Cysteine 64 of Ref-1 is not essential for redox regulation of AP-1 DNA binding

Abstract

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.

FIG. 1.

(A) Diagram of gene-targeted mutagenesis strategy. See the text for details. ref-1^C64ANeo, ref-1^C64A with neomycin resistance cassette. (B) Southern blot screen for gene-targeted ES cell clones. DNA from neomycin-resistant ES cell clones was digested with SacI and BglII and hybridized to an external probe as shown in panel A. The NeoTK cassette includes a SacI site, allowing detection of homologous recombination by the presence of a 9.1-kb fragment in addition to the wild-type 10.8-kb fragment. Lane 6 represents a gene-targeted clone. (C) Confirmation of gene targeting by PCR followed by restriction enzyme digestion. Exon 2 was PCR amplified from ES cell DNA, and amplicons were digested with PvuII. Lanes: 1, DNA ladder; 2, wild-type undigested amplicons; 3, wild-type PvuII-digested amplicons; 4 and 6, ref-1^C64A/+ undigested amplicons; 5 and 7, ref-1^C64A/+ PvuII-digested amplicons. Arrows mark bands amplified from wild-type alleles and digested bands amplified from ref-1^C64A alleles as indicated. (D) Confirmation of the endogenous ref-1^C64A mutation by genomic sequencing. Ref-1 exon 2 was PCR amplified from genomic DNA extracted from wild-type and ref-1^C64A/C64A primary embryonic fibroblasts, and amplicons were sequenced. Codons are labeled by mouse Ref-1 amino acid number and identity. Cysteine/alanine 64 is underlined. Sequencing verified the TG-GC mutation at codon 64 and the silent TC-AG mutation at codon 65. (E) Western blot analysis of Ref-1 protein in wild-type and ref-1^C64A/C64A cells. Lanes: 1, 20 μg of wild-type embryonic fibroblast nuclear extract; 2, 20 μg of ref-1^C64A/C64A embryonic fibroblast nuclear extract. Western blot analysis was performed with a polyclonal antibody specific for Ref-1 (43).

FIG. 2.

The C64A mutation does not affect Ref-1 AP endonuclease activity. Supercoiled pBluescript II plasmid was mock-treated or acid-treated to induce AP lesions as indicated. Plasmids were reacted with 50 ng of bovine serum albumin (BSA; lanes 2 and 12), 50 ng of recombinant human Ref-1 (rRef-1; lanes 3 and 13), 50 ng of recombinant human C65A Ref-1 (rC65A; lanes 4 and 14), nuclear extract from wild-type primary embryonic fibroblasts (10, 50, and 100 ng [lanes 5 to 7 and 15 to 17, respectively]), or nuclear extract from ref-1^C64A/C64A primary embryonic fibroblasts (10, 50, and 100 ng [lanes 8 to 10 and 18 to 20, respectively]). Ref-1 AP endonuclease activity was assayed by the conversion of supercoiled plasmid (SC) to an open circular form (OC).

FIG. 3.

Ref-1^C64A/C64A tissues retain normal Fos/Jun-reducing activity. Oxidized recombinant Fos and Jun peptides were incubated to allow heterodimerization. Extracts were added and Fos/Jun DNA binding was determined by EMSA by using a radiolabeled oligonucleotide probe containing an AP-1 DNA binding site. Lanes: 1, dilution buffer alone; 2, 10 mM DTT; 3 to 5, 10, 25, and 50 ng of wild-type lung extract, respectively; 6 to 8, 10, 25, and 50 ng of ref-1^C64A/C64A lung extract, respectively; 9 to 11, 10, 25, and 50 ng of wild-type heart extract, respectively; 12 to 14, 10, 25, and 50 ng of ref-1^C64A/C64A heart extract, respectively. Free probe is indicated by an arrow.

FIG. 4.

