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Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis¹

Takehiko Ueyama^*, Tomoko Kusakabe^*, Satoshi Karasawa^,,, Takumi Kawasaki^*, Aya Shimizu^*, Jeonghyun Son^*, Thomas L. Leto^¶, Atsushi Miyawaki and Naoaki Saito^2,*

^* Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan; Laboratory of Cell Function and Dynamics, Brain Science Institute, RIKEN, Saitama, Japan; Amalgaam, Tokyo, Japan; Medical and Biological Laboratories, Nagoya, Japan; and ^¶ Molecular Defenses Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
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 p40^phox interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47^phox 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 p67^phox with phagosomes involving adaptor proteins, p47^phox and p40^phox, during FcR-mediated phagocytosis. Although p67^phox is not targeted to phagosomes by itself, p47^phox functions as an adaptor for the ternary complex (p47^phox-p67^phox-p40^phox) in early stages of phagocytosis before phagosome closure, while p40^phox functions in later stages after phagosomal closure. Interestingly, a mutated "open" form of p40^phox linked p47^phox to closed phagosomes and prolonged p47^phox and p67^phox 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.

	Introduction

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In phagocytic cells, reactive oxygen species (ROS)³ are produced by NADPH oxidase, also known as the Nox2 system. The enzyme is a multiprotein complex assembled from the membrane-spanning flavocytochrome b₅₅₈ (composed of gp91^phox (Nox2) and p22^phox) and four cytosoplasmic components (p47^phox, p67^phox, p40^phox, and Rac) (1, 2). In unstimulated phagocytes, the oxidase is dissociated and inactive: the flavocytochrome b₅₅₈ is stored on the membranes of intracellular granules (3, 4), in particular, secondary (specific) granules, tertiary (gelatinase) granules, and secretary vesicles (5); Rac is maintained in a GDP-bound cytoplasmic complex dimerized with Rho-GDI (6); and the other phox proteins associate in a separate ternary cytoplasmic complex (p47^phox-p67^phox-p40^phox) (7) in a dephosphorylated state (8, 9, 10). During phagocyte activation, intracellular granules containing flavocytochrome b₅₅₈ fuse to newly forming phagosomes, and the ternary cytoplasmic complex and Rac bind to theses membranes by independent mechanisms: p47^phox is phosphorylated, thereby inducing conformational changes in p47^phox that promote the interaction of the ternary cytoplasmic complex with the flavocytochrome b₅₅₈. We recently showed that p40^phox also undergoes conformational changes by disruption of the intramolecular PX-PB1 domain interaction to enable the ternary cytoplasmic complex to bind to PI(3)P-enriched membranes (11). Finally, Rac translocates to phagosomes (12, 13) in a GTP-dependent manner (14, 15), resulting in generation of superoxide anion by the transfer of electrons from cytoplasmic NADPH to molecular oxygen.

Chronic granulomatous disease (CGD), characterized by defective microbial killing by phagocytic cells, is caused by defects or deficiencies in any one of four oxidase components: Nox2, p22^phox, p47^phox, or p67^phox. An essential role for Rac in Nox2 activation was also demonstrated in an oxidase-deficient patient who expressed mutated Rac2 (16, 17) and in mice rendered genetically deficient in Rac2 or in Rac1 plus Rac2 (18, 19). However, there have been no reports of p40^phox defects or deficiencies resulting in CGD. Rac and p67^phox together are minimum essential cytoplasmic components regulating electron flow through the flavocytochrome b₅₅₈ through GTP-dependent interactions (20, 21), hence, p67^phox is called an "activator" component. In contrast, p47^phox is called an "adaptor" or "organizer" component because it binds to membrane lipids (PI(3,4)P₂, phosphatidic acid (PA)) through its PX domain (22), is tethered to the flavocytochrome b₅₅₈ through direct interactions between p22^phox and its Src homology 3 (SH3) domain, and linked other cytoplasmic phox proteins to this complex (23, 24). CGD patients who lack p47^phox show impaired translocation of p67^phox to the particulate or membrane fraction, whereas CGD patients who lack p67^phox show normal translocation of p47^phox to the particulate fraction (25, 26, 27). Although p40^phox was shown to act as an oxidase inhibitory factor in some reconstituted systems (28, 29, 30, 31), studies in p40^phox-deficient mice (32), in p40^phoxR58A/– transgenic mice (33), or in FcIIAR-reconstituted cells (34) indicate p40^phox functions as an essential positive regulator of the Nox2 system. p40^phox also has a PX domain that specifically binds to PI(3)P (35, 36), a phospholipid enriched in phagosomes during phagocytosis (37) and in early endosomes which fuse to phagosomes (38). Thus, p40^phox was suggested to serve as an "adaptor" component that recruits p67^phox and p47^phox to membranes (39). In recent work, we showed that both p47^phox and p40^phox function as "adaptor" or "carrier" proteins of p67^phox using arachidonic acid (AA) as a stimulus in the RAW264.7 model (11).

In this study, we developed a new complementation reporter system that can detect protein-protein interactions at cellular levels under confocal fluorescence microscopy based on fusion proteins of fragments of monomeric coral fluorescent reporter protein (monomeric Kusabira-Green (mKG)). The efficacy and specificity of this system was confirmed when used to detect the interaction of leucine zipper proteins produced as various fusion constructs. We used this system to demonstrate the recently reported PX-PB1 intramolecular interaction within p40^phox (11) in whole live cells. Furthermore, we examined intramolecular interactions within p47^phox with this system, and propose a new model for the autoinhibited state of p47^phox in which a structure encompassing the tandem SH3 domains bound to the autoinhibitory region (AIR; SuperSH3/AIR), and sequence within residues 341–360 are suggested to interact with its N-terminal PX domain. Finally, we examined the adaptor functions of p47^phox and p40^phox during FcR-mediated phagocytosis, in which targeting of p67^phox to phagosomes depends on both of these proteins. p47^phox functions as an early stage adaptor protein of the ternary cytoplasmic complex, while p40^phox functions as a late stage adaptor protein of the complex that links p47^phox to closed phagosomes and prolonged retention of p47^phox and p67^phox on phagosomes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Materials

Goat polyclonal Ab against human p47^phox or p67^phox and rabbit polyclonal Ab against human p40^phox were described previously (28, 40). Rabbit polyclonal Ab against GST and mouse mAb against (His)₆ were obtained from Santa Cruz Biotechnology and Amersham Biosciences, respectively. Mouse mAb (1E6B5) against the N-terminal fragment of mKG (mKG(N)) was a gift from Medical & Biological Laboratories. Mouse polyclonal Ab against early endosome Ag-1 (EEA1) and rabbit polyclonal Ab against EEA1 were obtained from BD Biosciences and Affinity BioReagents, respectively.

Cell culture

RAW264.7 macrophages (13) were maintained in DMEM (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated FBS (Invitrogen) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37°C in 5% CO₂. HEK293 cells (American Type Culture Collection) were maintained in EMEM (Wako Pure Chemical Industries) containing 10% heat-inactivated FBS (Invitrogen), 100 µM nonessential amino acids (Invitrogen), and antibiotics at 37°C in 5% CO₂.

Immunoprecipitation, immunoblotting, immunocytochemistry

Immunoprecipitation and immunoblotting were performed, as described previously (11). HEK293 cells were transfected using Fugene 6 (Roche). Forty-eight hours after the transfection, cells were lysed in homogenizing buffer. For detection of protein expression, the total cell lysates were subjected to SDS-PAGE and immunoblotting. For detection of protein-protein interaction, the total cell lysates were centrifuged at 800 x g for 5 min at 4°C, the supernatants were incubated with anti-p67^phox Ab for 2 h at 4°C, and then with protein G-Sepharose 4B (Amersham Biosciences) for an additional 12 h at 4°C. The precipitates were washed three times and the aliquots of precipitates were subjected to SDS-PAGE followed by immunoblotting using anti-p40^phox Abs (1/1000, room temperature (RT) for 2 h). For immunocytochemical studies, cells (transfected or untransfected) were fixed and permeabilized, as described (41), and were stained using primary Abs at RT for 2 h. Primary Abs were visualized by confocal microscopy using Alexa 488-conjugated anti-rabbit IgG (1/2000, 0.5 h at RT; Molecular Probes).

Engineering mKG fluorescent protein and a complementation-based interaction detecting system using mKG

To get a monomeric form of the Kusabira-Green protein (mKG; GenBank No. AB359188; excitation maximum at 494 nm, emission maximum at 507 nm), we introduced 7 mutations (Lys⁴⁹Glu, Cys⁶⁵Ala, Pro⁷⁰Val, Lys¹⁸⁵Glu, Lys¹⁸⁸Glu, Ser¹⁹²Asp, Ser¹⁹⁶Gly) into monomeric Kusabira-Orange protein (excitation maximum at 548 nm, emission maximum at 559 nm) (42) using semirandom mutagenesis as described (43). Proteins were expressed in Escherichia coli strain JM109(DE3), purified, and characterized spectroscopically as previously described (44). Fluorescence quantum yields were determined using enhanced GFP as a standard. To develop a complementation method using separate fragments of mKG fluorescent protein to detect protein-protein interactions under a confocal fluorescence microscope, we chose the position between Gly¹⁶⁸ and Gly¹⁶⁹ of mKG as a cleavage point for constructing fusion-proteins (mKG(N): 168 aa, mKG(C): 51 aa), because it is predicted to form a flexible loop structure within this region in mKG according to a previous report (42, 45). To systemically use these protein fragments to detect protein-protein interactions, we constructed four plasmids (see Fig. 1A): a and b, in which the divided mKG protein fragments, mKG(N) and mKG(C), are fused to N-terminal portions of interacting proteins of interest, and c and d, in which the divided mKG protein fragments are fused to C-terminal portions of interacting proteins. The efficacy of this system was examined using the well-characterized heterodimeric complex of leucine zipper acidic protein (LZA; AQLEKELQALEKENAQLEWELQALEKELAQK) and leucine zipper basic protein (LZB; AQLKKKLQALKKKNAQLKWKLQALKKKLAQK) as a positive interacting control. All four fusion-protein combinations (a + b, c + d, a + d, and b + c; see Fig. 1B) were shown to interact, as reflected in their ability to complement each other in reconstituting detectable green fluorescence. However, other transfected proteins or combinations exhibited no detectable fluorescence ([c alone, d alone, c + mKG(C), and mKG(N) + d] (see Fig. 1B) [a alone, b alone, a + mKG(C), mKG(N) + b, mKG(N) + mKG(C)] (data not shown)).

View larger version (56K): [in this window] [in a new window]   FIGURE 1. Detection of protein-protein interaction by confocal fluorescence microscopy using the mKG reporter system in transfected cells. A, Plasmids engineered for expression of N-terminally (a and b) or C-terminally (c and d) mKG fragment-tagged protein. Shown are the cDNA insert maps cloned into the BamHI/XhoI sites of pcDNA3. B, All combinations of complementary fusion proteins (left half: a + b, c + d, a + d, and b + c) are capable of reconstituting mKG fluorescence in HEK293 cells when fused to interacting LZA and LZB. Negative controls of mKG system in the absence of fused interacting modules (right half). Bar, 50 µm.

All cDNA fragments were cloned into BamHI/EcoRI site of LZA-mKG(N) or LZB-mKG(C), thereby replacing the leucine zipper cDNA sequences, using forward and reverse primers that provide these restriction sites during cDNA PCR amplification. The cDNA of the PX domain of p40^phox(1–167 aa) was amplified by PCR and cloned into LZB-mKG(C), designated as p40^phox(PX)-mKG(C). Since we (residues 318–328, Ref. 11) and others (E259, D269, and F320, Ref.46) have reported residues required for the intramolecular PX-PB1 domain interaction in p40^phox, p40^phox(PB1:237–339) and p40^phox(PB1:259A/269A/318–321:4A) were amplified by PCR and cloned into LZA-mKG(N) and/or LZB-mKG(C), designated as p40^phox(PB1)-mKG(N), p40^phox(PB1)-mKG(C), p40^phox(PB1:2A+4A)-mKG(N), respectively. PCR-amplified mDsRed with KpnI/SacI sites and p40^phox(PX) with SacI/EcoRI sites were cloned into KpnI/EcoRI sites of LZA-mKG(N), designated as mDsRed-p40^phox(PX)-mKG(N). PCR-amplified cDNAs of p40^phox adapted with NotI sites and mKG(N) adapted with NotI/XhoI sites were cloned into NotI/XhoI sites of mKG(C)-LZB and designated as mKG(C)-p40^phox-mKG(N). mKG(C)-p40^phox(F320A)-mKG(N) was made using the QuikChange II XL site-directed mutagenesis kit (Stratagene), and designated as mKG(C)-p40^phox(320A)-mKG(N).

The PX domain (1–128 aa) and two SH3 domains of p47^phox (151–285) were amplified by PCR and cloned into LZB-mKG(C), designated as p47^phox(PX)-mKG(C) and p47^phox(SH3n-SH3c)-mKG(C), respectively. p47^phox (151–285), p47^phox (286–340), p47^phox (151–340), p47^phox (151–360) were amplified by PCR and cloned into LZA-mKG(N), designated as p47^phox(SH3n-SH3c)-mKG(N), p47^phox(AIR)-mKG(N), p47^phox(SH3n-AIR)-mKG(N), p47^phox(SH3n-360)-mKG(N), respectively. p47^phox(SH3n-360,S303D/S304D/S328D)-mKG(N) and p47^phox(SH3n-360,W193R)-mKG(N) were made using the QuikChange II XL site-directed mutagenesis kit, and designated as p47^phox(SH3n-360,3D)-mKG(N), and p47^phox(SH3n-360,193R)-mKG(N), respectively. Residues 341–360 of p47^phox were cloned into LZA-mKG(N), and designated p47^phox (341–360)-mKG(N). All modified expression vectors were sequenced to confirm their identities.

