Role of the diacylglycerol kinase α-conserved domains in membrane targeting in intact T cells
Role of the diacylglycerol kinase α-conserved domains in membrane targeting in intact T cells
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DOI：
10.1074/jbc.m702085200
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发表时间：
2007-11-30
影响因子：
4.8
通讯作者：
Merida, Isabel
中科院分区：
文献类型：
--
作者：
Merino, Ernesto;Sanjuan, Miguel A.;Merida, Isabel
二酰基甘油激酶（DGK）将二酰基甘油磷酸化为磷脂酸，改变这两种脂质介质的细胞水平。在高等生物中发现了10种DGK亚型，分为5种亚型。它们都含有一个保守的C-末端结构域和至少两个功能未知的富含半胱氨酸的基序。DGK α是一种I型酶，在T细胞活化期间作为基于二酰基甘油的信号的负调节剂。在这里，我们研究了DGK α结构域的功能作用，使用突变分析来研究完整细胞中的膜结合。我们发现，这两个非典型的C1结构域是必不可少的质膜靶向的蛋白质在完整的细胞，但不必要的催化活性。我们还确定的C-末端序列的蛋白质作为膜结合所必需的磷脂酸依赖性的方式。最后，我们证明，在钙结合结构域的情况下，受体依赖性易位的截短的蛋白质的酪氨酸（335）的磷酸化调节。这项功能研究提供了新的见解，这个脂质激酶家族的所谓的保守结构域的作用，并证明存在额外的结构域，赋予特定的质膜定位到这个特定的亚型。
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGK alpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGK alpha domains using mutation alanalysis to investigate membrane binding in intact cells. We show that the two atypical C1domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.