Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system.
BACKGROUND
The transcription factor Nrf2 and its negative regulator Keap1 play important roles in transcriptional induction of a set of detoxifying and anti-oxidant enzymes. To gain an insight into our present enigma as to how cells receive oxidative and electrophilic signals and transduce them to Nrf2, we have developed a zebrafish model system for molecular toxicological studies.


RESULTS
We systematically cloned zebrafish cytoprotective enzyme cDNAs and found their expression to be efficiently induced by electrophilic agents. We consequently identified the presence of Nrf2 and Keap1 in zebrafish. Both loss- and gain-of-function analyses demonstrated that Nrf2 is the primary regulator of a subset of cytoprotective enzyme genes, while Keap1 suppresses Nrf2 activity in zebrafish. An ETGE motif, critical for the Nrf2-Keap1 interaction, was identified in the Neh2 domain of Nrf2 by reverse two-hybrid screening and found to be indispensable for the regulation of Nrf2 activity in zebrafish.


CONCLUSIONS
Taken together, these results indicate that the Nrf2-Keap1 system is highly conserved among vertebrates and that the interface between Nrf2 and Keap1 forms an important molecular basis of this regulatory system.
Kobayashi
Makoto
M
The Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8577, Japan.
Itoh
Ken
K
Suzuki
Takafumi
T
Osanai
Hitoshi
H
Nishikawa
Keizo
K
Katoh
Yasutake
Y
Takagi
Yaeko
Y
Yamamoto
Masayuki
M
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D006873:Hydroquinones / Q000494:pharmacology; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D015027:Zebrafish; D029961:Zebrafish Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D006873:Hydroquinones; C117301:Keap1 protein, mouse; C527640:Keap1a protein, zebrafish; D000072019:Kelch-Like ECH-Associated Protein 1; D008840:Microfilament Proteins; D051267:NF-E2-Related Factor 2; C495636:Nfe2l2 protein, mouse; D015534:Trans-Activators; D029961:Zebrafish Proteins; C495638:nfe2l2a protein, zebrafish; C018855:2-tert-butylhydroquinone