HEK293 cells were transfected by a pair of mKG constructs using Fugene 6. Twenty-four hours after the transfection, fluorescence-positive cells with nuclei staining by Hoechst 33258 (1:5000, 0.5 h, 37°C; Ref. 47) were observed and counted under a LSM 510 invert (Carl Zeiss) confocal laser scanning fluorescence microscope (x40 oil) using 385–470 nm (for Hoechst 33258) and 505–550 nm (for mKG) narrow band-pass barrier filters with the same microscopic conditions and settings (lens, power of exitation laser, pinhole (optical slice), scan time, scan averaging, scan magnification, detection gain, etc.). Transfection efficacies were confirmed using a pair of p40^phox(PB1)-mKG(N) and p40^phox(PX)-mKG(C) as a positive control. Green fluorescence-positive cells/Hoechst 33258-positive cells were used for statistical analysis. Imaging experiments using the mKG system were performed at least in duplicate and were repeated in at least three independent transfection experiments (n 6).

GST-based in vitro-binding (pull-down) assay

The purified (His)₆-p40^phox(PX:1–167 aa) and the purified GST-p40^phox(PB1:237–339 aa) were described previously (11). p40^phox(PB1:259A/269A,318–321(4A)), in which residues 259, 269, and 318–321 are replaced by alanine residues, in pGEX-6P-1 (GE Healthcare) was made using the QuikChange II XL site-directed mutagenesis kit, and designated as GST-p40^phox(PB1:2A+4A). PCR-amplified cDNA of the PX domain of p47^phox(aa 1–128) was cloned into BamHI and EcoRI sites of pGEX-6P-1, and designated GST-p47^phox(PX). All constructs were sequenced to confirm their identities. The purified GST-p40^phox(PB1:2A+4A) and the purified GST-p47^phox(PX) were obtained, as described previously (11).

To detect the interaction between p40^phox(PX) and each p40^phox(PB1) protein, the purified (His)₆-p40^phox(PX) (300 nM) was mixed with each purified GST-tagged fragment of p40^phox (300 nM) in 500 µl of buffer (11). Glutathione-Sepharose-4B beads were added to the solution, and rotated for 1 h at 4°C. The precipitates were washed three times with the same buffer and the aliquots of precipitates were subjected to SDS-PAGE and followed by immunoblotting using anti-(His)₆ Ab (1/1000, RT for 2 h).

To detect direct interactions between p47^phox(PX) and other fragments of p47^phox, the purified GST-p47^phox(PX) (300 nM) was mixed with the lysates (in the presence of 0.1% Triton X-100 and protease inhibitors) of HEK293 cells that were transfected with various plasmids encoding mKG(N)-tagged fragments of p47^phox, and rotated for 2 h at 4°C. Glutathione-Sepharose-4B beads were added to the solution, rotated for 8 h at 4°C, and then washed three times with the same buffer. The material absorbed to beads was eluted with 10 mM glutathione, and the eluants were subjected to SDS-PAGE, followed by immunoblotting using anti-mKG(N) Ab (1/1000, RT for 2 h).

Construction of plasmids for imaging studies of phagocytosis

GFP-p47^phox, GFP-p67^phox, GFP-p67^phox(AD), which is lacking the activation domain (AD: residues 199–212), GFP-p67^phox(AD,K355A), GFP-p40^phox, GFP-p40^phox(D289A), and p40^phox(PX) were described previously (11, 48). p40^phox(D289A) and p67^phox(K355A) were reported as mutations that disrupt interactions between p40^phox and p67^phox (39). p40^phox-internal ribosomal entry site (IRES)-DsRed2, p40^phox(D289A)-IRES-DsRed2, and p47^phox-IRES-GFP-p67^phox(AD) were described previously (11). The fragments encoding p67^phox(AD) and GFP-p47^phox were amplified by PCR, cloned into BglII/EcoRI sites of MCS of pIRES2-DsRed2 (BD Clontech) and BstXI and XbaI sites of pIRES2-DsRed2 in place of DsRed2, respectively, and designated p67^phox(AD)-IRES-GFP-p47^phox. The pIRES2-DsRed2 plasmids containing p47^phox-IRES- GFP-p67^phox(AD,355A), p47^phox(PR)-IRES-GFP-p67^phox(AD), p47^phox(PR)-IRES-GFP-p67^phox(AD,355A), or p67^phox(AD,355A)-IRES-GFP-p47^phox were made using the QuikChange II XL site-directed mutagenesis kit. We confirmed that GFP-p47^phox and GFP-p67^phox behave like wild-type proteins, at least with regard to its interactions and assembly with other phox proteins and its ability to support ROS production in the Nox2 system (11, 48). p40^phox(318–321:4A), in which residues 318–321 are replaced by four alanine residues, p40^phox(318–321:4A,R105K), and p40^phox(318–321:4A,D289A) in pEGFP-C1 (BD Clontech) were made using the QuikChange II XL site-directed mutagenesis kit, and designated GFP-p40^phox(4A), GFP-p40^phox(4A,105K), and GFP-p40^phox(4A,289A), respectively. p40^phox(318–321:4A) and p40^phox(318–321:4A,D289A) in pIRES2-DsRed2 were made using the QuikChange II XL site-directed mutagenesis kit, and designated p40^phox(4A)-IRES-DsRed2 and p40^phox(4A,289A)-IRES-DsRed2. All modified expression vectors (listed in Table I) were sequenced to confirm their identities.

IgG opsonized phagocytosis targets (BIgG) were prepared using 2-µm glass beads, as described previously (48). A total of 1.0 x 10⁵ RAW264.7 cells were seeded on 35-mm glass-bottom dishes (MatTek Chambers) and transfected using Fugene 6. Twenty-four to 32 hours after the transfection, the culture medium was replaced with HBSS⁺⁺ (49). After HBSS⁺⁺ containing BIgG (five targets per cell) was added to each plate, images were collected at 5-s intervals for 10 min using a confocal laser-scanning fluorescence microscope with a heated stage and objective as described previously (48). The time of addition of BIgG was chosen as time 0. The accumulation of GFP-tagged phox proteins was evaluated based on intensity profiles collected around BIgG.

All imaging experiments were performed in triplicate and were repeated in at least three independent transfection experiments (n 9). All imaging experiments were very reproducible (80%).

Online supplemental material⁴

Supplemental video 1 shows the vesicular localization and the accumulation of GFP-p67^phox(AD) on phagosomes by coexpression with p40^phox(4A) during FcR-mediated phagocytosis in RAW264.7 cells (total time 295 s). Supplemental video 2 (total time 295 s) shows the vesicular localization and the accumulation of GFP-p67^phox(AD) both on phagosomal cups and phagosomes by coexpression with p47^phox and p40^phox(4A). Supplemental video 3 (total time 430 s) shows vesicular localization and the accumulation of GFP-p47^phox both on phagosomal cups and phagosomes by coexpression with p67^phox and p40^phox(4A). Supplemental video 4 (total time 295 s) shows cytoplasmic localization and the limited (not prolonged) accumulation of GFP-p47^phox on phagosomal cups/phagosomes by coexpression with p67^phox(AD,355A) and p40^phox(4A), in which p67^phox can interact with p47^phox, but not p40^phox.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Detection of the intramolecular PX-PB1 interaction in p40^phox using the mKG system

Our previous studies suggested the PX domain of p40^phox is hindered from binding to membranes and that mutations or deletions within C-terminal regions of the PB1 domain of p40^phox enable direct interactions of p40^phox with early endosomes, as seen with the isolated PX domain (11). To confirm and extend these findings suggesting that the intramolecular contacts within p40^phox prevent its interaction with membranes in resting cells, we used the newly developed mKG fluorescent protein to detect protein-protein interactions between the p40^phox PX domain and the p40^phox PB1 domain in whole cells. Preliminary experiments exploring the efficacy and specificity of the mKG system with interacting LZA and LZB proteins indicated that separate N- and C-terminal portions of the mKG protein fused with LZA and LZB can associate to form fluorescent mKG reporter complexes, regardless of whether the fusions occur at N- or C-terminal ends of the leucine zipper proteins or the mKG fragments (Fig. 1). Cotransfection of p40^phox(PB1)-mKG(N) and p40^phox(PX)-mKG(C) fusion proteins in HEK293 cells reconstituted green cellular fluorescence (Fig. 2A). In contrast, cotransfection of p40^phox(PB1:2A+4A)-mKG(N) and p40^phox(PX)-mKG(C) resulted in only faint green fluorescence (Fig. 2A). Cytoplasmic localization of p40^phox(PB1)-mKG(N) and p40^phox(PB1:2A+4A)-mKG(N) was confirmed by indirect immunofluorescence using p40^phox Ab (Fig. 2B, upper panel). Furthermore, comparable expression of p40^phox(PB1)-mKG(N) with p40^phox(PX)-mKG(C) and 40^phox(PB1:2A+4A)-mKG(N) with p40^phox(PX)-mKG(C) was confirmed by Western blotting using p40^phox Ab (Fig. 2B, lower panel).

View larger version (65K): [in this window] [in a new window]   FIGURE 2. Detection of the p40phox PX-PB1 domain interaction in transfected HEK293 cells using the mKG system. A, Interaction between p40phox(PB1)-mKG(N) and p40phox(PX)-mKG(C) (left), but not p40phox(PB1:2A+4A)-mKG(N) and p40phox(PX)-mKG(C) (right). All imaging data are representative of at least three independent transfection experiments. Bar, 50 µm. B, Subcellular localization of p40phox(PB1)-mKG(N) and p40phox(PB1:2A + 4A)-mKG(N) stained by anti-p40phox Ab (upper panel). Immunoblotting showing comparable expression of p40phox(PB1)-mKG(N) with p40phox(PX)-mKG(C) and 40phox(PB1:2A+4A)-mKG(N) with p40phox(PX)-mKG(C) (lower panel). Bar, 10 µm. C, GST-based pull-down assays of interactions between purified (His)6-p40phox(PX) and GST-p40phox(PB1) or GST-p40phox(PB1:2A+4A). Complexes bound to glutathione-Sepharose-4B beads were immunoblotted with anti-(His)6 Ab (upper panel). Representative of three independent experiments. D, Vesicular (condensed) localization of mDsRed-p40phox(PX)-mKG(N) is observed in cells lacking reconstituted mKG green fluorescence (asterisks), but not in cells with high mKG green fluorescence (marked with circles). Immunoblotting detects expression of mDsRed-p40phox(PX)-mKG(N) and p40phox(PB1)-mKG(C). Bar, 10 µm. E, Colocalization of mDsRed-p40phox(PX)-mKG(N) and EEA1. Bar, 10 µm. F, Interaction between mKG(C) tagged to the N terminus of full-length p40phox with mKG(N) tagged to the C terminus of p40phox, expressed as a single fusion protein, by intramolecular interaction in p40phox. Transfected mKG(C)-p40phox(320A)-mKG(N) exhibits faint mKG fluorescence, reflecting disruption of the intramolecular PX-PB1 domain interaction. Immunoblotting reveals comparable expression of mKG(C)-p40phox-mKG(N) and mKG(C)-p40phox(320A)-mKG(N). Bar, 50 µm.

Detection of intramolecular PX domain interactions in p47^phox involving residues 341–360 using the mKG system

It has been reported that p47^phox exists in an autoinhibited conformation due to intramolecular interactions in the resting state in which the tandem SH3 domains (SH3n and SH3c) and the PX domain are inaccessible to bind p22^phox and membrane lipids, respectively (reviewed in Ref. 50). The first autoinhibited model proposed that two distinct inhibitory interactions occur: between a PXXP motif in the PX domain and the SH3c domain and between the AIR and the SH3n domain (51). Subsequently, the SuperSH3 model was proposed in which the binding surfaces for p22^phox formed by both tandem SH3 domains are masked entirely by the AIR in the resting state (52, 53). Although the two interactions (PX domain-SH3c and SuperSH3-AIR) are mutually exclusive in structural terms, the latter interaction is considerably stronger (51, 52); thus, the basis for the proposed masking of the PX domain that prevents membrane lipid interactions remains unclear.

To examine intramolecular interactions within p47^phox that mask binding of the PX domain to lipids in a resting state, we used the mKG system. First, we tested the SuperSH3 model by cotransfection of p47^phox(AIR)-mKG(N) and p47^phox(SH3n-SH3c)-mKG(C), and observed cells with green fluorescence (Fig. 3A). We then examined whether the PX domain could interact with the autoinhibited structure constructed from the tandem SH3 domains and the AIR (SuperSH3/AIR). Cotransfection of p47^phox(SH3n-AIR)-mKG(N) and p47^phox(PX)-mKG(C) revealed cells with green fluorescence; however, cotransfection of p47^phox(PX)-mKG(C) with p47^phox(SH3n-SH3c)-mKG(N) or with p47^phox(AIR)- mKG(N) revealed significantly fewer fluorescence-positive cells (Fig. 3B). These results indicate that the PX domain interacts with the autoinhibited structure constructed from both the SuperSH3 domain and the AIR (SuperSH3/AIR), but not with the SuperSH3 domain nor the AIR alone. We compared interactions of p47^phox(SH3n-AIR)-mKG(N) and p47^phox(PX)-mKG(C) with p47^phox(SH3n-360)-mKG(N) and p47^phox(PX)-mKG(C). The number of green fluorescence-positive cells observed with cotransfection of p47^phox(SH3n-360)-mKG(N) and p47^phox(PX)-mKG(C) was even greater than that seen with p47^phox(SH3n-AIR)-mKG(N) and p47^phox(PX)-mKG(C) (Fig. 3B). Furthermore, the number of fluorescence-positive cells observed with cotransfection of p47^phox(SH3n-360)-mKG(N) and p47^phox(PX)-mKG(C) was dramatically decreased by the substitution with p47^phox(SH3n-360,3D)-mKG(N) or p47^phox(SH3n-360,193R)-mKG(N) (Fig. 3B). Finally, cotransfection of p47^phox (341–360)-mKG(N) and p47^phox(PX)-mKG(C) also revealed cells with green fluorescence (Fig. 3B). Expression of p47^phox(PX)-mKG(C) and comparable expression of mKG(N)-tagged proteins were confirmed by immunoblotting (Fig. 3C). The number of green fluorescence-positive cells reflecting the strength of protein-protein interactions is statistically analyzed in Fig. 3D. Furthermore, we validated the performance of the mKG system and the accuracy of quantifying detectable interactions by comparison with independent pull-down assays using purified GST-p47^phox(PX) and lysates of HEK293 cells transfected with various plasmid encoding mKG(N)-tagged fragment of p47^phox. Consistent with previous reports (51, 52), p47^phox(SH3n-SH3c)-mKG(N) and p47^phox(AIR)-mKG(N) bound weakly to GST- p47^phox(PX), while p47^phox(SH3n-360)-mKG(N) was strongly bound to GST- p47^phox(PX) in comparison to p47^phox(SH3n-AIR)-mKG(N) (Fig. 3E). These experiments confirmed the following rank order in affinity for binding to p47^phox-PX using both methods: p47^phox(SH3n-SH3c)-mKG(N) < p47^phox(AIR)-mKG(N) << p47^phox(SH3n-AIR)-mKG(N) < p47^phox(SH3n-360)-mKG(N). Comparable amounts of input proteins are confirmed by anti-p47^phox (GST proteins) and by anti-mKG(N) (HEK293 cells lysates) blotting, respectively (Fig. 3E). These results suggest that the PX domain interacts with the inhibited SuperSH3/AIR structure and with downstream residues 341–360, and that this interaction is disrupted by changes that open the SuperSH3/AIR structure, such as those mimicking phosphorylation of at least residues S303/S304/S328 or by the W193R mutation in SH3n (Fig. 3E) (52). We also examined phosphorylation-mimicking or -blocking effects at S345/S348/S359 using p47^phox(341–360,3D) and p47^phox(341–360,3A) in which residues S345/S348/S359 were substituted by three aspartates or alanines; however, these changes did not influence the interaction with the PX domain (data not shown). Taken together, these results show that the p47^phox PX domain does not interact directly with p47^phox SH3 domains, rather it interacts only with the closed SuperSH3/AIR structure and sequence directly downstream (residues 341–360). This proposed model explains how the exposure of membrane-binding SH3 and PX domains are linked together to phosphorylation of the AIR.

View larger version (58K): [in this window] [in a new window]   FIGURE 3. Detection of intramolecular interactions that mask the PX domain in p47phox using the mKG system in HEK293 cells. A, Interaction of p47phox(AIR)-mKG(N) and p47phox(SH3n-SH3c)-mKG(C). All imaging data are representative of at least three independent transfection experiments. B, p47phox(PX)-mKG(C) interactions are examined using various mKG(N)-tagged p47phox fragment. Bar, 50 µm. C, Immunoblotting of protein expression of p47phox(PX)-mKG(C) and comparable expression of mKG(N)-tagged proteins. D, Percent of mKG fluorescence-positive cells/Hoechst 33258-positive cells observed in B. Data are obtained from experiments performed in duplicate and repeated in at least three independent set of transfection experiments (n 3), and expressed as means ± SD. E, GST-based pull-down assays between purified GST-p40phox(PX) and HEK293 cell lysates expressing mKG(N)-tagged fragment of p47phox. Complexes bound to Glutathione-Sepharose-4B beads were immunoblotted with -mKG(N) Ab. Representative of three independent experiments. F, Proposed model of intramolecular interactions within p47phox in the resting state, in which the interaction of the PX domain with the SuperSH3/AIR structure along with residues 341–360. In active state, unmasked SuperSH3 and unmasked PX domain can bind to the PR motif of p22phox, and PI(3,4)P2 and PA, respectively. The PR motif of p47phox binds to SH3c of p67phox.

Accumulation of p67^phox by p47^phox at phagosomes during FcR-mediated phagocytosis

In this study, GFP-p67^phox(AD) was used instead of GFP-p67^phox, because overexpression of all three active phox proteins leads to death of transfected RAW264.7 cells, as previously noted (11). During FcR-mediated phagocytosis, GFP-p47^phox accumulated at phagosomal cups and on phagosomes with an accumulation time of 60 s (detected at T = 155–220 (Fig. 4A) and see Fig. 7C), similar to a previous report (48). In contrast, GFP-p67^phox(AD) (Fig. 4B), as well as GFP-p67^phox (data not shown), showed no accumulation at the phagosomal cup nor on phagosomes in the absence of other transfected cytosolic phox proteins. However, in the case when GFP-p67^phox(AD) was coexpressed with p47^phox using p47^phox-IRES-GFP-67^phox(AD), GFP-p67^phox(AD) accumulated at phagosomal cups and on phagosomes with accumulation times 60 s (detected at T = 180–245) (Fig. 4C), as seen with GFP-p47^phox. GFP-67^phox(AD) did not accumulate at phagosomal cups or phagosomes when coexpressed with p47^phox using p47^phox(PR)-IRES-GFP-67^phox(AD), which does not interact with GFP-67^phox(AD) (Fig. 4D). The accumulation of GFP-tagged phox protein was further confirmed based on fluorescence intensity profiles transversing the phagosomal cup/phagosome (Fig. 4, A–D, right panel of each). These results demonstrated that p47^phox functions as a "carrier" for p67^phox to phagosomal cups or phagosomes during FcR-mediated phagocytosis, consistent with earlier studies on p47^phox-deficient CGD neutrophils (27). The protein expression of plasmid (GFP-p47^phox, GFP-p67^phox(AD), p47^phox-IRES-GFP-p67^phox(AD), and p47^phox(PR)-IRES-GFP-p67^phox(AD)) was confirmed by immunoblotting (Fig. 4E).

View larger version (57K): [in this window] [in a new window]   FIGURE 4. p47phox-mediated accumulation of p67phox on early phagosomes in RAW264.7 cells. A, Accumulation of GFP-p47phox on phagosomal cups (arrow) and phagosomes (arrowhead) during FcR-mediated phagocytosis. Right panel showing the intensity profile of GFP-tagged protein detected along the red arrow transversing the phagosomal cup/phagosome. IgG-opsonized glass beads (2 µm) were added at time 0, although phagocytosis of each bead begins at different time points. All imaging data are representative of at least three independent transfection experiments. *, Phagosome. B, No accumulation of GFP-p67phox(AD) on phagosomal cups (arrow) or phagosomes (arrowhead) when expressed alone. C, Accumulation of GFP-p67phox(AD) on phagosomal cups (arrow) and phagosomes (arrowhead) when coexpressed with p47phox. D, No accumulation of GFP-p67phox(AD) on phagosomal cups (arrow) or phagosomes (arrowhead) when coexpressed with p47phox(PR). E, Immunoblotting confirms protein expression by plasmids transfected into HEK293 cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 7. Prolonged retention of GFP-p47phox on phagosomes in RAW264.7 cells coexpressing p67phox and active p40phox. A, Subcellular localization of GFP-p47phox by coexpression of p67phox(AD) and p40phox(4A) (before stimulation), and accumulation of GFP-p47phox at phagosomal cups (arrow) and late phagosomes (arrowhead) during phagocytosis of BIgG. Cell exhibiting both GFP-p47phox and diffuse DsRed2 fluorescence also produces untagged p67phox(AD) and untagged p40phox(4A). A movie is available in supplemental video 3. IgG-opsonized glass beads (2 µm) were added at time 0, although phagocytosis of each bead begins at different time points. All imaging is representative of at least three independent transfection experiments. B, Subcellular localization of GFP-p47phox by coexpression of p67phox(AD,355A) and p40phox(4A). Cells exhibiting both GFP-p47phox and diffuse DsRed2 fluorescence also produce untagged p67phox(AD) and untagged p40phox(4A). Time-lapsed image during phagocytosis is shown in supplemental video 4. C, Comparison of retention times of GFP-p47phox on phagosomes by coexpression of p67phox(AD) plus mock vector or p40phox(4A,289A), or p40phox(4A). Data were obtained from experiments performed in triplicate and repeated in at least three independent transfection experiments, and expressed as means ± SD. D, Immunoblotting confirming protein expression by plasmids (HEK293 cells) used in Fig. 7.

Accumulation of p67^phox by p40^phox at phagosomes during FcR-mediated phagocytosis

To clarify the function of p40^phox as a "carrier" protein for p67^phox, we used p40^phox(4A), which has an "open conformation" resulting from disruption of the intermolecular PX-PB1 interaction (11). GFP-40^phox was localized in cytoplasm (Fig. 5A) and transient vesicular accumulation of GFP-p40^phox was observed occasionally, which fuses with newly forming phagosomes during FcR-mediated phagocytosis (11). In contrast, GFP-p40^phox(4A) was localized predominately on vesicular structures (Fig. 5B, left), was colocalized with a marker of early endosomes, EEA1 (Fig. 5B), and accumulated on phagosomes during FcR-mediated phagocytosis (data not shown). The early endosome localization of GFP-p40^phox(4A) was dramatically decreased with GFP-p40^phox(4A,105K) (Fig. 5C), in which 105K disrupts PI(3)P binding. GFP-p40^phox(289A) translocated to early endosomes in response to AA (11), while GFP-p40^phox(4A,289A) was also localized at vesicular structures (Fig. 5D). Consistent protein expression using these plasmids (GFP-p40^phox, GFP-p40^phox(4A), GFP-p40^phox(4A,105K), and GFP-p40^phox(4A,289A)) was confirmed by immunoblotting (Fig. 5E). GFP-p67^phox(AD) was localized at vesicular structures when coexpressed with p40^phox(4A)-IRES-DsRed2, which identifies cotransfected cells expressing no tagged p40^phox(4A) by DsRed2 fluorescence (Fig. 5F). GFP-p67^phox(AD) localized at vesicular structures accumulated at phagosomes after phagosomal closure (at least >80 s (T = 175–255); Fig. 5F and supplemental video 1). The retention time of GFP-p67^phox(AD) on phagosomes was longer than 100 s, as seen with GFP-p40^phox(PX) (11). Similar results were obtained using another p40^phox mutation that induces the active, opened conformation, p40^phox(F320A) (46) (data not shown). GFP-p67^phox(AD) was localized only in the cytoplasm when coexpressed with p40^phox(4A,289A)-IRES-DsRed2 (Fig. 5G). GFP-p67^phox(AD,355A) was also localized only in cytoplasm even when coexpressed with p40^phox(4A)-IRES-DsRed2 (Fig. 5H). In these cases, neither GFP-p67^phox(AD) nor GFP-p67^phox(AD,355A) showed any accumulation on phagosomes (data not shown). p40^phox(4A) (expressed by p40^phox(4A)-IRES-DsRed2) interacted with GFP-p67^phox(AD) (Fig. 5I, right), as seen with wild-type p40^phox (expressed by p40^phox-IRES-DsRed2) (Fig. 5I, left). This interaction was disrupted by the D289A mutation in p40^phox (expressed by p40^phox(4A,289A)-IRES-DsRed2) or by the K355A mutation in GFP-p67^phox(AD) (Fig. 5I). Consistent protein expression using these plasmids (p40^phox- IRES-DsRed2, p40^phox(289A)-IRES-DsRed2, p40^phox(4A)-IRES-DsRed2, p40^phox(4A,289A)-IRES-DsRed2, and GFP-p67^phox(AD,355K)) was also confirmed in Fig. 5I. These results demonstrate that p40^phox also functions as a "carrier" for p67^phox to phagosomes during FcR-mediated phagocytosis.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. Accumulation of p67phox on mature phagosomes in RAW264.7 cells expressing active (open) p40phox. A, Cytoplasmic localization of GFP-40phox. All imaging data are representative of at least three independent transfection experiments. B, Colocalization of GFP-p40phox(4A) and a maker of early endosomes, EEA1. C, Disappearance of the early endosome localization of GFP-p40phox(4A) by R105K mutation. D, Vesicular localization of GFP-p40phox(4A,289A). E, Immunoblotting showing protein expression by plasmids transfected into HEK293 cells. F, Subcellular localization of GFP-p67phox(AD) when coexpressed with p40phox(4A) (before stimulation), and accumulation of GFP-p67phox(AD) at phagosomes (arrowhead) after phagosomal closure. Cell exhibiting diffuse DsRed2 fluorescence expresses untagged p40phox(4A). Asterisk, circle, and triangle show the same phagosome, respectively. A movie is available in supplemental video 1. IgG-opsonized glass beads (2 µm) were added at time 0, although phagocytosis of each bead begins at different time points. G, Subcellular localization of GFP-p67phox(AD) by coexpression of p40phox(4A,289A). Cells exhibiting diffuse DsRed2 fluorescence express no tagged p40phox(4A,289A). H, Subcellular localization of GFP-p67phox(AD,355A) when coexpressed with p40phox(4A). Cells exhibiting diffuse DsRed2 fluorescence express untagged p40phox(4A). I, Interaction of GFP-p67phox(AD) both with wild-type p40phox (left panel) and p40phox(4A) (right panel) in HEK293 cells. Representative of at least three independent transfection experiments.

Sequential accumulation of p67^phox by p47^phox and p40^phox at phagosomes during FcR-mediated phagocytosis

To study the kinetics of p67^phox localization during FcR-mediated phagocytosis where both carrier proteins exist, we expressed p47^phox and an active, opened confirmation p40^phox with GFP-p67^phox(AD) using p47^phox-IRES-p67^phox(AD) and p40^phox(4A)-IRES-DsRed2. GFP-p67^phox(AD) was localized in vesicular structures and the cytoplasm before stimulation, and accumulated at phagosomal cups and on phagosomes during phagocytosis of BIgG (at least 85 s (T = 160–245); Fig. 6A and supplemental video 2). Substitution of p40^phox(4A) by p40^phox(4A,289A) abolished the vesicular localization pattern of GFP-p67^phox(AD) (Fig. 6B). These results were confirmed in complementary experiments using p47^phox-IRES-GFP-p67^phox(AD,355A) and p40^phox(4A)-IRES-DsRed2 (data not shown). Furthermore, disruptions of both interactions between p47^phox and p67^phox, and between p40^phox and p67^phox using p47^phox(PR) and p67^phox(AD,355K), respectively, resulted in no accumulation of GFP-p67^phox(AD) at phagosomal cups or on phagosomes (Fig. 6C). These results were further confirmed in complementary experiments using p47^phox(PR)-IRES-GFP-p67^phox(AD) and p40^phox(4A,289A)-IRES-DsRed2 (data not shown). These results suggest that p67^phox accumulates at phagosomal cups and on phagosomes through interactions both with p47^phox and with p40^phox, in which the early stage accumulation involves p47^phox (at phagosomal cups and phagosomes (60 s); see Fig. 4C) and the late-stage accumulation involves p40^phox (after phagosomal closure; see Fig. 5F). Expression of all three phox proteins by two plasmids (p47^phox-IRES-GFP-p67^phox(AD) + p40^phox(4A)-IRES-DsRed2, p47^phox-IRES-GFP-p67^phox(AD) + p40^phox(4A,289A)-IRES-DsRed2, and p47^phox(PR)-IRES-GFP-p67^phox(AD,355K) + p40^phox(4A)-IRES-DsRed2) was confirmed by immunoblotting (Fig. 6D).

View larger version (35K): [in this window] [in a new window]   FIGURE 6. Sustained accumulation of GFP-p67phox on phagosomal cups and phagosomes in RAW264.7 cells, involving interactions with p47phox and p40phox. A, Subcellular localization of GFP-p67phox(AD) by coexpression of p47phox(AD) and p40phox(4A) (before stimulation), and accumulation of GFP-p67phox(AD) at phagosomal cups (arrow) and phagosomes (arrowhead) during phagocytosis of BIgG. Cell exhibiting both GFP-p67phox(AD) and diffuse DsRed2 fluorescence also produces untagged p47phox and untagged p40phox(4A). A movie is available in supplemental video 2. IgG-opsonized glass beads (2 µm) were added at time 0, although phagocytosis of each bead begins at different time points. All imaging data are representative of at least three independent transfection experiments. B, Subcellular localization of GFP-p67phox(AD) when coexpressed with p47phox and p40phox(4A,289A). Cell exhibiting both GFP-p67phox(AD) and diffuse DsRed2 fluorescence also produces untagged p47phox and untagged p40phox(4A,289A). C, No accumulation of GFP-p67phox(AD) on phagosomal cups (arrow) or phagosomes (arrowhead) following disruptions of interactions between GFP-p67phox(AD) and p47phox and between GFP-p67phox(AD) and p40phox(4A). Cell exhibiting both GFP-p67phox(AD,355K) and diffuse DsRed2 fluorescence also produces untagged p47phox(PR) and untagged p40phox(4A). D, Immunoblotting showing protein expression by plasmids (HEK293 cells) used in Fig. 6.

Prolonged accumulation of p47^phox by p40^phox at phagosomes during FcR-mediated phagocytosis

Although we demonstrate that both p47^phox and p40^phox function as "carrier" proteins for p67^phox, the carrier function of p40^phox appears to be stronger than that of p47^phox when mutated opened form of p40^phox is expressed. To confirm this observation, GFP-p47^phox kinetics during FcR-mediated phagocytosis were monitored with coexpression of p67^phox(AD) and p40^phox(4A), using p67^phox(AD)-IRES-GFP-p47^phox and p40^phox(4A)-IRES-DsRed2. GFP-p40^phox was localized at vesicular structures and the cytoplasm before stimulation, and during phagocytosis of BIgG GFP-p47^phox accumulated at phagosomal cups and phagosomes (at least >105 s (T = 210–315); Fig. 7A and supplemental video 3). The vesicular structures also fused to phagosomes (supplemental video 3). Substitution of p67^phox(AD) with p67^phox(AD,355A) revealed no vesicular localization pattern of GFP-p47^phox (Fig. 7B); however, it still showed the accumulation at phagosomal cups (limited (not prolonged) accumulation; supplemental video 4). This result was further confirmed in complementary experiments using p67^phox(AD)-IRES-GFP-p47^phox and p40^phox(4A,289A)-IRES-DsRed2 (data not shown). These results indicate that p40^phox functions as a "carrier" even for p47^phox through p67^phox interactions. To verify the carrier function of p40^phox for p47^phox, we examined the retention times of GFP-p47^phox at phagosomal cups and on phagosomes during phagocytosis of BIgG. The retention of GFP-p47^phox (n = 8, 63.8 ± 13.8 s) was markedly prolonged (n = 13, 220.0 ± 40.6 s) by coexpression with p40^phox(4A), and this prolonged accumulation time was abolished (n = 9, 62.8 ± 12.0 s) by the substitution of p40^phox(4A) with p40^phox(4A,289A) (Fig. 7C). Expression of all three phox proteins by two plasmids (p67^phox(AD)-IRES-GFP-p47^phox + p40^phox(4A)-IRES-DsRed2, p67^phox(AD,355K)-IRES-GFP-p47^phox p40^phox(4A)-IRES-DsRed2, and p67^phox(AD)-IRES-GFP-p47^phox + p40^phox(4A,289A)-IRES-DsRed2) was confirmed by immunoblotting (Fig. 7D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In this report, we engineered a method using complementary fragments of the monomeric coral fluorescent reporter protein (mKG) to detect protein-protein interactions between fusion proteins at the cellular level by direct visualization under a confocal fluorescence microscope. There are several reports using the complementation-based method to detect protein-protein interaction (54, 55, 56, 57, 58, 59). The first report of the complementation method using fluorescent protein was based on GFP in E. coli (60). Subsequently, methods using YFP, BFP, or CFP in mammalian cells were also reported (45, 61). Our newly developed mKG system has two distinct advantages from these conventional methods: 1) the background signal observed related to nonspecific binding is very low and 2) because mKG has sharper emission spectra than BFP/CFP (data not shown), the mKG system would be better suited for simultaneous visualization of multiple protein interactions within the same cell, if we can engineer different color fluorescent proteins by through mutations in the N-terminal fragment of mKG. The complementation-based method is a very easy way for screening protein-protein (domain-domain) interaction in real time, compared with conventional methods such as immunoprecipitation, in vitro-binding assays with purified proteins, the yeast two-hybrid system, detection of surface plasmon resonance using BIACORE, and fluorescence resonance energy transfer analysis. The mKG system has high efficacy and specificity for detecting real-time protein-protein interactions in intact cells. As shown in Fig. 2, the specificity of this system was confirmed two ways: 1) in vitro pull-down assays using purified proteins and 2) inhibition of the specific binding of p40^phox(PX) to early endosomes by intramolecular contacts between the PB1 and PX domains of p40^phox. As shown in Fig. 2D, the mKG system is also very reliable, because the system is based on a GFP and the specific protein-protein interaction could be confirmed by direct visualization of red fluorescent protein. However, the mKG system cannot evaluate the strength of binding affinity among different sets of interacting proteins, because the observed fluorescence intensity of complemented mKG protein depends on the varying intrinsic stabilities and levels of the expressed complementary tagged proteins. Thus, we cannot compare fluorescence intensity values as a direct indication of relative binding affinities among the different interacting partners studied in Figs. 1, 2, 3A, and 3B.

