S-Nitrosylation of p47^phox enhances phosphorylation by casein kinase 2

Figures & data

Figure 1. S-Nitrosylation of p47^phox. (A) Immunoblot analysis of recombinant p47^phox. After incubating p47^phox with various concentrations of SNAP or PN for 30 minutes at room temperature, the samples were subjected to SDS–PAGE and immunoblotting with the anti-nitrocysteine IgG antibody. (B) Identification of the S-nitrosylation target cysteine in p47^phox by using site-directed mutagenesis. The S-nitrosylation of wild type and p47^phox C→A mutants was detected using the DAN reagent. The fluorescence signal of the untreated control protein was subtracted as background. Values correspond to three independent assays with the standard deviation indicated. *P < 0.01 versus wild type, C98A, C111A, and C396 p47^phox mutant proteins. (C) After incubation with 1 mM SNAP or 1 mM PN for 30 minutes at room temperature, the wild type and the p47^phox C196A mutant were subjected to SDS–PAGE, followed by immunoblotting with the anti-nitrocysteine IgG antibody.

Figure 2. Effect of S-nitrosylation on in vitro phosphorylation of recombinant p47^phox by CK2. (A) Recombinant p47^phox (1 μg) was incubated with various concentrations of SNAP or PN (30 minutes at room temperature), followed by incubation with [gamma-³²P]ATP and CK2. The labeled proteins were separated by SDS–PAGE. Proteins were visualized using the anti-p47^phox IgG antibody, and ³²P incorporation (p-p47^phox) was monitored by autoradiography. (B) Effect of 1 mM DTT and degraded SNAP or PN on the phosphorylation of S-nitrosylated p47^phox by CK2. (C) SNAP- or PN-treated wild type and p47^phox C196A mutant samples were phosphorylated by CK2 with [gamma-³²P]ATP. The labeled proteins were separated by SDS–PAGE, and ³²P incorporation was monitored by autoradiography. (D) Recombinant p47^phox (1 μg) was incubated with various concentrations of PN (30 minutes at room temperature) and then with [gamma-³²P]ATP and PKC. The labeled proteins were separated by SDS–PAGE. Proteins were visualized with anti-p47^phox IgG, and ³²P incorporation was monitored by autoradiography.

Figure 3. Conformational change in p47^phox protein by S-nitrosylation. (A) Recombinant wild type and SH3 domains of p47^phox (1 μg) were incubated with [gamma-³²P]ATP and CK2. The labeled proteins were separated by SDS–PAGE. Proteins were visualized using the anti-p47^phox IgG antibody, and ³²P incorporation was monitored by autoradiography. (B) The SH3 domain of p47^phox (1 μg) was incubated with PN (30 minutes at room temperature) and then with [gamma-³²P]ATP and CK2. The labeled proteins were separated by SDS–PAGE. Proteins were visualized using the anti-p47^phox IgG antibody, and ³²P incorporation (p-SH3) was monitored by autoradiography. (C) Changes in p47^phox structure following S-nitrosylation. Steady-state emission spectra of intrinsic fluorescence of native p47^phox and SH3 domains treated with SNAP or PN were analyzed with a spectrophotometer at an excitation wavelength of 278 nm. The maximum protein fluorescence intensity was determined at 338 nm. Data are representative of three experiments. *P < 0.01 versus untreated p47^phox. (D) Visualization of binding of CK2-beta subunit to S-nitrosylated p47^phox using dot blot analysis.

Figure 4. Proposed model of S-nitrosylation-mediated enhancement of CK2 phosphorylation of p47^phox. See text for details.