Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomer-α-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.

中文翻译：

---

使用蛋白质肉豆蔻酰化和细胞磷脂酸传感器检测细胞中的二酰基甘油激酶 η 活性

二酰基甘油激酶 (DGK) 磷酸化二酰基甘油以产生磷脂酸 (PtdOH) 并调节两种脂质第二信使：二酰基甘油和 PtdOH 之间的平衡。多项证据表明 DGK 的 η 同工酶参与双相情感障碍的发病机制。然而，调节病理生理功能的详细分子机制仍不清楚。原因之一是难以检测 DGKη 的细胞活性。为了克服这一困难，我们利用蛋白质豆蔻酰化和细胞 PtdOH 传感器，即 α-突触核蛋白 (α-Syn-N) 的 N 端区域。尽管在 COS-7 细胞中表达的 DGKη 广泛分布在细胞质中，但肉豆蔻酰化 (Myr)-AcGFP-DGKη 和 Myr-AcGFP-DGKη-KD（失活（激酶死亡）突变体）基本上位于质膜中。而且，DsRed 单体-α-Syn-N 与 Myr-AcGFP-DGKη 显着共定位，但不与 Myr-AcGFP-DGKn-KD 在质膜上共定位。当 COS-7 细胞受到渗透压冲击时，所有 DGKη 构建体都专门转移到渗透压冲击反应颗粒 (OSRG)。DsRed 单体-α-Syn-N 在 OSRG 处仅与 Myr-AcGFP-DGKη 显着共定位，并且在未受刺激的 COS-7 细胞（2.5）的质膜处表现出比 Myr-AcGFP-DGKη 更高的信号/背景比（3.4），表明 α-Syn-N 更有效地检测 OSRG 中的 Myr-AcGFP-DGKη 活性。因此，这些结果表明肉豆蔻酰化和 PtdOH 传感器的组合有效地检测细胞中的 DGKη 活性，并且这种方法可以方便地检查 DGKη 的分子功能。此外，该方法将有助于开发靶向 DGKη 的药物。此外，