Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
J Cell Sci. 2009 Dec 15;122(Pt 24):4452-64. doi: 10.1242/jcs.058537. Epub 2009 Nov 17.
Antioxidants cause dissociation of nuclear factor erythroid 2-related factor 2 (Nrf2) from inhibitor of Nrf2 (INrf2) and so Nrf2:INrf2 can serve as a sensor of oxidative stress. Nrf2 translocates to the nucleus, binds to antioxidant response element (ARE) and activates defensive gene expression, which protects cells. Controversies exist regarding the role of antioxidant-induced modification of INrf2 cysteine 151 or protein kinase C (PKC)-mediated phosphorylation of Nrf2 serine 40 in the release of Nrf2 from INrf2. In addition, the PKC isoform that phosphorylates Nrf2S40 remains unknown. Here, we demonstrate that antioxidant-induced PKC-delta-mediated phosphorylation of Nrf2S40 leads to release of Nrf2 from INrf2. This was evident from specific chemical inhibitors of PKC isoenzymes in reporter assays, in vitro kinase assays with purified Nrf2 and PKC isoenzymes, in vivo analysis with dominant-negative mutants and siRNA against PKC isoforms, use of PKC-delta(+/+) and PKC-delta(-/-) cells, and use of Nrf2S40 phospho-specific antibody. The studies also showed that antioxidant-induced INrf2C151 modification was insufficient for the dissociation of Nrf2 from INrf2. PKC-delta-mediated Nrf2S40 phosphorylation was also required. Nrf2 and mutant Nrf2S40A both bind to INrf2. However, antioxidant treatment led to release of Nrf2 but not Nrf2S40A from INrf2. In addition, Nrf2 and mutant Nrf2S40A both failed to dissociate from mutant INrf2C151A. Furthermore, antioxidant-induced ubiquitylation of INrf2 in PKC-delta(+/+) and PKC-delta(-/-) cells occurred, but Nrf2 failed to be released in PKC-delta(-/-) cells. The antioxidant activation of Nrf2 reduced etoposide-mediated DNA fragmentation and promoted cell survival in PKC-delta(+/+) but not in PKC-delta(-/-) cells. These data together demonstrate that both modification of INrf2C151 and PKC-delta-mediated phosphorylation of Nrf2S40 play crucial roles in Nrf2 release from INrf2, antioxidant induction of defensive gene expression, promoting cell survival, and increasing drug resistance.
抗氧化剂导致核因子红细胞 2 相关因子 2（Nrf2）与 Nrf2 抑制剂（INrf2）解离，因此 Nrf2:INrf2 可以作为氧化应激的传感器。Nrf2 易位到细胞核，与抗氧化反应元件（ARE）结合并激活防御性基因表达，从而保护细胞。关于抗氧化剂诱导的 INrf2 半胱氨酸 151 修饰或蛋白激酶 C（PKC）介导的 Nrf2 丝氨酸 40 磷酸化在 Nrf2 从 INrf2 中释放中的作用存在争议。此外，磷酸化 Nrf2S40 的 PKC 同工型仍不清楚。在这里，我们证明抗氧化剂诱导的 PKC-δ介导的 Nrf2S40 磷酸化导致 Nrf2 从 INrf2 中释放。这从报告基因测定中的 PKC 同工酶的特定化学抑制剂、用纯化的 Nrf2 和 PKC 同工酶进行的体外激酶测定、用显性负突变体和针对 PKC 同工型的 siRNA 进行的体内分析、使用 PKC-δ(+/+)和 PKC-δ(-/-)细胞以及使用 Nrf2S40 磷酸化特异性抗体来证明。研究还表明，抗氧化剂诱导的 INrf2C151 修饰不足以使 Nrf2 从 INrf2 中解离。PKC-δ 介导的 Nrf2S40 磷酸化也是必需的。Nrf2 和突变 Nrf2S40A 都与 INrf2 结合。然而，抗氧化剂处理导致 Nrf2 而不是 Nrf2S40A 从 INrf2 中释放。此外，Nrf2 和突变 Nrf2S40A 都不能从突变 INrf2C151A 中解离。此外，在 PKC-δ(+/+)和 PKC-δ(-/-)细胞中发生了抗氧化剂诱导的 INrf2 泛素化，但在 PKC-δ(-/-)细胞中 Nrf2 未能释放。Nrf2 的抗氧化剂激活减少了依托泊苷介导的 DNA 片段化并促进了 PKC-δ(+/+)细胞的存活，但在 PKC-δ(-/-)细胞中没有。这些数据共同表明，INrf2C151 的修饰和 PKC-δ 介导的 Nrf2S40 磷酸化都在 Nrf2 从 INrf2 中的释放、抗氧化诱导防御性基因表达、促进细胞存活和增加药物抗性中起关键作用。
