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

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.

Figure 1. Nrf2 is phosphorylated in vivo at multiple sites by MAPKs.

(A) HEK293T cells were cotransfected with expression vectors for HA-tagged Nrf2 and the indicated MAPKs. Cells were lysed in denaturing conditions. Cell lysates were diluted and immunoprecipitated with anti-HA antibodies. The immunoprecipitated protein was analyzed by immunoblot with antibodies specific for phosphorylated serine or threonine residues adjacent to a proline (pS/TP) (Abcam ab9344). p-Nrf2: phosphorylated Nrf2; Tub: tubulin. (B–F) Identification of distinct phosphorylation sites by tandem mass spectrometry analysis. Nrf2 proteins were purified through immunoprecipitation with anti-HA antibodies from HEK293T cells overexpressing HA-tagged Nrf2. The Nrf2 protein was size-separated by SDS-PAGE and visualized by Coomassie blue staining. The gel containing the Nrf2 protein was isolated and subjected to mass spectrometry analysis. m/z: mass to charge ratio.

Figure 2. Combined alanine substitution on all phosphorylation sites causes only a moderate decrease in the transcriptional activity of Nrf2.

(A) MDA-MB-231 cells were cotransfected with expression vectors for the GSTA1-ARE-dependent firefly luciferase, the TK-Renilla luciferase, and the indicated Nrf2 mutant. Luciferase reporter gene activities were analyzed using the Promega dual-luciferase reporter gene assay system. WT: wild-type; TK: thymidine kinase promoter; Inr: initiator. Relative luciferase activities and standard deviations were calculated from three independent assays. * p<0.05 when compared to WT. (B) Reporter gene assay was performed as described above using NQO1-ARE-dependent luciferase reporter gene construct in MDA-MB-231 cells. (C) Total cell lysates from one of the reporter gene assays were subjected to immunoblot analysis with anti-HA antibodies for detection of Nrf2.

Figure 3. Genetic disruption of Nrf2 phosphorylation slightly attenuates Nrf2 activities without affecting its protein levels.

(A) HEK293T cells were cotransfected with expression vectors for HA-tagged Nrf2 and JNK2. Cell lysates were immunoprecipitated with anti-HA antibodies. The immunoprecipitated protein was analyzed by immunoblot with antibodies specific for phosphorylated serine or threonine residue adjacent to a proline (pS/TP) (Abcam ab9344). wt: wild-type; 5A: the mutant with combined alanine substitution on all five phosphorylation sites. (B–C) HEK293T cells overexpressing either Nrf2-WT or Nrf2-5A were subjected to immunoblot (B) and qRT-PCR analysis (C). The error bars indicate the standard deviations calculated from triplicate samples. * p<0.05 when compared to wt. (D) MDA-MB-231 cells were cotransfected with expression vectors for the GSTA1-ARE-dependent luciferase reporter gene, Keap1, and the indicated Nrf2. Cells were treated with several Nrf2 inducers for 16 hrs prior to reporter gene analysis. tBHQ: tert-butylhydroquinone (50 µM); SF: sulforaphane (20 µM); PGJ2: 15-Deoxy-Δ-12,14-prostaglandin J2 (10 µM); As: sodium arsnite (20 µM); Orid: Oridonin (10 µM); Cd: cadmium chloride (20 µM); H₂O₂: hydrogen peroxide (500 µM). Error bars indicate standard deviations calculated from three independent assays. * p<0.05 when compared to wt in the same treatment group. (E) Total cell lysates from the above reporter gene assay were analyzed by immunoblot.

Figure 4. 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.