Chu Guoxiang, Kranias Evangelia G
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert B. Sabin Way, Cincinnati, OH 45267-0575, USA.
Basic Res Cardiol. 2002;97 Suppl 1:I43-8. doi: 10.1007/s003950200028.
Dephosphorylated phospholamban (PLB) is an inhibitor of the affinity of the sarcoplasmic reticulum (SR) Ca2+ pump (SERCA2) for Ca2+. Phosphorylation of PLB relieves its inhibitory effects on SERCA2, with subsequent acceleration of Ca2+ transport into the SR lumen, which has been suggested to underlie the positive inotropic and lusitropic actions of beta-adrenergic agonists in the mammalian heart. PLB can be phosphorylated at Ser16 by cAMP-dependent protein kinase (PKA) and Thr17 by Ca2+-calmodulin-dependent protein kinase (CaMKII) during beta-agonist stimulation. However, the interrelationship and relative contribution of dual site phosphorylation to the cardiac stimulatory effects are not clear. The recent availability of the PLB knockout mouse, in combination with mutagenesis and transgenic technologies, have provided excellent model systems for expression of each of the phosphorylation site-specific PLB mutants in the heart and elucidation of the functional interplay between PKA- and CaMKII-dependent pathways of PLB phosphorylation. Transgenic mice expressing similar levels of the wild-type, S16A, or T17A mutant PLB in the null background were generated and they were characterized in parallel. Our results indicate that 1) reinsertion of PLB into the knockout mouse heart reverses the hyperdynamic cardiac function associated with PLB deficiency, 2) phosphorylation of Ser16 in PLB is sufficient to mediate its maximal cardiac contractile responses to beta-adrenergic stimulation, and 3) Ser16 phosphorylation is a prerequisite for Thr17 phosphorylation in vivo during beta-agonist stimulation, but Thr17 can be phosphorylated independently of Ser16 in vitro. Thus, these studies revealed novel insights into the interdependence and physiological significance of PKA (Ser16) and CaMKII (Thr17) pathways of PLB phosphorylation during beta-adrenergic stimulation in the mammalian heart.
去磷酸化的受磷蛋白（PLB）是肌浆网（SR）Ca2+泵（SERCA2）对Ca2+亲和力的抑制剂。PLB的磷酸化可解除其对SERCA2的抑制作用，随后加速Ca2+转运至SR腔，这被认为是β-肾上腺素能激动剂在哺乳动物心脏中产生正性肌力和变时性作用的基础。在β-激动剂刺激过程中，PLB可在Ser16位点被环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）磷酸化，在Thr17位点被Ca2+ - 钙调蛋白依赖性蛋白激酶（CaMKII）磷酸化。然而，双位点磷酸化之间的相互关系及其对心脏刺激作用的相对贡献尚不清楚。最近，PLB基因敲除小鼠的出现，结合诱变和转基因技术，为在心脏中表达每个磷酸化位点特异性的PLB突变体以及阐明PKA和CaMKII依赖性PLB磷酸化途径之间的功能相互作用提供了出色的模型系统。我们构建了在基因敲除背景下表达相似水平的野生型、S16A或T17A突变体PLB的转基因小鼠，并对它们进行了平行表征。我们的结果表明：1）将PLB重新导入基因敲除小鼠心脏可逆转与PLB缺乏相关的心脏高动力功能；2）PLB中Ser16的磷酸化足以介导其对β-肾上腺素能刺激的最大心脏收缩反应；3）在β-激动剂刺激过程中，Ser16磷酸化是体内Thr17磷酸化的先决条件，但在体外Thr17可独立于Ser16被磷酸化。因此，这些研究揭示了在哺乳动物心脏β-肾上腺素能刺激过程中，PKA（Ser16）和CaMKII（Thr17）途径的PLB磷酸化之间的相互依赖性和生理意义的新见解。
