Phosphorylation of P47 Directs Phox Homology Domain from SH3

Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation

Tetsuro Ago*†, Futoshi Kuribayashi*†, Hidekazu Hiroaki‡, Ryu Takeya*†, Takashi Ito§, Daisuke Kohda‡, and Hideki Sumimoto*†¶

*Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; †Department of Molecular and Structural Biology, Kyushu University Graduate School of Medical Science, Fukuoka 812-8582, Japan; ‡Department of Structural Biology, Biomolecular Engineering Research Institute, Osaka 565-0874, Japan; and §Division of Genome Biology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan

Edited by Bernard M. Babior, The Scripps Research Institute, La Jolla, CA, and approved February 7, 2003 (received for review September 18, 2002) Protein–phosphoinositide interaction participates in targeting pro- p47phox PX structure reveals a positively charged deep pocket (8) teins to membranes where they function correctly and is often that is supposed to interact with a negatively charged small modulated by phosphorylation of lipids. Here we show that molecule. Indeed it has been uncovered recently that PX do- protein phosphorylation of p47phox, a cytoplasmic activator of the mains function as a phosphoinositide-binding module (9–14). microbicidal phagocyte oxidase (phox), elicits interaction of However, it is presently unknown how the lipid-binding activity p47phox with phosphoinositides. Although the isolated phox ho- of PX domains is regulated. mology (PX) domain of p47phox can interact directly with phos- The phagocyte oxidase, dormant in resting cells, becomes phoinositides, the lipid-binding activity of this protein is normally activated during phagocytosis to produce superoxide, a precur- suppressed by intramolecular interaction of the PX domain with sor of microbicidal oxidants, in consumption with NADPH (6, the C-terminal Src homology 3 (SH3) domain, and hence the 15–18). The significance of the NADPH oxidase in host defense wild-type full-length p47phox is incapable of binding to the lipids. is exemplified by recurrent and life-threatening infections that The W263R substitution in this SH3 domain, abrogating the inter- occur in patients with chronic granulomatous disease, the phago- action with the PX domain, leads to a binding of p47phox to cytes of which lack the superoxide-producing system (15–18). phosphoinositides. The ﬁndings indicate that disruption of the The catalytic core of the enzyme is a membrane-integrated intramolecular interaction renders the PX domain accessible to the flavocytochrome, namely cytochrome b558, comprised of the two lipids. This conformational change is likely induced by phosphor- subunits gp91phox and p22phox. The activation of the oxidase ylation of p47phox, because protein kinase C treatment of the requires the proteins p47phox and p67phox and the small GTPase wild-type p47phox but not of a mutant protein with the S303͞ Rac, which exist in the cytoplasm of resting phagocytes and 304͞328A substitution culminates in an interaction with phos- translocate after cell stimulation to membranes to interact with phoinositides. Furthermore, although the wild-type p47phox the cytochrome, leading to superoxide production (6, 15–19). translocates upon cell stimulation to membranes to activate the It is well established that p47phox plays a central role in oxidase, neither the kinase-insensitive p47phox nor lipid-binding- membrane translocation of cytosolic factors, an event that is defective proteins, one lacking the PX domain and the other essential for activation of the NADPH oxidase: In chronic carrying the R90K substitution in this domain, migrates. Thus the granulomatous disease patients with p47phox deficiency, p67phox protein phosphorylation-driven conformational change of p47phox fails to migrate, whereas p47phox becomes targeted to membranes enables its PX domain to bind to phosphoinositides, the interaction in stimulated phagocytes from p67phox-defective patients (20). phox of which plays a crucial role in recruitment of p47 from the The resting form of p47phox is likely in a closed inactive confor- cytoplasm to membranes and subsequent activation of the phago- mation where its two SH3 domains are masked via an intramo- cyte