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J. Biol. Chem., Vol. 277, Issue 6, 4512-4518, February 8, 2002

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The p40^phox and p47^phox PX Domains of NADPH Oxidase Target Cell Membranes via Direct and Indirect Recruitment by Phosphoinositides^*

Yong Zhan, Joseph V. Virbasius, Xi Song, Darcy P. Pomerleau, and G. Wayne Zhou

From the Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication, October 2, 2001, and in revised form, November 28, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Phox homology (PX) domain has recently been reported to bind to phosphoinositides, and some PX domains can localize to endosomes in vivo. Here we show data to support the conclusion that the p40^phox PX domain binds to phosphatidylinositol 3-phosphate specifically in vitro and localizes to endosomes in intact cells. In addition, its Y59A/L65Q mutant, which has decreased affinity for phosphatidylinositol 3-phosphate in vitro, fails to target EGFP-p40-PX to endosomes. However, unlike published results, we find that the p47^phox PX domain weakly binds to many phosphoinositides in vitro showing slightly higher affinity for phosphatidylinositol 3,4,5-trisphosphate. Moreover, we show for the first time that upon insulin-like growth factor-1 stimulation of COS cells, the p47^phox PX domain is localized to the plasma membrane, and this subcellular localization is dependent on PI 3-kinase activity. Unexpectedly, its R42Q mutant that loses in vitro phosphoinositide-binding ability can still target EGFP-p47-PX to the plasma membrane. Our data suggest that the translocation of p47^phox PX domain to the plasma membrane does involve 3'-phosphoinositide(s) in the process, but the phosphoinositide-binding of p47^phox PX domain is not sufficient to recruit it to the plasma membrane. Therefore, the p40^phox and p47^phox PX domains can target subcellular membranes via direct or indirect recruitment by phosphoinositides, while both are under the control of phosphatidylinositol 3-kinase activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Phox homology (PX)¹ domain is a module with about 130 amino acids that is present in the p40^phox and the p47^phox subunit of NADPH oxidase as well as in some other proteins such as Mvp1p, Vps5p, and Grd19p in yeast, and phospholipase D1, PI 3-kinase, CISK, and sorting nexin proteins in mammalian cells (1-8). It represents a widely distributed module with potential functions in regulation of vesicular trafficking, growth factor-receptor degradation in lysosomes, signal transduction mechanisms, and NADPH oxidase activity. Recently, PX domains have been shown to interact with different phosphoinositides and target the PX domain-containing proteins to specific subcellular locations (see Refs. 2-8; for reviews, see Refs. 10 and 11). Several PX domains, including those of Vam7p, SNX3, CISK, and p40^phox, specifically interact with PI(3)P. They can also target those proteins to endosomes in vivo (2-5, 7, 8). In addition, 14 out of 15 PX domains from yeast bind to PI(3)P with different affinity (40). However, the PX domain of CPK PI 3-kinase interacts with PI(4,5)P₂ (6), while the p47^phox PX domain has been reported to preferentially interact with PI(3,4)P₂ (5). Moreover, the PX domain of p47^phox shows binding to its C-terminal SH3 domain (9).

