Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression.
Keap1 is a negative regulator of Nrf2, a bZIP transcription factor that mediates adaptation to oxidative stress. Previous studies suggested this negative regulation is a consequence of Keap1 controlling the subcellular distribution of Nrf2. We now report that Keap1 also controls the total cellular level of Nrf2 protein. In the RL34 non-transformed rat liver cell line, Nrf2 was found to accumulate rapidly in response to oxidative stress caused by treatment with sulforaphane, and the accumulation resulted from inhibition of proteasomal-mediated degradation of the bZIP protein. By heterologously expressing in COS1 cells epitope-tagged Nrf2 and an Nrf2DeltaETGE mutant lacking the Keap1-binding site, in both the presence and absence of Keap1 we demonstrate that Nrf2 is subject to ubiquitination and proteasomal degradation independently of both Keap1 and the redox environment of the cell. In oxidatively stressed cells, this is the sole mechanism responsible for Nrf2 degradation. However, under homeostatic conditions Nrf2 is subject to a substantially more rapid mode of proteasomal degradation than it is in oxidatively stressed cells, and this rapid turnover of Nrf2 requires it to interact with Keap1. Within Nrf2, the N-terminal Neh2 domain is identified as the redox-sensitive degron. These data suggest that Keap1 negatively regulates Nrf2 by both enhancing its rate of proteasomal degradation and altering its subcellular distribution.
McMahon
Michael
M
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom. m.j.m.mcmahon@dundee.ac.uk
Itoh
Ken
K
Yamamoto
Masayuki
M
Hayes
John D
JD
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015151:Immunoblotting; D017879:Isothiocyanates; D009097:Multienzyme Complexes / Q000378:metabolism*; D051267:NF-E2-Related Factor 2; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010957:Plasmids / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D013347:Subcellular Fractions; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
D000975:Antioxidants; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D000939:Epitopes; D017879:Isothiocyanates; D009097:Multienzyme Complexes; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D012333:RNA, Messenger; D013454:Sulfoxides; D013861:Thiocyanates; D015534:Trans-Activators; D025801:Ubiquitin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C016766:sulforaphane