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

http://www.ncbi.nlm.nih.gov/pubmed/15572695

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 RIS Download

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

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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

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