Functional relationship between sphingomyelin synthase‐related protein and diacylglycerol kinase δ
Functional relationship between sphingomyelin synthase‐related protein and diacylglycerol kinase δ
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鞘磷脂合酶相关蛋白与二酰甘油激酶δ之间的功能关系
DOI：
10.1096/fasebj.2020.34.s1.02289
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Sakane Fumio
中科院分区：
文献类型：
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作者：
Murakami Chiaki;Hoshino Fumi;Sakai Hiromichi;Hayashi Yasuhiro;Yamashita Atsushi;Sakane Fumio
二酰甘油激酶（DGKδ）的δ同工酶通过将二酰甘油（DG）转化为磷脂酸（PA）而在脂质信号转导中起重要作用。有趣的是，我们最近证明，DGKδ优选代谢棕榈酸（16：0）和/或棕榈油酸（16：1）的DG分子种类，以响应成肌细胞中的高葡萄糖刺激。然而，目前尚不清楚含16：0和/或16：1的DG分子种类来自何处，尽管另一种含花生四烯酸（20：4）的DG来源于磷脂酰肌醇（PI）周转。鞘磷脂合酶（SMS）1和SMS相关蛋白（SMSr）分别通过将磷酸胆碱/磷酸乙醇胺转移至神经酰胺来合成鞘磷脂和神经酰胺-磷酸乙醇胺。这些也被认为是DG生成酶。SMS 1、SMSr和DGKδ都含有一个不育的α基序结构域（SAM），是一个推测的蛋白质相互作用模块。有趣的是，我们发现DGKδ通过它们的自组装膜直接与SMSr相互作用。结果与结论我们首次利用LC-MS/MS研究了过表达DGK δ和/或SMSr的COS-7细胞中PA分子种类的变化。我们发现SMSr和DGKδ的过表达显著增加了COS-7细胞中含有16：0-和/或16：1-的PA种类（如16：0/16：1-、16：0/16：0-、16：0/18：1-和16：0/18：2-PA）的产生。DGKδ和SMSr可能在功能上相连，SMSr通过提供16：0-和/或16：1-DG在DGKδ的上游途径中起作用。为了支持这种可能性，我们测定了SMSr过表达细胞中的DG水平。与对照细胞相比，SMSr-过表达细胞中的总DG水平显著增加。特别是，含有16：0-和/或16：1-的DG种类，如14：0/16：0（30：0）-、16：0/16：1（32：1）-、16：1/18：2（34：3）-和16：1/18：1（34：2）-DG显著增加。这些结果进一步说明了DGKδ和SMSr之间的函数关系（SMSr向DGKδ提供DG）。为了研究SMSr-DGK δ异聚复合物的形成是否影响DGKδ活性，我们使用DG和ATP作为底物在体外测量了纯化的DGKδ活性。有趣的是，当存在纯化的SMSr和纯化的DGKδ时，DGK活性显著增加。但SMSr的SAM缺失并不能显著提高DGKδ活性。综上所述，这些结果表明SMSr是DGKδ上游DG-提供酶的候选者之一，SMSr通过其自组装膜形成异聚体复合物来增强DGKδ活性。支持或资助信息本工作部分得到了MEXT/JSPS KAKENHI的赠款（资助号：JP 18 J20003 to C.M.）;千叶银行（致C.M.）;千叶大学创业实验室（致C.M.）;千叶大学全球杰出研究所国际交流项目公开招聘（至C.M.）;和美国生物化学和分子生物学学会（ASBMB 2019研究生/博士后旅行奖给C.M）
IntroductionThe δ isozyme of diacyglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). Intriguingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐ and/or palmitoleic acid (16:1)‐containing DG molecular species in response to high glucose stimulation in myoblasts. However, it is still unclear where 16:0‐ and/or 16:1‐containing DG molecular species come from, although another species arachidonic acid (20:4)‐containing DGs are derived from phosphatidylinositol (PI) turnover. Sphingomyelin synthase (SMS) 1 and SMS‐related protein (SMSr) synthesize the sphingomyelin and ceramide‐phosohoethanolamine by the transfer of phosphocholine/phosphoethanolamine to ceramide, respectively. These are also assumed to function as DG‐generating enzymes. SMS1, SMSr and DGKδ contain a sterile α motif domain (SAM), which is a putative protein interaction module. Interestingly, we found that DGKδ interacted with SMSr via their SAMs directly. In the present study, we investigated the functional relationship between DGKδ and SMSr.Results and ConclusionWe first investigated the changes in the amounts of PA molecular species in COS‐7 cells overexpressing DGKδ and/or SMSr using LC‐MS/MS. We found that the overexpression of SMSr and DGKδ significantly enhanced the production of 16:0‐ and/or 16:1‐containing PA species such as 16:0/16:1‐, 16:0/16:0‐, 16:0/18:1‐ and 16:0/18:2‐PA in COS‐7 cells. It is possible that DGKδ and SMSr are functionally linked and that SMSr acts in an upstream pathway of DGKδ by providing 16:0‐ and/or 16:1‐DG. To support the possibility, we determined DG levels in SMSr‐overexpressing cells. Compared with control cells, total DG levels in SMSr‐overexpressing cells were significantly increased. In particular, 16:0‐ and/or 16:1‐containig DG species, such as 14:0/16:0 (30:0)‐, 16:0/16:1 (32:1)‐, 16:1/18:2 (34:3)‐, and 16:1/18:1 (34:2)‐DG were significantly increased. These results further suggest the functional relationship (SMSr supplies DG to DGKδ) between DGKδ and SMSr. To examine whether forming SMSr‐DGKδ heteromeric complex affects DGKδ activity, we measured purified DGKδ activityin vitrousing DG and ATP as substrates. Interestingly, when presence of purified SMSr with purified DGKδ, DGK activity was significantly increased. However, the deletion of SAM of SMSr did not significantly augment the DGKδ activity. Taken together, these results suggest that SMSr is one of the candidates of upstream DG‐providing enzymes of DGKδ and that SMSr enhances DGKδ activity by forming heteromeric complex via their SAMs.Support or Funding InformationThis work was supported in part by grants from MEXT/JSPS KAKENHI (Grant Numbers: JP18J20003 to C.M.); The Chiba Bank (to C.M.); the Chiba University Venture Business Laboratory (to C.M.); Chiba University Open Recruitment for International Exchange Program at the Institute for Global Prominent Research (to C.M.); and the American Society for Biochemistry and Molecular Biology (ASBMB 2019 Graduate/Postdoctoral Travel Award to C.M)