Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization

Abstract

Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys 588 and Lys 591 of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) is a key oxidative stress response modifier that induces transcription of a variety of genes through binding to the antioxidant response element (ARE) 4 in target gene promoters (1-3). Nrf2-dependent activation of ARE-driven gene promoters is generally understood to lead to induction of cytoprotective proteins, which enable cells to combat oxidative insult (2-4). However, overexpression of Nrf2 in cancerous cells may actually be deleterious because it may enable them to sustain growth and become chemo-resistant to various prooxidant chemotherapeutic drugs (5-7).In nonstressed cells, Nrf2 activity is thought to be repressed by Keap1 (Kelch-like ECH-associated protein 1) (8 -10), a cytoskeleton-associated protein that, when complexed with Nrf2, promotes ubiquitin-mediated degradation of Nrf2. In one model for the activation of Nrf2 in stressed cells, electrophileor reactive oxygen species-induced release of Nrf2 is proposed to involve covalent modifications of Keap1 and/or Nrf2 in the cytoplasm. Such modifications include oxidation of key cysteine residues in Keap1 (11), phosphorylation of Nrf2 at Ser 40 by protein kinase C (12, 13), and switching of Cullin-3...

Nuclear Factor–Erythroid-2–Related Factor 2 in Aging and Lung Fibrosis

“…Keap1-independent mechanisms of Nrf2 activation involve several reported post-translational modifications, including phosphorylation, 15 acetylation, 69,70 and interactions with cofactors, such as Maf, 71 ATF4, 72 and CREB. 73 Kinasemediated phosphorylation of Ser-40 residue on Nrf2 is reported as a requisite for the activation and nuclear translocation of Nrf2.…”

“…79 Acetylation and deacetylation of Nrf2 promote nuclearcytoplasmic shuttling and regulation of its transcriptional activity. 69 Acetylation of lysine residues in Nrf2 enhances Nrf2-DNA binding and transcription of target genes. The acetylation of lysine residues in the Neh1 domain of Nrf2 by p300/CBP augments promoter-specific DNA binding in response to arsenite-mediated oxidative stress.…”

“…70 Mutating Lys-588 and Lys-591 of Nrf2 impairs Nrf2-dependent gene transcription by distorting the transcription-activating effect of CREB-binding protein. 69 Age-related changes in activities of protein kinases or acetyltransferases/ deacetylases may lead to impaired Nrf2 regulation. Agerelated changes in the expression and activity of PKC, 80 and phosphatidylinositol 3-kinase, 81 have been reported in both aging animal models and humans.…”

“…122 The mechanisms by which resveratrol activates Nrf2 are not well defined, but are unlikely to involve activation of sirtuins. 69 Curcumin, a polyphenol obtained from rhizome of Curcuma Longa, has been shown to be an effective Nrf2 activator and inducer of Nrf2-dependent antioxidant and anti-inflammatory genes. 123 In human monocytes, curcumin mediates the up-regulation of antioxidant defensive genes through Nrf2-ARE signaling via PKC and p38 mitogen-activated protein kinase.…”

SirT1 and STAT3 protect retinal pigmented epithelium cells against oxidative stress

“…SirT1 is also able to inhibit certain transcription factors, which regulate cellular redox balance. For example, inhibition of the transactivation capacity of nuclear factor-like 2 (Nrf2) in HepG2 cells (16). However the detailed mechanisms underlying the function of SirT1 against oxidative stress, particularly in RPEs, remain to be elucidated.…”