Huang H-C, Nguyen Truyen, Pickett Cecil B
Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
J Biol Chem. 2002 Nov 8;277(45):42769-74. doi: 10.1074/jbc.M206911200. Epub 2002 Aug 26.
Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II 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.
Nrf2是一种碱性亮氨酸拉链转录因子，它通过被称为抗氧化反应元件（ARE）的调控序列，成为编码抗氧化酶和II期解毒酶的基因协同转录的重要激活因子。最近我们报道了蛋白激酶C（PKC）参与磷酸化Nrf2并在氧化应激反应中触发其核转位的证据。我们在此表明，模拟潜在PKC位点之一的合成肽可阻断PKC催化亚基对纯化大鼠Nrf2的磷酸化。因此，在氨基酸40处具有丝氨酸到丙氨酸突变（S⁴⁰A）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，在报告基因检测中，当Keap1过表达时，它部分损害了ARE驱动转录的Nrf2激活。体外转录/翻译的Keap1可与Nrf2共免疫沉淀。PKC对野生型Nrf2的磷酸化促进了它与Keap1的解离，而Nrf2-S⁴⁰A突变体仍保持结合。这些发现与我们之前的研究共同表明，PKC催化的Nrf2在丝氨酸40处的磷酸化是导致ARE介导的细胞抗氧化反应的关键信号事件。
