Zhu D M, Tekle E, Chock P B, Huang C Y
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0340, USA.
Biochemistry. 1996 Jun 4;35(22):7214-23. doi: 10.1021/bi952471h.
The role of reversible phosphorylation in histamine-induced Ca2+ oscillations in HeLa cells has been investigated by using various activators and inhibitors of protein kinases and phosphatases. Electroporation was employed to introduce impermeable materials into single cells, which proved to be a useful and convenient tool. Of the kinases examined, cAMP-dependent kinase, protein kinase C, and calmodulin-dependent kinase II (CaMK II), only CaMK II was essential. When added during oscillations, both W-7, a calmodulin antagonist, and KN-62, a specific CaMK II inhibitor, caused one large Ca2+ spike before halting the process. Introduction of the Ca2+/calmodulin-independent catalytic domain of CaMK II into the cells forestalled their response to histamine. These results show that intracellular Ca2+ cannot oscillate when CaMK II is locked in either the inactive or the stimulated state. External Ca2+ electroporated into cells preloaded with the catalytic domains was quickly removed (but not when the cells were pretreated with the endoplasmic reticulum Ca(2+)-ATPase inhibitor, tapsigargin), indicating that the ATP-driven Ca2+ pump was somehow activated by CaMK II. Protein phosphatase inhibitors calyculin A and okadaic acid abolished ongoing oscillations and, when added at low concentrations, prolonged the interspike interval. Immunoprecipitation experiments with 32P(i)-labeled cells provided the first evidence that inositol 1,4,5-trisphosphate receptor (IP3R) was phosphorylated by CaMK II in vivo. The extent of phosphorylation was increased in the presence of histamine, significantly enhanced by calyculin A, and greatly reduced by W-7. Our observations are consistent with the concept that repetitive phosphorylation-dephosphorylation cycles regulating IP3R and Ca2+ pumps are a controlling factor for sustained Ca2+ oscillations in HeLa, and possibly other, cells.
通过使用蛋白激酶和磷酸酶的各种激活剂和抑制剂，研究了可逆磷酸化在组胺诱导的HeLa细胞Ca2+振荡中的作用。采用电穿孔法将不可渗透的物质导入单细胞，这被证明是一种有用且便捷的工具。在所检测的激酶中，环磷酸腺苷依赖性激酶、蛋白激酶C和钙调蛋白依赖性激酶II（CaMK II），只有CaMK II是必需的。在振荡过程中添加时，钙调蛋白拮抗剂W-7和特异性CaMK II抑制剂KN-62都会在终止该过程之前引起一个大的Ca2+峰值。将CaMK II的Ca2+/钙调蛋白非依赖性催化结构域导入细胞可预先阻止其对组胺的反应。这些结果表明，当CaMK II锁定在非活性或受刺激状态时，细胞内Ca2+无法振荡。电穿孔导入预先加载有催化结构域的细胞中的外部Ca2+被迅速清除（但当细胞用内质网Ca(2+)-ATP酶抑制剂毒胡萝卜素预处理时则不会），这表明ATP驱动的Ca2+泵以某种方式被CaMK II激活。蛋白磷酸酶抑制剂花萼海绵诱癌素A和冈田酸消除了正在进行的振荡，并且在低浓度添加时延长了峰间间隔。用32P(i)标记细胞进行的免疫沉淀实验首次证明，肌醇1,4,5-三磷酸受体（IP3R）在体内被CaMK II磷酸化。在组胺存在的情况下，磷酸化程度增加，花萼海绵诱癌素A显著增强，而W-7则大大降低。我们的观察结果与以下概念一致，即调节IP3R和Ca2+泵的重复磷酸化-去磷酸化循环是HeLa细胞以及可能其他细胞中持续Ca2+振荡的控制因素。
