p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation

Abstract

The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.

FIGURE 1.

Release of ROS in K562-phox model is decreased by mutations in p47^phox PX domain. A, structural motifs of p47^phox and the proposed interactions of p47^phox/PI(3,4)P₂ and p47^phox/p22^phox. B, K562-gp91/p67Cherry cells were co-transfected with p40^phox and p47YFP WT or PX module or SH3A domain mutants with Amaxa V (Amaxa) under the T-16 program. 15 μg of cell lysate was loaded in each lane. Immunoblot analysis of p47YFP, p67Cherry, p40^phox, gp91^phox, and p22^phox was performed using anti-GFP polyclonal antibody, anti-DsRed polyclonal antibody, which also recognizes Cherry protein, anti-p40^phox polyclonal antibody, anti-gp91^phox 54.1, and anti-p22^phox NS2, respectively. Blots are representative of three independent experiments. Shown is ROS production in K562 cells in response to PMA (C) and hIgG-latex beads (D) in the presence of isoluminol and HRP. E, ROS production in K562 cells (n = 3). Assays were performed in triplicate, and mean values ± S.D. (error bars) are shown. **, p < 0.01. PRR, proline-rich region.

FIGURE 2.

NADPH oxidase activity of p47^phox WT and p47^phox Arg⁹⁰ mutants in p47^phox KO mouse PMNs. A and D, Western blotting. 15 μg of cell lysate was used in immunoblotting. A vertical line was added when the sample was loaded in a non-continuous lane. Shown is PMA-stimulated (B and E) or fMLF-stimulated (C and F) ROS production in p47^phox KO mouse PMNs or PMNs transduced with p47YFP, p47YFP-R90A, p47^phox, or p47-R90K. Bar graphs represent four or five independent experiments. **, p < 0.01. Error bars, S.D.

FIGURE 3.

Extracellular and intracellular ROS production of p47^phox WT and p47^phox Arg⁹⁰ mutants in p47^phox KO mouse PMNs. A, extracellular and intracellular ROS production was induced by hIgG-latex beads (n = 4), SOZ (n = 5), serum-opsonized S. aureus (n = 4), or hyphae (n = 3). ♦, p47YFP-transduced p47^phox KO neutrophils; ♢, p47YFP-R90A-transduced p47^phox KO neutrophils; *, p47^phox KO neutrophils. The percentage of extracellular and intracellular ROS of p47^phox R90A mutant (B) or p47^phox R90K mutant (C) compared with p47^phox WT was normalized with the protein expression level in response to different particulate stimuli from 3–5 independent experiments. *, p < 0.05; **, p < 0.01. Error bars, S.D.

FIGURE 4.

Translocation of p47YFP and p47YFP-R90A during SOZ phagocytosis in p47^phox KO mouse PMNs. A, time lapse confocal microscopy was used to monitor SOZ phagocytosis by p47^phox KO mouse PMNs expressing p47YFP or p47YFP-R90A. B, percentage of positive phagosomes during SOZ phagocytosis. 226 cups and 816 internalized phagosomes were analyzed for PMNs expressing p47YFP, and 141 cups and 483 internalized phagosomes were analyzed for PMNs expressing p47YFP-R90A. C, the relative fluorescence intensity on the phagosomal membrane compared with the cytosol was determined in the p47YFP WT or mutant-transduced p47^phox KO mouse PMNs at the indicated stages and is shown in the graph as mean ± S.E. (error bars) (n = 3). 11, 9, and 7 phagosomes were analyzed for p47YFP, p47YFP-R90A, and p47YFP-W193R, respectively. The arrows indicate the cup of phagosomes, and asterisks indicate the internalized phagosomes. Bar, 5 μm.

FIGURE 5.

CGD-associated p47^phox R42Q mutant expressed in K562 cell model. A, YFP fusion protein expression in K562 cells was measured by flow cytometry. B, Western blot analysis of Triton X-100-soluble and -insoluble fractions. A vertical line was added when the sample was loaded in a non-continuous lane. C, distribution of p47YFP WT and R42Q in K562 cells stably expressing gp91^phox and p67Cherry. PMA-stimulated (D) or hIgG-latex-stimulated (E) ROS production in K562-gp91/p67Cherry cells co-transfected with p40^phox. Insets show the ROS production measured in the R42Q mutant. F, percentage of ROS production in K562 cells (n = 3). Error bars, S.D.

FIGURE 6.

CGD-associated p47^phox R42Q mutant expressed in p47^phox KO mouse PMNs. A, YFP fusion protein expression in p47^phox KO mouse PMNs was measured by flow cytometry. B, phox protein expression in p47^phox KO mouse PMN transduced with p47YFP WT or R42Q mutant. 15 μg of protein was loaded in each lane, except 30 μg of protein from R42Q mutant-transduced PMNs was loaded for probing p47^phox. C, phox protein expression in p47^phox KO mouse PMN transduced with p47^phox WT or R42Q mutant under puromycin selection. A vertical line was added when the sample was loaded in a non-continuous lane. D, distribution of p47YFP and p47YFP-R42Q in resting p47^phox KO mouse PMNs. N, nucleus. E, live image of p47^phox KO mouse PMNs expressing p47YFP-R42Q during SOZ phagocytosis. The arrow indicates the cup of phagosomes, and asterisks indicate the internalized phagosomes. Bar, 5 μm. Intracellular ROS production induced by hIgG-latex beads (F), serum-opsonized S. aureus (G), or hyphae (H), was measured in p47^phox KO mouse PMNs transduced with p47YFP or p47YFP-R42Q. The inset shows the intracellular phagosome ROS production measured in p47YFP-R42Q mutant induced by serum-opsonized S. aureus. ♦, p47YFP-transduced p47^phox KO neutrophils; ♢, p47-YFP R42Q-transduced p47^phox KO neutrophils; *, p47^phox KO nutrophils (n = 3).