Proc. Nadl. Acad. Sci. USAVol. 91, pp. 10650-10654, October 1994Biochemistry

Assembly of the phagocyte NADPH oxidase: Binding of Srchomology 3 domains to proline-rich targets

(ch c granulomatous dea/cytocxroime bsss/p47-pkox/p67-pkox)

THOMAS L. LETO*, ANTHONY G. ADAMS, AND ISABELLE DE MENDEZLaboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

Communicated by Vincent T. Marchesi, June 27, 1994 (received for review April 11, 1994)

ABSTRACT The NADPH oxidase responsible for genera-tio of superoxide anion and related microbicidal oxidants byphagocytes Is assembled from at least five distinct proteins.TWo are cytosolic components (p47-phox and p67-phox) thatcontain Src homology 3 (SH3) domine and associate with atransmembrane cytochrome bss5 upon activation. We showhere that the SH3 do of p47-phox bind to proine-richsequences in p47-phx Itself and the p22-phox subunit ofcytochrome b5ss. Binding of the p47-phox SH3 domains top22-phox was abolished by a mutation in one proline-richsequence (Pro'S' -* Gin) noted in a distinct form of chronicgranulomatous disease and was inhibited by a short proline-rich synthetic peptide corresonding to resIdues 149-162 ofp22-phox. Expression of mutated p22-phox did not restoreoxidase activity to p22-phox-defclent B cells and did not enablep22-phox-dependent translocation of p47-phox to membranesin phorbol ester-stimulated cells. We also show that the cyto-solic oxidase components associate with one another throughthe C-terminal SH3 domain of p67-phox and a proline-richC-terminal sequence in p47-phox. These SH3 target sitesconform to consensus features deduced from SH3 binding sitesin other systems. We propose a model in which the oxidasecomplex assembles through a mechanism involving SH3 do-mains of both cytosolic proteins and cognate proline-richtargets in other oxidase components.

Neutrophils respond to a number of stimuli with a burst ofoxygen consumption and production of superoxide. This"respiratory burst" is attributed to NADPH oxidase, anenzyme whose importance is evident in patients with chronicgranulomatous disease (CGD) (1), who suffer from increasedsusceptibility to bacterial and fungal infections due to defi-cient production ofmicrobicidal oxidants by phagocytes. TheNADPH oxidase is composed of five essential components,four that are affected in different genetic types of CGD (2-6)and a fifth component, the Ras-related small GTPase p21,that bestows guanine nucleotide sensitivity to the enzymecomplex (7-10). The functional center of the oxidase isflavocytochrome b558 (11, 12), composed of two subunits(gp9l-phox and p22-phox) that accept electrons from cyto-solic NADPH and donate them to molecular oxygen. Twocytosolic factors, p47- and p67-phox, translocate to themembrane and associate with the cytochrome upon activa-tion (13, 14), although their precise functions remain unclear.Both factors contain two Src homology 3 (SH3) domains (3,4) shown to be critical for assembly of these proteins into theactive, membrane-bound oxidase complex (15, 16).SH3 domains are conserved 60-aa sequences found in a

variety of intracellular signaling and cytoskeletal proteins inorganisms ranging from yeast to man (17, 18). Their occur-rence in the cytosolic oxidase components implied roles in

targeting these proteins to the membrane-based cytoskeletonor linking them to GTP-dependent regulatory elements im-plicated in other systems that utilize this motif. Structuralstudies on SH3 domains from several proteins revealed adomain consisting largely of 3-sheets, with many conservedresidues on one surface that represents a common bindingsite (ref. 19 and references therein). Several SH3 bindingsequences have been identified within putative SH3 targetproteins or peptides selected from biased combinatorial li-braries. These target sites were characterized as short pro-line-rich sequences with limited similarities (19-24).

In light of recent observations showing that SH3 domainsof both p67- and p47-phox are essential for oxidase assembly(15, 16), we examined the roles of these domains in theinteractions of several oxidase components. In this report wedescribe specific SH3 binding sites in both p47-phox andp22-phox; these sites exhibited features common to otherproline-rich SH3 targets in unrelated systems. Furthermore,a mutation in one of these sites, corresponding to a p22-phoxdefect associated with CGD (25), disrupts the interactionbetween p22-phox and the SH3 domains of p47-phox andabrogates translocation of p47-phox to membranes in cellsstimulated with phorbol 12-myristate 13-acetate (PMA).Based on these findings, we suggest a model for NADPHoxidase activation involving multiple SH3-mediated interac-tions within the assembled complex.

MATERIALS AND METHODSRecombinant Proteins. Methods for production of recom-

binant p47-phox and p67-phox from baculovirus-infectedcultured Sf9 insect cells were described elsewhere (16, 26).For production of glutathion S-transferase (GST) fusionproteins in Escherichia coli, DNA fragments encoding vari-ous domains of p47-, p67-, and p22-phox were prepared fromtheir respective cDNAs (2, 4) by polymerase chain reactions(PCRs) using oligonucleotide primers directed to appropriatedomain boundaries. Primers were designed with BamHI orEcoRI cleavage sites at their 5' ends for ligation in frame withGST in pGEX-3X or pGEX-2T expression vectors (27).Fusion proteins of SH3 domains contained the followingsequences: p47/SH3A-B, both SH3 domains of p47-phox (aa151-284); p47/SH3A and p47/SH3B, first (aa 151-214) andsecond (aa 227-284) SH3 domains of p47-phox, respectively;p67/SH3A and p67/SH3B, first (aa 241-304) and second (aa458-526) SH3 domains of p67-phox, respectively. The cyto-plasmic domain of p22-phox (aa 127-195) was fused to GSTin a similar manner. A Pro156 -+ Gin point mutation wasintroduced into p22-phox by two-stage amplification, where

Abbreviations: CGD, chronic granulomatous disease; EBV, Ep-stein-Barr virus; GST, glutathione S-transferase; PMA, phorbol12-myristate 13-acetate; SH3, Src homology 3.*To whom reprint requests should be addressed at: Laboratory ofHost Defenses, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health, Building 10, Room 11N106,9000 Rockville Pike, Bethesda, MD 20892.

10650

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

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codon 156 (CCG) was replaced with CAG in the primers usedin the first amplification step. Full-length p47 cDNA [EcoRIfiagment, clone 8A (2)] was also subcloned into pGEX-3X.All constructs were confirmed by DNA sequencing.The GST fusion proteins were induced for 2 hr from

logarithmic-phase E. coli DH5a transformants and affinitypurified on glutathione-Sepharose (Pharmacia), as described(27).

Binding Assays. For biotinylation of probes, affinity-purified GST-SH3 fusion proteins were conjugated at %1mg/ml with 2 mM sulfosuccinimidobiotin (Pierce) for 60 minat room temperature and dialyzed to remove unbound biotin.After confirmation that they contained similar amounts ofbiotin, the GST-SH3 conjugates were used to probe nitro-cellulose blots of proteins previously blocked in 5% nonfatmilk. Methods for probing, washing, and detection of boundbiotinylated probes were as described (24), using the bioti-nylated probes (3 Mg/ml) for 2 hr at 40C.For solution binding studies, lysates of recombinant p67-

phox proteins (16) from baculovirus-infected cells (2.5 x 104cell equivalents per assay) were incubated for 1 hr at 40C with100 A1 of GST-p47-phox bound to glutathione-Sepharosebeads. Bound proteins were washed three times in 15 volumesof ice-cold 100mM KCI/3 mM NaCl/3.5 mM MgCl2/0.15 mMphenylmethanesulfonyl fluoride/10mM Pipes (pH 7.5), elutedwith 1% SDS, and analyzed by SDS/PAGE followed byimmunoblotting with anti-p67-phox, as described (26).

Peptide Studies. Peptides were synthesized by the BiologicResources Section (National Institute of Allergy and Infec-tious Diseases) and confirmed by mass spectrometry, se-quencing, and amino acid composition analysis. For compe-tition studies, peptides (250 gM) were added to biotinylatedSH3 probes and incubated for 1 hr at room temperaturebefore blots were probed, as above. All blots were probedand developed (1 min) under identical conditions.

mmalian Expression Vectors. Full-length cDNAs forp47-phox and p22-phox were constructed in the episomalexpression vector pREP4 (Invitrogen) by strategies that willbe described elsewhere (T.L.L. and I.d.M., unpublishedwork). The CGD mutation Pro156 -* Gln was introduced intop22-phox by replacement of the 3' portion, from the sole NotI site, with the mutated sequence generated in pGEX (de-scribed above). Methods for electroporation and selection oftransfected Epstein-Barr virus (EBV)-transformed B cellswere recently described (16). Cotransfection of K562 cellswith p47- and p22-phox cDNAs required 20 pg of eachplasmid. Production of recombinant p22-phox in hygromy-cin-resistant lines was verified by Western blotting (5 x 106cell equivalents of membranes per lane on SDS/4-20opolyacrylamide gels) using p22-phox goat antiserum (1:500dilution) raised against aa 176-195 of p22-phox (gift fromM. E. Kleinberg and H. L. Malech, National Institute ofAllergy and Infectious Diseases).

Functional Studies in Transfected Cells. Whole-cell super-oxide production in transfected EBV-transformed B cells inresponse to PMA (2 pg/ml) was determined by a superoxidedismutase-inhibitable chemiluminescence assay (16). Mem-brane translocation experiments used PMA-stimulated (2pg/ml) K562 cells cotransfected with p47- and p22-phox(CGD mutant or wild-type) cDNAs. The distribution ofp47-phox was compared between membrane (80 pg) andcytosolic (20 ptg) fractions, prepared as described (16). Pro-teins were separated by SDS/8-16% PAGE, immunoblotted,and developed for anti-p47-phox reactivity with ECL re-agents (Amersham).

RESULTS AND DISCUSSIONBinding studies were conducted to detect SH3 binding siteswithin NADPH oxidase components and explore their role inoxidase assembly. Using a biotinylated probe constructed

from the SH3 domains ofp47-phox or p67-phox fused to GST,we obtained evidence for SH3 binding sites in both p22-phoxand p47-phox (Fig. 1). A probe comprising both SH3 domainsof p47-phox bound to p47-phox itself and to a 34-kDa bandcorresponding to the proline-rich C-terminal cytoplasmicdomain ofp22-phox (aa 127-195) fused to GST. Interestingly,one of the proline-rich segments of p22-phox (aa 149-162)was noted for a mutation (Pro'56 -* Gln) in a patient withCGD; the effects of this mutation were not known, thoughnormal cytochrome b558 content was observed in this pa-tient's neutrophils (25). Our results indicated that the p22-phox fusion protein containing this mutation was unable tobind to this .p47-phox-derived probe (Figs. 1 and 2). Incontrast to the specificity exhibited by both p47-phox SH3domains together, probes comprising single SH3 domainsfrom p47-phox or p67-phox bound less avidly to p47- andp22-phox. Moreover, the binding to p22-phox by these probeswas not greatly affected by the CGD-associated mutation.This weaker binding required longer development times toobtain comparable signals, which was evident from the lowerstaining intensities relative to a 21-kDa streptavidin-bindingprotein detected in all crude E. coli lysates. Under longerdevelopment conditions p67-phox was also detected, thoughthis binding was not considered significant in light of otherbinding experiments described below.

Several proline-rich synthetic peptides derived from p22-phox and p47-phox were compared as inhibitors of theinteraction between p22-phox and the two SH3 domains ofp47-phox (Fig. 2B). Their sequences were chosen on the basisof similarities to a number of known SH3 domain-binding

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FIG. 1. Binding of SH3 domains to p22-phox and p47-phox.Proteins from the following sources were separated by SDS/10%PAGE and blotted to nitrocellulose: p47 and p67 (1 ag), purep47-phox and p67-phox from baculovirus-infected Sf9 cells; GST-p22, crude lysate from pGEX-2T-transformedE. coliexpressingGSTfused to p22-phox (aa 127-195); GST-p22A, lysate from transformantexpressing mutated GST-p22-phox (Pro"56 - Gln) noted in CGD(25); control, lysate from uninduced pGEX-2T-transformed bacteria.Replicate blots were probed with biotinylated GST-SH3 fusionproteins indicated below each panel: p47/SH3A-B, both SH3 domainsof p47-phox; p47/SH3A and p47/SH3B, first and second SH3 do-mains of p47-phox, respectively; p67/SH3A and p67/SH3g, first andsecond SH3 domains of p67-phox, respectively. The 21-kDa bandwas a streptavidin-binding protein detected in all E. coli lysates (20,24). Staining with fast green reveals all proteins. Size markers (kDa)are at right. Arrowheads, p47 and GST-p22 protein bands.

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FIG. 2. Role of proline-rich sequences in binding of p47-phoxSH3 domains to p22-phox. (A) Alignment of proline-rich sequencesin p47-phox and p22-phox and comparison with other SH3 bindingsequences (single letter code). In the Abl-SH3 binding consensus(24), denotes hydrophobic residues and X indicates nonconservedpositions. Conserved prolines (shaded) were aligned with essentialresidues identified in 3BP1 (24). Consensus for p85 SH3-bindingpeptides was deduced from peptides selected from a biased combi-natorial library (19). Sequences of dynamin and SOS are proposedbinding sites for SH3 domains ofp85 and Grb2, respectively (21-23).(B) Inhibition of binding of p47-phox SH3 domains to p22-phox byproline-rich synthetic peptides. Proline-rich peptides (250 ,uM) de-fined in A (listed above each blot) were incubated with biotinylatedp47/SH3A-B prior to probing blots of GST-p22 proteins. Anti-p22-phox serum revealed comparable loading ofGST-p22-phox (P22) andmutated (Pro'56'- Gln) GST-p22-phox (P22A).

sequences, including proteins that bound to SH3 domains ofAbl, Grb2, and the p85 subunit of phosphatidylinositol 3-ki-nase (19-24) (Fig. 2A). Most sequences aligned with threemutation-sensitive proline residues described in 3BP1 (24);one of the prolines aligned with the mutated site in p22-phox(Pro'56) associated with CGD (25). Of six oxidase-relatedpeptides tested, only a peptide spanning aa 149-162 ofp22-phox effectively inhibited binding of the SH3 domains ofp47-phox to the cytoplasmic tail of p22-phox (Fig. 2B). Therelated peptide with the Pro'56 -> Gln CGD mutation did notcompete effectively, confirming the specificity of this pro-line-rich binding site and the effects of this CGD mutation onbinding of p47-phox SH3 domains to p22-phox.To explore the effects of this CGD-associated mutation on

assembly of the oxidase in whole cells, we compared thefunction of wild-type and CGD-mutant p22-phox in trans-fected cells. The expression vector pREP4 was used toproduce recombinant p22-phox in CGD patient B cells spe-cifically lacking this protein. Using these constructs, wedemonstrated expression of wild-type and mutant p22-phoxat levels comparable to those in normal B cells (Fig. 3A),though correction ofoxidase activity to a normal B-cell rangewas observed only with wild-type p22-phox (Fig. 3B). Toexplore further the p22-phox-p47-phox interaction in wholecells, we examined the translocation of p47-phox to mem-branes that occurs during oxidase activation. In PMA-stimulated neutrophils, assembly of the oxidase on mem-branes involves a movement of l10%ot of cytosolic p47-phox

FIG. Effects of the p22-phox CGD Pro6 Gln on

PMA-stimulated oxidase activity and p47-phox membrane transloca-tion in pREP4-transfected cells. (A) Anti-p22-phox Western blotting ofp22-phox-deficient (CGD) EBV-transformed B cells transfected withwild-type (wt, lane 1) or mutated (A P Q, lane 2) pREP4 p22-phoxconstructs. Lane 3, absence of p22-phox noted in the same untrans-fected CGD line; lane 4, endogenous p22-phox detected in normalEBV-transformed B cells. (B) Superoxide production by PMA-stimulated B cells described in A, expressed as relative chemilumines-cence units (RLU) in a luminol-based detection system (16). Resultsrepresent the mean and SD of duplicate reactions from three separatetransfection experiments (1-3) or six different normal B-cell lines (4).(C) Western blotting ofrecombinant wild-type (wt) and mutated (A P-Q) p22-phox in cotransfected K562 cells, compared with endogenousp22-phox in neutrophils (polymorphonuclear cells, PMN) and untrans-fected control K562 cells. (D) Membrane translocation ofrecombinantp47-phox in PMA-activated K562 cells (analyzed in C) coexpressingwild-type or mutated p22-phox. p47-phox content of membrane andcytosolic fractions was compared in unstimulated (-) or PMA-stimulated (10 min, 2 pg/ml) (+) cells.

to an active, membrane-bound complex (13, 14). Due to thelow abundance of oxidase proteins in EBV-transformed Bcells, translocation experiments were conducted in K562erythroleukemia cells, which were more easily transfectedand produced higher levels of oxidase proteins. This cell lineproduces high levels of superoxide when cotransfected withseveral oxidase genes (I.d.M. and T.L.L., unpublished ob-servations). By cotransfection with pREP4 constructs ofwild-type p22-phox along with p47-phox, we observed ap22-phox-dependent translocation ofp47-phox to membranesin PMA-stimulated K562 cells (Fig. 3D). In contrast, expres-sion ofsimilar levels ofmutated p22-phox did not result in anymovement of p47-phox to membranes after PMA activation.Thus, in this transfected cell model the significance ofthe lossof SH3 domain-binding activity, observed in vitro with thisCGD-associated (p22-phox Pro'56 Gln) mutation, was

confirmed by abrogation ofp47-phox translocation. Previouswork with CGD neutrophils showed that p47-phox membranetranslocation depended on cytochrome b558 (14, 28), thoughit was unclear which subunit recruited p47-phox to themembrane. Reports showing inhibitory effects of a peptide

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from the C terminus of gp91-phox proposed interactions ofp47-phox with the large subunit (29, 30). Our results withPMA-stimulated transfected K562 cells demonstrate p22-phox-dependent translocation of p47-phox, even in the ab-sence ofgp91-phox, and confirm the role for this SH3 bindingsite of p22-phox in translocation.SH3 domain-binding sites in p47-phox were also mapped by

probing a series of p47-phox fragments fused to GST (Fig. 4).The SH3 domains from p47-phox bound both N- and C-ter-minal segments ofp47-phox, but not the SH3 domains orGSTalone. Binding sites were defined within two regions contain-ing proline-rich segments (aa 70-83 and 358-390; Fig. 4, lanes9 and 10). These sequences conform somewhat to consensussequences derived from several SH3-binding sequences (Fig.2A). Binding to fragments derived from a proteolyticallysensitive region of p47-phox (aa 280-337) largely devoid ofprolines was also observed (Fig. 4, lanes 5 and 6); this bindingwas attributed to a high content of basic residues (net charge,+ 19). Surprisingly, a third proline-rich segment of p47-phox(aa 338-352) did not bind the SH3 probes, despite similaritiesto other SH3 binding sequences, though the correspondingsequence in murine p47-phox is markedly divergent and lacksthree of the four proline residues in the human protein (31).

Isolated SH3 domains from p67-phox also bound to the samesites in p47-phox (data not shown). To explore these interac-tions further, the effects ofp67-phox SH3-domain deletions onp47-phox binding were examined (Fig. 5). Deletion forms ofp67-phox were compared for their abilities to bind to a GST-p47-phox fusion protein immobilized on glutathione-Sepharose (Fig. SC) or to p47-phox and a series of truncatedp47-phox fusion proteins blotted onto nitrocellulose (Fig. SD).Our data indicate that binding of p67-phox to p47-phox in-volves only the C-terminal SH3 domain of p67-phox. The firstSH3 domain of p67-phox (aa 247-295) was neither necessarynor sufficient for binding to p47-phox. Removal ofthis domaindid not abolish p47-phox binding, whereas removal of theC-terminal domain resulted in no detectable binding in eitherassay. The binding site for intact p67-phox within p47-phox(Fig. SD) was more specific than those detected with the

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p47-phox aa 1-150, 151-284, 280-390, 280-338, 338-390, 338-352,358-390, and 70-84, respectively. (B) Binding of biotinylated p47-SH3A-B to blotted proteins (1.5 jg per lane) described inA [p67-phoxSH3 probes gave the same results (data not shown)]. (C) Anti-p47-phox blot of the same proteins.

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FIG. 5. Interaction of the C-terminal SH3 domain of p67-phoxwith the C terminus ofp47-phox. (A) Schematic ofp67-phox deletionproteins lacking SH3 domains (filled bars), produced in baculovirus-infected Sf9 cells (16). (B) SDS/PAGE and immunoblot analysis ofp67-phox proteins (5 x 103 cell equivalents per lane) described in A.(C) Analysis of the same preparations shown in B that bound toGST-p47-phox immobilized on glutathione-Sepharose. (D) Bindingof p67-phox proteins to the C terminus of p47-phox. Replicate blotsof truncated p47-phox fusion proteins (described in Fig. 3) wereprobed with the indicated p67-phox proteins (5 yg/ml at 4°C, 2 hr),which were then detected with anti-p67-phox (26).

isolated GST-SH3 domains (Fig. 4). p67-phox did not bind tothe N-terminal domain ofp47-phox, nor did it detect the highlybasic region ofp47-phox (aa 280-338) or any ofthe proteolyticproducts from this region that bound to the GST-SH3 probes.All of the p47-phox fusion proteins that bound p67-phox (orp67A&1) shared a common C-terminal sequence (aa 358-390)(Fig. SD, lanes 5, 7, and 9). Thus, the association of the twocytosolic components appears to involve a preferential inter-action of the C-terminal SH3 domain of p67-phox with theC-terminal segment of p47-phox.Based on the delineation of several specific interactions,

defined initially in vitro and explored further in transfectedcells, we propose a model for NADPH oxidase assembly inwhich SH3 domains play a central role (Fig. 6). We proposethat the two cytosolic components, p47- and p67-phox, containSH3 motifs that direct their translocation by binding to specifictargets in other oxidase components. The isolated SH3 do-mains of p47- and p67-phox exhibited similar binding patternsfor several proline-rich sequences in p47-phox and p22-phox;the specificity for preferred sites becomes apparent whenthese interactions are examined in the context of other do-mains within these proteins. The C-terminal SH3 domain of

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p67-phox exhibited a preference for binding the C-terminalproline-rich sequence ofp47-phox, while the two SH3 domainsof p47-phox together bound preferentially to p22-phox, per-haps through cooperative interactions with more than oneproline-rich segment in this domain. The p47-phox-p22-phoxinteraction was disrupted by the CGD-associated Pro56 mu-tation in p22-phox, both in vitro and in transfected cells, andwas inhibited by a short proline-rich peptide, indicating that aa149-162 ofp22-phox comprise a critical site in this interaction.The two proposed SH3 binding sites in p22-phox (aa 149-162)and p47-phox (aa 358-371) conform to the consensus sequence(XPXXPPPIXP, where T is a hydrophobic residue) deducedfrom several proline-rich targets of Abl-SH3 (24) and alsocontain the PPRP motif, a sequence common to several p85SH3-binding peptides, shown recently by NMR to bind di-rectly to this SH3 domain (19).The proposed model for oxidase assembly in Fig. 6 is

consistent with studies on CGD cells, showing that p67-phoxtranslocation requires p47-phox, whereas p47-phox transloca-tion depends on the cytochrome, but not on p67-phox (14, 28).It is supported by work showing that the two pure cytosolicfactors interact with each other (26) and recent observationsshowing that the C-terminal SH3 domain ofp67-phox is criticalfor translocation during oxidase assembly in activated cells(15, 16). This model extends work suggesting that both sub-units ofthe cytochrome interact with p47-phox (30), in that wehave shown the proline-rich tail of p22-phox participatesdirectly in translocation of p47-phox in PMA-activated cells.The importance of intramolecular interactions also observedwithin p47-phox, between its SH3 domains and proline-richsequences, relative to p22-phox binding is unclear. Thesepotentially competing interactions may account for the differ-ence between cytosolic (resting) and membrane-bound (ac-tive) forms of p47-phox; oxidase activation may requirechanges in p47-phox that would favor the p22-phox interac-tion, such as the multiple phosphorylation events that accom-pany membrane translocation (28). Such changes in p47-phoxstructure are analogous to those shown to affect the activity ofpp6(W', which also assumes an inactive conformation throughintramolecular interactions involving both its SH2 and SH3domains (32). These interactions within the NADPH oxidaseillustrate a number of ways in which proteins utilize theconserved SH3 motif to assemble multiprotein complexes.

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ActvaiActivation p67-phox

67-phox

FIG. 6. Proposed model forNADPH oxidase activation, showingspecific SH3-mediated interactions between assembled components.Proline-rich SH3 target sites were identified in both p22-phox andp47-phox; one of these sites is affected in CGD (circled) (25).Disruption of intramolecular interactions within p47-phox duringactivation may enable binding ofp47-phox SH3 domains to p22-phox.Cytosolic factors interact similarly through their C-terminal do-mains, possibly both in the active and in the resting states. A fifthessential oxidase component, Rac, is not shown because sites for itsinteraction are unknown.

These observations also provide insights on a mutation thathas been attributed to a rare form ofCGD. To our knowledge,NADPH oxidase is the first system in which an altered SH3binding site has been associated with human disease.

We thank Drs. J. I. Gallin, H. L. Malech, and H. F. Rosenberg forhelpful discussions. We also thank Drs. Gallin, Malech, and D.Kuhns for providing the B cells used in this study.

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