Modulation of p47 ^PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

Abstract

T he antimicrobial activity of phagocytes depends in part on the cells' ability to reduce oxygen to reactive microbicidal oxidants by means of an NADPH oxidase. In resting phagocytes, the NADPH oxidase is in a dormant state, but exposure of the cell to any of a variety of stimuli can activate the enzyme, causing it to release large amounts of O 2 Ϫ by reducing oxygen at the expense of NADPH. The oxidase is activated by the phosphorylation of one of its cytosolic subunits, p47 PHOX , on particular serines (1). In whole cells, the stimulation of an appropriate receptor activates PKC and phosphatidylinositol 3-kinase (PI3-kinase) (2-4). Although NADPH oxidase activation by PKC has been well characterized (5, 6), the association between PI3-kinase and NADPH oxidase activation remains to be established. A possible connection is through the activation of Akt (7), whose role in oxidase activation is suggested by the finding that Akt is activated rapidly when neutrophils are treated with oxidase-activating agents (8) and by the finding that oxidase activation is inhibited by wortmannin (9). The experiments described here show that Akt is able to activate the oxidase by phosphorylating p47 PHOX on serines S304 and S328. Experimental ProceduresMaterials. Active Akt was obtained from Upstate Biotechnology (Lake Placid, NY) or from J.-H.L., whose recombinant material was 95% pure by SDS͞PAGE. [␥-32 P]ATP was purchased from New England Nuclear. Phosphorylated peptides were obtained from Sigma.Isolation and Fractionation of Neutrophils. Neutrophils were obtained from normal subjects by dextran sedimentation and Ficoll͞Hypaque fractionation of freshly drawn citrateanticoagulated blood (10). After treatment on ice for 10-20 min with 5 l of 0.54 M diisopropyl fluorophosphate, the neutrophils, suspended at 10 8 cells per ml in a modified relaxation buffer (0.1 M KCl͞3 mM NaCl͞3.5 mM MgCl 2 ͞10 mM Pipes buffer, pH 7.3), were subjected to nitrogen cavitation. Membranes and cytosol were separated by centrifugation through a Percoll gradient. Aliquots of membrane and cytosol were stored at Ϫ70°C until use.Recombinant Oxidase Components. Recombinant fusion proteins composed of an upstream GST linked to a downstream p47 PHOX , p67 PHOX , or Rac2 were isolated from Escherichia coli transformed with pGEX-6P3 plasmids containing cDNA inserts encoding the downstream proteins. The fusion proteins were isolated by the addition of a prewashed 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) in PBS (133 mM NaCl͞16 mM Na 2 HPO 4 ͞2.7 mM KCl͞1.5 mM KH 2 PO 4 , pH 7.3). The tube was rotated end-over-end for 1 h at 4°C and then microcentrifuged for 3 min at 200 ϫ g to sediment the glutathione-Sepharose beads. The beads were washed four times with 10 bead volumes of PBS, and the bound fusion proteins were eluted by incubating for 30 min at 4°C with three 1-ml washes of 50 mM Tris⅐HCl, pH 8.0͞20 mM glutathione͞0.2 M NaCl. Excess glutathione was removed from the purified recombinant protein by dialysis against relaxation buffer and conce...

Inactivation of Host Akt/Protein Kinase B Signaling by Bacterial Pore-forming Toxins

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Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

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The coordination of signaling during Fc receptor-mediated phagocytosis

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