title: Absolute Amounts and Status of the Nrf2-Keap1-Cul3 Complex within Cells.
abstract: The transcription factor Nrf2 (NF-E2-related-factor 2) is essential for the oxidative and electrophilic stress responses. Keap1 (Kelch-like-ECH-associated-protein 1), an adaptor for a cullin-3 (Cul3)-based ubiquitin ligase, regulates Nrf2 activity through proteasomal degradation, and acts as a sensor for oxidative and electrophilic stresses. The Keap1-Cul3 complex is a critical regulator of the cellular Nrf2 level, and yet quantitative information regarding their endogenous intracellular concentrations in homeostatic conditions and during stress responses is unknown. We analyzed the absolute amounts of the Nrf2, Keap1, and Cul3 proteins in five murine cell lines by comparison with serial dilutions of purified recombinant protein standards in combination with quantitative immunoblot analyses. In the basal state, the amount of Nrf2 was maintained at lower levels than those of Keap1 and Cul3 proteins, whereas the electrophilic agent diethylmaleate dramatically increased Nrf2 to a level greater than that of Keap1 and Cul3, resulting in the accumulation of Nrf2 in the nucleus. In contrast, Keap1 and Cul3 did not display any changes in their abundance, subcellular localization, or interaction in response to electrophilic stimuli. Our results demonstrate that the regulation of the Nrf2 protein level during stress responses is mediated by the activity but not the composition of the Nrf2-Keap1-Cul3 complex.
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Iso
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan taka23@med.tohoku.ac.jp masiyamamoto@med.tohoku.ac.jp.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Liam
authors.initials: L
authors.lastname: Baird
authors.affiliation: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan taka23@med.tohoku.ac.jp masiyamamoto@med.tohoku.ac.jp.|Tohoku Medical-Megabank Organization, Sendai, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D044842:Cullin Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D000072019:Kelch-Like ECH-Associated Protein 1 / Q000378:metabolism*; D008298:Maleates / Q000494:pharmacology; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D018384:Oxidative Stress; D000067996:RAW 264.7 Cells; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: C503646:Cul3 protein, mouse; D044842:Cullin Proteins; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D008298:Maleates; D046912:Multiprotein Complexes; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D011994:Recombinant Proteins; C014476:diethyl maleate