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Molecular and Cellular Biology, February 2006, p. 1235-1244, Vol. 26, No. 4
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.4.1235-1244.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1

Shih-Ching Lo and Mark Hannink*

Department of Biochemistry, Life Science Center, University of Missouri—Columbia, Columbia, Missouri 65212

Received 30 August 2005/ Returned for modification 23 September 2005/ Accepted 4 December 2005

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. Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to repress steady-state levels of Nrf2 and Nrf2-dependent transcription. Cullin-dependent ubiquitin ligase complexes have been proposed to undergo dynamic cycles of assembly and disassembly that enable substrate adaptor exchange or recycling. In this report, we have characterized the importance of substrate adaptor recycling for regulation of Keap1-mediated repression of Nrf2. Association of Keap1 with Cul3 was decreased by ectopic expression of CAND1 and was increased by small interfering RNA (siRNA)-mediated knockdown of CAND1. However, both ectopic overexpression and siRNA-mediated knockdown of CAND1 decreased the ability of Keap1 to target Nrf2 for ubiquitin-dependent degradation, resulting in stabilization of Nrf2 and activation of Nrf2-dependent gene expression. Neddylation of Cul3 on Lys 712 is required for Keap1-dependent ubiquitination of Nrf2 in vivo. However, the K712R mutant Cul3 molecule, which is not neddylated, can still assemble with Keap1 into a functional ubiquitin ligase complex in vitro. These results provide support for a model in which substrate adaptor recycling is required for efficient substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes.

* Corresponding author. Mailing address: Department of Biochemistry, Life Science Center, M121 Medical Sciences Building, University of Missouri—Columbia, Columbia, MO 65212. Phone: (573) 882-7971. Fax: (573) 884-9395. E-mail: hanninkm{at}missouri.edu.

Molecular and Cellular Biology, February 2006, p. 1235-1244, Vol. 26, No. 4
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.4.1235-1244.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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