Physiological significance of reactive cysteine residues of Keap1 in determining Nrf2 activity.
Keap1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. Keap1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keap1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant Keap1(C273A) and/or Keap1(C288A) protein in Keap1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keap1(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keap1(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.
Yamamoto
Tae
T
Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan.
Suzuki
Takafumi
T
Kobayashi
Akira
A
Wakabayashi
Junko
J
Maher
Jon
J
Motohashi
Hozumi
H
Yamamoto
Masayuki
M
D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000375:Aging / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005804:Genes, Lethal / Q000235:genetics; D006706:Homeostasis; D006873:Hydroquinones / Q000494:pharmacology; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D006873:Hydroquinones; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C018855:2-tert-butylhydroquinone; D003545:Cysteine