Multiple Nuclear Localization Signals Function in the Nuclear Import of the Transcription Factor Nrf2

Melanie Theodore,Yumiko Kawai,Jianqi Yang,Yuliya Kleshchenko,Sekhar P Reddy,Fernando Villalta,Ifeanyi J Arinze

doi:10.1074/jbc.m709040200

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

Highlights

Nuclear factor erythroid 2-related factor 2 (Nrf2),4 in association with the cytoskeleton-associated Kelch-like protein Keap1, functions as a sensor of oxidative and electrophilic stress in cells [1,2,3,4]
We demonstrate that importin ␣5 and importin ␤1 are involved in the nuclear import of Nrf2
Nrf2 Mutated at the NLS1 or NLS3 Does Not Inhibit Promoter-inducing Activity of Wild-type Nrf2 or Stimulus-induced Nuclear Translocation of Wild-type Nrf2—Because mutation in any of the nuclear localization signal (NLS) motifs in Nrf2 resulted in its failure to localize to the nucleus as well as in its failure to transactivate antioxidant response element(s) (AREs)-driven reporter gene constructs, we considered the possibility that such mutations may confer a dominant negative property on the mutant species

Summary

The abbreviations used are

Nuclear factor erythroid 2-related factor 2; ARE, antioxidant response element; GFP, green fluorescent protein; EGFP, enhanced GFP ( known as red-shifted variant of wild-type GFP); Keap, Kelch-like ECH-associated protein 1; NLS, nuclear localization signal; Pck, coding sequence of rat liver cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK-C); tBHQ, tert-butylhydroquinone; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; NFAT, nuclear factor of activated T-cells; EMSA, electrophoretic mobility shift assay. A number of reports have indicated that phosphorylation at Ser of Nrf (in the cytoplasm) by protein kinase C, which appears to be concomitant with its activation, is not necessary for its nuclear import [24, 25] Those results, do not rule out the involvement of phosphorylation at other sites, by other kinases, or phosphorylation of potential accessory protein(s) that might impact its nuclear translocation. Nrf is not a direct target of MAPK (i.e. ERK), Zipper and Mulcahy [26] showed that MAPK-directed phosphorylation is a requirement for nuclear localization of Nrf during pyrrolidine dithiocarbamate-induced expression of glutamate cysteine ligase They suggest a model involving ERK-mediated phosphorylation of some type of accessory protein that might be required for Nrf nuclear translocation. We demonstrate that importin ␣5 and importin ␤1 are involved in the nuclear import of Nrf

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION