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. 2013 Feb 15;288(7):4755-62.
doi: 10.1074/jbc.M112.413492. Epub 2013 Jan 10.

RegB kinase activity is repressed by oxidative formation of cysteine sulfenic acid

Jiang Wu  1 , Zhuo Cheng, Khalilah Reddie, Kate Carroll, Loubna A Hammad, Jonathan A Karty, Carl E Bauer
Affiliations

RegB kinase activity is repressed by oxidative formation of cysteine sulfenic acid

Jiang Wu et al. J Biol Chem. .

Abstract

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.

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Figures

FIGURE 1.
FIGURE 1.
Non-reducing SDS-PAGE analysis of the oligomeric states of RegB″. A, oligomeric states of untreated, DTT-treated, and potassium ferricyanide-treated RegB″ on non-reducing SDS-PAGE. Aliquots of air-oxidized RegB″ were untreated, treated with 10 mm DTT or 1 mm ferricyanide at 22 °C for 15 min, respectively. Resultant samples were then separated by non-reducing SDS-PAGE and quantified by ImageJ. B, oligomeric state of the isolated RegB″ dimer on non-reducing SDS-PAGE after prolonged exposure to atmospheric oxygen. RegB″ dimer was analyzed by non-reducing SDS-PAGE right after isolation by size exclusion chromatography. After exposure to air at 4 °C for 5 days, the same sample was analyzed by non-reducing SDS-PAGE again to observe changes in oligomeric state.
FIGURE 2.
FIGURE 2.
Oxidation state of Cys-265 in air-oxidized RegB″ observed by LC-ESI-MS/MS. Air-oxidized RegB″ was first treated with iodoacetamide and DAz-2. After separation by non-reducing SDS-PAGE, RegB″ monomer and tetramer were excised from the gel and in-gel digested with chymotrypsin with the digested peptides subsequently subjected to LC-ESI-MS/MS analysis. A, EIC, mass spectrum (inset), and CID spectrum of intermolecular Cys-265 disulfide-bonded peptide ion at m/z 750.8. The peptide sequence and fragment ion assignments are listed on the figure. B, EIC, mass spectrum (inset) and CID spectrum of iodoacetamide-labeled (CAM) Cys-265-containing peptide ion at m/z 520.27. The peptide sequence and fragment ion assignments are listed on the figure. C, EIC, mass spectrum (inset), and CID spectrum of the peptide ion at m/z 424.4 containing DAz-2-labeled Cys-265. The peptide sequence and fragment ion assignments are listed on the figure.
FIGURE 3.
FIGURE 3.
In vitro DAz-2 labeling of RegB″. A, in vitro labeling of wild type RegB″ and RegB″ C265A. 2000 ng of wild type RegB″ and RegB″ C265A were incubated with 250 μm DAz-2 or DMSO at 37 °C for 2 h. 250 μm p-biotin then was added and incubated at 37 °C for 1 h. Labeled samples were resolved by SDS-PAGE and analyzed by streptavidin-HRP Western blot. The equal loading was confirmed by anti-STag-HRP Western blot. B, in vitro labeling of reduced and air oxygen-treated RegB″. RegB″ was first reduced by 10 mm DTT at 22 °C for 30 min in anaerobic hood. After removing DTT, 2000 ng of reduced RegB″ were exposed to air oxygen at room temperature for 30 min. Treated samples were labeled and analyzed as in A.
FIGURE 4.
FIGURE 4.
Analysis of free and modified thiols in RegB″ in vitro. Free thiol content in air-oxidized RegB″, air-oxidized RegB″ treated with 10 mm DTT at 22 °C for 1 h, and air-oxidized RegB″ treated with 20 mm sodium arsenite at 37 °C for 1 h were determined by chloromercuribenzoate (p-CMB) titration.
FIGURE 5.
FIGURE 5.
Formation of sulfenic acid at Cys-265 in vivo. A, formation of sulfenic acid at Cys-265 in aerobically cultured R. capsulatus. Aerobically cultured R. capsulatus cells carrying RegB-Flag and RegBC265A-flag were incubated with 5 mm DAz-2 or DMSO at 4 °C for 3 h. RegB-Flag and RegBC265A-flag were then enriched with ANTI-FLAG M2 affinity gel, reacted with 250 μm p-biotin, and analyzed by Streptavidin-HRP Western blot. Equal loading was confirmed by anti-Flag-HRP Western blots. B, formation of sulfenic acid at Cys-265 in aerobically cultured and anaerobically cultured R. capsulatus cells. Aerobically cultured and anaerobically cultured R. capsulatus cells were incubated with DAz-2 or DMSO, and the sulfenic acid formation in RegB-Flag was analyzed as in A.
FIGURE 6.
FIGURE 6.
Effect of the reduction of the sulfenic acid at Cys-265 on RegB kinase activity. 10 μm isolated RegB″ dimer was kept at 22 °C for 15 min in the absence (solid line with open circles) or presence (solid line with solid circles) of 10 mm DTT before [γ-32P]ATP was added to initiate the autophosphorylation reaction. Aliquots of reactions were removed at 0.5,1, 2, 4, 8 min and quenched in SDS-PAGE loading buffer, followed by SDS-PAGE separation, and phosphorimaging data analysis.
FIGURE 7.
FIGURE 7.
Model for the regulation of RegB by oxidative modifications of Cys-265 and ubiquinone binding. A, sulfenic acid mediated regulation. Under anaerobic conditions, the thiol group of Cys-265 exists as a free thiol and RegB is active. Under aerobic conditions, free thiol at Cys-265 could be oxidized to sulfenic acid that inhibits RegB kinase activity. B, disulfide bond mediated regulation. Under aerobic conditions, ∼20% thiols at Cys-265 will form disulfide bonds. Two disulfide bonds can form between two RegB dimers to convert active dimer to inactive tetramer. C, ubiquinone binding mediated regulation. RegB contains a ubiquinone binding site in the transmembrane domain that interacts with both reduced and oxidized ubiquinone. Under aerobic conditions, the ubiquinone pool in the membrane is predominantly reduced. Binding of reduced ubiquinone does not affect RegB kinase activity and RegB is active. Under aerobic conditions, the ubiquinone pool in the membrane is predominantly oxidized. Binding of oxidized ubiquinone can inhibit RegB kinase activity.
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