In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to target Nrf2 for proteosome-mediated degradation under normal culture conditions. Upon exposure to chemical inducers of Nrf2-dependent transcription, Keap1-dependent ubiquitination of Nrf2 is inhibited, leading to accumulation of Nrf2 and allowing subsequent activation of Nrf2-dependent transcription. Mutation of a single cysteine residue in the BTB domain of Keap1 markedly reduces inhibition of Keap1-dependent ubiquitination of Nrf2 by oxidative stress or sulforaphane. The ability of Keap1 to function as a redox-sensitive substrate adaptor protein for an E3 ubiquitin ligase complex constitutes a novel mechanism by which cells are able to sense and respond to electrophilic chemicals and oxidative stress.
Keap1 is one of more than 50 human proteins that share an N-terminal BTB domain, a central linker domain, and a C-terminal Kelch domain. BTB-Kelch proteins appear to have diverse biological roles in the regulation of the cytoskeleton (7
). In Drosophila melanogaster
, the Kelch protein, which is the founding member of the BTB-Kelch family, binds actin and regulates the cross-linking of actin filaments at ring canals that form between cells in the D. melanogaster
). In humans, mutations within the GAN1 gene cause giant axonal neuropathy, an autosomal recessive disease characterized by defects in intermediate filament organization in sensorimotor neurons (6
). Our results provide direct biochemical evidence that Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1. Several other BTB-Kelch proteins are able to associate with Cul3, including GAN1 (19
). Furthermore, residues within the BTB domain of Keap1 that are conserved in other BTB-Kelch proteins, including a highly conserved serine residue within the BTB domain, S104, are required for ubiquitination of Nrf2 (S.-C. Lo and M. Hannink, unpublished data) and repression of Nrf2-dependent transcription (62
). A glycine substitution at the corresponding residue in GAN1 has been reported in a patient with giant axonal neuropathy (6
). We suggest that the ability of BTB-Kelch proteins to function as substrate adaptors for Cul3-dependent E3 ubiquitin ligase complexes reflects a conserved biochemical function that underlies their diverse biological functions.
The Skp1 protein, which functions as a linker between Cul1 and F-box substrate adaptor proteins, contains a BTB domain fold (61
). The crystal structure of the Skp1-Cul1-Rbx1 complex has been used to predict amino acids required for association of MEI-26, a C. elegans
BTB domain protein, with the C. elegans
Cul3 protein (58
). Therefore, we constructed two mutant Keap1 proteins that contained alanine substitutions in place of the corresponding residues in Keap1. To our surprise, these mutant Keap1 proteins displayed an increased ability to associate with Cul3 and Rbx1. Our results suggest that Keap1 interacts with Cul3 in a manner distinct from the way in which Skp1 interacts with Cul1. In other experiments, we find that the BTB domain of Keap1 is not sufficient to associate with Cul3 (data not shown), in agreement with a recent report by Yamamoto and coworkers suggesting that the linker domain of Keap1 is critically required for association with Cul3 (31
). Nevertheless, the BTB domain of Keap1 is required for efficient down-regulation of steady-state levels of Nrf2 (31
). Furthermore, mutations within the BTB domain of Keap1, as reported in the present study, can result in increased association with Cul3 and increased levels of autoubiquitination. Importantly, these mutant Keap1 proteins are impaired in their ability to efficiently target Nrf2 for ubiquitination and subsequent proteosome-mediated degradation. These results suggest that the balance between ubiquitination of substrate (Nrf2) and substrate adaptor (Keap1) may contribute to regulation of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex, perhaps by regulating steady-state levels of Keap1.
Substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes is often tightly regulated by changes in cell physiology induced by environmental signals or cell cycle progression. For example, the well-characterized F-box protein βTrCP, which functions as a substrate adaptor protein for Cul1, only recognizes substrate proteins that are phosphorylated on two serine residues embedded within a conserved sequence motif of DSG
). As a result, the activity of the SCF1βTrCP
E3 ubiquitin ligase towards its substrates, which include IκBα and β-catenin, is regulated at the level of substrate binding. We find that inducers of Nrf2, including sulforaphane and quinone-induced oxidative stress, result in accumulation of Nrf2 but do not abolish the ability of Nrf2 to bind to Keap1. Thus, the Keap1-dependent E3 ubiquitin ligase complex, in contrast to other cullin-dependent E3 ubiquitin ligase complexes typified by the SCF1βTrCP
complex, is not regulated at the level of substrate binding.
