Regulation of function and protein expression of noradrenaline transporter by protein kinases

在内源性表达NA转运蛋白的PC12细胞中，研究了钙调素相关蛋白激酶对去甲肾上腺素转运蛋白（NAT）功能的调控。1. 我们确定了大鼠NAT cDNA的完整序列，并发现了Ca^<2+>/钙调素依赖性（CaM）激酶II磷酸化的五个一致位点。用纯化的脑CaM激酶II.2磷酸化大鼠NAT胞内cooh末端区域的合成肽。转染GFP-NAT融合蛋白的PC12细胞质膜广泛的nat样免疫反应性或荧光在与KN-93孵育或缺乏Ca^<2+>时消失。3. 在过表达CaM激酶II α亚基的PC12细胞克隆细胞系中，与野生型细胞相比，[^3H]NA的摄取、NAT mRNA和蛋白的表达均显著增加。在CaM激酶II α-过表达的细胞系中，磷酸化的转录因子CREB的含量也增加了。长期给药可显著抑制n -93的摄取增加和mRNA表达。这些结果表明，Ca^<2+>/钙调素依赖性蛋白激酶可能通过直接磷酸化NAT的cooh末端结构域和/或通过刺激NAT向质膜的易位促进NA的摄取，并且CaM激酶II的激活可能会长期增强NAT的表达。

Regulation of noradrenaline transporter (NAT) function by calmodulin-related protein kinases was studied in PC12 cells, which endogenously express NA transporters. 1. We determined a full sequence of rat NAT cDNA and found five consensus sites for phosphorylation by Ca^<2+>/calmodulin-dependent (CaM) kinase II.A synthetic peptide whose sequence was contained in the intracellular COOH-terminal domain of rat NAT was phosphorylated by purified brain CaM kinase II.2. Extensive NAT-like immunoreactivity or fluorescence of transfected GFP-NAT fusion protein in plasma membrane of PC12 cells disappeared after incubation with KN-93 or in the absence of Ca^<2+>. 3. In clonal cell lines of PC12 cells which overexpressed CaM kinase II α subunit, [^3H]NA uptake as well as expression of NAT mRNA and protein was markedly increased compared with wild type cells. Content of phosphorylated CREB, an active form of a transcription factor was also increased in CaM kinase II α-overexpressed cell lines. The increased uptake and mRNA expression were significantly suppressed by chronic treatment of KN-93. These results suggest that NA uptake might be facilitated by Ca^<2+>/calmodulin-dependent protein kinases through direct phosphorylation of COOH-terminal domain of NAT and/or through stimulated translocation of NAT to plasma membrane, and that activation of CaM kinase II might chronically enhance expression of NAT.