Molecular mechanism of human Nrf2 activation and degradation: Role of sequential phosphorylation by protein kinase CK2

Laboratory of Comparative Carcinogenesis, NCI at NIEHS, NIH, Research Triangle Park, NC 27709, USA.
Free Radical Biology and Medicine (Impact Factor: 5.74). 07/2007; 42(12):1797-806. DOI: 10.1016/j.freeradbiomed.2007.03.001
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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(r) 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) Ca(2+)-free media (cold/Ca(2+)-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/Ca(2+)-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.

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Available from: Jie Liu, Oct 05, 2015
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    • "In unstressed condition, Nrf2 is retained by Keap1 (Kelch-like ECH-associated protein1) in the cytoplasm and committed to proteosomal degradation (Kansanen et al., 2013). In contrast, phosphorylation of Nrf2 by several cytosolic kinases (Pi et al., 2007) stabilizes the protein, which then can translocate into the nucleus where binds to antioxidant-response elements (AREs) in genes encoding for antioxidative enzymes (Nguyen et al., 2003). In accordance with other authors (Hassane et al., 2013; Sen et al., 2013), we advanced the hypothesis that activation of mTOR induced by PN can be responsible for phosphorylation and translocation of Nrf2. "
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    ABSTRACT: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 06/2015; 230(6). DOI:10.1002/jcp.24863 · 3.84 Impact Factor
    • "through cysteine thiol modification of Keap1 (Zhang and Hannink, 2003) or via phosphorylation of its serine/threonine residues by upstream kinases, such as protein kinase C (PKC) (Bloom and Jaiswal, 2003), extracellular signal-regulated kinase (ERK) (Yu et al., 2000), casein kinase-2 (Pi et al., 2007), cAMP-activated protein kinase-a (AMPKa) (Mo et al., 2013), and Akt (Martin et al., 2004). Thymoquinone (2-Isopropyl-5-methylbenzo-1,4-quinone; TQ) is an active constituent of black cumin (Nigella sativa), also known as black seed, which is widely consumed as a condiment and has long been used in Ayurvedic medicine. "
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    ABSTRACT: Thymoquinone (TQ), an active constituent of Nigella sativa, possesses anti-inflammatory and anticancer properties. Multiple lines of evidence suggest that the induction of heme oxygenase-1 (HO-1) suppresses inflammation and carcinogenesis. In the present study, we examined the effect of TQ on HO-1 expression in human keratinocytes (HaCaT) and elucidated its underlying molecular mechanisms. TQ induced the expression of HO-1 in HaCaT cells in a concentration- and time-dependent manner. Treatment with TQ increased the localization of nuclear factor (NF)-erythroid2-(E2)-related factor-2 (Nrf2) in the nucleus and elevated the antioxidant response element (ARE)-reporter gene activity. Knockdown of Nrf2 abrogated TQ-induced HO-1 expression and the ARE luciferase activity. TQ induced the phosphorylation of extracellular signal-regulated kinase (ERK), Akt and cyclic AMP-activated protein kinase-α (AMPKα). Pharmacological inhibition of Akt or AMPKα, but not that of ERK, abrogated TQ-induced nuclear localization of Nrf2, the ARE-luciferase activity and the expression of HO-1. TQ also generated reactive oxygen species (ROS) and pretreatment with N-acetyl cysteine (NAC) abrogated TQ-induced ROS accumulation, Akt and AMPKα activation, Nrf2 nuclear localization, the ARE-luciferase activity, and HO-1 expression in HaCaT cells. Taken together, TQ induces HO-1 expression in HaCaT cells by activating Nrf2 through ROS-mediated phosphorylation of Akt and AMPKα.
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    • "One of the best characterized include phosphorylation by PKC (Huang et al., 2000), which is necessary for liberating NRF2 from KEAP1 (Bloom and Jaiswal, 2003) and thus promoting transport to the nucleus. Protein kinase CK2 can also phosphorylate NRF2, which promotes transport of NRF2 into the nucleus (Pi et al., 2007). Similar activating effects on NRF2 translocation to the nucleus have been observed for phosphatidylinositide 3-kinases (PI3K) (Nakaso et al., 2003), c-Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK) (Xu et al., 2006) and PERK (Cullinan et al., 2003). "
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    ABSTRACT: The nuclear factor erythroid 2 related factor 2 (NRF2) is a key regulator of endogenous inducible defense systems in the body. Under physiological conditions NRF2 is mainly located in the cytoplasm. However, in response to oxidative stress, NRF2 translocates to the nucleus and binds to specific DNA sites termed "anti-oxidant response elements" or "electrophile response elements" to initiate transcription of cytoprotective genes. Acute oxidative stress to the brain, such as stroke and traumatic brain injury is increased in animals that are deficient in NRF2. Insufficient NRF2 activation in humans has been linked to chronic diseases such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. New findings have also linked activation of the NRF2 system to anti-inflammatory effects via interactions with NF-κB. Here we review literature on cellular mechanisms of NRF2 regulation, how to maintain and restore NRF2 function and the relationship between NRF2 regulation and brain damage. We bring forward the hypothesis that inflammation via prolonged activation of key kinases (p38 and GSK-3β) and activation of histone deacetylases gives rise to dysregulation of the NRF2 system in the brain, which contributes to oxidative stress and injury.
    Neuropharmacology 11/2013; 79. DOI:10.1016/j.neuropharm.2013.11.004 · 5.11 Impact Factor
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