Molecular mechanism of human Nrf2 activation and degradation: Role of sequential phosphorylation by protein kinase CK2
Molecular mechanism of human Nrf2 activation and degradation: Role of sequential phosphorylation by protein kinase CK2
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DOI：
10.1016/j.freeradbiomed.2007.03.001
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发表时间：
2007-06-15
影响因子：
7.4
通讯作者：
Waalkes, Michael P.
中科院分区：
文献类型：
--
作者：
Pi, Jingbo;Bai, Yushi;Waalkes, Michael P.
Nrf 2是细胞对氧化应激反应中的关键转录因子。在这项研究中，我们确定了两种磷酸化形式的内源性人类Nrf 2化学诱导的氧化应激后，并提供证据表明，蛋白激酶CK 2介导的顺序磷酸化在Nrf 2的激活和降解中起着潜在的作用。人Nrf 2的预测分子量为66 kDa。然而，免疫印迹显示，在98和118 kDa的两个条带，这被确定为磷酸化的形式，增加响应Nrf 2诱导剂。此外，发现人Nrf 2是CK 2的底物，CK 2介导两个磷酸化步骤，导致两种形式的Nrf 2以不同的M迁移，在98 kDa（Nrf 2 -98）和118 kDa（Nrf 2 -118）。我们的研究结果支持钙调素结合调节CK 2活性的作用，因为冷（25 ℃）无钙培养基（冷/无钙）降低了细胞钙水平和CK 2-钙调素结合并诱导Nrf 2 -118形成，后者被CK 2特异性抑制剂阻止。凝胶位移分析表明，在冷/无钙条件下产生的Nrf 2 -118不结合抗氧化反应元件，表明Nrf 2 -98具有转录活性。相比之下，Nrf 2 -118更容易降解。这些结果为CK 2磷酸化作为Nrf 2介导的细胞抗氧化反应的关键控制因子提供了证据。(c)2007年爱思唯尔公司All rights reserved.
Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we identify two phosphorylated forms of endogenous human Nrf2 after chemically induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential roles in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing M, at 98 kDa (Nrf2-98) and 118 kDa (Nrf2-118). Our results support a role in which calmodulin binding regulates CK2 activity, in that cold (25 degrees C) Ca2+-free media (cold/Ca2+-free) decreased both cellular calcium levels and CK2-calmodulin binding and induced Nrf2-118 formation, the latter of which was prevented by CK2-specific inhibitors. Gel shift assays showed that the Nrf2-118 generated under cold/Ca2+-free conditions does not bind to the antioxidant response element, indicating that Nrf2-98 has transcriptional activity. In contrast, Nrf2-118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response. (c) 2007 Elsevier Inc. All rights reserved.