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Phosphotyrosine 1173 Mediates Binding of the Protein-tyrosine Phosphatase SHP-1 to the Epidermal Growth Factor Receptor and Attenuation of Receptor Signaling

Phosphotyrosine 1173 Mediates Binding of the Protein-tyrosine Phosphatase SHP-1 to the Epidermal... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 38, Issue of September 18, pp. 24839 –24846, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Phosphotyrosine 1173 Mediates Binding of the Protein-tyrosine Phosphatase SHP-1 to the Epidermal Growth Factor Receptor and Attenuation of Receptor Signaling* (Received for publication, April 7, 1997, and in revised form, June 14, 1998) Heike Keilhack‡, Tencho Tenev‡, Elke Nyakatura§, Jasminka Godovac-Zimmermann§, Lene Nielsen¶, Klaus Seedorf¶, and Frank-D. Bo ¨ hmer‡i From the ‡Research Unit “Molecular Cell Biology,” Medical Faculty, Friedrich Schiller University, D-07747 Jena, Germany, §Institute of Molecular Biotechnology e.V., Beutenbergstrasse 11, D-07745 Jena, Germany, and ¶Hagedorn Research Institute, Niels Steensen Vej 6, DK-2820 Gentofte, Denmark The protein-tyrosine phosphatase SHP-1 binds to and depending on the particular cell type or the activation state of cells. The PTPases with SH2 domains, SHP-1 and SHP-2 (8 – dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important 10), are prone for interaction with tyrosine-phosphorylated pro- for this interaction (Tenev, T., Keilhack, H., Tomic, S., teins and have been shown to bind to multiple receptor species Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, including transmembrane tyrosine kinases (11–15), cytokine A. V., Ullrich, A., and Bo ¨ hmer, F. D. (1997) J. Biol. Chem. receptors (16 –18), antigen receptors (19 –22), and adhesion 272, 5966 –5973). We mapped the EGFR phosphotyrosine molecules (23, 24). SHP-2 is a ubiquitously expressed enzyme 1173 as the major binding site for SHP-1 by a combina- and, as largely derived from genetic evidence for the Drosoph- tion of phosphopeptide activation, phosphopeptide ila SHP-2 homologue Csw, seems to present an essential com- competition, and receptor YF mutant analysis. Muta- ponent of a positive signaling cassette of multiple receptors (25, tional conversion of the EGFR sequence 1171–1176 AEY- 26), although negative modulation of receptor signaling by LRV into the high affinity SHP-1 binding sequence LEY- SHP-2 has also been reported (27, 28). SHP-1 is highly ex- LYL of the erythropoietin receptor (EpoR) led to a pressed in hematopoietic cells, and another closely related iso- highly elevated SHP-1 binding to the mutant EGFR form of SHP-1 is expressed from an alternative promotor in ) and in turn to an enhanced dephos- (EGFR 1171–1176EpoR epithelial cells (29). SHP-1 has been convincingly shown to phorylation of the receptor. SHP-1 expression inter- negatively regulate signaling of multiple receptors in hemato- fered with EGF-dependent mitogen-activated protein poietic cells, including Kit/SCF-receptor (30, 31), interleukin 3 kinase stimulation, and this effect was more pro- receptor (14), CSF-1 receptor (32), and the EpoR (17, 33). . Reduced SHP-1 nounced in case of EGFR 1171–1176EpoR SHP-1 binds to Kit/SCF receptor (14) but not to the CSF-1 binding to the EGFR Y1173F mutant resulted in a re- receptor (32) and may thus intercept with receptor signaling by duced receptor dephosphorylation by coexpressed different mechanisms. In case of the EpoR, SHP-1 is recruited SHP-1 and less interference with EGF-dependent mito- via its N-terminal SH2 domain to the receptor subsequent to gen-activated protein kinase stimulation. The effects of receptor mutations on SHP-1 binding were, however, EpoR phosphorylation by Jak2 kinase (17). A high affinity stronger than those on receptor dephosphorylation by SHP-1 binding site at Tyr-429 of EpoR has been characterized, SHP-1. Therefore, receptor dephosphorylation may be and its lack in respective YF mutants of EpoR or truncated the result of the combined activity of receptor-bound receptors leads to an enhanced EpoR signaling activity (17). SHP-1 and SHP-1 bound to an auxiliary docking protein. Negative modulation of EpoR signaling by SHP-1 appears to involve Jak2 dephosphorylation (17). In addition, direct bind- ing of SHP-1 to Jak2 and dephosphorylation of Jak2 by SHP-1 Activated and subsequently tyrosine-phosphorylated growth has also been demonstrated (34). factor receptors are rapidly dephosphorylated, a process that is The functional role of SHP-1 in epithelial cells is much less believed to negatively modulate signaling activity (1–7). The understood. SHP-1 can bind to the EGF receptor (11, 15, 35) PTPases involved in receptor dephosphorylation are fre- and to HER2 (13) and can dephosphorylate both in transient quently unknown. It is possible that multiple PTPases act in coexpression systems (13, 15) and in stably transfected epithe- concert on a given receptor, the identity of which may vary lial cells (36) . Thus, SHP-1 may negatively regulate HER family receptor signaling in epithelial cells. Both SH2 domains of SHP-1 are important for binding of SHP-1 to the EGFR and * This work was supported by grants from the Max-Planck Society (to for receptor dephosphorylation, although the N-terminal SH2 F. D. B.) and Deutsche Forschungsgemeinschaft (Bo 1043/3-1 to F. D. B. and J. G.-Z.; SFB197/A9). The costs of publication of this article domain contributes to a larger extent to the interaction (35). were defrayed in part by the payment of page charges. This article must The catalytic domain of SHP-1 provides the specificity for therefore be hereby marked “advertisement” in accordance with 18 EGFR dephosphorylation and cannot be replaced by the cata- U.S.C. Section 1734 solely to indicate this fact. lytic domain of SHP-2 in SHP-1/SHP-2 chimeras (35). To fur- To whom correspondence should be addressed: Research Unit “Mo- lecular Cell Biology,” Medical Faculty, Friedrich Schiller University, ther clarify the mechanism of SHP-1 EGFR interaction, we Drackendorfer Str. 1, D-07747 Jena, Germany. Tel.: 149 (3641) 304468; investigated the importance of different EGFR autophospho- Fax: 149 (3641) 304462; E-mail: [email protected]. rylation sites for the interaction with SHP-1. Activation of The abbreviations used are: PTPase, protein tyrosine phosphatase; purified recombinant SHP-1 by EGFR sequence-derived phos- EGF, epidermal growth factor; EGFR, EGF receptor; EpoR, erythropoi- etin receptor; HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; Fmoc, N-(9-fluorenyl) methoxycarbonyl; WGA, wheat germ T. Tenev, T. Bittorf, T. Beckers, and F.-D. Bo ¨ hmer, unpublished agglutinin. data. This paper is available on line at http://www.jbc.org 24839 This is an Open Access article under the CC BY license. 24840 EGF Receptor SHP-1 Interaction GST fusion proteins were eluted with 1 ml of buffer E (50 mM Tris-HCl, phopeptides, competition of phosphopeptides with binding of pH 8, 5 mM reduced glutathione) with end-over-end rotation for 30 min autophosphorylated EGFR to immobilized SHP-1 SH2 do- at 4 °C. The beads were pelleted by centrifugation at 1000 3 g and 4 °C mains, and binding of SHP-1 to a panel of EGFR YF mutants for 1 min, and the supernatant was collected. This elution step was was employed to identify Tyr(P)1173 as the main binding site repeated twice, and the supernatants were combined (3 ml). The eluate for SHP-1 on the EGFR. Introduction of the EpoR high affinity was dialyzed against Xa buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 2 SHP-1 binding site into the EGFR strongly increases SHP-1 mM CaCl ,1mM dithiothreitol). The protein concentration was deter- mined using the Bradford method. To remove the GST tag, GST-SHP-1 binding and also increases receptor dephosphorylation by was digested with Factor Xa. For this the protein concentration of the SHP-1. Introduction of a Y1173F mutation reduces but does not fusion protein was adjusted to 1 mg/ml using Xa buffer, and Factor Xa abrogate receptor dephosphorylation by coexpressed SHP-1. was added with a ratio of protease:fusion protein of 1:200. The mixture Thus, SHP-1 activity toward EGFR is modulated but not ex- was incubated for 16 h at 4 °C. The proteolytic reaction was stopped by clusively dependent on receptor binding. adding 5 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mM dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone (final MATERIALS AND METHODS concentrations). The mixture was applied to a 1-ml glutathione-Sepha- Chemicals and Reagents—EGF was purchased from Pepro Tech, Inc., rose 4B column equilibrated with buffer A (50 mM Tris-HCl, pH 8, 5 mM (Rocky Hill, NJ). Polyclonal anti-phosphotyrosine antibodies and mono- dithiothreitol) to remove free GST and uncleaved fusion protein. The clonal anti-pan-extracellular signal-regulated kinase antibodies were unbound proteins were eluted with buffer A by collecting 250 ml frac- obtained from Transduction Laboratories (Lexington, KY). Monoclonal tions. The fractions were analyzed by SDS-PAGE and Coomassie stain- anti-EGFR antibody 425 was a kind gift from Dr. Luckenbach (Merck ing. The positive fractions were combined and loaded on a MonoQ AG, Darmstadt, Germany). Polyclonal anti-EGFR and anti-SHP-1 an- HR5/5 column (Amersham Pharmacia Biotech) connected to a fast tibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Acti- protein liquid chromatography system. The bound proteins were eluted vated MAP kinase was detected with a polyclonal anti-active MAP with a linear NaCl gradient (80 –200 mM within 20 ml). The fractions kinase antibody (Promega Corporation, Madison, WI). Anti-HA 12CA5 were analyzed by SDS-PAGE and silver staining. Positive fractions monoclonal antibody was purchased from Babco (Berkeley, CA), myelin were pooled and concentrated using 10-kDa centrifugal filters (Milli- basic protein was from Sigma. For introduction of point mutations in pore Corporation, Bedford, MA). 20% glycerol was added, and the pro- the EGFR cDNA, an M13 mutagenesis kit (Bio-Rad) was used. tein solution was stored at 270 °C. The purity of the preparation was [g- P]ATP was purchased from NEN Life Science Products. Factor Xa 95%; 300 mg of SHP-1 could be obtained from a 500-ml bacterial culture. was obtained from Boehringer Mannheim. Dansyl-L-glutamyl-glycyl-L- A431 Cell Treatment and Binding of SHP-1 to the EGFR—A431 cells arginine chloromethyl ketone was from Calbiochem-Novabiochem. were grown in 94-mm dishes to about 70% confluency in Dulbecco’s Cloning of SHP-1 and SH2 Domain Mutants to pGEX Expression modified Eagle’s medium supplemented with 10% fetal calf serum. The Vector—The SHP-1 construct in pRK5RS-SHP-1 (35), which contains cells were starved for 16 h using serum-free Dulbecco’s modified Eagle’s the originally cloned cDNA, was sequentially treated with XhoI, Pfu, medium and subsequently stimulated with 100 ng/ml EGF for 5 min and EcoRI, and the obtained SHP-1 DNA was cloned into SmaI/EcoRI- and lysed in 700 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM digested pBluescriptKS. The 39-untranslated sequences were removed NaCl, 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 1 mM phenylmeth- by replacing the poly(A)-containing Eco47III-XbaI fragment in this ylsulfonyl fluoride, 10 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mM construct with an analogous fragment without poly(A) from the earlier sodium orthovanadate). The lysates were centrifuged at 25,000 3 g for 2 2 1 described chimera SH2 hg CAT (35). 59-Untranslated sequences were 20 min. To investigate whether the interaction of SHP-1 and EGFR is removed by replacing the EcoRI-BamHI fragment of original SHP-1 direct or not, a sequential immuno- and affinity precipitation approach cDNA with an analogous fragment produced by polymerase chain re- was used (37). A431 cell lysates were prepared as described above. action with SHP-1 cDNA in pBluescriptKS as template using primers EGFR was immunoprecipitated using the monoclonal anti-EGFR anti- 1C-E, 59AAGAATTCCCCTACAGAGAGATGCTGTCC-39, and 2C, 59- body 425. The precipitates were denatured in 100 ml of SI buffer (50 mM CCGGAATTCGGCGCCTGAGGCCTCCTCAAT-39. The modified SHP-1 Hepes, pH 7.5, 1% SDS, 1% b-mercaptoethanol) for 5 min at 95 °C. 100 DNA was removed from pBKS using EcoRI/NotI and cloned in pGEX- ml of denatured protein solution was mixed with 900 ml of lysis buffer. 