Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2

Abstract

Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

FIG. 1.

Assay system to examine the degradation mechanism of Nrf2. (A) Keap1 promotes Nrf2 degradation in the in vivo degradation system. An Nrf2 expression vector (2 μg) was transfected into Cos7 cells (90% confluent) with or without the Keap1 expression vector (1.5 μg). At 24 h after transfection, the cells were treated with dimethyl sulfoxide (DMSO) (lanes 1, 2, 4, 6, and 8) and 2 μM MG132 (lanes 3, 5, 7, and 9) for 12 h and directly lysed in sodium dodecyl sulfate sample buffer. (Upper panel) Whole-cell extracts were subjected to immunoblot analysis with an anti-Nrf2 antibody. (Lower panel). The expression level of cotransfected EGFP was used as an internal control. (B) Proteasome-specific inhibitors stabilize the Nrf2 protein. Transfected cells were treated with DMSO (lane 4), 2 μM MG132 (lane 5), 2 μM clasto-lactacystin β-lactone (lane 6), and E64 (lane 7) for 12 h. Immunoblot analysis was performed as described above. (C and D) The Nrf2 expressed in this system was rapidly degraded in a Keap1-dependent manner. Nrf2 and ΔETGE mutant were transfected into cells along with Keap1. At 36 h after transfection, the cells were treated with 10 μM cycloheximide (CHX) per ml for the periods indicated. (Upper panel) Whole-cell extracts were subjected to immunoblot analysis with an anti-Nrf2 antibody. (Lower panel). The expression level of EGFP was used as an internal control. The averages of the relative band intensities of Nrf2 (open squares) and ΔETGE mutant (closed circles) represent two independent experiments performed in duplicate.

FIG. 2.

Rapid turnover of Nrf2 requires its association with Keap1. (A) Schematic presentation of Nrf2 deletion mutants. ΔC lacks the C terminus including the NLS of wild-type Nrf2. ΔC/ETGE lacks both this C terminus and the ETGE motif, which is crucial for association with Keap1. (B) Cytoplasmic localization of ΔC and ΔC/ETGE mutants in Cos7 cells (b and c). These mutant proteins were stained by an immunohistochemical method with anti-Nrf2 (C4) antibody. Nuclei were stained with DAPI (d to f). (C and D) Deletion of the ETGE motif abolished the degradation of Nrf2 by Keap1. This suggests that Keap1 positively regulates the degradation of Nrf2 through its association. The experimental procedure was described in the legend to Fig. 1. The averages of the band intensities of ΔC and ΔC/ETGE mutants represent two independent experiments done in duplicate.

FIG. 3.

BTB and IVR domains contribute to degradation of Nrf2 by Keap1. (A) Schematic presentation of Keap1 deletion mutants. Keap1 contains mainly three characteristic domains, the BTB, IVR, and DGR domains. (B) Deletion of the BTB and IVR domains abolished Nrf2 degradation in the in vivo degradation assay (top). The experimental procedure is described in the legend to Fig. 1. The expression of cotransfected EGFP was used as an internal control (bottom). (C) The expression of Keap1 deletion mutants was also monitored by immunoblot analysis with two anti-Keap1 antibodies against the C-terminal and N-terminal ends (lanes 1 to 4 and lanes 5 to 7, respectively). wt, wild type.

FIG. 4.

Keap1 associates specifically with Cul3 in vivo and in vitro. (A) Complex formation of Keap1 and Cul3 in 293T cells. Endogenous Keap1 was precipitated with anti-Keap1 antibody and protein G beads (IP). The immunocomplex was subjected to immunoblot analysis with anti-Cul3 antibody. Whole-cell extracts of 293T cells expressing human Cul3 were used as a control (lanes 3 and 4). (B) Association between Keap1 and Cul3 in a transient-expression system. Whole-cell extracts prepared from 293T cells transfected with expression plasmids of HA-tagged Keap1 (1 μg) and 3xFlag Cul3 (1 μg) were subjected to immunoprecipitation (IP) with anti-Flag (M2) beads and immunoblot analysis with anti-HA antibody (IB). Analyses of cells expressing 3xFlag Cul3 with or without HA-Keap1 (lanes 1 and 2) are shown. Lane 3 is loaded with cell extracts expressing HA-Keap1 alone. (C) Among the Cul family proteins, Cul3 specifically interacts with Keap1. Expression plasmids (1 μg each) of Cul1 (lanes 1 and 6), Cul2 (lanes 2 and 7), Cul3 (lanes 3 and 8), Cul4A (lanes 4 and 9), and Cul5 (lanes 5 and 10) were transfected into 293T cells in the presence (lanes 1 to 5) or absence (lanes 6 to 10) of Flag-fused Keap1. Immunoprecipitation and immunoblot analyses were performed as described above (top). The asterisk indicates a nonspecific band. The expression levels of Cul proteins and Flag-Keap1 were verified by immunoblot analysis with anti-Myc and anti-Keap1 antibodies (middle and bottom, respectively)

FIG. 5.

