Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription
Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription
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DOI：
10.1074/jbc.m206911200
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发表时间：
2002-11-08
影响因子：
4.8
通讯作者：
Pickett, CB
中科院分区：
文献类型：
--
作者：
Huang, HC;Nguyen, T;Pickett, CB
NRF2是一种碱性亮氨酸拉链转录因子，通过被称为抗氧化反应元件(ARE)的调节序列，对编码抗氧化酶和11相解毒酶的基因的协调转录起着重要的激活作用。最近，我们报道了蛋白激酶C(PKC)参与磷酸化Nrf2并触发其核转位以响应氧化应激的证据。我们在这里表明，PKC催化亚基对纯化的大鼠Nrf2的磷酸化被模拟其中一个潜在的PKC位点的合成肽阻断。因此，具有S40A突变的Nrf2不能被PKC磷酸化。S40A突变不影响Nrf2/MafK与ARE的体外结合。然而，在报告基因实验中，当Keap1过表达时，它部分地损害了ARE驱动的转录的Nrf2激活。体外转录/翻译的Keap1可与Nrf2免疫共沉淀。PKC对野生型Nrf2的磷酸化促进了其与Keap1的解离，而Nrf2-S40A突变体仍保持联系。这些发现结合我们以前的研究表明，PKC催化的磷酸化。Nrf2在Ser-40的表达是导致ARE介导的细胞抗氧化反应的关键信号事件。
Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase 11 detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation. of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response.