Department of Medicine, Division of Neurology, University of British Columbia, Vancouver, BC V6T 2B5, Canada.
Department of Pediatrics and Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
Mol Biol Cell. 2020 May 1;31(10):1047-1059. doi: 10.1091/mbc.E20-01-0014. Epub 2020 Mar 18.
phosphocholine cytidylyltransferase-alpha (CCTα) and CCTβ catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.
磷酸胆碱胞苷转移酶-α（CCTα）和 CCTβ 催化磷脂酰胆碱（PC）生物合成的限速步骤。CCTα 通过其α-螺旋 M 结构域与核膜的结合而被激活，而这种结合受到相邻 P 结构域磷酸化的负调控。为了了解磷酸化如何调节 CCT 活性，我们针对 P 结构域的 N 端和 C 端的 pS319 和 pY359+pS362 分别开发了磷酸化特异性抗体。培养细胞中的油酸盐处理触发 CCTα 向核膜（NE）和核脂滴（nLDs）的易位，并迅速使 pS319 去磷酸化。去除油酸盐会导致 CCTα 与 NE 的解离，并增加 S319 的磷酸化。细胞中的胆碱耗竭也会导致 CCTα 向 NE 的易位和 S319 的去磷酸化。相反，在添加和去除油酸盐期间，Y359 和 S362 一直处于磷酸化状态，并且 CCTα-pY359+pS362 易位到油酸盐处理细胞的 NE 和 nLDs。突变分析表明，S319 的磷酸化受 Y359+S362 的调控，并且 CCTα-S315D+S319D 在定位于 NE 方面存在缺陷。我们的结论是，由于 S319 的去磷酸化和可能其他脯氨酸导向位点的负电荷极化以及 Y359 和 S362 磷酸化的保留，P 结构域发生了变化，并且 S319 和 S315 的去磷酸化参与了 CCTα 向核膜的募集。
