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.
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, 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
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, 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
Mol. Cell. Biol.
Date: Dec. 01, 2004
PubMed ID: 15572695
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