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
Source: PubMed


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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    • "The 98 kDa Nrf2 is considered the transcriptionally active form [3]. This form is upregulated in RAW 264.7 cells and diminished in HepG2. "

    • "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
    • "The identities of Nrf2 bands were established in that work by treating cells with the canonical Nrf2 activators sulforaphane (SFN) and tert-butyl hydroquinone (tBHQ), which allow Nrf2 to escape from ubiquitination and degradation, leading to accumulation of Nrf2 protein levels. The slower migrating band of the two appears to be due to phosphorylation (Apopa et al., 2008; Pi et al., 2007). The anomalously high migration and the occurrence of two bands are both resolved to a large extent by utilizing Bis–Tris gradient gels (Lau et al., 2013). "

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