Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells
Inhibition of deubiquitinating activity of USP14 decreases tyrosine hydroxylase phosphorylated at Ser19 in PC12D cells
复制标题
抑制 USP14 的去泛素化活性会降低 PC12D 细胞中 Ser19 磷酸化的酪氨酸羟化酶
DOI：
10.1016/j.bbrc.2016.03.022
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发表时间：
2016
影响因子：
3.1
通讯作者：
Akira Ota
中科院分区：
文献类型：
--
作者：
Akira Nakashima;Syuhei Ohnuma;Yu Kodani;Yoko S. Kaneko;Hiroshi Nagasaki;Toshiharu Nagatsu;Akira Ota
酪氨酸羟化酶(TH)是儿茶酚胺生物合成的限速酶，其稳定性是维持细胞中儿茶酚胺水平的根本因素。然而，由降解途径决定的细胞内稳定性仍不清楚。在这项研究中，我们研究了TH的磷酸化影响蛋白酶体途径的机制。在PC12D细胞中，MG-132对蛋白酶体的抑制使其Ser19、Ser31和/或Ser40处的TH分子在总TH蛋白中的磷酸化百分比在24小时内增加到约70%，而在基础条件下磷酸化的TH分子的百分比约为20%。此外，高特异性的环氧米星对蛋白酶体的抑制主要增加了其Ser19位的TH分子的磷酸化数量。Ser19的磷酸化通过一种称为分级磷酸化的过程增强了Ser40在细胞中的磷酸化。因此，蛋白酶体抑制可能导致所有3种磷酸化TH形式水平的增加，从而使数据解释复杂化。相反，作为USP14脱泛素化活性的抑制剂，Iu-1激活蛋白酶体降解，仅减少其Ser19处的TH分子的磷酸化数量，但不减少其Ser31和Ser40处的TH分子的磷酸化数量或TH分子的数量。这些结果表明，TH N-末端Ser19的磷酸化是通过泛素-蛋白酶体途径降解该酶的关键。
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its stability is a fundamental factor to maintain the level of the catecholamines in cells. However, the intracellular stability determined by the degradation pathway remains unknown. In this study, we investigated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway.