Christopher Butcher$, Paul Bennett$,. Michael D. ..... Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D.. (1993) Cell 75, 25-36. 13.5225- ...
Communication
THEJUURNALOF B I U L ~ ~ I C CHEMISTRY AL Vol. 269,No. 19,Issue of May 13,pp. 13752-13755, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
An SH3 Domain and Proline-rich Sequence Mediate an Interaction between Two Components of the Phagocyte NADPH Oxidase Complex* (Received for publication, February 24, 1994, and in revised form, March 11, 1994)
Peter Finant§, Yazuaki Shimizum, Ivan Goutli, Justin Hsuanll, Oanh 'Ikuongjl, Christopher Butcher$, Paul Bennett$, Michael D. Waterfieldll**, and Stuart Kelliet From the SYamanouchi Research Institute, Littlemore Hospital, Oxford OX4 4xN, United Kingdom, the (Ludwig Institutefor Cancer Research, 91 Riding House Street, London W l P 8Bl: United Kingdom, and the **Department of Biochemistry and Molecular Biology, University College, London WClE 6Bl: United Kingdom
SH3 domain-binding proteins have been reported. The first of these to be isolated, 3BP-1, binds the to SH3 domainof c-ab1 in vitro and contains a region with sequence homology to the GTPase-activating protein forthe monomeric GTPase Rho(4). In addition, an association between the GTPase dynamin and selected SH3 domains has also been described (5). The SH3 domains of src-related tyrosine kinases have been shown to associate with phosphatidylinositol 3"kinase p85 subunit (6, 71, and the signaling pathway from cell-surface receptors to Ras utilizes an adaptor protein Grb2 that couples to activated receptors through an SH2 domain and interacts with a downstream signaling component,Sos, via at least oneSH3 domain (8-12). The association of SH3 domains with cytoskeletalcomponents has also been reported (13, 14). The roles of SH2 and SH3 domains have been predominantly investigatedin proliferation-related signaling in adherent cells such as fibroblasts or nonadherent cells such as lymphocytes;however, in somecellsSH2 and SH3 domain-
containing proteins may also be important regulatory molecules in responses unrelated to cell division. In particular, two components of the phagocyte NADPH oxidase,p47p"" Neutrophils possess a multicomponent NADPH oxi- and p67Phox,each possess two SH3 domains and are normally dase system capable of producing large quantities of cytosolic proteins.Activation of the oxidase is precededby superoxide in a processknown as the respiratory burst translocation of p47phoxa n d p67p"" to the plasma membrane to (1). Upon stimulationof a phagocyticcell, two cytosolic form a complex with cytochrome b,,, and a small GTP-binding components of the oxidase, p67phmand p47phm,associate protein (15, 16). It is therefore possible thatthese protein-prob and a small tein interactions are mediated by SH3 domains. We report with a membrane-bound flavocytochrome GTP-binding proteinto form a functional enzyme com- here the identification of p47P"" as a protein that binds to the src homol- carboxyl-terminal SH3 domain of p67ph0"via a novel prolineplex. Each of the Phox proteins contains two ogy 3 (SH3) domains, which are of unknown function rich motif.
but are potential mediatorsof protein-protein interactions between components of the activated oxidase. We EXPERIMENTALPROCEDURES have isolated a 47-kDa protein from lysates of differenReagents-The bacterial expression vector pGEX-4T-1 was purthe car- chased from Pharmacia LKB Biotechnology Inc. Glutathione-agarose tiated HL60 cells thatspecificallyboundto boxyl-terminal SH3 domain ofp67Pho" and not to any beads were obtained from Sigma. Synthetic peptides were synthesized other SH3 domain tested. This protein was identified as by Zinsser Analytic (Maidenhead,United Kingdom)and Alta Bioscience p47Ph"', and the putative SH3 domain binding site was (Birmingham, United Kingdom). Rabbit polyclonal antisera raised to located to a carboxyl-terminal proline-rich region. Pro-recombinant p47ph" expressed as a GST' fusion protein in Escherichia line-rich synthetic peptides based on this carboxyl-ter- coli was a gift from A. W. Segal. Cell Culture-HL6O cells werecultured in RPMI 1640 and 10%heatminalregionspecificallyinhibited the binding of inactivated fetal calf serum containing 2 mM L-glutamine at 37 "C in 5% ~ 4 7 to ~ the ~ - carboxyl-terminal SH3 domain of p67pho", CO,. Cells were differentiated down the neutrophilic pathway by the and sequential truncation defined a unique minimal se- addition of Me,SO to a final concentration of 1.25% for5 days. quence,which,althoughsimilar,doesnotmatchthe GZutathione S-ZkansferaseFusion Proteins-DNA sequences encodconsensus sequencedefinedforotherSH3-binding ing the NH,-terminal SH3 domains of ~ 4 7 (amino ~ ~ " acids 157-215) and proteins. ~ 6 7 (amino ~ ~ " acids 237-398) were amplified by polymerase chain re-
The role of SH2 domains in the assembly of protein complexes containing phosphorylated tyrosine residues has been firmly established (2); however, the function of SH3 domains remains more elusive. Like SH2 domains, SH3 domains are thought to be involved in mediating protein-protein interactionsbetweensignalingcomponentsdownstream of membrane-bound receptors (3), and recently, a number of potential
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solelv to indicate this fact. 6 Towhom correspondence should be addressed. Tel.: 865-747100; F&: 865-748974. ll Present address: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba-Shi, Ibaraki 305, Japan.
action from a human neutrophil phage library (Clontech) and cloned into the pGEX-4T-1expression vector. Polymerase chain reaction primers corresponding toeach end of the SH3 domains contained EcoRI and Sal1 sites(p47N-SH3)or EcoRI and XhoI sites (p67N-SH3) tofacilitate cloning. The pGEX constructs were sequenced using the Sequenase system (U. S. Biochemical Corp.).The remaining pGEX-SH3 constructs wereproduced as describedpreviously (5). Constructs were transformed into E. coliXL1 and expression of GST fusion proteins performed as described previously (17). GSTSH3 Binding Assays-AEnity matrices were prepared by immobilizing 25 pg of fusion protein on 50 1.11of glutathione-agarose beads (Sigma). Differentiated HL60 cells were pelleted and washed twice in phosphate-buffered saline. Cell pellets were solubilized in lysis buffer (50 mM Tris, pH 7.5,5 mM EGTA, 2% (v/v) TritonX-100,75 mM NaC1,0.5 mM phenylmethylsulfonyl fluoride) and clarified by centrifugation at 14,000 x g for 15 min at 4 "C. Cell lysate was mixed with the GST-SH3 The abbreviations used are: GST, glutathione S-transferase; PAGE, polyacrylamide gelelectrophoresis.
13752
Function of NADPH Oxidase SH3 Domain
13753
68-
68-
-
45-
45-
FIG.3. In vitro binding ofp47phmto the COOH-terminal SH3 of p67ph". SH3 domain-binding proteins werepurified a s described under "Experimental Procedures" and in Fig. 1. Following SDS-PAGE, proteins were transferred to nitrocellulose and probed withantibodies raised to p47Phor.Gel lanes are labeled asfor Fig. 1. FIG.1.In v i t r o binding of detergent-solubleproteins from differentiatedHL60 cells to immobilized GST-SH3 fusion proteins. HL60 cell lysateswereincubatedwithglutathione-agarosebeads (Beads) and beads bound with glutathione S-transferase (GST)or GST fusion proteins containing the carboxyl-terminal SH3 domain of p67"h"x (p67C),amino-terminal SH3 domainof ~ 6 7 ~( p~6' 7"N ) ,carboxyl-terminal SH3 domain of p47p"'" (p47C), amino-terminalSH3domain of p47ph"" ( p 4 7 N ) , phospholipase Cy SH3 domain (PLCy), c-fgr SH3 domain (fgr), SH3 domain of the p85a subunit of phosphatidylinositol 3-kinase (P85).or the SH3 domain of c-src (src). Bound proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE (10% polyacrylamide), and visualizedby silver staining.
p
-68 "
"
-45
MGDTFIRHIALUjFEKRFVPSOHYVYMFLVKWQDLSEKWYRR~EIYEFHKTLK FTEIYEFHK[PepZ] HIALLGFEK(Pepl] 1
56
EMFPIEAGAINPENRIIPHLPAPKWFDGQRAAENRPGTLTEYCSTLMSLPTKISR
111
CPHLLDFFKVRPDDLKLPTDNQTKKPETYLMPKDGKSTATDITGPIILQTY~IA
IIPHLPA(Pep31
166
-200 -116 -95
-29
..
.
0
*
FIG.4. Inhibition of p47P"-p67P"C-SH3 domain binding by the p47Phar-derived proline-rich peptide,P2. Binding of ~ 4 to 7 ~ 2 7 6 PSMYLQKSGQDVSQAQRQIKRGAPPRRSSIRNAHSIHQRSRKRLSQDAYRRNSVR the COOH-terminal SH3 domain of ~67'"'"' was determined in the presence of synthetic peptides derived from proline-rich sequences of dynamin, peptide P1 (lane 4 ) ;p47Ph"', peptide P2 (lane5);~ 6 7 ~ ~pep'", 331 FUlQRRRQARPGPQSPGSPLEEERQTQRSKPOPAVPPRPSADLILNRCSESTKRK tide P3 (lane 6 ) ; cytochrome b,,,a, peptide P4 (lane 7 ) ; phosphotyrosine protein phosphatase (PTPase) lB, peptide P5 (lane8); vinculin 386 LASAV peptide P6 (lane 9); or in the absence of any peptide (lane 3). Lane 1 FIG.2. Alignment of tryptic peptide sequences derived from shows p67Ph"C-SH3 domain without cell lysate or peptide, and lane 2 the 47-kDa protein that bound to p67Ph"C-SH3 domain with hu- shows control GST affinity matrix plus cell lysate. The full peptide seman ~ 4 7 sequence. ~ ~ " Peptide sequences obtained from the tryptic quences and inhibitory activities are listed in Table I. HL60 cell lysate peptides (PEPI, PEPZ, PEP3) are indicated in bold below the appro- was incubated with the p67Ph"C-SH3 domain affinity resin inthe prespriate sequenceof p47ph". The proline-rich sequencethat isa potential ence of the proline-rich peptides a t a concentration of 750 PM. The SH3-binding region is underlined, and a homologous peptide, P2, was beads were washed and SDS-PAGE samples prepared as described in synthesized and used in further studies. Fig. 1. Proteins were resolved by SDS-PAGE and visualized by silver staining. affinity matrices for 3 h a t 4 "C. The beads were then washed extensively in wash buffer (50 mM Tris, pH 7.5, 0.1% (v/v) Triton X-100,10% RESULTS AND DISCUSSION (v/v) glycerol). Binding proteins were eluted by boiling in SDS-PAGE To investigate the role of these SH3 domains in phagocytic sample buffer, resolved by SDS-PAGE, and visualized by silver staining. In addition, binding assays were performed in the presence of proline- cells, we investigated whether proteins from such cells could rich synthetic peptides at concentrations of 750 and 400 PM, a s indicated specifically bind to SH3 domains. Affinity matrices were prein the relevant figure legends. pared by immobilizing various GST-SH3 domain fusion proProtein Sequencing-A 47-kDa polypeptide that boundtothe teins on glutathione-agarose beads. These matrices were used COOH-terminal SH3 domain of p67ph" was purified from 5 x lo9 differentiated HL60 cells by GST-SH3 affinity chromatography as de- to isolate proteins from detergent extracts of HL60 cells that scribed under "GST-SH3 Binding Assays." The protein sample(300 pl) had been differentiated down the neutrophilic pathway. Sevwas concentrated by centrifugal filtration and applied to SDS-PAGE. eral leucocyte proteins bound specifically to matrices consisting The 47-kDa polypeptide was identified by Coomassie Blue staining, of immobilized GST-SH3 fusion protein, but not to immobilized excised, and digested with trypsin in the gel slice. Peptides were ex- GST or glutathione-agarose alone (Fig. 1).In particular, a 68tracted from the gel using detergents and separated by tandem ion-exkDa polypeptide bound to the SH3domains of c-src and phoschange and reverse phase high performance liquid chromatography using a Hewlett-Packard 1090M chromatograph and diode array de- pholipase Cy, and the NH,-terminal SH3 domain of ~ 4 7 " ~ ' ~ investigation. tection. Purified peptides were sequenced using fast cycle, automated bound several proteins that are currently under Edman chemistry on an Applied Biosystems 477A sequenator, modi- In addition, a polypeptide of about 47 kDaspecifically bound to fied a s described (18). the COOH-terminal SH3 domain of p67"*" (p67"'W-SH3) but Western Blotting-Following protein sequencing, the identity of the not to any of the other SH3 domains tested (Fig. 1, asterisk). 47-kDa SH3 domain-binding protein was confirmed by Western blotThis polypeptide was isolated from 5 x lo9 cells by binding to ting. SH3 domain-bindingproteinswerepurified as previouslydescribed (under"GST-SH3 Binding Assays") and were resolved by SDS- the p67phoxC-SH3domain as described and identified by amino acid sequencing of tryptic peptides. Sequences derived from PAGE, transferred to nitrocellulosefilters, andincubatedwithan three different peptides from the 47-kDa protein showed 100% antibody against p47Phox(a gift from A.W. Segal). 221
Function of NADPH Oxidase SH3 Domain
13754
TABLEI Summary ofthe abilityof different proline-rich peptides to inhibit binding ofp47 to p67C-SH3 domain Binding studies were carried out asdescribed for Fig. 4. Peptide no.
Protein
P1 Dvnamin (360-373) KPQPAVPPRPSADL p47~hox P2 (218-235) APLQPQAAEPPPRPKTPE p67phm P3 P4 Cytochrome b,,a PTPase 1B P5 Vinculin P6
A
Inhibition of ~47:~67C-SH3 binding
Sequence (residue no.)
PAVPPAFtPRGSGPAPGPPPAG (786-806) KQPPSNPPPRPPAEA (148-162) DLEPPPEHIPPPPRPPKR (298-316) APPKPPLPEGEVPPPRPPPPE (858-878) TABLEI1 Summary ofthe ability of sequentiallytruncated peptides to inhibit binding ofp47 to p67C-SH3 domain Binding studies were carried out asdescribed for Fig. 5. Peptide
KPQPAVPPRPSADL PQPAVPPRPSADL QPAVPPRPSADL PAVPPRPSADL AVPPRPSADL VPPRPSADL PPRPSADL PRPSADL RPSADL KPQPAVPPRPSAD KPQPAVPPRPSA KPQPAVPPRPS QPAVPPRPS QPAVPPRP QPAVPPR QPAVPPKPS
Code
P2 P2-N1 P2-N2 P2-N3 P2-N4 P2-N5 P2-N6 P2-N7 P2-N8 P2-c1 P2-C2 P2-C3 P2-N2C3 P2-N2C4 P2-N2C5 P2-N2C3K
Inhibitory activity
+ + + +/-
+ + + + +
-
rich region of p47ph" was used in competition experiments. This peptide, with the sequence KPQPAVPPRPSADL (peptide P2) inhibited the binding of p47phoXto the p67phoxC-SH3domain at a concentration of 750 w (Fig. 4). Peptides corresponding to proline-rich sequences in p67Ph", the a subunit of cytochrome b,,,, PTPase lB, vinculin, and dynamin all failed to compete at RG.5. Characterization of the p67PhmC-SHSbinding site on the same concentration (Fig. 4, Table I), although of these prop4'7Phm.A, the effect of truncations at either the NH, or COOH termi- teins only dynamin has been shown to bind SH3 domains nus of peptide P2 on the ability to inhibit ~ 4 7 ~binding ~ " to ~ 6 7 ~ ~ " C -in vitro (5). Thus the interaction between the proline-rich SH3. P2a and P2b are different batches of peptide P2. P2-N indicates sequence ofp47ph" and p67PhmC-SH3appears to be highly NH,-terminal truncations, and P2-C indicates COOH-terminal truncations as listed in Table 11. B, effect of double truncations or amino acid specific. From dose-response studies, the half-maximal concensubstitutions on the ability of peptide P2 to inhibitthe binding of p47ph" tration for inhibition of binding was calculated to be approxito p67Ph"C-SH3 domain. The full peptide sequences and inhibitory ac- mately 100 w (data not shown). tivities are listed in Table 11. Binding of p47Ph0rto immobilized ~67~"CAseries of truncated variants of the synthetic peptide P2 was SH3 domain was assayed as described in Figs. 1 and 4. Solubilized protein was incubated with the p67phoxC-SH3 domain affinity resin in tested to further characterize the putative SH3domain binding the presence of the proline-rich peptides a t a concentration of 400 p. site (Fig. 5, A and B; Table 11).Truncations at either the NH,or Bound proteins were resolved by SDS-PAGE and visualized by silver COOH terminus led to the identification of a minimal active staining. core sequence of QPAVPPRPS(Fig. 5A). This was refined using peptides truncated a t both the NH, and COOH terminus to a identity withthe published amino acid sequence of p47ph" (Fig. minimal inhibitory sequence of QPAVPPRP (Fig. 5B 1. The pep2). In addition, the 47-kDa polypeptide was recognized by an- tide sequences and activities are summarized in Table 11. In tibodies raised to p47ph" (a gift from A.W. Segal) and Western addition, a peptide QPAVPPKP, in which arginine was substiblotting confirmed that p47phoX did not associate with any other tuted for lysine, had no inhibitory activity. This indicates that SH3 domain tested (Fig. 3). It should be noted that a 46-kDa the arginine is supplying more than just a charge for activity polypeptide that bound to the p47PhO"N-SH3domain (Fig. 1)did and thatrequirements other than proline sequence or spacing not cross-react with anti-p47Phoxantibodies. These data demon- are essential for SH3 domain binding. The SH3 binding motif of strate a specific interaction between p47Pbx and the COOH- 3BP-1 has been previously identified as APTMPPPLPP, with terminal SH3domain of p67PhoX. the proline residues at positions 2, 7, and 10 appearing to be Previous reports have suggested that proline-rich sequences crucial for binding to the cabl SH3 domain. Similarly, the are important for the recognition of SH3 domains (4-10, 19). corresponding sequence of a second SH3 domain-binding proAnalysis of the p47Ph" sequence revealed the presence of a tein, 3BP2, was identified as PPAYPPPPVP and theconsensus proline-rich motif near the COOH terminus of the protein. "0 sequence XPZMPPPZXP (where X is any amino acid and Z is a investigate the role of this site in thebinding to the p67ph"C- hydrophobic amino acid) was proposed (19).However, recently SH3 domain, a synthetic peptide corresponding to the proline- the p85a subunit of phosphatidylinositol 3"kinase has been
Function
of
NADPH Oxidase
shown t o associate with SH3domain of p59ry"and pp60v-src, and two inhibitory peptides NERQPAPLPPK andKPRPPRPLPVA have been described (11, 12) that do not fit this consensus. Furthermore, sequences that mediate binding of dynamin to several SH3domains have been identified as containing PAVPPARP (5). The binding sitefor p67PhoxC-SH3,although prolinerich, also does not match the consensus sequence, and it is likely that as more SH3 domain-binding proteins are characterized the consensus binding motif will be specific to individual SH3 domains. Both p47ph" and p67ph" translocate from the cytosol t o the plasma membrane after stimulation, and there is evidence to suggest that within thecytosol these components may exist as a 240-kDa complex (20).At present we donot know whether the association between p47phoXand the COOH-terminal SH3 domain of p67phoX occurs within thiscytosolic complex or mediates protein-protein interactions following oxidase activation. It is possible that the interactions of other proteins withinthe NADPH oxidase enzyme complex may be mediated by SH3 domains; for example, both p67phoX and thecytochrome b,,, a subunit contain putativeSH3-binding motifs, and another component of the oxidase, p40ph"",has recently been cloned and found to contain an SH3domain (21). The regulation of SH3-mediated interactions isat present unknown. However, distinct phosphorylation events occur both prior t o translocation of the Phox components and afterassociation with the cytochrome (22,23). It ispossible that phosphorylation of one or more of the oxidase components may alter protein structure to expose either SH3 domains or binding sites on the components of the oxidase. The present study demonstrates that~ 4 7 and ~ ~p67ph"x ' ~ interact witheach other via an SH3domain and a unique prolinerich sequence. Whether this contact forms the sole interaction between the proteins remains to be determined. Elucidation of the interactions that occur in the NADPH oxidase will lead to a greater understanding of the regulation of phagocyte re-
SH3 Domain
13755
sponses to inflammatory stimuli and should allow a more rational approach to thedevelopment of inhibitors of tissue-damaging free radicals release which occurs in a variety of diseases such as ischemia and arthritis. REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9.
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21. 22. 23.
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x.,
