GROWTH FACTOR-INDUCED PHOSPHORYLATION AND ACTIVATION OF AORTIC SMOOTH-MUSCLE NA+/CA2+ EXCHANGER
GROWTH FACTOR-INDUCED PHOSPHORYLATION AND ACTIVATION OF AORTIC SMOOTH-MUSCLE NA+/CA2+ EXCHANGER
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DOI：
10.1074/jbc.270.15.8996
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发表时间：
1995-04-14
影响因子：
4.8
通讯作者：
SHIGEKAWA, M
中科院分区：
文献类型：
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作者：
IWAMOTO, T;WAKABAYASHI, S;SHIGEKAWA, M
虽然Na+/Ca2+交换器是可兴奋组织中主要的Ca2+挤出系统之一，但对其通过蛋白磷酸化的调控知之甚少。我们现在提出的证据表明，Na+/Ca2+交换器在静止和生长因子刺激的培养主动脉平滑肌细胞中被磷酸化。用特异性多克隆抗体免疫沉淀法从p -32标记的细胞中分离出Na+/Ca2+交换剂。在血小板衍生生长因子- ee （PDGF-BB）、α -凝血酶或phorbol 12-肉豆蔻酸13-醋酸酯（PMA）的作用下，交换器的磷酸化程度增加了1.7倍。然而，血管紧张素II并没有显著增强磷酸化。磷酸化的程度似乎与生长因子诱导的细胞1,2-二酰基甘油的增加有关。磷酸化交换器的胰蛋白酶肽图谱分析检测到至少4个磷酸肽（P1至P4），表明磷酸化发生在多个位点。PDGF-BB和PMA增加了相同磷酸肽的磷酸化（特别是P1）。在静止和受刺激的细胞中磷酸化的氨基酸都是丝氨酸残基。我们发现生长因子增强了Na+/Ca2+交换活性，并且生长因子诱导的磷酸化刺激与交换活性之间存在良好的相关性。pdgf - bb诱导的交换剂的诱导活化被PMA长期处理的细胞所消除。这些结果表明，在血管平滑肌细胞中，Na+/Ca2+交换器被蛋白激酶c依赖性磷酸化激活，以响应生长因子。
Although the Na+/Ca2+ exchanger is one of the major Ca2+ extrusion systems in excitable tissues, little is known about its regulation via protein phosphorylation. We now present evidence that the Na+/Ca2+ exchanger is phosphorylated in quiescent and growth factor-stimulated cultured aortic smooth muscle cells. The Na+/Ca2+ exchanger was isolated from P-32-labeled cells by immunoprecipitation with a specific polyclonal antibody. Phosphorylation of the exchanger was increased by up to 1.7-fold in response to platelet-derived growth factor-EE (PDGF-BB), alpha-thrombin, or phorbol 12-myristate 13-acetate (PMA). However, angiotensin II did not enhance the phosphorylation significantly. The extent of phosphorylation appeared to correlate with the growth factor-induced increase in cell 1,2-diacylglycerol. At least four phosphopeptides (P1 to P4) were detected by tryptic phosphopeptide map analysis of the phosphorylated exchanger, suggesting that phosphorylation occurred at multiple sites. PDGF-BB and PMA increased phosphorylation of the same phosphopeptides (in particular P1). Phosphorylated amino acids were exclusively serine residues in both quiescent and stimulated cells. We found that growth factors enhanced Na+/Ca2+ exchange activity and that there was a good correlation between the growth factor-induced stimulations tions of phosphorylation and exchange activity. PDGF-BB-induced induced activation of the exchanger was abolished by prior long treatment of cells with PMA. These results suggest that the Na+/Ca2+ exchanger is activated by protein kinase C-dependent phosphorylation in response to growth factors in vascular smooth muscle cells.