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.
Zhang
Donna D
DD
Department of Biochemistry, University of Missouri-Columbia, M121 Medical Sciences Building, Columbia, MO 65212, USA.
Lo
Shih-Ching
SC
Cross
Janet V
JV
Templeton
Dennis J
DJ
Hannink
Mark
M
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001943:Breast Neoplasms / Q000473:pathology; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; D000072019:Kelch-Like ECH-Associated Protein 1; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D051267:NF-E2-Related Factor 2; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D016588:Anticarcinogenic Agents; D000975:Antioxidants; C503645:CUL3 protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; D006873:Hydroquinones; D047908:Intracellular Signaling Peptides and Proteins; D017879:Isothiocyanates; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D011506:Proteins; D013454:Sulfoxides; D013861:Thiocyanates; D015534:Trans-Activators; D014452:Ubiquitins; D012694:Serine; C018855:2-tert-butylhydroquinone; D008156:Luciferases; D044767:Ubiquitin-Protein Ligases; C016766:sulforaphane; D008239:Lysine