Structure-activity relationship of diacylglycerol kinase θ
Structure-activity relationship of diacylglycerol kinase θ
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DOI：
10.1016/j.bbalip.2003.11.008
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发表时间：
2004-03-22
影响因子：
4.8
通讯作者：
van Blitterswijk, WJ
中科院分区：
文献类型：
--
作者：
Los, AP;van Baal, J;van Blitterswijk, WJ
甘油二酯激酶(DGK)将第二信使甘油二酯(DAG)磷酸化为磷脂酸(PA)。在已确定的9种哺乳动物同工型中，DGKtheta是唯一一种在其N端调节区具有三个富含半胱氨酸结构域(CRD)的基因。我们以前报道过DGKtheta与活性RhoA结合并受到活性RhoA的负调控。我们现在报道，RhoA强烈结合到C-末端催化结构域，这可以解释它对DGK活性的抑制。为了帮助寻找DGKtheta的生理功能，我们进一步测定了它在体外的活性，作为15种不同的截断和一级结构中的点突变的函数。这些变化中的大多数分布在整个蛋白质中，使酶失活，这表明催化活性取决于它的所有保守结构域。大部分C-末端的CRD由15个氨基酸组成，在DGK亚型中高度保守。突变分析显示，该区域存在许多对酶活性至关重要的残基。我们认为，这种CRD延伸在蛋白质的正确折叠和/或底物呈现到蛋白质的催化区域中起着至关重要的作用。(C)2004爱思唯尔B.V.保留所有权利。
Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid (PA). Among the nine mammalian isotypes identified, DGKtheta is the only one with three cysteine-rich domains (CRDs) (instead of two) in its N-terminal regulatory region. We previously reported that DGKtheta binds to and is negatively regulated by active RhoA. We now report that RhoA strongly binds to the C-terminal catalytic domain, which would explain its inhibition of DGK activity. To help finding a physiological function of DGKtheta, we further determined its activity in vitro as a function of 15 different truncations and point mutations in the primary structure. Most of these alterations, located throughout the protein, inactivated the enzyme, suggesting that catalytic activity depends on all of its conserved domains. The most C-terminal CRD is elongated with a stretch of 15 amino acids that is highly conserved among DGK isotypes. Mutation analysis revealed a number of residues in this region that were essential for enzyme activity. We suggest that this CRD extension plays an essential role in the correct folding of the protein and/or in substrate presentation to the catalytic region of the protein. (C) 2004 Elsevier B.V. All rights reserved.