Recent experiments suggest that E3 ubiquitin ligase complexes that assemble around cullin scaffolds undergo cycles of assembly and disassembly that enable exchange of the core cullin-Rbx1 complex between different substrate adaptor proteins (12
). Substrate adaptor exchange is likely to be an important mechanism by which a new substrate molecule is brought into the complex. This substrate adaptor exchange model provides an attractive paradigm for understanding how Keap1-dependent ubiquitination of Nrf2 is regulated by oxidative stress or sulforaphane. Our results demonstrate that both sulforaphane and quinone-induced oxidative stress result in reduced association between Keap1 and Cul3. In contrast, association between Cul3 and the Keap1-C151S mutant protein, which is markedly resistant to inhibition by both sulforaphane and quinone-induced oxidative stress, is not significantly perturbed by either inducer of Nrf2. We propose that exposure of cells to sulforaphane or oxidative stress, by altering the redox state of Cys 151, reduces the ability of Nrf2-bound Keap1 proteins to associate with the Cul3-Rbx1 core complex. As a result, fewer Nrf2 molecules will be targeted for ubiquitination and subsequent degradation, leading to increased accumulation of Nrf2.
Nrf2 must also escape Keap1-mediated cytoplasmic sequestration in order to accumulate in the nucleus and activate gene expression. Keap1 binds to actin via its Kelch repeat domain and pharmacological disruption of the actin cytoskeleton enables Nrf2 to escape Keap1-mediated sequestration in the cytoplasm (29
). Inducers of Nrf2 may perturb the ability of Keap1 to associate with the actin cytoskeleton and thus enable release of Nrf2 into the nucleus. However, our results indicate that neither sulforaphane nor quinone-induced oxidative stress results in quantitative release of Nrf2 from Keap1. An alternative possibility is suggested by the observation that ongoing protein synthesis is required for accumulation of Nrf2 in the nucleus (24
). We propose that, under normal conditions, a single Keap1 protein is able to target multiple Nrf2 proteins for destruction. However, when the ability of Keap1 to efficiently target Nrf2 proteins for degradation is inhibited, each Keap1 protein (or Keap1 dimer) (62
), is only able to sequester a single Nrf2 protein. Thus, newly synthesized Nrf2 proteins will no longer be bound by Keap1 proteins and, instead, accumulate in the nucleus following transport from the cytoplasm. Consistent with this saturation model, overexpression of a Neh-GFP fusion protein enables nuclear accumulation of endogenous Nrf2 (5
The ability of structurally diverse chemicals to activate Nrf2-dependent gene expression correlates closely with their reactivity toward thiols (17
). Talalay and coworkers have identified four cysteine residues in Keap1 (Cys 257, Cys 273, Cys 288, and Cys 297) that are preferentially labeled following in vitro exposure of purified Keap1 to a cysteine-reactive alkylating agent (16
). Mutant Keap1 proteins containing serine substitutions for two of these residues (Cys 273 and Cys 288) are impaired in their ability to target Nrf2 for ubiquitination and to repress Nrf2-dependent gene expression in transfected cells (52
). In a previous study, members of our laboratory demonstrated that Cys 151 is required for both a novel redox-dependent alteration in Keap1 in cells exposed to oxidative stress and the ability of oxidative stress to activate Nrf2-dependent gene expression (60
). In the present work, we demonstrate that Cys 151 is required for inhibition of Keap1-dependent ubiquitination of Nrf2 by both sulforaphane and oxidative stress. Taken together, our results and those reported by Talalay's group suggest that multiple cysteine residues in Keap1 are capable of undergoing redox-dependent alterations. Identification of redox-dependent biochemical modifications that occur on the endogenous Keap1 protein will further our understanding of how cells sense the presence of reactive molecules and activate an Nrf2-dependent transcription program that protects sensitive biological molecules from chemical and oxidative damage.