Diacylglycerol kinases (DGKs) are lipid kinases that mediate the phosphorylation of diacylglycerol (DAG) leading to the production of phosphatidic acid (PtdOH). To examine the role of phosphorylation on DGK-θ, we first identified the phosphorylated sites on endogenous DGK-θ from mouse brain and found four sites: S15, S17, which we refer to phosphomotif-1 sites, and S22 and S26 which we refer to as phosphomotif-2 sites. This study focused on the role of these phosphorylated sites on enzyme activity, membrane binding, thermal stability, and cellular half-life of DGK-θ. After generating a construct devoid of all non-catalytic phosphorylation sites (4A), we also generated other constructs to mimic phosphorylation of these residues by mutating them to glutamate (E). Our data demonstrate that an increase in membrane affinity requires the phosphorylation of all four endogenous sites as the phosphomimetic 4E but not other phosphomimietics. Furthermore, 4E also shows an increase in basal activity as well as an increase in the Syt1-induced activity compared to 4A. It is noteworthy that these phosphorylations had no effect on the thermal stability or cellular half-life of this enzyme. Interestingly, when only one phosphorylation domain (phosphomotif-1 or phosphomotif-2) contained phosphomimetics (S15E/S17E or S22E/S26E), the basal activity was also increased but membrane binding affinity was not increased. Furthermore, when only one residue in each domain mimicked an endogenous phosphorylated serine (S15E/S22E or S17E/S26E), the Syt1-induced activity as well as membrane binding affinity decreased relative to 4A. These results indicate that these endogenous phosphorylation sites contribute differentially to membrane binding and enzymatic activity.

中文翻译：

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DGK-θ N 端磷酸化的作用

二酰甘油激酶 (DGK) 是介导二酰甘油 (DAG) 磷酸化从而产生磷脂酸 (PtdOH) 的脂质激酶。为了检查磷酸化对 DGK-θ 的作用，我们首先鉴定了小鼠大脑内源性 DGK-θ 上的磷酸化位点，并发现了四个位点：S15、S17（我们称之为磷酸基序 1 位点）以及 S22 和 S26（我们称之为磷酸化位点）。作为磷酸基序2位点。本研究重点关注这些磷酸化位点对 DGK-θ 的酶活性、膜结合、热稳定性和细胞半衰期的作用。在生成没有所有非催化磷酸化位点的构建体（4A）后，我们还生成了其他构建体，通过将这些残基突变为谷氨酸来模拟这些残基的磷酸化（E）。我们的数据表明，膜亲和力的增加需要所有四个内源位点磷酸化为磷酸模拟物 4E，而不是其他磷酸模拟物。此外，与 4A 相比，4E 还显示出基础活性的增加以及 Syt1 诱导的活性的增加。值得注意的是，这些磷酸化对该酶的热稳定性或细胞半衰期没有影响。有趣的是，当只有一个磷酸化结构域（磷酸基序1或磷酸基序2）含有磷酸模拟物（S15E/S17E或S22E/S26E）时，基础活性也增加，但膜结合亲和力没有增加。此外，当每个结构域中只有一个残基模拟内源性磷酸化丝氨酸（S15E/S22E 或 S17E/S26E）时，Syt1 诱导的活性以及膜结合亲和力相对于 4A 降低。这些结果表明这些内源磷酸化位点对膜结合和酶活性的贡献不同。