Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes.
Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.
Kang
Moon-Il
MI
Center for Tsukuba Advanced Research Alliance and Japan Science and Technology Agency-Exploratory Research for Advanced Technology Environmental Response Project, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan.
Kobayashi
Akira
A
Wakabayashi
Nobunao
N
Kim
Sang-Geon
SG
Yamamoto
Masayuki
M
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019610:Cytoprotection / Q000235:genetics*; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism*; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D006801:Humans; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D051267:NF-E2-Related Factor 2; D041681:NIH 3T3 Cells; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C117301:Keap1 protein, mouse; D000072019:Kelch-Like ECH-Associated Protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; C495636:Nfe2l2 protein, mouse; D016877:Oxidants; D015534:Trans-Activators; D045647:DNA Glycosylases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C473642:NEIL2 protein, human