oxidase. lecular interaction with the C-terminal region of this protein (7, 21). Upon cell stimulation, p47phox becomes phosphorylated at ne of the most dominant themes in current cell biology is the C-terminal quarter, which causes a conformational change Oacute and sophisticated targeting of proteins to new cellular that leads to exposure of the SH3 domains (7, 21, 22). The locations, e.g., to membranes, the nucleus, and so forth. Re- unmasked SH3 domains directly bind to p22phox, an interaction cruitment of proteins to cell membranes is often triggered by that is required for membrane translocation of p47phox and phosphorylation of the lipid phosphatidylinositol (PtdIns), which resultant activation of the oxidase (21, 23). Thus the SH3 can create targeting sites for proteins (1, 2). The phosphorylation domains of p47phox participate in the interaction with p22phox, or hydrolysis of inositol-containing lipids in cell membranes is whereas the C-terminal region negatively regulates the translo- currently known to orchestrate numerous complex cellular cation of p47phox via the intramolecular interaction with the SH3 events (3, 4). A variety of protein modules such as pleckstrin domains. However, the role of the N-terminal PX domain has homology and FYVE domains recognize specific phospho- remained to be elucidated. inositides (phosphorylated forms of PtdIns) to recruit proteins Here we show that the PX domain of p47phox exhibits a to appropriate cell membranes (1, 2). phosphoinositide-binding activity that is normally suppressed by The phagocyte oxidase (phox) homology (PX) domain (5), interacting intramolecularly with the C-terminal SH3 domain. also known as the phox and Bem1p 2 (PB2) domain (6, 7), occurs in the phox proteins p47phox and p40phox in mammals, the polarity establishment protein Bem1p in budding yeast, and a variety of This paper was submitted directly (Track II) to the PNAS ofﬁce. eukaryotic proteins involved in membrane trafficking. We have Abbreviations: PtdIns, phosphatidylinositol; phox, phagocyte oxidase; PX, phox homology; determined the NMR structure of the PX domain of p47phox and SH3, Src homology 3; PtdIns(4)P, PtdIns 4-monophosphate; PtdIns(4,5)P2, PtdIns 4,5- bisphosphate; PtdIns(3)P, PtdIns 3-monophosphate; PtdIns(3,4)P2, PtdIns 3,4-bisphosphate; demonstrated that it interacts with the C-terminal Src homology PMA, phorbol 12-myristate 13-acetate. 3 (SH3) domain of this protein (8). The p47phox PX domain ␤ ¶To whom correspondence should be addressed at: Medical Institute of Bioregulation, consists of an antiparallel -sheet formed by three strands and Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: four helices, and an inspection of the molecular surface of the [email protected].

4474–4479 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0735712100 Downloaded by guest on September 27, 2021 Stimulus-induced phosphorylation of p47phox disrupts the in- Activation of the NADPH Oxidase in a Whole-Cell System. We trans- tramolecular interaction to render the PX domain in a state duced both gp91phox and p67phox genes into the leukemic cell line accessible to phosphoinositides, which promotes membrane K562 cells as described (7). The pREP4 vector encoding the translocation of this protein and thus plays a crucial role in wild-type full-length p47phox (amino acids 1–390), a full-length activation of the phagocyte NADPH oxidase. This work provides mutant p47phox carrying the R90K or S303͞304͞328A substitu- a previously unknown example of protein phosphorylation elic- tion, or a PX-truncated p47phox (p47-⌬PX, amino acids 129–390) iting protein–phosphoinositide interaction for its recruitment to was transfected by electroporation to the doubly transduced membranes. K562 cells. The K562 cells stably expressing the wild-type or mutant p47phox were selected, and the stable expression in Ͼ99% Experimental Procedures of cells used was confirmed by flow cytometer (FACScan, Plasmid Construction and Expression and Purification of Recombinant Becton Dickinson) using anti-p47phox antibody (Transduction Proteins. The DNA fragment encoding the full-length p47phox or Laboratories, Lexington, KY). Superoxide production by the ϫ 5 ͞ PX domain (p47-PX; amino acids 1–128) was