NADPH oxidase, consisting of both membrane-associated subunits and cytosolic subunits, plays a very important role in the immune defense system of neutrophils and other phagocytic cells (for a recent review, see Ref. 12). Defects in those subunits will result in dysfunction of the enzyme and clinically will cause chronic granulomatous disease. Patients with chronic granulomatous disease suffer severe, recurrent bacterial and fungal infections. In the resting state, the subunits of NADPH oxidase are differentially localized to the membrane (gp91^phox and p22^phox) and to the cytoplasm (p40^phox, p67^phox, and p47^phox). In response to invasive microorganisms, the cytosolic subunits of NADPH oxidase translocate to the membrane and fuse with the membrane-bound subunits to form an activated enzyme complex. The core part of the active enzyme consists of the cytosolic subunit p67^phox, a GTPase Rac1/2, the membrane subunits gp91^phox and p22^phox, while p47^phox is required for the assembly of the active NADPH oxidase complex acting as an adaptor for p67^phox (13-15), and the role of p40^phox is likely to be a regulator in the translocation of p67^phox (16-18). p40^phox has also been shown to be critical for the function of the active enzymatic complex (4). However, little is known about the functions of the two cytosolic PX domain-containing subunits p40^phox and p47^phox in the activation of NADPH oxidase, especially how they are activated and translocated to form a functional complex with the membrane-bound subunits. Recently, two reports have indicated that the PX domains in p40^phox and p47^phox bind to different phosphoinositides (4, 5). In addition, the p40^phox PX domain can localize to endosomes in vivo by binding to PI(3)P. Here we report data that support the above conclusion regarding the p40^phox PX domain (4, 5). Furthermore, we find that the phosphoinositide-binding profile of the p47^phox PX domain differs from the published data (5). We also show that the p47^phox PX domain can localize to the plasma membrane upon IGF-1 activation of COS cells and that the membrane targeting is sensitive to PI 3-kinase activity. However, this targeting function is not dependent on the phosphoinositide-binding ability of the p47^phox PX domain. Our data suggest that the PX domains in NADPH oxidase can target subcellular membranes via direct or indirect recruitment by phosphoinositides, controlled by PI 3-kinase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- All phospholipids used for the liposome binding assays were purchased and prepared as reported before (6). Nitrocellulose membranes spotted with phospholipids from 100 to 1.56 pmol/spot (PIP-Array^TM) were purchased from Echelon Research Laboratories.

Construction of Plasmids and Mutagenesis-- DNA fragments encoding the p40^phox PX domain (amino acids 9-140) and the p47^phox PX domain (amino acids 2-125) were amplified from a human cDNA library (CLONTECH, from human spleen) and cloned into pXL1a vector (modified from pET15b) with the cleavage of NheI and SalI enzymes. For green fluorescent protein fusion, the coding sequence of p40^phox PX domain was subcloned into pEGFP-C1 vector (CLONTECH) by the PCR-mediated addition of XhoI and PstI sites to p40-PX sequence (amino acids 13-133). EGFP-p47-PX was similarly generated by the addition of XhoI and BamHI sites to p47-PX (amino acids 2-116). The Y59A/L65Q double mutant of the p40^phox PX domain and the R42Q mutant of the p47^phox PX domain were made using the QuikChange^TM XL site-directed mutagenesis kit (Stratagene) following the manufacturer's protocol. The P73A/P76A double mutant and the R42Q/P73A/P76A triple mutant of p47-PX were similarly generated by using the wild type EGFP-p47-PX or EGFP-p47-PX R42Q mutant as the template. All cloned plasmids were confirmed by DNA sequencing.

Expression and Purification of Fusion Proteins-- The recombinant pXL1a plasmids encoding the corresponding PX domains were transformed into Escherichia coli BL21(DE3). Cells were grown at 37 °C to A₆₀₀ 0.5-0.6 and induced with 0.1 mM isopropyl -D-thiogalactoside for 6 h at 18 °C. Cells were lysed by sonication in extraction buffer (25 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 10 mM -mercaptoethanol, and protease inhibitors 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and then cleared by centrifugation. The supernatant was incubated with Ni²⁺-nitrilotriacetic acid-agarose beads (Qiagen) at 4 °C. After serial wash, the fusion protein was finally eluted from the beads with elution buffer (25 mM Tris·HCl, 150 mM NaCl, 125-250 mM imidazole, pH 7.5) and then buffer-exchanged to liposome binding buffer.

Liposome Binding Assay-- A published liposome binding assay protocol (6) was used for the binding experiments with minor modification. The phospholipid mixture (100 µg/reaction) was dried in a SpeedVac (Savant). The dried mixture was then resuspended in 100 µl of liposome buffer (25 mM Tris·HCl, 150 mM NaCl, pH 7.5); sonicated in a bath sonicator for 15 min; and spun for 10 min at 14,000 rpm at 4 °C. The liposomes were resuspended in 100 µl of binding buffer (25 mM Tris·HCl, 150 mM NaCl, 1 mM MgCl₂, pH 7.5) containing 10 µg of purified protein and incubated for 15 min at room temperature and then centrifuged as before. The supernatant was saved, and the pellet was resuspended in 100 µl of binding buffer. Then 25 µl of both fractions was analyzed by SDS-PAGE and Coomassie Blue staining. The intensity of the stained bands was quantitatively determined with an LKB Ultroscan laser densitometer.

Protein-Lipid Overlay Assay-- Protein-lipid overlay assays were performed as previously described (19), except that His₆-tagged proteins were used in this study. Various phospholipids were dissolved in a mixture of methanol and chloroform in 1:1 ratio and then spotted onto nitrocellulose membranes (Hybond-C; Amersham Biosciences) and air-dried. The membrane with various phospholipids was blocked in 3% fatty acid-free bovine serum albumin (Sigma) in TBST buffer (50 mM Tris·HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h at room temperature. The membrane was then incubated overnight at 4 °C in the same solution with 0.2 µg/ml His-tagged protein. After wash with TBST buffer, the membrane was incubated for 1 h at room temperature with 1:1,000 dilution of the anti-His monoclonal antibody (Novagen). The membrane was washed as before and then incubated with a 1:5,000 dilution of alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Bio-Rad). Finally, the membrane was washed with TBST buffer, and the His-tagged protein binding to the phospholipids was detected by enhanced chemiluminescence (Bio-Rad). Protein-lipid overlay assays with PIP-Array^TM were performed while following the same procedure as above.

Cell Transfection and Fluorescence Microscopy-- COS cells were maintained and transfected by the calcium phosphate method. After 16 h, cells were changed to serum-free medium for 3 h before further treatment. For transferrin labeling, cells were incubated for 15 min with 5 µg/ml rhodamine-conjugated human transferrin (Molecular Probes, Inc., Eugene, OR) and then fixed in 4% formaldehyde in PBS. For actin staining, cells were fixed in 4% formaldehyde following treatment with wortmannin (Sigma) and/or IGF-1 (Calbiochem). Cells were then permeabilized in PBS with 1% fetal bovine serum and 0.5% Triton X-100 and then incubated for 30 min with 1 unit/ml rhodamine-conjugated phalloidin (Molecular Probes) in the same buffer. Cells were washed in the same buffer and mounted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The p40^phox PX Domain Specifically Binds to PI(3)P-- We generated the p40^phox PX domain as the His-tagged protein expressed in bacterial cells and investigated its binding affinity for phosphoinositides using a protein-lipid overlay assay. The purified protein was incubated with a membrane spotted with various phospholipids, and bound protein was detected with anti-His antibody. As shown in Fig. 1A, p40-PX exhibited high affinity for PI(3)P, low affinity for PI(4)P, PI(5)P, and PI(3,5) P₂, but no binding to other phosphoinositides. When lipid membrane spotted with serially diluted phospholipids (PIP-Array^TM) was employed, p40-PX bound to PI(3)P at low concentrations and bound to PI(4)P and PI(5)P at relatively high concentrations, whereas binding to PI(3,5)P₂ was only detected at the 100-pmol spot (Fig. 1B).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The PX domain of p40phox selectively binds to PI(3)P. A and B, binding of either wild-type p40-PX (WT) or p40-PX (Y59A/L65Q) mutant to immobilized phospholipids. Nitrocellulose membranes spotted with 100 pmol of different phospholipids (A) or with serially diluted phospholipids (B) were incubated with either wild-type or mutant p40-PX proteins. Then the anti-His antibody was applied to detect the proteins binding to specific phospholipids by enhanced chemiluminescence. C and D, liposome binding assays demonstrate the binding selectivity of p40-PX for PI(3)P. C, binding percentage of p40-PX to liposomes containing 5% phosphoinositides. Liposomes were composed of 50 µg of PC and 50 µg of PE (designated as CE) or of 47.5 µg of PC, 47.5 µg of PE, and 5 µg of the tested phosphoinositides. The purified p40-PX protein was incubated with liposomes for 15 min. After centrifugation, liposome pellets (P) and supernatants (S) were analyzed by SDS-PAGE and Coomassie Blue staining. D, comparison of wild-type p40-PX to its Y59A/L65Q mutant in binding to PI(3)P. Liposomes were composed of equal amount of PC and PE plus the indicated percentages of PI(3)P. The graph shows the binding percentage of the wild-type p40-PX and its Y59A/L65Q mutant to liposomes containing different amounts of PI(3)P. The results are the average of three independent experiments, whereas the gels (insets) show a representative result. 3P, phosphatidylinositol 3-phosphate; 4P, phosphatidylinositol 4-phosphate; 5P, phosphatidylinositol 5-phosphate; 34P2, phosphatidylinositol 3,4-bisphosphate; 35P2, phosphatidylinositol 3,5-bisphosphate; 45P2, phosphatidylinositol 4,5-bisphosphate; P3, phosphatidylinositol 3,4,5-trisphosphate.

We further studied the phosphoinositide-binding selectivity of the p40^phox PX domain using the liposome binding assay. p40-PX displayed remarkably selective binding to liposomes that contain 5% of PI(3)P (Fig. 1C). About 30% p40-PX protein was observed in the liposome pellets with PI(3)P, while less than 8% of p40-PX protein was observed in the liposome pellets with other phosphoinositides. To further characterize its binding affinity for PI(3)P, we quantified the percentage of p40-PX protein present in the supernatants and in the liposome pellets after incubation with liposomes that contain between 1 and 20% of PI(3)P (Fig. 1D). The PI(3)P binding of p40-PX was observed in liposome pellets with as little as 1% of PI(3)P and increased with increasing concentrations of PI(3)P in the liposomes until it reached saturation for liposomes containing more than 15% of PI(3)P. All of the results suggest that the p40^phox PX domain selectively interacts with PI(3)P, consistent with the published reports (4, 5).

The Y59A/L65Q Double Mutant of the p40^phox PX Domain Remarkably Reduces Its Binding Affinity for PI(3)P-- A double mutation (Y42A/L48Q) within the PX domain of Vam7p results in vacuolar trafficking defects in yeast (20) caused by the loss of its ability to bind to PI(3)P (2, 6). Since these two amino acids are conserved in the PX domains of p40^phox and Vam7p, we generated the corresponding double mutant (Y59A/L65Q) of p40-PX and examined its binding affinity for PI(3)P, using both the protein-lipid overlay assay and the liposome binding assay. In protein-lipid overlay assays, binding of the mutant to PI(3)P could be seen only at relatively high concentrations (Fig. 1B). The overall binding affinity was reduced to a level similar to PI(4)P and PI(5)P (Fig. 1, A and B). The liposome binding assay also showed that the double mutant lost its ability to bind to liposomes containing PI(3)P. Less than 5% of total mutated protein was observed in the liposome pellets containing 15 or 20% of PI(3)P (Fig. 1D). Therefore, the double mutant of the p40^phox PX domain selectively decreases its binding ability to PI(3)P.

The p40^phox PX Domain Is Sufficient for Endosome Localization in Vivo-- We tested the binding ability of the isolated p40^phox PX domain to PI(3)P in vivo by fusing it to the C terminus of enhanced green fluorescent protein and transfecting the EGFP-p40-PX expression vector into COS cells. The EGFP-p40-PX protein was found to localize to numerous cytoplasmic vesicles concentrated in the perinuclear region (Fig. 2A). This pattern of staining overlapped almost completely with the pattern of fluorescence obtained upon loading cells with rhodamine-conjugated transferrin (Fig. 2, B and C), indicating that the p40^phox PX domain binds to endosomal membranes in vivo, where PI(3)P has been known to reside in intact mammalian cells (21-23). Upon the addition of the PI 3-kinase inhibitor wortmannin, vesicular staining with EGFP-p40-PX was lost; instead, EGFP-p40-PX appeared as diffuse cytoplasmic staining with some localization to the nucleus (Fig. 2D). Likewise, incorporation of the double mutation, which diminishes PI(3)P binding in vitro, resulted in a similar diffuse pattern in vivo (Fig. 2E). These results are consistent with the notion that the p40^phox PX domain is capable of targeting to endosomes in vivo based on its ability to bind selectively and strongly to PI(3)P.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   The p40phox PX domain colocalizes with an endosomal marker in COS cells dependent on PI 3-kinase activity. COS cells were transfected with EGFP-p40-PX (A-D) or the PI(3)P binding-defective Y59A/L65Q mutant of EGFP-p40-PX (E). Transfected cells were also labeled with rhodamine-conjugated transferrin for 15 min (A-C) or treated with 100 nM wortmannin for 15 min (D).

