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Molecular and Cellular Biology, April 2008, p. 2758-2770, Vol. 28, No. 8
0270-7306/08/$08.00+0     doi:10.1128/MCB.01704-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Physiological Significance of Reactive Cysteine Residues of Keap1 in Determining Nrf2 Activity{triangledown}

Tae Yamamoto,1,{dagger} Takafumi Suzuki,1,2,{dagger} Akira Kobayashi,3 Junko Wakabayashi,1 Jon Maher,1 Hozumi Motohashi,2,3 and Masayuki Yamamoto1,2,3*

Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan,1 Environmental Response Project, Exploratory Research for Advanced Technology-Japan Science and Technology Corporation, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan,2 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan3

Received 15 September 2007/ Returned for modification 18 October 2007/ Accepted 4 February 2008

Keap1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. Keap1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keap1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant Keap1(C273A) and/or Keap1(C288A) protein in Keap1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keap1(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keap1(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.

* Corresponding author. Mailing address: Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan. Phone: 022-717-8084. Fax: 022-717-8090. E-mail: masi{at}mail.tains.tohoku.ac.jp

{triangledown} Published ahead of print on 11 February 2008.

{dagger} T.Y. and T.S. contributed equally to this work.

Molecular and Cellular Biology, April 2008, p. 2758-2770, Vol. 28, No. 8
0270-7306/08/$08.00+0     doi:10.1128/MCB.01704-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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