amplified by PCR K562 cells (1 10 cells) in response to 200 ng ml of phorbol using a cloned human p47phox cDNA. Mutations leading to the 12-myristate 13-acetate (PMA) was determined as superoxide indicated amino acid substitutions were introduced by PCR- dismutase-inhibitable chemiluminescence detected with an mediated site-directed mutagenesis (7). The DNAs were ligated enhancer-containing luminol-based detection system to pGEX-2T (Amersham Biosciences) and͞or pREP4 (Invitro- (DIOGENES, National Diagnostics) as described (7, 28). gen) and sequenced for confirmation of their identities. Proteins phox fused to glutathione S-transferase (GST) were expressed in Membrane Translocation of p47 . The K562 cells expressing the phox Escherichia coli strain BL21 and purified by glutathione- wild-type or mutant p47 protein were suspended at a con- centration of 1 ϫ 107 cells per ml in PBS (137 mM NaCl͞2.68 mM Sepharose 4B (Amersham Biosciences) as described (7, 24). ͞ ͞ ͞ ͞ KCl 1 mM CaCl2 1 mM MgCl2 5 mM glucose 8.1 mM ͞ Na2HPO4 1.47 mM KH2PO4, pH 7.4) and stimulated for 10 min Binding of PX Domain to Liposomes. The in vitro liposome-binding ͞ assay was carried out according to the methods of Patki et al. (25) at 37°C with PMA (200 ng ml). After centrifugation, cells were resuspended in relaxation buffer (100 mM KCl͞3 mM NaCl͞3.5 and Takeuchi et al. (26) with minor modifications (14). Briefly, ͞ ͞ ␮ mM MgCl2 1.25 mM EGTA 20 M p-amidinophenylmethane- liposomes were prepared by mixing phosphatidylcholine, PtdIns ␮ ͞ ␮ ͞ 4-monophosphate [PtdIns(4)P], PtdIns 4,5-bisphosphate sulfonyl fluoride/80 g/ml leupeptin 20 g/ml pepstatin A 20 ␮g/ml chymostatin͞10 mM Hepes, pH 7.4) and lysed by three [PtdIns(4,5)P2], PtdIns 3-monophosphate [PtdIns(3)P], or PtdIns 3,4-bisphosphate [PtdIns(3,4)P ] (purchased from Sigma rounds of 5-sec sonication. The sonicates were centrifuged at 2 ϫ g or Matreya, Pleasant Gap, PA) at the proportions indicated, 10,000 for 10 min, and the supernatant was overlaid on a 10% (wt͞vol) sucrose cushion followed by ultracentrifugation at drying the mixture under nitrogen, and resuspending in a sample 150,000 ϫ g for 30 min. The high-speed supernatant was used as buffer (100 mM NaCl͞10 mM MgCl ͞20 mM Hepes, pH 7.2). 2 the cytosolic fraction, whereas the high-speed pellet was washed Liposomes (100 ␮M) were incubated with the indicated GST- three times with relaxation buffer, suspended in Laemmli sample fusion proteins (100 pmol) in 50 ␮l of the sample buffer for 10 buffer, and used as the membrane fraction. Proteins were min at room temperature and collected by ultracentrifugation at analyzed by Western blot with the anti-p47phox antibody and 100,000 ϫ g for 45 min. The supernatant was removed carefully, developed by using ECL-plus (Amersham Biosciences). and the liposome pellet was resuspended in 50 ␮l of the sample buffer. Samples were analyzed by 10% SDS͞PAGE and stained Results with Coomassie brilliant blue. To estimate the amounts of The p47phox PX Domain Is Normally Inaccessible to Phosphoinositides proteins on the gel, densitometric analysis was carried out by via Intramolecular Interaction with the C-Terminal SH3 Domain. We using NIH IMAGE software. and others have reported recently that the PX domain of p47phox interacts with various phosphoinositides including PtdIns(4)P, Phosphorylation of Recombinant p47phox by Protein Kinase C (PKC). BIOCHEMISTRY phox PtdIns(3,4)P2, PtdIns(4,5)P2, and PtdIns(3)P (11, 14), whereas Phosphorylation of recombinant GST–p47 was carried out by that of p40phox specifically binds to PtdIns(3)P (11, 12, 14). The using human recombinant PKC␤2 (Calbiochem) as described phox phox presence of p47 in the cytoplasm before cell stimulation (22). Phosphorylation of p47 was confirmed by a retarded phox ͞ suggests that interaction of the p47 PX domain with mem- mobility of the protein on SDS PAGE and by incorporation of brane phospholipids does not occur in resting cells. Indeed, the ␥ 32 the radioactivity from [ - P]ATP as described (22). The pro- full-length wild-type p47phox was incapable of binding to phos- tein-phosphatase inhibitor NaF (50 mM) was added to the phoinositides such as PtdIns(4)P (Fig. 1A) and PtdIns(3,4)P2 solution of phosphorylated protein, which was used for the (data not shown), whereas an isolated p47phox PX domain did liposome-binding assay. effectively bind to these lipids (Fig. 1A). Thus the PX domain of p47phox seems to be normally inaccessible to phosphoinositides. 