The p47^phox PX Domain Shows Promiscuity in Phosphoinositide Binding-- We also generated the His-tagged p47^phox PX domain and examined its binding affinity for different phosphoinositides by using protein-lipid overlay assays (Fig. 3, A and B). When the same amounts of p40-PX and p47-PX proteins were used, p47-PX exhibited much weaker binding to all of the phosphoinositides tested, compared with that of p40-PX (Fig. 1B). Among the seven phosphoinositides, p47-PX showed relatively more affinity for PI(3)P, PI(5)P, and PI(3,4,5)P₃, less affinity for PI(3,5)P₂, PI(4,5)P₂, and PI(4)P, and no detectable binding to PI(3,4)P₂ (Fig. 3, A and B).

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Characterization of p47-PX binding to different phosphoinositides. A and B, binding of either wild-type (WT) p47-PX or p47-PX (R42Q) mutant to immobilized phospholipids. Protein-lipid overlay assays were performed as described under "Experimental Procedures" and also in Fig. 1. Nitrocellulose membranes were spotted with 100 pmol of different phosphoinositides (A) or different phosphoinositides (B) from 1.56 to 100 pmol. C and D, liposome binding assays demonstrate the binding of wild-type p47-PX to different phosphoinositides. Liposomes were composed of 5, 10, or 15 µg of different phosphoinositides, while PC and PE in equal amounts comprised the rest in each liposome containing 100 µg of phospholipid in total. E, comparison of wild-type p47-PX with its R42Q mutant in binding to different PIPs. It was performed with 5% liposomes, which contained 47.5 µg of PC, 47.5 µg of PE, and 5 µg of different PIPs. The gels (shown as insets) were quantitatively analyzed by densitometry, and data were the average of three separate experiments. 3P, phosphatidylinositol 3-phosphate; 4P, phosphatidylinositol 4-phosphate; 5P, phosphatidylinositol 5-phosphate; 34P2, phosphatidylinositol 3,4-bisphosphate; 35P2, phosphatidylinositol 3,5-bisphosphate; 45P2, phosphatidylinositol 4,5-bisphosphate; P3, phosphatidylinositol 3,4,5-trisphosphate.

We also examined the percentage of p47-PX bound to liposomes that contain from 5 to 15% of different phosphoinositides. Fig. 3C shows the SDS-PAGE gels of p47-PX in the supernatants and in the liposome pellets containing different amounts of phosphoinositides, whereas Fig. 3D shows the binding percentage. In liposomes containing 5% tested phosphoinositides, about 40% of p47-PX protein was found to bind to PI(3,4)P₂ or PI(3,5)P₂, whereas less than 25% of p47-PX protein bound to liposomes containing other phosphoinositides. However, the difference in binding affinity among all phosphoinositides tended to be less at higher phosphoinositide concentrations (Fig. 3D).

Both the protein-lipid overlay assay and the liposome binding assay showed that the p47^phox PX domain bound to most phosphoinositides. However, they differ on the order of binding affinity. For example, the PI(3,4)P₂ binding of p47-PX was not observed, and the PI(3,5)P₂ binding was very weak in protein-lipid overlay assays, but it bound to both of them with a relatively high percentage in the liposome binding assays. The binding of p47-PX to the two phosphoinositides has also been reported before (5). Meanwhile, p47-PX showed consistent binding to PI(3,4,5)P₃, in both the protein-lipid overlay and the liposome binding assay.