The IVR domain of Keap1 associates with the N-terminal end of Cul3. (A) Whole-cell extracts prepared from 293T cells transfected with various expression plasmids of HA-tagged Keap1 deletion mutants (1 μg) and 3xFlag tagged Cul3 (1 μg) were subjected to an immunoprecipitation (IP) assay with anti-Flag antibody beads and immunoblot (IB) analysis with anti-HA antibody (top). The expression levels of Keap1 deletion mutants and 3xFlag Cul3 were verified by immunoblot analysis with anti-HA and anti-Flag antibodies (middle and bottom, respectively). Analyses of cell lysates coexpressing 3xFlag Cul3 and HA-Keap1 (lane 2), ΔBTB (lane 3), ΔIVR (lane 4), or ΔDGR (lane 5) are shown. Lane 1 is loaded with cell extract expressing only 3xFlag Cul3. (B) Cysteine residues in the IVR domain are not crucial for the association between Keap1 and Cul3. The IVR contains seven cysteine residues (from Cys226 to Cys297), renumbered arbitrarily from 1 to 7. The association between Cul3 and Keap1 Cys point mutants (27) was examined by immunoprecipitation (upper panel) as described above. The expression levels of Cys mutants were verified by immunoblot analysis with anti-Keap1 antibody (lower panel). (C) Schematic presentation of Cul3 deletion mutants. (D) The N-terminal sequence of Cul3 is crucial for its association with Keap1. Whole-cell extracts of 293T cells transfected with expression plasmids of Cul3 deletion mutants (1 μg) and Keap1 (1 μg) were prepared and subjected to immunoprecipitation with anti-Flag (M2) beads and immunoblotting with anti-Keap1 antibody. Analyses of cell lysates expressing 3xFlag Cul3 (lanes 1 and 3), N280 (lanes 4 and 5), or ΔN280 (lanes 6 and 7) in the presence (lanes 2, 3, 5, and 7) or absence (lanes 1, 4, and 6) of Keap1 are shown (top). The expression levels of Keap1 and Cul3 deletion mutants were verified by immunoblot analysis with anti-Keap1 and anti-Flag antibodies (middle and bottom, respectively)

FIG. 6.

The BTB domain of Bach1 does not bind Cul3. Whole-cell extracts prepared from 293T cells transfected with expression plasmids (3 μg) in the combinations indicated were subjected to immunoprecipitation (IP) with anti-Flag antibody-conjugated beads followed by immunoblot (IB) analysis with an anti-Myc antibody (top). The expression level of each protein was monitored by immunoblot analysis with anti-Myc (middle) and anti-Flag (bottom) antibodies, respectively.

FIG. 7.

Ubiquitination of Nrf2 by Keap1 and Cul3 in vivo. Nrf2 (1 μg) was expressed in 293T cells, along with several combinations of Keap1 (0.5 μg) and Cul3 (1.5 μg)-Roc1 (1 μg), as indicated in the figure, in the presence of His-tagged ubiquitin (HisUb; 1 μg). As a control, the ΔETGE mutant was also transfected. Whole-cell extracts were prepared and subjected to affinity purification with Ni²⁺ resin. Precipitates (ppt) were eluted by boiling in sodium dodecyl sulfate sample buffer and subjected to immunoblot (IB) analysis (upper panel) with anti-Nrf2 antibody. The expression level of Nrf2 in the whole-cell extracts was also verified by immunoblot analysis with anti-Nrf2 antibody (lower panel).

FIG. 8.

Schematic model of the Keap1-Cul3 complex function as an E3 ligase. The cytoplasmic factor Keap1, bound on actin filaments, acts as a sensor for oxidative and electrophilic stress through two cysteine residues in the IVR domain. In the absence of stimuli, Keap1 sequesters the transcription factor Nrf2, a major regulator of the oxidative stress response, in the cytoplasm. In addition, Keap1 functions as an adaptor of the Cul3-based E3 ligase. This E3 ligase conjugates ubiquitin to Nrf2 and promotes rapid degradation of Nrf2 by proteasome in order to inhibit the expression of oxidative stress response genes under normal conditions.