The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about where these DG molecular species come from. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In the present study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro. Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

中文翻译：

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二酰基甘油激酶δ和鞘磷脂合酶相关蛋白通过它们的无菌α基序结构域在功能上相互作用。

通过将二酰基甘油（DG）转化为磷脂酸（PA），二酰基甘油激酶（DGKδ）的δ同工酶在脂质信号传导中起着关键作用。先前我们证明DGKδ优选磷酸化含有棕榈酸（16：0）和/或棕榈油酸（16：1）的DG分子种类，而不是将花生四烯酸（20：4）的DG种类磷酸化，后者被认为是DGK在高葡萄糖刺激的成肌细胞中，磷脂酰肌醇转换产生的底物。但是，对于这些DG分子种类的来源知之甚少。DGKδ和两种产生DG的酶鞘磷脂合成酶（SMS）1和SMS相关蛋白（SMSr）包含一个无菌的α基序域（SAMD）。在本研究中，我们发现SMSr-SAMD与DGKδ-SAMD共同免疫沉淀，而不是SMS1-SAMD。全长DGKδ与全长SMSr的共沉淀比与SMS1共同的强。但是，SAMD缺失的SMSr和DGKδ变体分别与全长DGKδ和SMSr仅弱相互作用。这些结果强烈表明，DGKδ通过它们各自的SAMD与SMSr相互作用。为了确定DGKδ和SMSr之间关系的功能结果，我们使用LC-MS / MS研究了COS-7细胞中DGKδ和/或SMSr的过表达是否改变了PA物种的水平。我们发现SMSr过表达显着增强了包含16：0-或16：1的PA物种（例如14：0/16：0-，16：0/16：0-，16：0/18：1-）的产生和/或16：1/18：1-PA在DGKδ过表达的COS-7细胞中。此外，SSMr通过其SAMDs增强了DGKδ活性。在一起