The R42Q Mutant of p47^phox PX Domain Eliminates Its Binding to Phosphoinositides-- Recently, a point mutation (R42Q) in the p47^phox PX domain was identified in patients with chronic granulomatous disease (24). To investigate the effects of the mutation on phosphoinositide binding, we generated the R42Q mutant of the p47^phox PX domain and applied it to both the protein-lipid overlay assay and the liposome binding assay. No binding was detected in the 5% liposome binding assay (Fig. 3E). Subsequently, the protein-lipid overlay assay also showed similar results. The binding to PI(3)P, PI(4)P, and PI(5)P was also reduced, whereas binding to PI(3,5)P₂, PI(4,5)P₂, PI(3,4,5)P₃, and PI(3,4)P₂ was totally undetectable (Fig. 3, A and B).

The p47^phox PX Domain Targets the Plasma Membrane upon IGF-1 Activation-- We fused the p47^phox PX domain to EGFP, transfected the EGFP-p47-PX to COS cells, and then examined its localization in IGF-1-stimulated or -unstimulated cells. In basal cells, EGFP-p47-PX was found throughout the cytoplasm as well as in the nucleus with little if any association with the plasma membrane (Fig. 4A). However, upon stimulation with IGF-1, EGFP-p47-PX appeared to collect at sites of membrane folding or protrusion (Fig. 4D). These are apparently sites of membrane ruffling, as indicated by the concentration of F-actin at these sites, detected by staining with rhodamine-conjugated phalloidin (Fig. 4E). Since growth factor-stimulated membrane-ruffling has been shown to coincide with PI 3-kinase products in several systems (25-27), we tested whether the localization of EGFP-P47-PX was sensitive to the PI 3-kinase inhibitor wortmannin. In the presence of wortmannin, EGFP-p47-PX failed to translocate to membrane ruffles (Fig. 4G), and no substantial colocalization with F-actin could be observed (Fig. 4, H and I). Therefore, the membrane-targeting of EGFP-p47-PX is regulated by the PI 3-kinase activity.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   The p47phox PX domain translocates to membrane ruffles dependent on PI 3-kinase activation. COS cells were transfected with wild-type EGFP-p47-PX. Cells were serum-starved and then left untreated (A-C), stimulated with 25 ng/ml IGF-1 for 5 min (D-F), or treated with 100 nM wortmannin (G-I) prior to IGF-1 stimulation. After fixing and permeabilization, cells were labeled with rhodamine-conjugated phalloidin.

Mutants of p47^phox PX Domain Still Possess Membrane-targeting Function-- To test whether the membrane-targeting function of the p47^phox PX domain is caused by the interactions between it and phosphoinositides, we generated the R42Q mutant of p47-PX, which had diminished binding to all PIPs in vitro, fused the mutant to EGFP, and examined its translocation and colocalization with F-actin on IGF-1 stimulation. Surprisingly, the mutant could still localize to the plasma membrane and colocalize with F-actin (Fig. 5, A-C). This suggests that the phosphoinositide binding ability is not sufficient to recruit the p47^phox PX domain to the plasma membrane.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   The p47phox PX domain mutants target the plasma membrane. COS cells were transfected with the EGFP-p47-PX R42Q mutant (A-C), R42Q/P73A/P76A triple mutant (D-F), or P73A/P76A double mutant (G-I); stimulated with 25 ng/ml IGF-1 for 5 min; and then labeled with rhodamine-conjugated phalloidin.

In the p47^phox subunit, the PX domain contains a PXXP motif that interacts with its C-terminal SH3 domain (9). We hypothesized that the membrane targeting of the p47^phox PX domain could be caused by the interaction between the polyproline motif in the PX domain and a SH3 domain-containing protein in the plasma membrane. To test this hypothesis, we generated both the P73A/P76A double mutant and the R42Q/P73A/P76A triple mutant of EGFP-p47-PX and examined their translocation in COS cells on IGF-1 stimulation. Unexpectedly, both mutants still possessed the ability to translocate to the plasma membrane (Fig. 5, D-I). These results suggest that the interactions, other than simple PX-SH3 interaction between the p47^phox PX domain and some other protein(s) in the plasma membrane, might contribute to the membrane-targeting function of p47^phox.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The subunits of NADPH oxidase are separately located at the membrane and cytosol in the resting state. Once activated by the invading microorganisms, the cytosolic subunits will translocate to the membrane and form an active enzyme complex with the membrane-bound subunits. However, the detailed mechanism for the assembly and activation of NADPH oxidase is not yet understood. Recently, the PX domains in p40^phox and p47^phox were reported to bind to different phosphoinositides and may potentially target the cytosolic subunits to specific membrane locations (4, 5). Our results support the published report about the p40^phox PX domain. It is shown that the p40^phox PX domain binds to PI(3)P specifically with high affinity, while the Y59A/L65Q double mutant almost abolishes its PI(3)P binding in vitro. We have also shown that the p40^phox PX domain is able to colocalize with rhodamine-conjugated transferrin, an organelle marker for endosomal membranes in intact cells. This PI(3)P-dependent subcellular localization was lost for the Y59A/L65Q double mutant or after the transfected cells were treated with wortmannin, a PI 3-kinase inhibitor.

We find that the p47^phox PX domain has varied phosphoinositide-binding patterns in different assays. In protein-lipid overlay assays, the p47^phox PX domain showed an order of preference of PI(5)P > PI(3)P = PI(3,4,5)P₃ > PI(4)P > PI(3,5)P₂ = PI(4,5)P₂, but it did not show binding to PI(3,4)P₂. However, in liposome binding assays, the p47^phox PX domain bound to most phosphoinositides, with an order of PI(3,4)P₂ = PI(3,5)P₂ > PI(3)P = PI(5)P = PI(3,4,5)P₃ = PI(4,5)P₂ > PI(4)P at low lipid concentration. In contrast, a previous report shows that the p47^phox PX domain binds with high affinity to PI(3,4)P₂ > PI(3,5)P₂ > PI(3)P = PI(3,4,5)P₃ > PI(4)P = PI(5)P = PI(4,5)P₂ (5). Our liposome binding data are consistent with the published data that suggest the p47^phox PX domain binds to PI(3,4)P₂ (5). However, the protein-lipid overlay assay shows no binding to PI(3,4)P₂ in about 10 different experiments of our study.

Unlike p40-PX, which distinctly interacts with PI(3)P, p47-PX shows conflicting evidence on its phosphoinositide binding from different binding assays. It seems that the p47^phox PX domain does not have clear selectivity in binding to phosphoinositides. It has been noticed that p47-PX is a highly positively charged (28) domain, and polyphosphorylation apparently gives phosphoinositides negative charge. The nonselectivity of p47-PX to phosphoinositides, as also seen for some PH domains, may be due to nonspecific electrostatic attraction between the positive face of the PX domain and negatively charged surfaces formed by acidic phospholipids (29). In order to address this point, we have tested the binding of p47-PX to PI and PS, which are both negatively charged phospholipids. As in the protein-lipid overlay assay, no binding of p47-PX to PI or PS was presented (Fig. 3, A and B). We then generated liposomes in which PI or PS comprised 80% of the total lipid and contained up to 16-fold the phosphoinositides in the previous liposome binding assays for p47-PX. However, less than 15% of p47-PX protein was found to bind to PS (data not shown), still lower than the percentage in the previous studies with most phosphoinositides, while no binding to PI was present. Hence, the nonspecific electrostatic interaction seems to exist in the liposome binding assays at higher concentrations but is unlikely to account for all of the phosphoinositide-binding affinity of p47-PX. Although we cannot rule out the possibility that nonspecific binding exists in protein-lipid overlay assays, it is likely that the PX domain of p47^phox possesses the ability to interact with multiple lipid ligands, since it displays preference for PI(3,4,5)P₃ and some affinity for PI(3)P and PI(3,5)P₂ in this study, while its binding to PI(3,4)P₂ has also been previously observed (5). All of these 3'-phosphoinositides are the products of PI 3-kinases.

