The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain
The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain
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DOI：
10.1042/bj20040681
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发表时间：
2004-09-15
影响因子：
4.1
通讯作者：
Sakane, F
中科院分区：
文献类型：
--
作者：
Imai, S;Kai, M;Sakane, F
DGK（二酰基甘油激酶）调节两种生物活性脂质，二酰基甘油和磷脂酸的浓度。DGKdelta 1或其PH（普列克底物蛋白同源性）结构域单独已显示在PMA处理的细胞中从细胞质易位至质膜。在本研究中，我们确定了PH结构域中的Ser-22和Ser-26作为DGKdelta 1的PMA和表皮生长因子依赖性磷酸化位点。体外和完整细胞实验表明，cPKC（常规蛋白激酶C）直接磷酸化这些Ser残基。令人困惑的是，DGKdelta 1的Ser-22和Ser-26处的丙氨酸/天冬酰胺突变体及其PH结构域仍然通过PMA处理持续易位，这表明PH结构域磷酸化不是酶易位的原因，并且易位是由PMA依赖性但不依赖于cPKC的过程引起的，该过程尚未被鉴定。有趣的是，天冬氨酸突变，模拟磷酸丝氨酸，在Ser-22或Ser-26，抑制全长DGKdelta 1和PH结构域的易位显着，这表明磷酸化负调控酶易位。我们的研究结果提供了证据的磷酸化DGKdelta 1 PH结构域的cPKC，并建议磷酸化参与控制DGKdelta 1的亚细胞定位。
DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.