(A) Serum induction does not affect Fos/Jun-reducing activity in wild-type or ref-1^C64A/C64A primary embryonic fibroblast extracts. Fos/Jun-reducing activity was determined by EMSA as described in the legend to Fig. 3. Primary embryonic fibroblasts were cultured in medium containing 0.5% FCS for 4 days and then stimulated with medium containing 20% FCS. Cells were harvested at various time points throughout the 24-h stimulation. Lanes: 1, dilution buffer alone; 2, 10 mM DTT; 3 to 11, 1-μg wild-type nuclear extracts harvested at 0, 0.2, 0.5, 1, 2, 3, 5, 10, and 24 h poststimulation, respectively; 12 to 20, 1-μg ref-1^C64A/C64A nuclear extracts harvested at 0, 0.2, 0.5, 1, 2, 3, 5, 10, and 24 h poststimulation, respectively. Free probe and recombinant Fos/Jun-bound probe (rFos/rJun) are indicated by arrows. (B) Serum-induced endogenous AP-1 DNA binding is not compromised in ref-1^C64A/C64A cells. Nuclear extracts were analyzed by EMSA as described for panel A except without added recombinant Fos and Jun peptides. Lanes are identical to those described for panel A. Free probe and recombinant Fos/Jun- or endogenous AP-1-bound probe are indicated by arrows. (C) Fos, Jun, and Ref-1 protein levels at selected time points during serum induction. Extracts described for panels A and B were subjected to Western analysis with antibodies for the indicated proteins. Lanes: 1 to 5, wild-type extracts at 0, 0.5, 1, 3, and 5 h poststimulation, respectively; 6 to 10, ref-1^C64A/C64A extracts at 0, 0.5, 1, 3, and 5 h poststimulation, respectively. Antibodies are indicated on the right of the panel. Molecular masses in kilodaltons are indicated on the left on the panel.

FIG. 5.

(A) Wild-type and ref-1^C64A/C64A embryonic fibroblasts are equally susceptible to H₂O₂ treatment. Primary embryonic fibroblast cultures were treated with H₂O₂ at concentrations ranging from 100 nM to 1 mM for 24 h. Mock-treated cultures and cultures treated with 1 μM and 10 μM H₂O₂ are shown at 5 and 24 h posttreatment, as indicated. (B) H₂O₂ treatment does not affect Fos/Jun-reducing activity in wild-type or ref-1^C64A/C64A primary embryonic fibroblast extracts. Recombinant Fos/Jun-reducing activity was determined by EMSA as described in the legend to Fig. 3. Lanes: 1, mock-treated sample; 2, 10 mM DTT; 3 to 8, wild-type embryonic fibroblast extracts at 0, 0.5, 1, 3, 6, and 24 h following 1 μM H₂O₂ challenge, respectively; 9 to 14, ref-1^C64A/C64A embryonic fibroblast extracts at 0, 0.5, 1, 3, 6, and 24 h following 1 μM H₂O₂ challenge, respectively; 15, extract dilution buffer alone. The recombinant Fos/Jun complex (rFos/rJun) and free probe are indicated by arrows. (C) H₂O₂-induced AP-1 DNA binding is enhanced in ref-1^C64A/C64A cells. Endogenous AP-1 DNA binding activity was determined as described in the legend to Fig. 3. Lanes: 1 to 6, wild-type embryonic fibroblasts assayed at 0, 0.5, 1, 3, 6, and 24 h following challenge with 1 μM H₂O₂; 7 to 12, ref-1^C64A/C64A embryonic fibroblasts assayed at 0, 0.5, 1, 3, 6, and 24 h following challenge with 1 μM H₂O₂. Free probe and endogenous AP-1-bound probe are indicated by arrows.

FIG. 6.

(A) All recombinant proteins were expressed as six-His-tagged fusion proteins and purified as described in Materials and Methods. Ten micrograms of each recombinant protein was run on a sodium dodecyl sulfate-polyacrylamide gel and stained with Coomassie blue. Lanes: 1, wbFos (1); 2, TK550 Jun (22); 3, wild-type Ref-1; 4, C65A Ref-1. (B) Recombinant C65A Ref-1 retains Fos/Jun-reducing activity. Recombinant Ref-1 proteins were assayed for redox activity by EMSA as described in the legend to Fig. 3. Recombinant protein stocks were adjusted to 10 mg/ml to ensure equal carryovers of storage buffer. Lanes: 1, dilution buffer alone; 2, 10 mM DTT; 3 to 5, 10, 25, and 50 ng of recombinant Ref-1, respectively; 6 to 8, 10, 25, and 50 ng of recombinant C65A Ref-1, respectively; 9 to 11, 10, 25, and 50 ng of recombinant mDab-1, respectively; 12 to 14, storage buffer diluted 1:1,000, 1:400, and 1:200, respectively. Free probe is indicated by an arrow.