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J. Biol. Chem., Vol. 283, Issue 14, 8984-8994, April 4, 2008

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

Melanie Theodore¹, Yumiko Kawai, Jianqi Yang², Yuliya Kleshchenko, Sekhar P. Reddy^¶, Fernando Villalta, and Ifeanyi J. Arinze³

From the School of Medicine and the School of Graduate Studies and Research, Meharry Medical College, Nashville, Tennessee 37208-3599, the Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, and the ^¶Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205

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 5 and β1 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 -β heterodimer nuclear import receptor system plays a critical role in the import process.

Received for publication, November 5, 2007 , and in revised form, January 31, 2008.

^* This work was supported in part by National Institutes of Health Grants SO6-GM08037 and HL-66109. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Center of Excellence Grant HRSA D34HP00003 (to the School of Medicine, Meharry Medical College), by Recruitment in Science Education Grant 5 R25 GM059994 (to the School of Graduate Studies and Research, Meharry Medical College), and by NHLBI, National Institutes of Health Grant T32HL007737.

² Supported by National Institutes of Health Metabolism Training Grant DK-07319.

³ To whom correspondence should be addressed. Tel.: 615-327-6586; Fax: 615-327-6442; E-mail: iarinze{at}mmc.edu.

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