15 phox NMR Spectroscopy. A uniformly N-labeled PX domain of p47 We have shown recently that the p47phox PX domain interacts (amino acids 1–128) was prepared by growing the cells in M9 intramolecularly with the C-terminal SH3 domain of this protein 15 ͞ minimal medium containing NH4Cl (1 g liter) and purifying as via the canonical SH3-target PXXP motif (RIIPHLP), and this described (8). NMR spectra were recorded at 25°C on a Bruker interaction is abrogated by the amino acid substitution of (Billerica, MA) DMX600 spectrometer. The NMR sample con- arginine for the conserved residue Trp-263 in the SH3 domain tained 0.2 mM p47-PX protein, 5 mM sodium-Mes buffer (pH (8). To elucidate the mechanism that regulates the phospho- 1 ͞ 2 1 15 phox 5.5), and 5 mM DTT in 95% H2O 5% H2O. The H N inositide-binding activity of the p47 PX domain, we tested heteronuclear sequential quantum correlation spectra were re- whether the intramolecular interaction prevents the p47phox PX corded in the presence of increasing concentrations of inositol domain from associating with phosphoinositides. The full-length 1,4,5-trisphosphate or inositol 1,4-bisphosphate up to 1.5 mM p47phox carrying the W263R substitution in the C-terminal SH3 (for detail, see Figs. 6 and 7, which are published as supporting domain was capable of binding to phosphoinositides such as information on the PNAS web site, www.pnas.org). The nor- PtdIns(4)P (Fig. 1B), PtdIns(3,4)P2 (Fig. 1B), and PtdIns(4,5)P2 malized chemical-shift changes were calculated according to the (data not shown), indicating that the p47phox PX domain in the equation ⌬␦ ϭ {⌬␦(1H)2 ϩ [⌬␦(15N)͞7]2}1/2. Figures were drawn resting state is likely inaccessible to phosphoinositides via the by using the program MOLMOL (27). intramolecular interaction with the C-terminal SH3 domain.

Ago et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4475 Downloaded by guest on September 27, 2021 Fig. 1. (A) Phosphoinositide-binding activity of the full-length p47phox (p47-F). GST–p47-F, GST–p47-PX, or GST alone was incubated with phospho- lipids͞liposomes containing phosphatidylcholine (90%) and PtdIns(4)P (10%). P and S, liposomal pellet and supernatant after centrifugation, respectively. Samples were analyzed by 10% SDS͞PAGE, stained with Coomassie brilliant blue, and quantitated by densitometric analysis. The actual bands of GST alone, GST–p47-F, and GST–p47-PX are running at different molecular masses of Ϸ27, 73, and 40 kDa, respectively. (B) Effect of the W263R substitution on phosphoinositide-binding activity of p47-F. GST–p47-F or GST–p47-F(W263R) was incubated with phospholipids͞liposomes containing 10% PtdIns(4)P or Fig. 2. Phosphorylation-induced conformational change of p47phox, which PtdIns(3,4)P2. Samples were analyzed as described for A. Each value represents the mean of data from more than three independent experiments, with bars leads to its interaction with phosphoinositides. (A) Membrane translocation of phox ͞ ͞ representing the standard deviation. the wild-type p47 (WT) and a mutant protein with the S303 304 328A substitution. The gp91phox and p67phox doubly transduced K562 cells were transfected with pREP4 vector or the vector encoding the full-length wild-type phox ͞ ͞ Phosphorylation of p47phox Seems to Cause a Conformational Change p47 or the one with the S303 304 328A substitution. After cell stimulation with PMA (200 ng͞ml), the cell lysates were fractionated by centrifugation, That May Render the PX Domain Capable of Binding to Phospho- and the cytosolic (C) and membrane (M) fractions were analyzed by immuno- inositides. To clarify the mechanism that disrupts the intramo- blot with anti-p47phox antibody. The actual bands of both wild-type and lecular interaction between the PX and C-terminal SH3 domains mutant proteins are running at 47 kDa. (B) Activation of the phagocyte to direct the PX domain toward phosphoinositides, we investi- NADPH oxidase by the wild-type p47phox and a mutant protein with the gated the effect of