Nrf2 Possesses a Redox-insensitive Nuclear Export Signal Overlapping with the Leucine Zipper Motif

Wenge Li,Mohit R Jain,Chi Chen,Xin Yue,Vidya Hebbar,Renping Zhou,A.-N Tony Kong

doi:10.1074/jbc.m410601200

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.

Highlights

Mammalian liver cells possess elaborate and highly efficient cytoprotective machinery
In the C terminus there is a canonical bipartite nuclear location signals (NLSs) localized in the basic region featuring 2 tandems of 3– 4 basic amino acids separated by 10 amino acids [33]
In searching for a nuclear export signal (NES) we found embedded in the leucine zipper domain a leucine-rich motif (537L*KKQLSTL*YL546) that conforms to the canonical NES motif of ⌽XXX⌽XX⌽X⌽ [34,35,36], where ⌽ stands for long chain hydrophobic amino acid residues, and X can be any amino acid

Summary

Introduction

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)1 [1] and ␥-glutamylcysteine synthetase and NAD(P)H:quinone oxidoreductase 1 [2,3,4] as well as phase II-detoxifying enzymes such as glutathione S-transferase (GST) [2, 3], quinone reductase [4], and UDP-glucuronosyltransferase [3]. Pivotal to this antioxidant response is a transcriptional factor Nrf (NF-E2-related factor 2) [5]. 5Ј-GCT GAA AAC TTC GGC ATA TGC GGT GCT GGC TTG TTT TTT CGC TAG GTG-3Ј

Methods

Results

Conclusion