A Regulated Adaptor Function of p40^phox: Distinct p67^phoxMembrane Targeting by p40^phoxand by p47^phox

Abstract

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47 phox , p67 phox , p40 phox , and Rac) translocate and associate with the membrane-spanning flavocytochrome b 558 , leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40 phox that regulate its translocation to membranes upon stimulation. GFP-p40 phox translocates to early endosomes, whereas GFP-p47 phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67 phox does not translocate to membranes when expressed alone, but it is dependent on p40 phox and p47 phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40 phox or GFP-p47 phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67 phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40 phox or p47 phox . Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40 phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40 phox and p47 phox function as diverse p67 phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms. INTRODUCTIONIn phagocytic cells, reactive oxygen species (ROS) are produced by NADPH oxidase (Nox2 system). The enzyme is a multiprotein complex assembled from a membrane-spanning flavocytochrome b 558 (composed of gp91 phox [Nox2] and p22 phox ) and four cytoplasmic components (p47 phox , p67 phox , p40 phox , and Rac) (Leto, 1999;Nauseef, 2004;Quinn and Gauss, 2004). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b 558 is stored on the membranes of intracellular granules (Jesaitis et al., 1990), Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-guanine nucleotide dissociation inhibitor (GDI) , and the other phox proteins associate in a separate ternary cytoplasmic complex (p47 phox -p67 phox -p40 phox ) in a dephosphorylated state (Bolscher et al., 1989;Rotrosen and Leto, 1990;Kuribayashi et al., 2002;Lapouge et al., 2002). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse with phagosomes; p47 phox is phosphorylated, thereby inducing conformational changes in p47 phox that promote the interaction of the cytoplasmic complex with the flavocytochrome b 558 ; and Rac dissociates from Rho-GDI and translocates independently to the membrane after exchange of GDP for GTP (Heyworth et al., 1994;Zhao et al., 2003), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.Chronic granulomatous disease...

Cooperation of p40 with p47 for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis

“…Human Nox2, p47 phox , p67 phox , and p40 phox in pcDNA3.1 were described previously (12,27,37). p67 phox (K355A), which does not bind p40 phox (29) in pcDNA3.1, was made by sitedirected mutagenesis using a QuikChange II XL site-directed mutagenesis kit (Stratagene).…”

“…p67 phox (K355A), which does not bind p40 phox (29) in pcDNA3.1, was made by sitedirected mutagenesis using a QuikChange II XL site-directed mutagenesis kit (Stratagene). GFP-p47 phox , GFP-p67 phox , GFPp40 phox , and GFP-p40 phox (PX) were also described previously (12,27,38 was made using the QuikChange. The purified GSTp40 phox (PB1) and full-length His 6 -p40 phox proteins were obtained as described previously (12).…”

“…The purified (His) 6 -p40 phox (PX) (100 nM) was mixed with the purified GST-p40 phox (PB1) (100 nM) in 400 l of the binding buffer (12). After 10 min of rotation at 4°C, 0.01 mM H 2 O 2 was added in the solution and incubated for 10 min at 4°C.…”

“…In unstimulated phagocytes, the oxidase is dissociated and inactive; the flavocytochrome b 558 is stored on the membranes of intracellular granules (4), and the other Phox proteins associate in a separate cytoplasmic ternary complex (p47 phox -p67 phox -p40 phox ) (5) in a dephosphorylated state (6 -8). During phagocyte activation, intracellular granules containing flavocytochrome b 558 fuse to newly forming phago-somes, and the cytoplasmic ternary complex binds to these membranes; p47 phox is phosphorylated (9,10), thereby inducing conformational changes that promote interaction of the ternary complex with p22 phox (11), and p40 phox also undergoes conformational changes by disruption of the intramolecular PX-PB1 domain interaction to enable the ternary complex to bind through the p40 phox PX domain to PI(3)P (12,13), which is enriched in phagosomes (14 -16).…”

“…phox functions as an early stage carrier and adaptor protein of the cytoplasmic ternary complex, whereas p40 phox functions as a late stage carrier or adaptor protein that links the cytoplasmic ternary complex to closed phagosomes and prolongs retention of the complex on phagosomes using PI(3)P binding during Fc␥R-mediated oxidative burst (12,27). Although mounting evidence suggested that p40 phox functions as an essential positive regulator of the Nox2-based NADPH oxidase, only recently was p40 phox deficiency described in a CGD patient, who has compound heterozygosity for a missense mutation predicting a R105Q substitution in the PX domain and a frameshift mutation at codon 52 (K52R) with a premature stop at codon 79 and exhibited a severe defect in Fc␥R-mediated oxidative burst but not in PMA-or fMLP-stimulated extracellular ROS release (28).…”