phosphorylation of this protein, because it is S303͞304͞328A substitution. Cells were stimulated with PMA as described for well established that after cell stimulation p47phox becomes A, and superoxide production was determined by superoxide dismutase- phosphorylated to translocate from the cytoplasm to membranes inhibitable chemiluminescence change, monitored with DIOGENES. (C) Effect of phosphorylation of p47phox on its phosphoinositide-binding activity. GST– (29–31). In addition, we have elucidated recently that three ͞ ͞ serine residues in the C-terminal quarter of p47phox (Ser-303, p47-F (WT) or GST–p47-F (S303 304 328A) was treated with PKC in vitro and incubated with phospholipids͞liposomes containing 10% PtdIns(4)P. Samples Ser-304, and Ser-328) are critical for the conformational change were analyzed as described for Fig. 1B. The actual bands of both GST-fusion leading to the oxidase activation (7, 22). Indeed, when cells were proteins are 74 kDa. (D) Effect of the S303͞304͞328D substitution of p47phox phox stimulated with PMA, a direct activator of PKC, a mutant p47 on its phosphoinositide-binding activity. GST–p47-F(WT) or GST–p47-F(S303͞ carrying the triple replacement of these serines with the kinase- 304͞328D) was incubated with phospholipids͞liposomes containing 10% insensitive alanines (S303͞304͞328A) failed to translocate to PtdIns(4)P. Samples were analyzed as described for Fig. 1. membranes and to activate the NADPH oxidase, whereas the wild-type p47phox became targeted to membranes (Fig. 2A) and fully induced superoxide production (Fig. 2B). interaction with phosphoinositides. To find critical residues of phox The phosphorylation-induced conformational change of the p47 PX domain involved in binding to phosphoinositides, p47phox causes the exposure of the SH3 domains, which are we analyzed its interaction with inositol phosphates by NMR. normally masked via an intramolecular interaction; the un- The solution structure of the p47phox PX domain, which we ␤ masked SH3 domains bind to p22phox, the small subunit of determined recently, consists of three -strands and four helices phox cytochrome b558 (7, 21, 22). It seems likely that this conforma- (8). An inspection of the molecular surface of the p47 PX tional change also increases the accessibility of the PX domain. domain structure reveals a positively charged deep pocket (Ϸ8 As expected, p47phox, which had been phosphorylated in vitro Å in depth), where the two strongly conserved residues in various with PKC, was capable of binding to phosphoinositides such as PX domains, Arg-90 and Phe-44, are exposed to the solvent (Fig. PtdIns(4)P (Fig. 2C). On the other hand, a mutant p47phox with 3A). Among the residues in the pocket, Arg-90 displayed the the S303͞304͞328A substitution could not interact with the lipid largest chemical-shift perturbation in the presence of the inositol even when treated with PKC (Fig. 2C). In addition, a mutant phosphate (Fig. 3 A and B), suggesting its direct involvement in p47phox carrying the simultaneous replacement of the three recognition of polar heads of phosphoinositides. To test this serines with aspartates (S303͞304͞328D), mimicking a phos- possibility, we prepared a mutant p47phox PX domain and phorylated form (7), was able to bind to PtdIns(4)P without examined its activity in binding to phosphoinositides by the treatment with PKC (Fig. 2D). Thus phosphorylation of liposome assay. We chose lysine for the substitution for the p47phox likely induces a conformational change, i.e., a domain arginine residue considering the possibility of bidentate nature rearrangement, which may render the PX domain accessible to of the hydrogen-bond interactions between the arginine side phosphoinositides. chain and the phosphate groups of inositol phosphates. This R90K substitution is more conservative than that of other amino Arg-90 Is Present in a Pocket of the p47phox PX Domain and Plays a acid residues, e.g., leucine as reported (11), because it does not Crucial Role in Binding to Phosphoinositides. We finally attempted change the electrostatic property of the molecular surface. To to answer the question of what the role for the phosphoinositide- clarify that the substitution does not affect the structural integ- binding activity of the p47phox PX domain in the phox activation rity of the domain, we measured the 1H 15N heteronuclear is. For this purpose, it is necessary to prepare a mutant p47phox sequential quantum correlation spectrum of the R90K mutant of protein with an amino acid substitution that leads to a defective the p47phox PX domain. The NMR spectrum was essentially the

