氨酸16突变为丙氨酸，因为突变型受磷蛋白形式在体外可作为钙调蛋白依赖性蛋白激酶的底物。这些发现表明，丝氨酸16磷酸化是受磷蛋白苏氨酸17磷酸化的先决条件，防止磷酸丝氨酸形成会导致哺乳动物心脏中β-激动剂刺激反应减弱。
