Cysteine 64 of Ref-1 is not essential for redox regulation of AP-1 DNA binding.
Ref-1 participates in DNA repair as well as in redox regulation of transcription factor function. The redox function of Ref-1 involves reduction of oxidized cysteine residues within the DNA binding domains of several transcription factors, including Fos and Jun. Reduction of these residues is required for DNA binding, providing a redox-dependent mechanism for regulation of target gene expression. Previous in vitro studies implicated cysteine 65 of human Ref-1 (cysteine 64 of mouse Ref-1) as the redox catalytic site. We analyzed the in vivo role of cysteine 64 in redox regulation of AP-1 activity by introducing a cysteine-to-alanine point mutation into the endogenous mouse Ref-1 gene (ref-1(C64A)). Unlike Ref-1 null mice, which die very early in embryonic development, homozygous ref-1(C64A) mice are viable, they survive to normal life expectancy, and they display no overt abnormal phenotype. Although Ref-1 provides the major AP-1-reducing activity in murine cells, ref-1(C64A) cells retain normal levels of endogenous AP-1 DNA binding activity in vivo as well as normal Fos- and Jun-reducing activity in vitro. These results demonstrate that Ref-1 cysteine 64/65 is not required for redox regulation of AP-1 DNA binding in vivo, and they challenge previous hypotheses regarding the mechanism by which Ref-1 regulates the redox-dependent activity of specific transcription factors.
Ordway
Jared M
JM
Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Eberhart
Derek
D
Curran
Tom
T
D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D006720:Homozygote; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D015533:Transcriptional Activation
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D004247:DNA; D006861:Hydrogen Peroxide; D019757:Carbon-Oxygen Lyases; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine; D010100:Oxygen