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) (537LKKQLSTLYL546) 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. 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) (537LKKQLSTLYL546) 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. Mammalian liver cells possess elaborate and highly efficient cytoprotective machinery. When challenged with oxidative or electrophilic stress, these cells respond with rapid and coordinated expression of antioxidant gene products, including heme oxygenase 1 (HO-1) 1The abbreviations used are: HO-1, heme oxygenase 1; ARE, antioxidant response element; bZIP, basic leucine zipper; CNC, cap-and-collar domain; DBD, DNA binding domain; DEM, diethyl maleate; GFP, green fluorescent protein; EGFP, enhanced GFP; GSH, reduced glutathione; GST, glutathione S-transferase; Keap1, Kelch-like ECH-associated protein 1; NAC, N-acetyl-l-cysteine; Neh2, Nrf2-ECH homology domain 2; Nrf2, NF-E2-related factor 2; NLS, nuclear location signals; NES, nuclear export signals; CRM1, chromosome region maintenance; LMB, leptomycin B; DAPI, 4′,6-diamidine-2′-phenylindole; IP, SUL, sulforaphane. (1.Alam J. Stewart D. Touchard C. Boinapally S. Choi A.M. Cook J.L. J. Biol. 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In the study we a canonical NES with the leucine zipper motif of Nrf2. This Nrf2-NES binds to and is to mutations the leucine in the NES nuclear export expression of this Nrf2-NES exclude the GAL4 DNA binding domain the is the Nrf2-NES to be redox-insensitive. this NES and the mechanisms nuclear localization of Nrf2 of Nrf2 and the antioxidant response. and and cells cells as a with and and cells as with and and and and and of Nrf2 by Kan and Chan of The ZIP domain of Nrf2 (amino acids 503–589) and into by The enhanced green fluorescent protein The GAL4 DNA binding domain and into by to a protein. also into as an to an of or as and as in a mutations the site-directed to the with and to leucine to The and by DNA in of of and with of of DNA and as 1 of and and by a The in the with the into cells The and by DNA and or cells in a of cells to of into of with of and of the of as C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). into of of in a to the of and The with with and cell to with to and an with and in a with and the a as N. PubMed Scopus Google Scholar). The of by and of to into the a of in the of The by cells with and in of and and into 1 of with The expression of by the of the cells by The in and 1 and by The cells 1 of to of and by The into an and with and protein with and and of GST and GST and into to a GST to their The into E. and in with with the cells and in of the cell with and into the with the GST proteins with the GST of GST or proteins with of to of protein and The with and 1 and by and cells in of or the in cell 1 1 and The protein of cell by the cell of proteins with of protein. 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This Nrf2-NES nuclear export by and site-directed When this Nrf2-NES could the nuclear distribution of into a cytosolic distribution show that the Nrf2-NES may a in the localization of Nrf2. is also that the of the NES with the leucine zipper motif of Nrf2. this Nrf2-NES to be redox-insensitive. these help of the mechanisms the transcriptional activation of Nrf2. the of Nrf2 activation mainly Keap1 A. T. Yamamoto M. 2004; PubMed Scopus Google Scholar). is Nrf2 nuclear is In study the of a NES in Nrf2 that Nrf2 is a or an In Nrf2 is Keap1 in the that may be nuclear export of Nrf2 to in the cytoplasm. This NES may to be to nuclear In other Keap1 may be the to Nrf2 in the cytoplasm. The NES motif we in Nrf2 to the canonical NES J.L. 82: Full Text PDF PubMed Scopus Google Scholar, U. J. 82: Full Text PDF PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). NES a motif as of to be acids such as and be J.L. 82: Full Text PDF PubMed Scopus Google Scholar, U. 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Full Text Full Text PDF PubMed Scopus Google Scholar). be to of Nrf2 a in nuclear treatment with protein or of with NES in the of in the this of the with a may help to this In study the distribution of appeared to be by treatment with oxidants and or reducing compounds and other redox compounds be to that this Nrf2-NES is redox-insensitive. other and NES in Nrf2 may also to be redox that the Nrf2-NES located in the ZIP domain is in to the that the nuclear export of protein by a redox-sensitive NES Kobayashi A. M. N. T. N. Yamamoto M. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and Kobayashi A. M. N. T. N. Yamamoto M. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the redox of the NES in to key The that is in Nrf2-NES or the of may the between Nrf2-NES and the the NES in the ZIP domain as well as other NES in Nrf2 Nrf2 may a activation Nrf2 when by is a activation is well that the the of Nrf2 Keap1 could be a redox-sensitive in the in Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus (524) Google Scholar, Itoh K. Wakabayashi N. Yamamoto M. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). the subsequent could the importance of the step as the rate-limiting step Nrf2 activation. This may also in the of the Keap1 to of of Nrf2 is in the mice N. Itoh K. Wakabayashi J. S. S. S. T. Harada T. J.D. Yamamoto M. PubMed Scopus Google Scholar). the redox of Nrf2 is a redox-sensitive in the basic region in the of the and DNA binding domain D. S. 2002; PubMed Scopus Google Scholar). of this the Keap1 retention and nuclear of Nrf2. the the DNA binding of Nrf2 D. S. 2002; PubMed Scopus Google Scholar). The and of redox of of Nrf2 in the may help to a of Nrf2 activation by oxidative The of a NES in Nrf2 help to the activation of Nrf2, may also help decipher the of of Nrf2. the to the activation of Nrf2, the of Nrf2 in the nucleus may also be of Nrf2 and a antioxidant response may be as to the to respond to Nrf2 a rapid The of Nrf2 is as as T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. E. S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that Nrf2 is in the cytoplasm T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, D. E. S. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), may also the nucleus K. Wakabayashi N. T. O'Connor T. Yamamoto M. PubMed Scopus Google Scholar). Nrf2 be and to rapid of the redox of the study Nrf2-NES as a the signal of Nrf2. In the of nuclear the Nrf2-NES could be by formation with the ZIP Nrf2 in the the in the to nuclear to be be an In the study a NES in Nrf2 in the leucine zipper binds to and is to could also exclude nuclear protein into cytoplasm. we also that this Nrf2-NES appeared to be Because the of transcription is the of a of and NES, the characterization of the NES in Nrf2 is the step to the mechanisms the nuclear and export of Nrf2, a the transcription of cytoprotective genes. Chan and Kan and in and the of the and of this