In this study, we aimed to clarify the molecular mechanisms governing rapid Nrf2 turnover and the contribution of Keap1 to this degradation pathway. We revealed that the rapid degradation of Nrf2 requires direct association with Keap1. We also found that the IVR domain of Keap1 specifically interacts with Cul3, a component of the E3 ligase complex. These results provide the first convincing evidence for proteasomal degradation of Nrf2 and the function of Keap1 as an adaptor for Cul3-based E3 ligase (Fig. ). The critical stress-responsive transcription factors IκB, Hif-1α, and Nrf2 have now been shown to share the ubiquitin-proteasome system in their rapid turnover and to use specific Cul-type E3 ligases, with IκB, Hif-1α, and Nrf2 using Cul1, Cul2, and Cul3, respectively. To our knowledge, Nrf2 and Keap1 are the first mammalian substrate and adaptor reported for the Cul3-based E3 ligase system.
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, (more ...)
While Cul3 in Caenorhabditis elegans
was reported to ubiquitinate the substrate MEI-1/katanin through MEL26 as an adaptor (20
), the molecular mechanisms of Cul3 activity remain to be elucidated. A subset of proteins harboring the BTB domain were recently reported to serve as an adaptor in the Cul3-based E3 ligase system (4
). Our present data indicate that the BTB domain of Keap1 is necessary for Keap1 to function as an accelerator of Nrf2 degradation. However, one unexpected finding in this study is that deletion of the BTB domain does not significantly affect the association of Keap1 with Cul3. Rather, Keap1 effectively associates with Cul3 through the IVR domain. The BTB domain of Bach1 does not bind to Cul3 either (Fig. ), demonstrating that not all BTB domains interact with Cul3.
The molecular mechanism by which the IVR domain interacts with Cul3 is unclear at present. Zhang and Hannink recently reported that mutation of the reactive cysteines Cys273 and Cys288 in the IVR domain repressed Nrf2 degradation due to an impaired ubiquitin pathway (31
). Based on their observation, they proposed the hypothesis that these two cysteines in the IVR domain are crucial for the complex formation between Keap1 and an unknown E3 ligase. Although our present data indicate that Cul3 recognizes the IVR domain, our data clearly show that Cul3 binds to Keap1 despite mutations in the two reactive cysteines, indicating that Cul3 recognizes an alternative motif in the IVR domain. Furthermore, alanine substitutions of both Cys273 and Cys288 did not affect the ubiquitination of Nrf2 (data not shown). Thus, the present results disagree with their report, so this point remains to be clarified.
We envisage that two lines of evidence may be pertinent in this regard. First, the electrophilic reagent Dex-mes binds to the two reactive cysteines in the IVR domain (3
) and liberates Nrf2 from Keap1. Second, Keap1 containing alanine substitutions of both Cys273 and Cys288 did not effectively repress the transactivation activity of Nrf2 in a cell culture system (27
). Based on these observations, we propose that the two reactive cysteine residues in the IVR regulate the association of Keap1 with Nrf2.
Our results in Fig. suggest that the BTB domain of Keap1 contributes to the turnover of the Nrf2 protein. Consistent with these data Zipper and Mulcahy (33
) showed that the BTB domain of Keap1 is crucial for its dimerization and negative regulation of Nrf2. Furthermore, Zhang and Hannink (31
) have recently reported that a C151S Keap1 mutation in the BTB domain significantly reduced Nrf2 activation in response to oxidants (31
). Thus, while our analysis indicates that Cys151 is not a direct target of Dex-mes conjugation (3
), it seems likely that this residue contributes to the function of Keap1. Further work to clarify the role of BTB in the Keap1/Nrf2 pathway is required.
Stresses on the endoplasmic reticulum were recently reported to activate the Nrf2-Keap1 system through direct phosphorylation of Nrf2 by pancreatic endoplasmic reticulum eukaryotic initiation factor 2α kinase (2
). This observation suggests that the Nrf2-Keap1 system can be activated by signals other than oxidative stress. The ubiquitin-like protein NEDD8 regulates the E3 ligase activity of Cul3 by covalent modification, which is essential for the association of Cul3 with E2 enzyme (8
). The Cul3 complex contains a COP9 signalosome that functions as a deneddylation enzyme (1
). Hence, one emerging hypothesis is that down-regulation of Cul3 activity by deneddylation may enable Nrf2 stabilization, by which signals can induce the expression of cytoprotective target genes. Supporting this notion, the addition of a proteasome inhibitor was reported to induce the expression of the GCL gene, which encodes the catalytic subunit of γ-glutamylcysteine synthetase (22
). Under our present experimental conditions, however, we could not detect the promotion of NEDD8 modification of Cul3 by oxidative stress (data not shown). Nonetheless, we feel that it is still of interest to explore this hypothesis.
The Neh2 domain of Nrf2 is ubiquitinated in a Keap1-dependent manner and possesses seven lysine residues, some of which might be conjugated with a polyubiquitin chain (Y. Kato, K. Itoh, and M. Yamamoto, submitted for publication). In addition, deletion of the C-terminal Neh1, Neh3, and Neh6 domains of Nrf2 renders the protein more stable than full-length Nrf2 (Fig. and , compare 7.5 and 25 min, respectively), suggesting that Nrf2 contains an alternative degradation signal in this C-terminal portion. We previously showed two modes of Nrf2 degradation, namely, Keap1-dependent degradation in the cytoplasm and Keap1-independent degradation in the nucleus (9
). The turnover of Nrf2 by the latter pathway is slower than that by the former. We surmise that Nrf2 degradation in the nucleus also uses the proteasome pathway but must be Cul3 independent. Therefore, the next important task would be to identify an E3 ligase complex that is essential for the nuclear degradation of Nrf2.
We exploited Cul3 double-stranded RNA (dsRNA) in a preliminary examination to assess whether endogenous Cul3 regulates the degradation of Nrf2 in collaboration with Keap1. Cul3 dsRNA was transfected into HeLa cells, and cyclin E was first analyzed as a control, since Cul3 was reported to determine the stability of cyclin E in the ubiquitin-proteasome system (24
). However, although transfection of Cul3 dsRNA significantly affected the expression of Cul3 mRNA, only a marginal accumulation of cyclin E was found (data not shown). Similarly, down-regulation of Cul3 did not affect the stability of endogenous Nrf2 (data not shown). One plausible explanation for this observation is that because the dsRNA did not completely abrogate the Cul3 protein, the residual small amount of Cul3 might have been enough for rapid degradation of Nrf2 to take place. Supporting this hypothesis, we found that expression of Keap1 alone significantly enhanced the ubiquitination of Nrf2 in an in vivo ubiquitin assay whereas further expression of Cul3 and Roc1 enhanced ubiquitination only marginally (Fig. ). These results suggest that Cul3 and Roc1 are abundant within living cells. An alternative explanation is that Keap1-independent degradation, as described above, might compensate for the loss of Cul3 function.
The function of Keap1 as an E3 ligase adaptor has similarity to the function of pVHL. It has been well documented that multiple human diseases are provoked by mutations in pVHL. These mutations usually affect the function of pVHL as an adaptor and cause aberrant stabilization of Hif-1α (11
). Thus, cellular homeostasis requires not only inducible activation of transcription factors in response to stress stimuli but also continuous inactivation of the transcription factors through their rapid degradation or subcellular compartmentalization during unstressed conditions. Interestingly, a subset of Cul proteins requires WD40 repeat proteins as an adaptor to target substrates. This WD40 repeat domain is known to form a β-barrel structure (28
). The DGR domain of Keap1 also forms a β-barrel structure. These observations further support our contention that Cul-based E3 ligases have common properties in both function and structure.