Iwamoto T, Pan Y, Nakamura T Y, Wakabayashi S, Shigekawa M
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.
Biochemistry. 1998 Dec 8;37(49):17230-8. doi: 10.1021/bi981521q.
We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.
我们比较了在几乎没有内源性活性的CCL39成纤维细胞中表达的三种哺乳动物钠钙交换体异构体（NCX1 - NCX3）的磷酸化依赖性调节。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）或血小板衍生生长因子 - BB可显著增强表达NCX1或NCX3的细胞对钠离子依赖的45钙离子摄取，但对表达NCX2的细胞无此作用，而用钙磷蛋白C预处理细胞或预先长时间暴露于PMA可消除这种增强作用。这表明NCX1或NCX3，而非NCX2，受到涉及蛋白激酶C（PKC）的信号通路刺激。使用[32P]正磷酸盐标记细胞的免疫沉淀实验表明，在未刺激的细胞中，NCX2和NCX3蛋白的磷酸化程度远低于NCX1蛋白，并且用PKC激活剂处理后磷酸化程度并未增加，尽管NCX1的磷酸化显著增强。通过定点诱变，我们确定在PMA刺激的细胞中，NCX1蛋白的三个磷酸化位点为Ser - 249、Ser - 250和Ser - 357，其中Ser - 250主要被磷酸化。我们发现，将这些丝氨酸残基替换为丙氨酸的NCX1突变体对PMA仍保持正常反应。相反，缺失大的中央胞质环的NCX1或NCX3突变体失去了对PMA的反应性。这些结果表明，PKC对NCX1或NCX3的调节需要中央胞质环，但不需要交换体的直接磷酸化。
