Oxidoreductive modification of two cysteine residues in paired domain by Ref-1 regulates DNA-binding activity of Pax-8.
We have reported reversible oxidoreductive regulation of DNA-binding activity of Pax-8: oxidation inhibits its binding and subsequent reduction restores the binding. Here, we show that Cys-45 and Cys-57 in the paired domain of rat Pax-8, which are conserved in all Pax members, are responsible for the redox regulation of its binding. Electrophoretic mobility shift assay using deletion mutants and mutants with substitution of cysteine with serine revealed that oxidation by diamide of either Cys-45 or Cys-57 loses the DNA binding of Pax-8. An intracellular oxidoreductive enzyme redox factor-1 (Ref-1) could reduce the oxidized Cys-45 or Cys-57 and restored the binding. Furthermore, reporter gene assay showed that transcriptional activity of wild-type Pax-8 was enhanced by co-expression of Ref-1. When the mutant with double substitutions of Cys-45 and Cys-57, which was insensitive to oxidation, was transfected, the basal transactivation level was much higher than that of wild-type Pax-8, while it was not enhanced by Ref-1. These results demonstrated that oxidoreductive modification of Cys-45 and Cys-57 via Ref-1 plays a role in redox regulation of Pax-8 in living cells.
Cao
Xia
X
Division of Molecular and Cellular Adaptation, Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Japan.
Kambe
Fukushi
F
Ohmori
Sachiko
S
Seo
Hisao
H
D000818:Animals; D016466:CHO Cells; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010084:Oxidation-Reduction; D000072636:PAX8 Transcription Factor; D051761:Paired Box Transcription Factors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000072636:PAX8 Transcription Factor; C083325:PAX8 protein, human; D051761:Paired Box Transcription Factors; C499497:Pax8 protein, rat; D015534:Trans-Activators; D004706:Endodeoxyribonucleases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; C470319:Apex1 protein, rat; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine