Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis

Abstract

We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40phox interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47phox and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341–360. Furthermore, we demonstrated sequential interactions of p67phox with phagosomes involving adaptor proteins, p47phox and p40phox, during FcγR-mediated phagocytosis. Although p67phox is not targeted to phagosomes by itself, p47phox functions as an adaptor for the ternary complex (p47phox-p67phox-p40phox) in early stages of phagocytosis before phagosome closure, while p40phox functions in later stages after phagosomal closure. Interestingly, a mutated “open” form of p40phox linked p47phox to closed phagosomes and prolonged p47phox and p67phox retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.

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

“…1A, center), as reported previously in RAW 264.7 macrophages (12) and PBL-985 granulocytes (28). We (12,27) and others (13,31) (Fig. 1B).…”

“…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).…”

“…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).…”

“…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).…”

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

“…WT and UNG −/− splenic B cells were stimulated for 3 d. Cell lysates were prepared using RIPA buffer supplemented with protease inhibitor. BiFC constructs of UNG were performed by fusing UNG with either N-or C-terminal fragments of Kusabira Geen Fluorescent protein (mKG) as described by Ueyama et al (57). Cotransfection of the constructs was performed in appropriate combinations in 293T cells, and cells were harvested after either 24 h or 48 h for FACS analysis.…”