Nrf2 Possesses a Redox-sensitive Nuclear Exporting Signal in the Neh5 Transactivation Domain

 Antioxidant response is an important cytoprotective reaction. When exposed to oxidative stress, mammalian cells can respond with a rapid and coordinated expression of diverse antioxidant genes such as heme oxygenase 1 (HO-1)3 (1), {gamma}-glutamyl-cysteine synthetase/ligase, NAD(P)H:quinone oxidoreductase 1 (24), and the phase II detoxifying enzymes such as glutathione S-transferase (GST) (2, 3) and UDP-glucuronosyltransferase (4). Pivotal to the antioxidant response is a transcription factor called Nrf2 (nuclear factor-erythroid 2-related factor 2) (5). Nrf2 knock-out mice display reduced constitutive and inducible expression of HO-1 and GST (69). Nrf2 null mice are also more susceptible to the chemical toxin treatments (10, 11). Nrf2 is a member of the basic leucine zipper (bZIP) transcription factor subfamily featuring a Cap `n Collar motif (5). Like many transcription factors, Nrf2 signaling is regulated by subcellular localization (12). Under homeostatic conditions, Nrf2 molecules are predominantly sequestered in the cytoplasm. When stimulated with oxidative or electrophilic compounds, Nrf2 proteins can quickly translocate to the nucleus and form heterodimers with bZIP proteins called small Maf (musculoaponeurotic fibrosarcoma) proteins (13, 14). Nrf2/Maf heterodimer formation can enhance the specificity and binding of Nrf2 to a cis-acting enhancer called antioxidant-responsive element (ARE) (15) located in the promoter of many antioxidant and phase II detoxification genes (1619).

 Until now, Nrf2 signaling is generally considered to be regulated by a cysteine-rich protein called Keap1 (Kelch-like ECH-associated protein 1) (20, 21). Nrf2 forms a dimer with Keap1 in vitro (22) and probably in vivo as well. An ETGE motif (17) and a DLG motif (23) of the Neh2 (Nrf2 ECH homology 2) domain of Nrf2 have been elucidated to mediate cooperative binding with the Kelch/double glycine repeat domain of Keap1 (19, 24). Keap1 is an actin-binding protein (19). Treatments with compounds to dissolve the cytoskeleton can result in Nrf2 nuclear accumulation (25). So the cytosolic sequestering of Nrf2 is attributed to Keap1 retention. Keap1 is also identified as a Cullin 3-dependent adaptor protein for ubiquitin ligase ubiquitin protein isopeptide ligase (2628). Therefore, Nrf2 molecules may not only be sequestered by Keap1 but also subjected to constant degradation. In vitro, the Nrf2/Keap1 dimer can only be formed under reducing conditions (22) and can be disrupted by the treatment of phyto-oxidant sulforaphane or the phenolic compound tert-butylhydroquinone (tBHQ) (22). Some cysteine residues in Keap1, such as Cys-151, Cys-273, and Cys-288, are found to be critical in Nrf2 retention and release (24, 29). Based on these observations, it is hypothesized that Keap1 functions as a redox-switch for Nrf2 signaling (22). Whereas reducing conditions favor Keap1 retention of Nrf2, oxidative signals can induce Keap1 to quickly release Nrf2, leading to nuclear translocation of Nrf2 (20).

 Although the Keap1 anchoring model seems to successfully explain the repression and activation of Nrf2 signaling in response to the changing redox conditions, some controversial observations are reported recently. It is reported that the cytosolic distribution of Keap1 is maintained by active nuclear export rather than cytoskeleton anchoring (30, 31). In certain cell lines, such as hepatoma HepG2 and H4IIEC3 cells, remarkably high amounts of endogenous Nrf2 are found in the cell nucleus at unstimulated conditions (32). Even if the Keap1 anchoring model is still applicable in these cells, the amount or activity of cytosolic Keap1 proteins may not be sufficient to sequester Nrf2. Furthermore, the basal expression of Nrf2-driven antioxidant genes such as HO-1, the modifier subunit of glutamyl-cysteine ligase, and Prdx1 (peroxiredoxin I) remained unchanged in the livers of Keap1 knock-out mice (33). Conceptually, the necessity of Keap1 in Nrf2 signaling is based on the assumption that Nrf2 is unable to maintain cytosolic segregation by itself under quiescent conditions. In addition, the redox-switching role of Keap1 assumes that Nrf2 per se lacks redox responsiveness, or the redox responsiveness Nrf2 is functionally irrelevant to its nuclear translocation. To date, only one reactive cysteine is characterized in the basic region of Nrf2 (34). Mutation of this cysteine to a serine only alters its DNA binding affinity but fails to alter the subcellular localization of Nrf2 (34). These data therefore seem to provide further support to those assumptions. However, with the rapid progress in Nrf2 studies, those assumptions may merely reflect our limited knowledge about Nrf2. Recent studies have identified a nuclear export signal (NES) in the ZIP domain of Nrf2 (NESzip) and a bipartite nuclear localization signal (NLS) in the basic region of Nrf2 (bNLS) (35, 36). These discoveries raise the question whether Nrf2 possesses other functional NES or NLS motifs. In this study, we identify a new functional NES located in the transactivation (TA) domain of Nrf2. The existence of multiple NES and NLS motifs in Nrf2 enables Nrf2 to maintain cytosolic segregation by itself under quiescent conditions. Furthermore, unlike the NESzip, this NESTA possesses a reactive cysteine residue (Cys-183). An enhanced green fluorescence protein (EGFP)-tagged Nrf2 segment (amino acids 162-295), called EGFP-NESTA, exhibited a dosage-dependent nuclear translocation when treated with sulforaphane. Therefore, Nrf2 can sense the redox signal and translocate to the nucleus. These discoveries suggest Nrf2 may be able to transduce redox signals in a Keap1-independent manner. Keap1, however, may provide an additional regulation of the quantity of Nrf2 both at basal and at inducible conditions.

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