Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1

Abstract

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.

FIG. 1.

Oxidative/electrophilic stress provokes the nuclear accumulation of de novo Nrf2 protein. Wild-type MEF cells were treated with tBHQ (lanes 2 to 9; final concentration, 100 μM) or DMSO (lanes 1 and 10 to 13) in the presence (lanes 6 to 13) or absence (lanes 1 to 5) of the protein synthesis inhibitor cycloheximide (10 μM) for different time points as indicated in the figure. Nuclear extracts were prepared and subjected to immunoblot analysis using anti-Nrf2 and anti-lamin B antibodies (top and bottom panels).

FIG. 2.

Keap1 requires Cys273 and Cys288 to suppress Nrf2 activity. (A) Schematic structures of the cysteine mutants of the IVR domain. (B) Keap1 repression activity was measured by luciferase assay. Expression plasmids for Nrf2 and Keap1 wild type (lanes 3 and 9) or cysteine mutants (lanes 4 to 6, 10, and 11) (90 ng and 10 ng, respectively) were transfected into NIH 3T3 cells (2 × 10⁴) along with pNQO1-ARE reporter plasmid (50 ng) and pRL-TK (50 ng) as an internal control. At 36 h after transfection, the luciferase activity was measured according to the manufacturer's instructions. Assays were performed twice in triplicate.

FIG. 3.

The Keap1-C273&288A mutant sequestrates but does not degrade Nrf2 in the cytoplasm. (A) Expression plasmids for Nrf2 and Flag-tagged Keap1 or Keap1-C273&288A mutant were transfected into Cos7 cells. At 36 h after transfection, the cells were subjected to immunohistochemical staining using anti-Nrf2 (a to d) and anti-Flag (M2) (e to h) antibodies. Nuclei were stained with DAPI (i to l). (B) The subcellular localization of Nrf2 was classified into three categories: predominantly cytoplasmic localization (C > N, blue bars), roughly equal localization between the cytoplasmic and nuclear compartments (C = N, yellow bars), and predominantly nuclear localization (C < N, red bars). The subcellular localization within 100 cells is shown. (C) Immunoblot analysis using fractionated cell extracts of transfected cells. The transfection method was performed as described above. The cells were subjected to cell fractionation into cytoplasmic (C) and nuclear (N) fractions and subsequent immunoblot analysis. Anti-lamin B and anti-α-tubulin antibodies were used as markers for the nuclear and cytoplasmic extracts, respectively.

FIG. 4.

Reduced Cys273 and Cys288 are crucial for the ubiquitin-dependent degradation of Nrf2. (A) Alanine mutation or oxidation of Cys273 and Cys288 impairs Keap1-mediated degradation of Nrf2. The degradation activity of Keap1 was monitored by an in vivo degradation assay. The expression plasmids for Nrf2 and wild type or C273&288A mutant Keap1 (2 μg and 1.5 μg, respectively) were transfected into Cos7 cells, as indicated in the figure. EGFP plasmid (50 ng) was cotransfected to verify the transfection efficiency. At 24 h after transfection, cells were treated with DMSO (lanes 1 to 3 and 5) or tBHQ (lane 4; final concentration, 100 μM) for 12 h. Whole-cell extracts were prepared and subjected to immunoblot analysis using anti-Nrf2 and anti-GFP antibodies (top and bottom panels, respectively). (B) Alanine mutation or oxidation of Cys273 and Cys288 impairs Keap1-mediated ubiquitination of the Neh2 domain. An in vivo ubiquitination assay was performed. A GFP-fused Neh2 domain that harbors the Keap1-dependent ubiquitination site was used in this assay (Neh2-GFP). The expression plasmids for Neh2-GFP and wild-type Keap1 or Keap1-C273&288A mutant were transfected into 293T cells, as indicated in the figure, along with a His-tagged ubiquitin (HisUb) plasmid. At 24 h after transfection, cells were treated with MG132 (final concentration, 2 μM) in the absence (lanes 1 to 3 and 5) or presence (lane 4; final concentration, 100 μM) of tBHQ for 12 h. Whole-cell extracts were prepared and subjected to Ni²⁺ affinity purification. Precipitates were visualized by immunoblot analysis with anti-Nrf2 antibody.

FIG. 5.

Oxidative/electrophilic stress does not cause dissociation of the Keap1-Nrf2 complex. (A) The effects of in vivo tBHQ treatment on the association between Keap1 and Nrf2 were examined by immunoprecipitation. Expression plasmids for Flag-tagged Nrf2 and Keap1 (2 μg and 1.5 μg, respectively) were transfected into 293T cells. At 24 h after transfection, cells were treated with tBHQ (lane 4, 100 μM final concentration) or the proteasome inhibitor MG132 (lane 5, 2 μM final concentration) for 12 h. Whole-cell extracts were prepared and subjected to immunoprecipitation experiments using anti-Flag antibody beads. Immunoprecipitates were visualized by immunoblot analysis with anti-Keap1 and anti-Nrf2 antibodies (top and middle panels, respectively). The expression levels of Keap1 in whole-cell extracts were monitored by immunoblot analysis with an anti-Keap1 antibody (bottom panel). (B and C) In vitro tBHQ treatment does not cause dissociation of the Keap1-Nrf2 complex. Transfection was performed as described above, and whole-cell extracts were prepared from the transfected 293T cells. The expression levels of Nrf2 and Keap1 mutant were monitored by immunoblot analysis using anti-Nrf2 and anti-Keap1 antibodies (B, top and bottom panels, respectively). Whole-cell extracts were treated with DMSO (C, lanes 4 and 8) or tBHQ (lanes 5 and 9, 10 μM final concentration; lanes 6 and 10, 50 μM final concentration; lanes 7 and 11, 500 μM final concentration) for 4 h at 4°C and subjected to immunoprecipitation with anti-Flag antibody beads. Precipitates were visualized by immunoblot analysis using anti-Keap1 and anti-Nrf2 antibodies (top and bottom panels, respectively). Lanes 1 to 3 were loaded with the immunoprecipitates of cells transfected with empty, wild-type Keap1, and C273&288A mutant Keap1 expression vectors, respectively.

FIG. 6.

Oxidative/electrophilic stress does not affect formation of the Keap1-Nrf2 complex. Electrophilic modification of Keap1 with biotinylated 15d-PGJ₂ does not affect formation of the Keap1-Nrf2 complex. 293T cells expressing Keap1 were treated with biotinylated 15d-PGJ₂ (Bio-PGJ₂, lanes 1, 4, and 5; 10 μM final concentration) for 1 h. Whole-cell extracts were prepared and mixed with whole-cell extracts of Nrf2-expressing (lanes 1, 3, and 5) or wild-type (lanes 2 and 4) cells. Biotinylated Keap1 was precipitated from the mixed cell extracts using avidin beads, and precipitates were subjected to immunoblot analysis with anti-Nrf2 and anti-Keap1 antibodies (top and bottom panels, respectively).

FIG. 7.

Schematic model of the Nrf2 activation mechanism that is tightly coupled to Keap1-mediated degradation of Nrf2. Under homeostatic conditions, Nrf2 is sequestered in the cytoplasm by the Keap1-Cul3 complex and rapidly degraded in the ubiquitin-proteasome dependent manner. This Keap1-mediated degradation activity requires two reactive cysteine residues (Cys273 and Cys288) of Keap1. After the oxidative/electrophilic stress challenge, modification of these cysteine residues of Keap1 inhibits ubiquitin conjugation to Nrf2 by the Keap1-Cul3 complex, thereby provoking opening of the Keap1 gate and resulting in the nuclear accumulation of Nrf2.