Regulation of the phagocyte NADPH oxidase activity: phosphorylation of gp91phox/NOX2 by protein kinase C enhances its diaphorase activity and binding to Rac2, p67phox, and p47phox

Abstract

Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47(phox), p67(phox), p40(phox), and Rac2) translocate to the membranes, where they associate with flavocytochrome b(558), which is composed of gp91(phox)/NOX2 and p22(phox), to form the active system. During neutrophil stimulation, p47(phox), p67(phox), p40(phox), and p22(phox) are phosphorylated; however, the phosphorylation of gp91(phox)/NOX2 and its potential role have not been defined. In this study, we show that gp91(phox) is phosphorylated in stimulated neutrophils. The gp91(phox) phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91(phox), which confirms that this phosphoprotein is gp91(phox). The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91(phox), and protein kinase C (PKC) phosphorylated the recombinant gp91(phox)- cytosolic carboxy-terminal flavoprotein domain. Two-dimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91(phox)-cytosolic tail on the same peptides that were phosphorylated on gp91(phox) in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91(phox) flavoprotein cytosolic domain and its binding to Rac2, p67(phox), and p47(phox). These results demonstrate that gp91(phox) is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91(phox)/NOX2 is a novel mechanism of NADPH oxidase regulation.

Figure 1.

Analysis of gp91^phox/NOX2 phosphorylation in PMA-activated human neutrophils. ³²Pi-labeled neutrophils (5×10⁷ cells/ml) were incubated in the absence (resting) or presence of PMA (200 ng/ml for 8 min). Cell lysates were prepared and immunoprecipitated with monoclonal anti-gp91^phox (IP anti-NOX2) and anti-p22^phox (IP anti-p22^phox) antibodies, as described in Materials and Methods. Proteins were subjected to SDS-PAGE, blotted on nitrocellulose, and detected by autoradiography (³²P-NOX2) and immunoblotting with anti-gp91^phox (NOX2) and anti-p22^phox antibodies (p22^phox). Data are representative of 4 experiments.

Figure 2.

Analysis of gp91^phox/NOX2 and p47^phox phosphorylation in neutrophils from healthy donors and an X°-CGD patient. A) Analysis of NADPH oxidase protein expression. Neutrophils were isolated from a healthy donor and from an X°-CGD patient. Cells (2×10⁶) were lysed and analyzed by SDS-PAGE and Western blotting using anti-gp91^phox/NOX2, anti-p67^phox, anti-p47^phox, and anti-p22^phox antibodies. B) Analysis of gp91^phox/NOX2 phosphorylation. ³²Pi-labeled neutrophils (2×10⁷ cells/ml) from a healthy donor and from a X°-CGD patient were incubated with PMA (200 ng/ml) for 8 min and lysed. Lysates were immunoprecipitated with anti-gp91^phox/NOX2 antibody, and immunoprecipitates were subjected to SDS-PAGE, blotted onto nitrocellulose, and detected by autoradiography (³²P-NOX2) and Western blotting (NOX2). C) Analysis of p47^phox phosphorylation. ³²Pi-labeled neutrophils (2×10⁷ cells/ml) from a healthy donor and from an X°-CGD patient were incubated with PMA (200 ng/ml) for 8 min and lysed. P47^phox was immunoprecipitated, analyzed by SDS-PAGE, blotted onto nitrocellulose, and detected by autoradiography (³²P-p47^phox) and Western blotting (p47^phox). D) Analysis of deglycosylated gp91^phox/NOX2. ³²Pi-labeled neutrophils (5×10⁷ cells/ml) were incubated with PMA (200 ng/ml) for 8 min and lysed. Lysates were immunoprecipitated with anti-gp91^phox/NOX2 antibody, and immunopreciptates were subjected to deglycosylation, as described. Control and deglycosylated samples were subjected to SDS-PAGE, blotted onto nitrocellulose, and detected by autoradiography (³²P-NOX2) and Western blotting (NOX2). Data are representative of 3 experiments.

Figure 3.

PMA, fMLP, and OPZ induce gp91^phox/NOX2 phosphorylation in human neutrophils. ³²Pi-labeled neutrophils were incubated with PMA (200 ng/ml for 8 min), fMLP (1×10⁻⁶ M for 1 min), and OPZ (0.5 mg/ml for 10 min), as indicated. Cells were lysed, and gp91^phox/NOX2 was immunoprecipitated, as described. Proteins were separated by SDS-PAGE and analyzed by autoradiography (³²P-NOX2) and Western blotting (NOX2). Data are representative of 3 experiments.

Figure 4.

