Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome.
Under homeostatic conditions, Nrf2 activity is constitutively repressed. This process is dependent on Keap1, to which Nrf2 binds through the Neh2 domain. Since the N-terminal subdomain of Neh2 (Neh2-NT) contains evolutionarily conserved motifs, we examined the roles they play in the degradation of Nrf2. In Neh2-NT, we defined a novel motif that is distinct from the previously characterized DIDLID motif and designated it DLG motif. Deletion of Neh2-NT or mutation of the DLG motif largely abolished the Keap1-mediated degradation of Nrf2. These mutations were found to enfeeble the binding affinity of Nrf2 to Keap1. The Neh2-NT subdomain directed DLG-dependent, Keap1-independent, degradation of a reporter protein in the nucleus. By contrast, mutation of DLG did not affect the half-life of native Nrf2 protein in the nucleus under oxidative stress conditions. These results thus demonstrate that DLG motif plays essential roles in the Keap1-mediated proteasomal degradation of Nrf2 in the cytoplasm.
Katoh
Yasutake
Y
Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan.
Iida
Katsuyuki
K
Kang
Moon-Il
MI
Kobayashi
Akira
A
Mizukami
Mio
M
Tong
Kit I
KI
McMahon
Michael
M
Hayes
John D
JD
Itoh
Ken
K
Yamamoto
Masayuki
M
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1; D007930:Leucine / Q000737:chemistry; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2; D041681:NIH 3T3 Cells; D018384:Oxidative Stress; D010957:Plasmids; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014452:Ubiquitins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D046988:Proteasome Endopeptidase Complex; D007930:Leucine