Kuo T H, Wang K K, Carlock L, Diglio C, Tsang W
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 1991 Feb 5;266(4):2520-5.
Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.
细胞中质膜Ca2+泵的调节对于维持钙稳态至关重要。由于已知蛋白激酶C通过直接磷酸化或诱导细胞蛋白的基因表达来调节其功能，我们研究了蛋白激酶C在质膜Ca2+泵调节中的可能作用。通过免疫沉淀从在不存在或存在佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA，蛋白激酶C的激活剂）的情况下培养的[32P]正磷酸盐标记的大鼠主动脉内皮细胞中分离Ca2+泵。用PMA处理细胞导致5分钟内磷酸化水平迅速增加（1.3倍），3小时后进一步增加至2.9倍。长时间的PMA处理还诱导了Ca2+泵mRNA的积累，随后泵蛋白水平增加。泵mRNA诱导的峰值水平出现在4小时，比未用PMA处理的对照培养物高8 - 20倍。Ca2+泵蛋白积累的速率较慢，6小时后达到最大值3.5倍。蛋白激酶C抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪和蛋白激酶C的下调抑制了泵mRNA的诱导。无活性的佛波酯4α-佛波二癸酸酯也未能模拟PMA的作用。这些结果表明Ca2+泵表达的诱导是由蛋白激酶C依赖性机制介导的。此外，由于当环己酰亚胺和PMA一起添加时Ca2+泵mRNA的诱导被阻断，这表明需要新合成的蛋白因子来产生mRNA诱导。我们的结果表明蛋白激酶C参与内皮细胞中Ca2+泵的调节。在蛋白水平上，它通过磷酸化修饰Ca2+泵，在基因水平上，它刺激其mRNA的表达，从而增加泵蛋白的量。
