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Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response.

Sun Z, Huang Z, Zhang DD - PLoS ONE (2009)

Bottom Line: Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation.These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity.We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.

ABSTRACT
The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.

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Phosphorylation of Nrf2 moderately enhances its nuclear accumulation without affecting the interaction between Nrf2 and Keap1.(A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
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pone-0006588-g004: Phosphorylation of Nrf2 moderately enhances its nuclear accumulation without affecting the interaction between Nrf2 and Keap1.(A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.

Mentions: Keum et al. have reported that phosphorylation of Nrf2 by p38 MAPKs increased the interaction between Nrf2 and Keap1 in an in vitro GST-pulldown experiment [33]. To test whether similar effects can be observed in vivo, HEK293T cells were co-transfected with an expression vector for either HA-tagged Nrf2-WT or Nrf2-5A, along with expression vectors for CBD-tagged Keap1, and the indicated MAPK. Cell lysates were pulled-down with chitin beads and the precipitated protein complexes were analyzed by immunoblot with anti-HA and anti-CBD antibodies (Figure 4A). The interaction between Nrf2 and Keap1 was not affected by the phosphorylation status of Nrf2, consistent with the finding that phoshorylation of Nrf2 does not affect its protein stability (Figure 3E).


Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response.

Sun Z, Huang Z, Zhang DD - PLoS ONE (2009)

Phosphorylation of Nrf2 moderately enhances its nuclear accumulation without affecting the interaction between Nrf2 and Keap1.(A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2719090&req=5

pone-0006588-g004: Phosphorylation of Nrf2 moderately enhances its nuclear accumulation without affecting the interaction between Nrf2 and Keap1.(A) HEK293T cells were cotransfected with expression vectors for the indicated MAPKs, HA-tagged Nrf2, and CBD-tagged Keap1. Cells were harvested and lysed in RIPA buffer. Cell lysates were pulled-down with chitin beads and analyzed by immunoblot with anti-HA and anti-CBD antibodies. CBD: chitin binding domain; wt: wild-type; 5A: mutant with combined alanine substitution on all five phosphorylation sites. (B) NIH3T3 cells were transfected with expression vectors for the indicated Nrf2 protein and Keap1. The subcellular localization of Nrf2 was determined by indirect immunofluorescence analysis with anti-HA antibodies. (C) Quantification analysis of the immunofluorescence assay. At least 100 positively stained cells were examined by fluorescence microscopy. Percentages of cells in which Nrf2 was localized predominantly in the cytosol, the whole cell, or the nucleus were presented as a bar graph.
Mentions: Keum et al. have reported that phosphorylation of Nrf2 by p38 MAPKs increased the interaction between Nrf2 and Keap1 in an in vitro GST-pulldown experiment [33]. To test whether similar effects can be observed in vivo, HEK293T cells were co-transfected with an expression vector for either HA-tagged Nrf2-WT or Nrf2-5A, along with expression vectors for CBD-tagged Keap1, and the indicated MAPK. Cell lysates were pulled-down with chitin beads and the precipitated protein complexes were analyzed by immunoblot with anti-HA and anti-CBD antibodies (Figure 4A). The interaction between Nrf2 and Keap1 was not affected by the phosphorylation status of Nrf2, consistent with the finding that phoshorylation of Nrf2 does not affect its protein stability (Figure 3E).

Bottom Line: Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation.These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity.We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.

ABSTRACT
The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.

Show MeSH
Related in: MedlinePlus