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

蛋白激酶对去甲肾上腺素转运蛋白功能和蛋白表达的调节

• 批准号: 11680763
• 负责人: KIUCHI Yuji
• 金额: $ 0.83万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680763/
• 关键词: Noradrenaline transporter; Noradrenaline reuptake; Ca^<2+>; calmodulin-dependent kinase II; Protein phosphorylation; Intracellular translocation; Regulation of expression; PC12 cells; Green fluorescent protein (GFP); 遺伝子発現; トランスロケーション

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.

在内源性表达去甲肾上腺素转运体的PC12细胞中，研究了钙调蛋白相关蛋白激酶对去甲肾上腺素转运体(NAT)功能的调节。1.测定了大鼠NAT基因的全长序列，发现了5个与Ca~(2+)/CaM依赖(CaM)依赖的(CaM)激酶II共同的磷酸化位点。2.用纯化的脑CaM激酶II磷酸化一个合成肽，该合成肽的序列位于大鼠NAT的COOH末端。经KN-93或无Ca~(2+)及Gt~(2+)处理后，PC12细胞质膜上广泛存在的GFP-NAT融合蛋白的NAT样免疫反应或荧光消失。3.与野生型细胞相比，高表达CaM激酶IIα亚基的克隆细胞对[~(3 H)]NA的摄取以及NAT基因和蛋白的表达均显著增加。在高表达的细胞系中，转录因子的活性形式--磷酸化的CREB的含量也增加。在α过表达的细胞系中，磷酸化的CREB的含量也增加。慢性应用KN-93可显著抑制KN-93的摄取增加和mRNA表达的增加。这些结果表明，钙/钙调素依赖的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转位来促进NA的摄取，而CaM激酶II的激活可能长期促进NAT的表达。

项目成果

Kubota N., et al.: "Regulation of serotonin transporter gene expression in human glial cells by growth factors."Eur.J.Pharmacol.. (in press).

Kubota N. 等人：“生长因子调节人神经胶质细胞中的血清素转运蛋白基因表达。”Eur.J.Pharmacol..（出版中）。

Kiuchi Y., et al. (co-author): "Pharmacology of Depression (in Japanese)"Shinko Igaku Shuppan, Tokyo (in press).

木内 Y.，等人。

Misa Yamada: "Identification of a novel splice variant of heat shock cognate protein 70 after chronic antidepressant treatment in rat frontal cortex"Biochem. Biophys. Res. Commun.. 261・2. 541-545 (1999)

Misa Yamada：“大鼠额叶皮质中热休克同源蛋白 70 的新型剪接变体的鉴定”Biochem.Res. 261-545 (1999)。

Kiuchi Y.: "Acute and chronic regulation of monoamine transporter functions (in Japanese)"Folia Pharmacol.Jpn.. 116, Suppl 1. 101-106 (2000)

Kiuchi Y.：“单胺转运蛋白功能的急性和慢性调节（日语）”Folia Pharmacol.Jpn.. 116，Suppl 1. 101-106 (2000)

木内祐二: "抗うつ薬の標的分子モノアミントランスポーターの機能調節"日本薬理学会雑誌. 116補冊1. 101-106 (2000)

Yuji Kiuchi：“单胺转运蛋白的功能调节，抗抑郁药的靶分子”日本药理学会杂志 116 增刊 1. 101-106 (2000)。