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Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

Pubmed ID: 15572695


  • Zhang DD
  • Lo SC
  • Cross JV
  • Templeton DJ
  • Hannink M


Molecular and cellular biology

Publication Data

December 1, 2004

Associated Grants

  • Agency: NIGMS NIH HHS, Id: 1R01 GM 59213
  • Agency: NCI NIH HHS, Id: CA 66134
  • Agency: NCI NIH HHS, Id: P50 CA 103130

Mesh Terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Anticarcinogenic Agents
  • Antioxidants
  • Breast Neoplasms
  • COS Cells
  • Cell Cycle Proteins
  • Cell Line, Tumor
  • Cercopithecus aethiops
  • Cullin Proteins
  • DNA-Binding Proteins
  • Genes, Reporter
  • Humans
  • Hydroquinones
  • Immunoblotting
  • Intracellular Signaling Peptides and Proteins
  • Isothiocyanates
  • Luciferases
  • Lysine
  • NF-E2-Related Factor 2
  • Oxidation-Reduction
  • Oxidative Stress
  • Precipitin Tests
  • Protein Structure, Tertiary
  • Proteins
  • Serine
  • Substrate Specificity
  • Thiocyanates
  • Trans-Activators
  • Ubiquitin-Protein Ligases
  • Ubiquitins