Effect of PMA and GF109203X on gp91^phox/NOX2 phosphorylation and ROS production in human neutrophils. A) Kinetics of PMA-induced gp91^phox/NOX2 phosphorylation in human neutrophils. ³²Pi-labeled neutrophils (5×10⁷ cells/ml) were incubated with PMA (200 ng/ml) for various times. Gp91^phox/NOX2 was immunoprecipitated, and samples were subjected to SDS-PAGE, blotted on nitrocellulose, and detected by autoradiography (³²P-NOX2) and Western blotting (NOX2). B) Kinetics of PMA-induced NADPH oxidase activation in human neutrophils. Human neutrophils (5×10⁵ cells/0.5 ml Hank’s buffer) were treated with buffer or PMA (200 ng/ml), and luminol-amplified chemiluminescence was measured. C) Effect of the PKC inhibitor GF109203X on gp91^phox/NOX2 phosphorylation in human neutrophils. ³²Pi-labeled neutrophils were incubated in the presence or absence of GF109203X (1–8 μM) for 15 min, stimulated with PMA (200 ng/ml) for 8 min, and gp91^phox/NOX2 was immunoprecipitated from lysates. Lysates were analyzed by autoradiography (³²P-NOX2) and immunoblotting (NOX2). Data are representative of 3 experiments.

Figure 5.

Phosphorylation of recombinant gp91^phox/NOX2 cytosolic flavoprotein domain by purified PKC in vitro and phosphoamino acid analysis. A) Recombinant NOX2 (291-570) was expressed in bacteria, and purified protein was analyzed by SDS-PAGE and Coomassie blue staining as described. B) Recombinant protein was incubated with [γ-³²P]-ATP in the presence of PKC, reactions were terminated at the indicated times, and proteins were separated by SDS-PAGE and analyzed by autoradiography and immunoblotting with the anti-NOX2 antibodies. C) Phosphorylated NOX2 was separated by SDS-PAGE, tranferred to PVDF, excised, and subjected to acid hydrolysis, as described. Phosphoamino acids were collected and mixed with or without standard markers (phosphoserine, phosphothreonine, phosphotyrosine) and separated by thin layer electrophoresis. Standard phosphoamino acids were visualized by 0.2% ninhydrin, and phosphorylated amino acids were detected by autoradiography. Duplicate samples are shown. D) Phosphorylated NOX2 (291-570) was separated by SDS-PAGE, tranferred to nitrocellulose, excised, and then incubated with 50 mg/ml CNBr in 70% (v/v) formic acid for 16 h at room temperature in the dark. Supernatant was dried in a speed-vac and lyophilized. Peptides were analyzed by Tris-Tricine SDS-PAGE followed by autoradiography. Data are representative of 4 experiments.

Figure 6.

Tryptic phosphopeptide mapping of phosphorylated gp91^phox/NOX2 isolated from PMA-activated neutrophils and recombinant gp91^phox/NOX2 (291-570) phosphorylated in vitro by PKC. ³²Pi-labeled neutrophils were incubated with or without PMA (200 ng/ml) for 8 min, and gp91^phox/NOX2 was immunoprecipitated. Recombinant NOX2 (291-570) was incubated with [γ-³²P]-ATP in the presence of PKC, reactions were terminated at the indicated times, and proteins were separated by SDS-PAGE and analyzed by autoradiography. ³²P-gp91^phox/NOX2 and ³²P- NOX2 (291-570) were recovered from nitrocellulose sheets and submitted to trypsin digestion. Peptides were subjected to first-dimension electrophoresis and second-dimension liquid chromatography, as described. Phosphorylated peptides were detected by autoradiography. Data are representative of 3 experiments.

Figure 7.

Effect of PKC-induced phosphorylation of gp91^phox/NOX2-cytosolic flavoprotein domain on interaction with p47^phox, p67^phox, and Rac2. GST-Rac2, GST-p67^phox, phosphorylated GST-p47^phox, and GST alone were incubated in the presence of 5 pmol of phosphorylated or nonphosphorylated recombinant NOX2 (291-570) and glutathione-Sepharose beads in interaction buffer for 1 h. After washing, the complex was eluted with reduced glutathione and analyzed by SDS-PAGE and Western-Blots using protein specific antibodies. Data are representative of 3 experiments.

Figure 8.

Effect of PKC-induced phosphorylation of gp91^phox/NOX2-cytosolic flavoprotein domain on INT-reductase activity in the absence or presence of p47^phox, p67^phox, and Rac2. Recombinant NOX2 (291-570) alone (A) or in the presence of p47phox, p67phox, and Rac2 (B) was incubated with or without PKC, and INT-reductase activity was measured. Activity was determined in a 500 μl volume of assay buffer containing 0.2 mM INT and 1 μg of the phosphorylated or nonphosphorylated NOX2 (291-570), which was preincubated with (+) or without (−) DPI (20 μM) for 15 min. Reaction was initiated by addition of 0.2 mM NADPH, and rate of INT reduction was monitored by measuring absorbance at 500 nm. Inserts: DPI-inhibitable INT-reductase activity. Data are representative of 6 experiments. *P < 0.05.