Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation.
Down-regulation of NOX2 activity in phagocytes mediated by ATM-kinase dependent phosphorylation.
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DOI：
10.1016/j.freeradbiomed.2017.09.007
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发表时间：
2017-12
影响因子：
7.4
通讯作者：
Stasia MJ
中科院分区：
文献类型：
--
作者：
Beaumel S;Picciocchi A;Debeurme F;Vivès C;Hesse AM;Ferro M;Grunwald D;Stieglitz H;Thepchatri P;Smith SME;Fieschi F;Stasia MJ
NADPH氧化酶（NOX）具有许多生物学作用，但其调控潜在毒性ROS分子产生的机制尚不清楚。先前确定的21个残基插入序列（称为NIS）影响NOX活性，其预测的灵活性使其成为提供控制NOX活性位点的动态开关的良好候选者。我们构建了NOX2嵌合体，其中NIS被删除或与其他nox （NIS1、3和4）的嵌合体交换。它们都含有功能性血红素，在分化的PLB-985细胞的质膜上表达正常。然而，NOX2-ΔNIS和NOX2- nis1既没有nadph氧化酶活性，也没有还原酶活性，并且p47phox和p67phox异常易位到吞噬体膜。这表明了NIS的功能作用。有趣的是，与野生型细胞相比，NOX2-NIS3细胞在激活后表现出超氧化物过量。矛盾的是，纯化的未受刺激的NOX2-NIS3的Vmax仅为WT-NOX2的三分之一。因此，我们假设翻译后事件调节NOX2活性，并且在NOX2- nis3和WT-NOX2之间存在差异。我们证明了位于NOX2的NIS （NOX2-NIS）的ataxia毛细血管扩张突变激酶（ATM激酶）的磷酸化靶点Ser486在纯化的细胞色素b558中被12-肉豆酸酯-13-乙酸（PMA）刺激后磷酸化。此外，ATM激酶抑制和NOX2 Ser486Ala突变增强了NOX活性，而Ser486Glu突变抑制了NOX活性。因此，NIS3中缺乏Ser486可以解释NOX2-NIS3突变体中超氧化物过量产生的原因。这些结果表明，pma刺激的NOX2- nis通过ATM激酶磷酸化导致NOX2活性失活的动态开关。我们假设这种下调在NOX2-NIS3突变体中是有缺陷的，因为缺乏Ser486。
NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.
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影响因子：
4.8
作者：
Grizot, S;Fieschi, F;Pebay-Peyroula, E
通讯作者：
Pebay-Peyroula, E
影响因子：
4.8
作者：
Li, XJ;Grunwald, D;Stasia, MJ
通讯作者：
Stasia, MJ
影响因子：
2.2
作者：
Lord, Connie I.;Riesselman, Marcia H.;Taylor, Ross M.
通讯作者：
Taylor, Ross M.
影响因子：
6.6
作者：
Frey RS;Ushio-Fukai M;Malik AB
通讯作者：
Malik AB
影响因子：
2.9
作者：
Durand, Dominique;Cannella, Dominique;Fieschi, Franck
通讯作者：
Fieschi, Franck