title: Nrf2 Possesses a redox-insensitive nuclear export signal overlapping with the leucine zipper motif.
abstract: Basic leucine zipper (bZIP) protein Nrf2 is a key transcription factor mediating the antioxidant response. Under homeostatic conditions Nrf2 is anchored to cysteine-rich Keap1 and sequestered in the cytoplasm. When challenged with oxidative stress, Keap1 functions as a redox-sensitive switch and releases Nrf2. Subsequently, Nrf2 translocates into the cell nucleus and binds to a cis-acting enhancer called the antioxidant response element located in the promoters of a battery of cytoprotective genes and initiates their transcription. In this study we identify a canonical nuclear export signal (NES) ((537)LKKQLSTLYL(546)) located in the leucine zipper (ZIP) domain of the Nrf2 protein. The enhanced green fluorescent protein-tagged ZIP domain of Nrf2 (amino acids 503-589) exhibited a CRM1-dependent cytosolic distribution that could be abrogated by site-directed mutations or treatment with the nuclear export inhibitor, leptomycin B. Ectotopic expression of the Nrf2-NES could also exclude the GAL4 DNA binding domain into the cytoplasm. This NES overlapped with the ZIP motif in Nrf2, suggesting that the formation of heterodimers between Nrf2 and other bZIP proteins may simultaneously mask the NES and attenuate Nrf2 nuclear export. The Nrf2-NES appeared to be redox-insensitive. Neither oxidants (sulforaphane and diethyl maleate) nor reducing compounds (N-acetyl-l-cysteine and reducing glutathione) could disrupt the cytosolic distribution of Nrf2zip. Because Nrf2 activation is generally redox-sensitive, the redox insensitivity of this Nrf2-NES indicates the importance of Keap1 retention as a key rate-limiting step in Nrf2 activation. The characterization of the Nrf2 NES may help decipher the mechanisms governing nuclear localization and subsequent transcriptional activation of Nrf2-mediated cytoprotective genes.
authors.affiliation: Department of Pharmaceutics, Ernest-Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, USA.
authors.forename: Wenge
authors.initials: W
authors.lastname: Li
authors.forename: Mohit R
authors.initials: MR
authors.lastname: Jain
authors.forename: Chi
authors.initials: C
authors.lastname: Chen
authors.forename: Xin
authors.initials: X
authors.lastname: Yue
authors.forename: Vidya
authors.initials: V
authors.lastname: Hebbar
authors.forename: Renping
authors.initials: R
authors.lastname: Zhou
authors.forename: A-N Tony
authors.initials: AN
authors.lastname: Kong
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016350:Leucine Zippers / Q000502:physiology*; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051267:NF-E2-Related Factor 2; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; C495637:Nfe2l2 protein, rat; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators