Iwamoto T, Pan Y, Wakabayashi S, Imagawa T, Yamanaka H I, Shigekawa M
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan.
J Biol Chem. 1996 Jun 7;271(23):13609-15. doi: 10.1074/jbc.271.23.13609.
The cardiac Na+/Ca2+ exchanger (NCX1) plays a major role in the extrusion of Ca2+ from cardiomyocytes. We studied the role of protein phosphorylation in the regulation of cardiac NCX1 using CCL39 stably overexpressing the canine cardiac NCX1 and rat neonatal cardiomyocytes. In both cell types, the NCX1 protein immunoprecipitated with a chicken anti-NCX1 antibody exhibited a significant basal phosphorylation that was further enhanced by treatment with endothelin-1, acidic fibroblast growth factor, phorbol 12-myristate 13-acetate, or okadaic acid. In contrast, calphostin C, K252a, or EGTA inhibited the phosphorylation. The phosphorylation occurred on two major tryptic phosphopeptides (P1 and P2) exclusively on serine residues. Evidence is presented suggesting that P2 was derived from an N-terminal half (amino acids 240-475) of the central cytoplasmic domain of NCX1 and was phosphorylated directly by protein kinase C (PKC). The agents that increased NCX1 phosphorylation significantly enhanced both the forward and reverse modes of Na+/Ca2+ exchange. This exchange activation exhibited a very good correlation with the NCX1 phosphorylation. In NCX1-transfected cells, PKC down-regulation following prolonged exposure to phorbol 12-myristate 13-acetate abolished the acidic fibroblast growth factor-induced activation of exchange activity. On the other hand, cell ATP depletion reduced the exchange activity and abolished the effects of the above agents on exchange activity. These results indicate that the cardiac NCX1 is up-regulated by PKC-catalyzed phosphorylation. The cardiac NCX1 thus could play an important role in the previously reported negative inotropic actions of phorbol esters and other PKC-activating agents.
心脏钠钙交换体（NCX1）在心肌细胞排出钙离子的过程中起主要作用。我们使用稳定过表达犬心脏NCX1的CCL39细胞和大鼠新生心肌细胞，研究了蛋白质磷酸化在心脏NCX1调节中的作用。在这两种细胞类型中，用鸡抗NCX1抗体免疫沉淀的NCX1蛋白均表现出显著的基础磷酸化，用内皮素-1、酸性成纤维细胞生长因子、佛波酯12-肉豆蔻酸13-乙酸酯或冈田酸处理后，这种磷酸化进一步增强。相反，钙调蛋白C、K252a或乙二醇双四乙酸抑制了磷酸化。磷酸化发生在两个主要的胰蛋白酶磷酸肽（P1和P2）上，且仅发生在丝氨酸残基上。有证据表明，P2来源于NCX1中央胞质结构域的N端半段（氨基酸240 - 475），并由蛋白激酶C（PKC）直接磷酸化。增加NCX1磷酸化的试剂显著增强了钠钙交换的正向和反向模式。这种交换激活与NCX1磷酸化表现出非常好的相关性。在NCX1转染的细胞中，长时间暴露于佛波酯12-肉豆蔻酸13-乙酸酯后PKC下调，消除了酸性成纤维细胞生长因子诱导的交换活性激活。另一方面，细胞ATP耗竭降低了交换活性，并消除了上述试剂对交换活性的影响。这些结果表明，心脏NCX1通过PKC催化的磷酸化上调。因此，心脏NCX1可能在先前报道的佛波酯和其他PKC激活剂的负性肌力作用中起重要作用。
