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Molecular and Cellular Biology, October 2004, p. 8477-8486, Vol. 24, No. 19
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.19.8477-8486.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Keap1-BTB Protein Is an Adaptor That Bridges Nrf2 to a Cul3-Based E3 Ligase: Oxidative Stress Sensing by a Cul3-Keap1 Ligase

Sara B. Cullinan,1 John D. Gordan,1 Jianping Jin,2 J. Wade Harper,2 and J. Alan Diehl1*

The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, Department of Cancer Biology, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania,1 Department of Pathology, Harvard Medical School, Boston, Massachusetts2

Received 10 May 2004/ Returned for modification 7 June 2004/ Accepted 1 July 2004

The Nrf2 transcription factor promotes survival following cellular insults that trigger oxidative damage. Nrf2 activity is opposed by the BTB/POZ domain protein Keap1. Keap1 is proposed to regulate Nrf2 activity strictly through its capacity to inhibit Nrf2 nuclear import. Recent work suggests that inhibition of Nrf2 may also depend upon ubiquitin-mediated proteolysis. To address the contribution of Keap1-dependent sequestration versus Nrf2 proteolysis, we identified the E3 ligase that regulates Nrf2 ubiquitination. We demonstrate that Keap1 is not solely a cytosolic anchor; rather, Keap1 is an adaptor that bridges Nrf2 to Cul3. We demonstrate that Cul3-Keap1 complexes regulate Nrf2 polyubiquitination both in vitro and in vivo. Inhibition of either Keap1 or Cul3 increases Nrf2 nuclear accumulation, leading to promiscuous activation of Nrf2-dependent gene expression. Our data demonstrate that Keap1 restrains Nrf2 activity via its capacity to target Nrf2 to a cytoplasmic Cul3-based E3 ligase and suggest a model in which Keap1 coordinately regulates both Nrf2 accumulation and access to target genes.

* Corresponding author. Mailing address: AFCRI, University of Pennsylvania Cancer Center, 454 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 746-6389. Fax: (215) 746-5511. E-mail: adiehl{at}mail.med.upenn.edu.

Molecular and Cellular Biology, October 2004, p. 8477-8486, Vol. 24, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.19.8477-8486.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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