p47 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, p47phox 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.The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytic leukocytes plays a key role in innate host defense against bacterial and fungal infections (1-3). The phagocyte NADPH oxidase is composed of membrane-integrated flavocytochrome b 558 (a heterodimer composed of gp91 phox (NOX2) and p22 phox ) and four cytosolic components: p47 phox , p67 phox , p40 phox , and Rac2 (2-4). Upon activation by either soluble or particulate stimuli, the cytosolic subunits translocate to flavocytochrome b 558 to form the activated NADPH oxidase complex, resulting in electron transfer from cytosolic NADPH through FAD and heme groups to extracellular or phagosome-located oxygen, from which superoxide is generated (2-4). Genetic defects in any of the five phox subunits of the NADPH oxidase complex result in chronic granulomatous disease (CGD), 2 which is characterized by absent or deficient NADPH oxidase activity, recurrent pyogenic infections, and granulomatous inflammation (1, 5, 6).The assembly of the NADPH oxidase complex is essential for activation of superoxide production, and p47 phox plays a central role in this assembly (2-4, 7-12). From the N terminus to the C terminus, p47 phox contains a Phox homology (PX) domain, two tandemly arranged Src homology 3 (SH3) domains, an autoinhibitory region (AIR), and a proline-rich region (PRR; Fig. 1A). In the resting state, p47 phox is autoinhibited via intramolecular interactions o...

Protein-tyrosine Phosphatase Shp2 Positively Regulates Macrophage Oxidative Burst

“…Phagocytosis Assays-Synchronized phagocytosis was performed as previously described (24). Briefly, 2 ϫ 10 6 macrophages were washed with PBS and resuspended in 3 ml of PBSG, added to 6-well plate, and kept on ice for 5 min, followed by adding 300 l of cold SOZ or zymosan (4.4 g/l in PBSG) for a final concentration of 400 g/ml.…”

“…An Lmax microplate luminometer (Molecular Devices, Sunnyvale, CA) was used to record luminescence as previously described (24,26). For some experiments, macrophages were preincubated at 37°C with Syk inhibitor, R406, NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), or Erk inhibitor for 30 min.…”

“…Retroviral Transduction of Bone Marrow and Macrophage Differentiation-Retroviral transduction of murine bone marrow low density mononuclear cells (C57Bl/6 background) with MSCV-WT Shp2-YFP, Shp2-R32K-YFP, or Shp2-C463A-YFP or with MIEG3-WT Shp2, Shp2-D61Y, or Shp2-E76K was performed as described (23,24). Transduced cells were sorted by fluorescence-activated cell sorting (FACSCalibur; BD Biosciences) prior to macrophage differentiation, as described (23).…”

“…ROS Detected by Chemiluminescence in Intact Cells-ROS production during synchronized phagocytosis of SOZ or zymosan stimulation was measured in the presence of 20 M luminol with 20 units/ml HRP (24,30,31). An Lmax microplate luminometer (Molecular Devices, Sunnyvale, CA) was used to record luminescence as previously described (24,26).…”

Mechanosensitive Regulation of Cortactin by p47 phox

“…20 Importantly, p47 phox possesses an actin-binding site, which is 1 of the strongest indicators that this protein interacts with actin/cytoskeletal proteins. 21,22 Numerous protein kinases have been implicated in the phosphorylation of p47 phox , including protein kinase C, Akt, extracellular signal-regulated kinase 1/2, Src, and p21-activated kinase. [23][24][25] Some kinases, such as protein kinase A and casein kinase II, influence p47 phox through a negative inhibitory effect.…”