4476 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0735712100 Ago et al. Downloaded by guest on September 27, 2021 Fig. 4. Role of the p47phox PX domain and its phosphoinositide-binding activity in activation of the phagocyte NADPH oxidase. (A) Arg-90 of the p47phox PX domain, which is involved in binding to phosphoinositides. GST– p47-PX or GST–p47-PX (R90K) was incubated with phospholipid͞liposomes containing 10% PtdIns(3,4)P2. Samples were analyzed as described for Fig. 1. (B) Membrane translocation of the wild-type p47phox (WT), a mutant protein lacking the PX domain (⌬PX), and the one with the R90K substitution and activation of the phagocyte NADPH oxidase in cells expressing these proteins. The gp91phox- and p67phox-transduced K562 cells were transfected with pREP4 vector or the vector to express the indicated form of p47phox.(Upper) After cell ͞ Fig. 3. NMR analysis of the interaction of the p47phox PX domain and inositol stimulation with PMA (200 ng ml), the cell lysates were fractionated by phosphate. The normalized chemical-shift changes were calculated after centrifugation, and the cytosolic fraction (C) and membrane fraction (M) were phox adding 1.5 mM inositol 1,4,5-trisphosphate to a sample of 0.2 mM 15N-labeled analyzed by immunoblot with anti-p47 antibody. The actual bands of phox phox phox ⌬ p47phox PX domain. The residues with 1H 15N crosspeaks that shifted Ͼ0.065 p47 -wild type, p47 -R90K, and p47 - PX are running at 47, 47, and ppm are shown in orange (Ile-9, Leu-11, Tyr-26, Phe-44, Thr-53, Lys-55, Glu-56, 45.5 kDa, respectively. (Lower) Superoxide production was determined by Trp-80, Ala-87, Gln-91, Gly-92, Thr-93, Leu-94, Glu-96, Tyr-97, and His-113), and superoxide dismutase-inhibitable chemiluminescence change. (C) Effect of those that shifted Ͼ0.150 ppm (maximum 0.227 ppm) are shown in red wortmannin on membrane translocation and activation of the oxidase. Cells phox (Phe-81, Gly-83, and Arg-90) on the stick drawing (A) and the molecular expressing p47 -wild type were preincubated in the presence or absence of ͞ surface (B) of the p47phox PX structure (PDB ID code 1GD5). The two conserved 100 nM wortmannin (wort) and stimulated with PMA (200 ng ml). Membrane phox residues Phe-44 and Arg-90 involved in the inositol phosphate binding are translocation of p47 (Left) and superoxide production (Right) were ana- drawn in cyan. (Left) Viewed in the same orientation. (Right) Rotated 110° (A) lyzed as described for B. and 180° (B) along the vertical axis. tion, neither translocation of the wild-type p47phox nor the same as that of the wild-type p47phox PX domain (data not oxidase activation was affected by the PtdIns 3-kinase inhibitors shown), confirming the unchanged overall conformation wortmannin (Fig. 4C) and LY294002 (data not shown), indicat- after the R90K substitution. The mutant p47phox PX domain ing that the lipid kinase does not regulate both events in BIOCHEMISTRY with the R90K substitution bound to PtdIns(3,4)P2 (Fig. 4A) PMA-stimulated K562 cells. and PtdIns(4,5)P (data not shown), but only weakly. 2 Discussion Thus, this conserved residue is directly involved in binding to phosphoinositides. In the present study we show that although the isolated PX domain of p47phox can interact directly with phosphoinositides, Phosphoinositide-Binding Activity of the p47phox PX Domain Is Essen- the lipid-binding activity of this protein is normally suppressed tial for Membrane Translocation of This Protein and Activation of the by intramolecular interaction of the PX domain with the C- Phagocyte NADPH Oxidase. Upon cell stimulation, p47phox migrates terminal SH3 domain. Stimulus-induced phosphorylation of from the cytoplasm to membranes, an event that is required for p47phox disrupts the intramolecular interaction to direct the PX activation of the phagocyte NADPH oxidase (15–18). To test the domain toward phosphoinositides. Resultant interaction of the role for the p47phox PX domain, especially the role for its PX domain with the phospholipids promotes membrane trans- phosphoinositide-binding activity, we expressed a p47phox lacking location of p47phox and thus plays an essential role in activation the PX domain and a full-length one with the R90K substitution of the phagocyte NADPH oxidase. Hence the protein phospho- in p47phox-deficient K562 cells (7). In response to PMA, an agent rylation-driven conformational change of p47phox regulates the that directly stimulates PKC and fully activates the oxidase in phosphoinositide-binding activity of the PX domain, which cells, the wild-type p47phox translocated to membranes and determines the localization of this protein, thereby serving as a induced superoxide production (Fig. 4B), whereas the PX- switch for the oxidase. truncated protein could not (Fig. 4B). Intriguingly, the R90K The PX domain is thus required but does not seem sufficient substitution also resulted in a severely impaired translocation for membrane translocation of p47phox, because the isolated and decreased superoxide production (Fig. 4B), indicating that p47phox PX domain neither binds to cell membranes in resting both events require the phosphoinositide-binding activity per se. cells nor translocates to cell membranes in PMA-stimulated cells Thus the p47phox PX domain plays an essential role via its (R.T. and H.S., unpublished data). This may be due to the fact phosphoinositide-binding activity in translocation of this protein that it only binds weakly to phosphoinositides compared with to membranes and activation of the NADPH oxidase. In addi- other PX domains (ref. 11 and R.T. and H.S., unpublished data).

Ago et al. PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4477 Downloaded by guest on September 27, 2021 residues (Ile-9, Leu-11, Thr-53, Lys-55, Glu-56, and His-113) distant from the binding pocket (Fig. 3), suggesting that a change of the PX domain conformation is induced by the inositol phosphate binding. Consistent with this, it is suggested that conformational changes of the PX domain of the yeast vacuole protein Vam7p accompany its interaction with PtdIns(3)P (9). Thus, the two PX ligands, i.e., inositol phosphates and the p47phox C-terminal SH3 domain, seem to mutually influence the other’s binding through conformational changes of the p47phox PX domain. A similar SH3 domain-mediated regulation may occur in other PX domain-containing proteins that also harbor SH3 domains such as Bem1p and p40phox (32). Phosphorylation-mediated membrane association is also observed in other PX-containing proteins such as mammalian phospholipase D1 (33, 34) and its yeast homologue Spo14p (35), Fig. 5. A proposed mechanism underlying the regulation of p47phox in raising the possibility that the translocation also may be medi- activation of the phagocyte NADPH oxidase. Human p47phox comprises 390 ated by the PX domain and regulated by protein phosphoryla- amino acid residues, and PX͞PB2 represents the PX domain (5), also called the tion. On the other hand, in some cases the interaction of the PX PB2 domain (6, 7). The C-terminal SH3 domain, containing Trp-263, interacts domain with phosphoinositides could be constitutive rather than intramolecularly with the PXXP motif of the PX domain. Stimulus-induced inducible. It has been shown that sorting nexins (36, 37) and the phosphorylation of p47phox causes a conformational change, by which both PX class II PtdIns 3-kinases (38) localized in the membrane fraction and SH3 domains become accessible to their membranous targets, phospho- without cell stimulation, suggesting that their PX domains may phox inositides and p22 , respectively. Cooperation of these two interactions, interact constitutively with phosphoinositides. each being indispensable, enables p47phox to form a stable complex with phox phox This study provides a previously unknown example that protein– cytochrome b558 (composed of the two subunit gp91 and p22 ), leading to activation of the phagocyte NADPH oxidase. phosphoinositide interaction is elicited by protein phosphorylation. It seems evident that changes in the levels of phosphoinositides such as PtdInsP3 and PtdInsP2 are not involved in the interaction of We have shown previously that the SH3 domains of p47phox are p47phox with phosphoinositides, at least in PMA-stimulated cells, masked in the resting state (21, 32) and that phosphorylation because PMA does not affect the levels of the lipids (39, 40), and causes a conformational change that renders the SH3 domains PtdIns 3-kinase inhibitors do not affect membrane translocation of capable of specifically interacting with p22phox, an interaction p47phox in PMA-stimulated cells (Fig. 4C). In contrast, in other that also is essential for membrane translocation of p47phox and known cases, a kinase or phosphatase for the inositol-containing thus for the oxidase activation (7, 22). Taken together with the lipids modulates interaction between lipids and proteins by pro- present findings, we propose a previously uncharacterized mech- ducing specific phosphoinositides (1, 2). Although the association of phox anism underlying the regulation of p47phox in activation of the p47 with phosphoinositides is thus primarily regulated by pro- phagocyte NADPH oxidase (Fig. 5). Stimulus-induced phos- tein phosphorylation, it is also possible that PtdIns 3-kinase can phorylation of p47phox causes a conformational change, by which enhance the membrane recruitment in neutrophils stimulated with both PX and SH3 domains become accessible to their membra- chemoattractants such as formyl-methionyl-leucyl-phenylalanine phox nous targets, phosphoinositides and p22phox, respectively. Coop- (fMLP): The PX domain of p47 is capable of binding to eration of these two interactions, each being indispensable but PtdIns(3,4)P2, a product of the lipid kinase, with high preference not sufficient, enables p47phox to form a stable complex with (11, 14). It is well known that fMLP efficiently activates PtdIns 3-kinase in neutrophils, and fMLP-elicited superoxide production cytochrome b558, leading to activation of the phagocyte NADPH is inhibited by PtdIns 3-kinase inhibitors (39–41). oxidase. phox phox It also should be noted that the PX domain of p40 prefer- The present findings indicate that the p47 PX domain in phox the resting state is inaccessible to phosphoinositides via its entially binds to PtdIns(3)P, whereas the p47 PX domain prefers intramolecular interaction with the C-terminal SH3 domain. other phosphoinositides such as PtdIns(4)P and PtdIns(3,4)P2 (11, This effect on the PX domain may be due to the fact that the SH3 12, 14). Because both proteins are tightly associated in the same domain directly masks the phosphoinositide-binding pocket or protein complex in resting cells and simultaneously migrate to that the SH3 domain attenuates the PX–phosphoinositide in- membranes upon cell stimulation (6, 15–19), the difference in teraction in an allosteric fashion. Data from our structural phosphoinositide specificity may suggest that the two PX domains analyses prefer the latter explanation. We reported recently that associate with distinct types of membranes to facilitate a membrane chemical-shift changes are observed throughout the p47phox PX fusion, which is expected to proceed during phagocytosis when the structure when the p47phox C-terminal SH3 domain interacts with NADPH oxidase becomes activated. This possibility should be the proline-rich segment of the PX domain, suggestive of an SH3 tested in future studies. domain-induced conformational change (8). In particular, Arg-90 and Phe-44 in the inositol phosphate-binding pocket are We thank Professor Masato Hirata (Kyushu University) and Dr. Kazuhisa Ota (Kanazawa University) for helpful comments, and Yohko Kage influenced by the interaction with the SH3 domain. In the (Kyushu University) for excellent technical assistance. This work was partly present study, we performed another NMR titration experiment supported by grants from the Ministry of Education, Culture, Sports, in which the addition of inositol phosphates to a solution of the Science, and Technology, Japan, ONO Medical Research Foundation, and p47phox PX domain caused chemical-shift changes of several the BIRD project of the Japan Science and Technology Corporation.

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