In order to examine the potential targeting function of the p47^phox PX domain, we fused it with EGFP and introduced it into COS cells. In unstimulated cells, EGFP-p47-PX was not localized to endosomes, despite the PI(3)P binding ability in vitro (Fig. 4A). However, when cells were treated with IGF-1, EGFP-p47-PX translocated to discrete plasma membrane sites, which corresponded to the membrane ruffles as evidenced by colocalization of EGFP-p47-PX with phalloidin-stained F-actin; pretreatment with wortmannin abolished this phenomenon (Fig. 4). Our data show for the first time that the p47^phox PX domain can target the plasma membrane, and the targeting function is dependent on the PI 3-kinase activity.

PI 3-kinase plays a very important role in the activation of NADPH oxidase (30, 31). IGF-1 or insulin can induce the activation of PI 3-kinase and increase the concentration of PI(3,4,5)P₃ in the plasma membrane (32-37). Furthermore, membrane ruffling induced by insulin or growth factors is found to correlate with the production of PI(3,4,5)P₃ (38, 39), while the induction of SHIP, a phosphatidylinositol 5'-phosphatase that dephosphorylates PI(3,4,5)P₃, causing more PI(3,4)P₂, eliminates this actin rearrangement (27). The observed colocalization of the transfected EGFP-p47-PX and membrane ruffles in COS cells on IGF-1 stimulation may suggest that translocation of the p47^phox PX domain and formation of membrane ruffles are either under similar regulation or actually in the same pathway, at least for PI 3-kinase activity. Also, the phosphoinositide-binding ability of p47-PX especially to PI(3,4,5)P₃ is seemingly critical for its membrane association. However, the R42A mutant of the p47^phox PX domain still possesses the ability of membrane targeting, while it completely loses phosphoinositide-binding ability in vitro. Thus, the p47^phox PX domain localizes to the plasma membrane in a mechanism other than simple phosphoinositide recruitment, although it is apparently downstream of PI 3-kinase activation. Moreover, the mutations in the PXXP motif within p47-PX cannot abolish its membrane association. This suggests that some interactions, other than a simple PX-SH3 interaction between the p47^phox PX domain and some other protein(s), play a role in its translocation to the plasma membrane.

In summary, we have shown that the two PX domains in NADPH oxidase bind to different phosphoinositides in vitro and target different subcellular locations in COS cells via different pathways controlled by PI 3-kinase. The targeting function of the p40^phox PX domain directly involves its binding to PI(3)P, while phosphoinositides are not sufficient to directly recruit the p47^phox PX domain to the plasma membrane. Interactions with some proteins are more likely to contribute to the membrane translocation of p47^phox PX domain.

	ACKNOWLEDGEMENTS

We thank Dr. Michael P. Czech for support and advice and Dr. Chuanyou Zhang and Dr. Kimmy Yuan for technical help. We also thank Dr. Zhen Y. Jiang and members in the Zhou laboratory for helpful discussion.

	FOOTNOTES

^* This work was supported by a research grant from the American Diabetes Association and an Annual Research Fund grant from the Worcester Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-6869; Fax: 508-856-1218; E-mail: wayne.zhou@umassmed.edu.

Published, JBC Papers in Press, November 29, 2001, DOI 10.1074/jbc.M109520200

	ABBREVIATIONS

The abbreviations used are: PX, Phox homology domain; p40-PX, the p40^phox PX domain; p47-PX, the p47^phox PX domain; PI 3-kinase, phosphatidylinositol 3-kinase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PI(3)P, phosphatidylinositol 3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(5)P, phosphatidylinositol 5-phosphate; PI(3, 4)P₂, phosphatidylinositol 3,4-bisphosphate; PI(3, 5)P₂, phosphatidylinositol 3,5-bisphosphate; PI(4, 5)P₂, phosphatidylinositol 4,5-bisphosphate; PI(3, 4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; IGF-1, insulin-like growth factor-1; EGFP, enhanced green fluorescent protein; SH3, Src homology 3.

	REFERENCES

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