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Molecular and Cellular Biology, December 2004, p. 10941-10953, Vol. 24, No. 24
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.24.10941-10953.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex

Donna D. Zhang,1 Shih-Ching Lo,1 Janet V. Cross,2 Dennis J. Templeton,2 and Mark Hannink1*

Department of Biochemistry, Life Sciences Center, University of Missouri—Columbia, Columbia, Missouri,1 Department of Pathology, University of Virginia, Charlottesville, Virginia2

Received 29 June 2004/ Returned for modification 1 August 2004/ Accepted 20 September 2004

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

* Corresponding author. Mailing address: Department of Biochemistry, University of Missouri—Columbia, M121 Medical Sciences Building, Columbia, MO 65212. Phone: (573) 882-7971. Fax: (573) 884-4597. E-mail: hanninkm{at}missouri.edu.

{dagger} D.D.Z. and S.-C.L. contributed equally to this work.

Molecular and Cellular Biology, December 2004, p. 10941-10953, Vol. 24, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.24.10941-10953.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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