Using the mKG system, we also evaluated interdomain interactions within p47^phox that affect the accessibility of its membrane-binding domains. Targeting of p47^phox to the phagosomal membrane requires two distinct interactions: binding of its PX domain to membrane phospholipids (both PI(3,4)P₂ and PA) and interactions between its tandem SH3 domains and a proline-rich (PR) motif on p22^phox. We have reported that disruption of either one of these interactions abolishes membrane targeting of p47^phox (11, 48). The SuperSH3 model specifying autoinhibitory AIR interactions responsible for masking of the tandem SH3 domains in the resting, unphosphorylated state has been well-recognized (52, 53). However, details on autoinhibitory interactions responsible for masking the PX domain of p47^phox remain unclear, and direct binding of the PX domain PXXP motif to SH3c (51) is incompatible with and would preclude the autoinhibitory SuperSH3/AIR interactions, because the two interactions would involve the same binding surface of SH3c. The present study indicates that the "closed" structure encompassing the tandem SH3 domains bound to the AIR (SuperSH3/AIR) interacts directly with the PX domain, and that this interaction is enhanced by residues 341–360. Recently, Durand et al. (62) reported that p47^phox in the resting state adopts an elongated (nonglobular) conformation detected by small-angle x-ray scattering, and suggested weak interactions are plausible between the PX domain and the SuperSH3/AIR structure and could also involve a sequence downstream of the AIR (62), consistent with the interdomain contacts we detected by the mKG reporter system. Furthermore, microcalorimetry titration experiments that suggest the PX domain contributes to or enhances the SuperSH3/AIR interaction (52) are consistent with our observations using the mKG system.

We recently showed using the AA-stimulated RAW264.7 cell model that both p40^phox and p47^phox function as regulated "carrier" proteins for p67^phox through distinct membrane-targeting mechanisms (11). Although neutrophils from CGD patients lacking p47^phox or p67^phox show impaired translocation of p40^phox to the membrane fraction by soluble stimulation (PMA or fMLP) (26), it is well-known that p40^phox functions in FcR-mediated phagocytosis (33, 34). We and others reported that p47^phox accumulates transiently on phagosomal cups and mature phagosomes during FcR-mediated phagocytosis and acts as a "carrier" for p67^phox (27, 48); however, little is known on how p40^phox functions in the assembly of the Nox2 complex during FcR-mediated phagocytosis. Here, we demonstrated that both p40^phox and p47^phox function as "carrier" proteins for p67^phox during FcR-mediated phagocytosis: p47^phox functions in early stages of phagocytosis during phagosome formation, while p40^phox functions in late stages of phagocytosis after phagosomal closure (dependent on PI(3)P generation). Furthermore, the accumulation or retention times of p47^phox on phagosomes are prolonged by coexpression of an active form of p40^phox, p40^phox(4A), which has an open conformation accessible to PI(3)P (63.8 S –220 s: Fig. 7C). Consistent with these observations, the specific affinity of the p40^phox PX domain for liposomes containing PI(3)P is 3.5 times higher than that of the p47^phox PX domain for PI(3,4)P₂ (22, 63), although addition of PA to PI(3,4)P₂ increases the affinity of the p47^phox PX domain (22). Although addition of high concentrations (20 µM) of PI(3,4)P₂ to the HEK293 cell alone promotes the plasma membrane translocation of GFP-p47^phox(PX) (63), we reported that both the SH3 domain (p47^phox)-PR motif (p22^phox) interaction and PX domain are required for p47^phox translocation to the plasma membrane (11). In contrast to p47^phox, the carrier function of p40^phox is dependent on PI(3)P binding of the PX domain. However, p47^phox is absolutely essential for stable binding of the ternary phox complex (p47^phox-p67^phox-p40^phox) to flavocytochrome b₅₅₈ through interaction with p22^phox even when the carrier function for the ternary phox complex is transferred from p47^phox to p40^phox, because mutations associated with this interaction (P156Q in p22^phox and W193R in p47^phox) completely abolish ROS production (48, 64, 65). We extend the model for Nox2 complex assembly by accounting for these spatiotemporal factors during the FcR-mediated respiratory burst: 1) in early stages, p47^phox functions as a carrier for translocation of the ternary phox complex to newly forming phagosomes (during and following phagosomal cup formation) both through PI(3,4)P₂ and PA specific-binding of the PX domain of p47^phox and through interaction with p22^phox; 2) in later stages following phagosomal closure, the carrier function of p47^phox is replaced by p40^phox, whereby the ternary phox complex interacts with sealed phagosomes and is retained at phagosomes through PI(3)P specific-binding of the PX domain of p40^phox. PA derived from PLD2 (66) and PI(3,4)P₂ derived from SHIP-1 (67) could play important roles in targeting p47^phox to the phagosomal cup. In the present study, we used the active, opened-conformation form of p40^phox, p40^phox(4A), to delineate sequential interactions of the ternary phox complex during FcR-mediated phagocytosis. A recent report also supports our proposed "carrier" function of p40^phox for the ternary phox complex (31); using fMLP and PMA as activators, it showed that disruption of the p40^phox PX domain interactions leads to dissociation of the active, membrane-bound oxidase complex. It is interesting that a severe defect in Staphylococcus aureus killing occurs in p40^phox-deficient mice (32), although there are no reports to date of CGD or CGD-like symptoms related to p40^phox deficiency. Further study is needed to address the questions of how and when p40^phox is activated to translocate and participate in assembly of the Nox2 complex during FcR-mediated phagocytosis (68).

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by a Grant-in-Aid for Scientific Research from the Global Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan; a Grant-in-Aid for Scientific Research on Priority Areas and (C) of Education, Culture, Sports, Science, and Technology in Japan; the SHISEIDO Grants for Scientific Research; a grant from Hyogo Science and Technology Association; and a grant from The Naito Foundation.

² Address correspondence and reprint requests to Dr. Naoaki Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. E-mail address: naosaito{at}kobe-u.ac.jp

³ Abbreviations used in this paper: ROS, reactive oxygen species; CGD, chronic granulomatous disease; PA, phosphatidic acid; SH3, Src homology 3; AA, arachidonic acid; mKG, monomeric Kusabira Green; AIR, autoinhibitory region; EEA1, early endosome Ag-1; RT, room temperature; LZA, leucine zipper acidic protein; LZB, leucine zipper basic protein; AD, activation domain; IRES, internal ribosomal entry site; PR, proline rich.

⁴ The online version of this article contains supplemental material.

Received for publication November 30, 2007. Accepted for publication April 29, 2008.

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