NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation
NOXO1 phosphorylation on serine 154 is critical for optimal NADPH oxidase 1 assembly and activation
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DOI：
10.1096/fj.12-216432
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发表时间：
2013-04-01
期刊：
影响因子：
4.8
通讯作者：
Dang, Pham My-Chan
中科院分区：
文献类型：
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作者：
Debbabi, Maya;Kroviarski, Yolande;Dang, Pham My-Chan
主要在结肠上皮细胞中表达的NADPH氧化酶1（NOX 1）产生活性氧（ROS）需要膜结合组分p22（PHOX）和胞质伴侣NOX组织者1（NOXO 1）、NOX激活剂1（NOXA 1）和Rac 1。与其吞噬细胞对应物NOX 2相反，NOX 1调节的分子基础尚不清楚。由于NOXO 1缺乏在其同源物p47（PHOX）中发现的磷酸化区域，因此目前的观点是NOX 1活化在没有NOXO 1磷酸化的情况下发生。然而，在这里，我们证明佛波醇肉豆蔻酸酯乙酸酯（PMA）刺激NOXO 1磷酸化在转染的人胚肾（HEK）293上皮细胞模型通过蛋白激酶C和确定Ser-154作为主要的磷酸化位点。来自T84结肠上皮细胞的内源性NOXO 1也被磷酸化，表明NOXO 1磷酸化是生理相关的。在转染的HEK-293细胞中，PMA诱导的Ser-154磷酸化增强了NOXO 1与NOXA 1（+97%）和p22（PHOX）C-末端区域（+384%）的结合，增加了NOXO 1与p22（PHOX）的共定位，并允许NOX 1产生最佳的ROS，这一点通过使用S154 A和S154 D突变体与野生型NOXO 1相比得到了证实（P
Reactive oxygen species (ROS) production by NADPH oxidase 1 (NOX1), which is mainly expressed in colon epithelial cells, requires the membrane-bound component p22(PHOX) and the cytosolic partners NOX organizer 1 (NOXO1), NOX activator 1 (NOXA1), and Rac1. Contrary to that of its phagocyte counterpart NOX2, the molecular basis of NOX1 regulation is not clear. Because NOXO1 lacks the phosphorylated region found in its homolog p47(PHOX), the current view is that NOX1 activation occurs without NOXO1 phosphorylation. Here, however, we demonstrate that phorbol myristate acetate (PMA) stimulates NOXO1 phosphorylation in a transfected human embryonic kidney (HEK) 293 epithelial cell model via protein kinase C and identify Ser-154 as the major phosphorylated site. Endogenous NOXO1 from T84 colon epithelial cells was also phosphorylated, suggesting that NOXO1 phosphorylation is physiologically relevant. In transfected HEK-293 cells, PMA-induced phosphorylation on Ser-154 enhanced NOXO1 binding to NOXA1 (+97%) and to the p22(PHOX) C-terminal region (+384%), increased NOXO1 colocalization with p22(PHOX), and allowed optimal ROS production by NOX1 as demonstrated by the use of S154A and S154D mutants compared with that by wild-type NOXO1 (P