5′-Nucleotidase domain-containing protein 2 (NT5DC2) has been revealed by genome-wide association studies (GWAS) as a gene implicated in neuropsychiatric disorders related to the abnormality of dopamine (DA) activity in the brain. Based on its amino acid sequence, NT5DC2 is assumed to be a member of the family of haloacid dehalogenase-type phosphatases; although there is no information about its function and structural conformation. We recently reported that NT5DC2 binds to tyrosine hydroxylase (TH) and that the down-regulation of NT5DC2 tended to increase DA synthesis. In this study, we investigated whether NT5DC2 could regulate the catalytic activity of TH, which converts tyrosine to DOPA, because the phosphorylation level of TH, controlled by protein kinases and phosphatases, is well known to regulate its catalytic activity. The down-regulation of NT5DC2 by siRNA increased mainly DOPA synthesis by TH in PC12D cells, although this down-regulation tended to increase the conversion of DOPA to DA by aromatic l-amino acid decarboxylase. The increased DOPA synthesis should be attributed to the catalytic activity of TH controlled by its phosphorylation, because Western blot analysis revealed that the down-regulation of NT5DC2 tended to increase the level of TH phosphorylated at its Ser residues, but not that of the TH protein. Moreover, the induction of kinase activity by forskolin markedly potentiated the phosphorylation of TH at its Ser40 in PC12D cells having down-regulated NT5DC2. Immunocytochemical analysis of PC12D cells demonstrated that NT5DC2, TH protein, and TH phosphorylated at its Ser40 were predominantly localized in the cytoplasm and that the localization of NT5DC2 and TH proteins partially overlapped. Collectively, our results indicate that NT5DC2 could work to inhibit the DOPA synthesis by decreasing the phosphorylation of TH at its Ser40. We propose that NT5DC2 might decrease this phosphorylation of TH by promoting dephosphorylation or by inhibiting kinase activity.

全基因组关联研究 (GWAS) 已揭示含有 5'-核苷酸酶结构域的蛋白 2 (NT5DC2) 作为与大脑中多巴胺 (DA) 活性异常相关的神经精神疾病有关的基因。根据其氨基酸序列，NT5DC2 被认为是卤酸脱卤酶型磷酸酶家族的成员；虽然没有关于其功能和结构构象的信息。我们最近报道了 NT5DC2 与酪氨酸羟化酶 (TH) 结合，并且 NT5DC2 的下调倾向于增加 DA 合成。在这项研究中，我们研究了 NT5DC2 是否可以调节将酪氨酸转化为多巴的 TH 的催化活性，因为众所周知，由蛋白激酶和磷酸酶控制的 TH 的磷酸化水平可以调节其催化活性。升-氨基酸脱羧酶。多巴合成的增加应归因于由其磷酸化控制的 TH 的催化活性，因为蛋白质印迹分析显示 NT5DC2 的下调倾向于增加其 Ser 残基处的 TH 磷酸化水平，但不会增加 TH 蛋白的磷酸化水平. 此外，毛喉素对激酶活性的诱导显着增强了 NT5DC2 下调的 PC12D 细胞中 TH 的 Ser40 磷酸化。PC12D 细胞的免疫细胞化学分析表明，NT5DC2、TH 蛋白和在其 Ser40 处磷酸化的 TH 主要位于细胞质中，并且 NT5DC2 和 TH 蛋白的定位部分重叠。总的来说，我们的结果表明 NT5DC2 可以通过降低 TH 在其 Ser40 处的磷酸化来抑制多巴合成。