Xu A, Hawkins C, Narayanan N
Department of Physiology, University of Western Ontario, London, Canada.
J Biol Chem. 1993 Apr 25;268(12):8394-7.
It is well known that phosphorylation of the membrane protein phospholamban by cAMP-dependent or Ca2+/calmodulin-dependent protein kinase results in the activation of the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR); such enzyme activation is thought to be due to the disruption of an inhibitory interaction of non-phosphorylated phospholamban with the ATPase. We describe here a novel mechanism for the regulation of the ATPase through direct phosphorylation of this enzyme by a Ca2+/calmodulin-dependent protein kinase (CaM kinase) associated with the SR membrane. It is shown that incubation of cardiac SR in the presence of Ca2+ and calmodulin results in the phosphorylation of the ATPase in addition to the previously recognized substrates of CaM kinase, viz. phospholamban and Ca2+ channel. The phosphorylated amino acid in the ATPase has been identified as serine. Phosphorylation of the membrane-bound ATPase is stimulated by exogenous CaM kinase. Furthermore, ATPase purified from cardiac SR is phosphorylated by exogenous CaM kinase and the phosphorylated enzyme displays 2-fold increase in catalytic activity without any appreciable change in its Ca2+ sensitivity. Thus, direct phosphorylation of the Ca(2+)-pumping ATPase by CaM kinase can stimulate its enzymatic activity and, therefore, Ca2+ transport function.
众所周知，环磷酸腺苷（cAMP）依赖性蛋白激酶或钙离子/钙调蛋白依赖性蛋白激酶对膜蛋白受磷蛋白的磷酸化作用会导致心肌肌浆网（SR）的钙离子泵ATP酶激活；这种酶激活被认为是由于未磷酸化的受磷蛋白与ATP酶之间的抑制性相互作用被破坏所致。我们在此描述了一种通过与SR膜相关的钙离子/钙调蛋白依赖性蛋白激酶（CaM激酶）对该酶进行直接磷酸化来调节ATP酶的新机制。结果表明，在钙离子和钙调蛋白存在的情况下孵育心肌SR，除了之前已识别的CaM激酶底物，即受磷蛋白和钙离子通道外，还会导致ATP酶的磷酸化。ATP酶中被磷酸化的氨基酸已被鉴定为丝氨酸。膜结合ATP酶的磷酸化受到外源性CaM激酶的刺激。此外，从心肌SR中纯化的ATP酶被外源性CaM激酶磷酸化，磷酸化后的酶催化活性提高了2倍，而其钙离子敏感性没有明显变化。因此，CaM激酶对钙离子泵ATP酶的直接磷酸化可刺激其酶活性，进而刺激钙离子转运功能。
