The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase

Abstract

The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.The Nrf2 transcription factor regulates the expression of antioxidant genes following cellular insults that induce oxidative stress (2, 3, 4, 13). Under homeostatic conditions, Nrf2 remains in an inactive cytoplasmic form through association with the bricabrac, tramtrack, and broad complex (BTB) domain-containing protein Keap1 (17). In response to endoplasmic reticulum stress or oxidative stress, Nrf2-Keap1 dissociation is triggered and Nrf2 accumulates in the nucleus, where it forms an active heterodimeric transcription factor, inducing the transcription of target genes involved in redox homeostasis (5,6,14,15,17).Although it is clear that Keap1 can maintain Nrf2 in the cytoplasm, accumulation of many transcriptional regulators is also suppressed through the action of the 26S proteasome. Although it was initially thought that Nrf2 activation was strictly regulated through inhibition of nuclear import, increasing evidence indicates that Nrf2 protein levels are maintained at low levels through proteasome-mediated degradation (18,20,21,24,28). The fact that Keap1 has been implicated in both Nrf2 cytoplasmic sequestration and proteolysis suggests a model in which the regulation of Nrf2 activity is tightly regulated by proteolysis in the cytoplasmic compartment. Similar modes of regulation have been documented for other critical cellular regulators, such as p53 (9, 22) and cyclin D1 (8).In general, proteins are targeted to the 26S proteasome through the covalent attachment of polyubiquitin chains. Ubiquitin conjugation is mediated by the sequential activities of an E1 enzyme, which mediates the ATP-dependent activation of ubiquitin, an E2 ubiquitin-conjugating enzyme (Ubc), and an E3 ubiquitin ligase; E2 and E3 function to coordinate the transfer of ubiquitin to the substrate protein. In addition to functioning in ubiquitin transfer, E3 generally drives substrate specificity and has thus been of intense interest.The SCF liga...

Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype

“…One of these siRNA, Target1, has been shown to increase Nrf2 protein in HaCaT keratinocyte cells, resulting in up-regulation of endogenous human antioxidant genes and an elevation in intracellular GSH. While the present study was being undertaken, two groups reported using plasmid-delivered short hairpin RNA against mouse Keap1 in 293T cells to demonstrate that Keap1 functions as an adaptor protein for the ubiquitination of Nrf2 by the Cul3-Roc1 ligase (21,22). In neither instance, however, was the effect of RNA interference on the expression of endogenous target genes investigated.…”

“…Humans possess 51 proteins containing both BTB͞POZ and Kelch repeat domains; Keap1 is a member of subclass 1 (19). Keap1 controls the half-life of Nrf2 by acting as a substrate adaptor protein for the cullin 3-Roc1 ubiquitin E3 ligase (20)(21)(22). Under homeostatic conditions, Keap1 promotes degradation of Nrf2 via the 26S proteasome, but this activity is lost during redox stress.…”

Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells

“…Nrf2 localizes in the cytoplasm in the absence of an activator, though a small fraction of Nrf2 mediates constitutive transcription of a number of target genes in the nucleus in uninduced cells. The cytoplasmic Nrf2 forms a complex with Keap1, which functions as a key regulator of Nrf2 [10,13,15,16]. According to this model, Keap1, a cytoplasmic protein, sequesters Nrf2 in the cytoplasm and serves as a substrate adaptor, bringing Nrf2 to the cullin 3 (Cul3) dependent E3 enzyme that catalyzes the ubiquitination of the Nrf2 protein.…”

Sites of alkylation of human Keap1 by natural chemoprevention agents

“…By binding to the 5=-upstream regulatory antioxidant response element (ARE) regions of cytoprotective genes, Nrf2 upregulates their transcription [2,3], thus protecting cells against damage by reactive oxygen intermediates or other electrophilic species [4]. Keap1 functions as a bridge between Nrf2 and the Cullin3-based E3-ligase ubiquitination complex, promoting ubiquitination and subsequent proteasomal degradation of Nrf2 [5][6][7], thus preventing nuclear accumulation of Nrf2. Without the intervention of Keap1, levels of Nrf2 are elevated in the nucleus causing upregulation of the ARE [8].…”