5X-1. To clone the SH2 domains only, an EcoRI/SmaI fragment of The partially renatured proteins were incubated with 50 mg of immo- modified SHP-1 was cloned into pGEX-5X-1. The SHP-1 SH2 domain bilized GST-SHP-1 or GST for2hat4 °C.The beads were washed three point mutants with inactivated N-terminal SH2 domain (R32K), with times with HNGT (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton inactivated C-terminal SH2 domain (R138K), or both inactivated SH2 X-100, 10% glycerol, 1 mM Na VO ), 60 ml23 Laemmli buffer was 3 4 domains (R32K, R138K) were subcloned from the corresponding full- added, and the beads were boiled for 5 min. Proteins were separated by length DNAs in pRK5 (35) to pGEX-5X-1 using EcoRI/XhoI. SDS-PAGE, and bound proteins were visualized by immunoblotting. Preparation of GST Fusion Proteins and of Recombinant SHP-1— Peptide Synthesis and Purification—Six peptides encompassing the pGEX-5X-1 SHP-1, pGEX-5X-1 SH2, and the SH2 domain point mu- following EGFR sequences, PQRY LVIQGD, DADEY LIPQQGFF, 954 992 tants in pGEX-5X-1 were used to transform Escherichia coli strain VPEY INQSVPK, NPVY HNQPLN, NPDY QQDFFPK, 1068 1086 1148 BL21(DE3). A single colony was inoculated into 50 ml of LB medium NAEY LRVAPQS, were synthesized using Fmoc chemistry on an containing 50 mg/ml ampicillin and cultured overnight. 25 ml of this ABI 433 synthesizer (Applied Biosystems, Foster City, CA) and esta- overnight culture were added to 500 ml of LB/ampicillin and grown at blished procedures in solid phase peptide chemistry (38, 39). We syn- 37 °C to A 5 0.8. Isopropyl-1-thio-b-D-galactopyranoside was thesized each peptide with and without phosphorylation of tyrosine 600nm added to a final concentration of 0.1 mM, and the bacteria were kept for using either Fmoc-Tyr(PO3H2)-OH (Novabiochem) or Fmoc-Tyr(t-bu- 16 h at 25 °C under shaking. Thereafter, the bacteria were pelleted at tyl)-OH (Applied Biosystems, Foster City, CA). The peptides were ini- 6,000 3 g and 4 °C for 10 min. The pellet was resuspended in 10 ml of tially purified on a SMART chromatographic system (Amersham Phar- a buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM macia Biotech) using an reverse phase column C2/C18 Sephacil 2.1/10 b-mercaptoethanol, 2 mM EGTA, 1 mM EDTA, 5 mM b-glycerophos- column in 0.1% trifluoroacetic acid, 5% acetonitrile with a gradient of phate, 2 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mg/ml bestatin, 1 mg/ml 0 –100% acetonitrile in 60 min at a flow rate of 100 ml/min. The antipain, 1 mg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. phosphopeptides were further purified by chelating chromatography The suspension was three times frozen and thawed, lysozyme was (40) and subsequently desalted using C18 Sep-Pak Cartridges (Waters added (0.5 mg/ml final concentration), and the mixture was incubated Corporation, Milford, MA). The eluted phosphopeptides were dried in a at 25 °C for 20 min. Subsequently EDTA and Triton X-100 were added vacuum concentrator and redissolved in water. The phosphopeptide in final concentrations of 20 mM and 0.5%, respectively, and the mixture concentrations were determined by complete dephosphorylation using was incubated again for 20 min at 25 °C. The bacterial lysate was GST-SHP-1 and determination of released inorganic phosphate (41). centrifuged at 50,000 3 g and 4 °C for 30 min, and the supernatant was The sequences and the purity of the peptides were verified by complete transferred to a fresh 15-ml polypropylene tube containing 1 ml of N-terminal protein sequencing using an Applied Biosystems Procise glutathione-Sepharose 4B (Amersham Pharmacia Biotech). This sus- 494 4-cartridge sequencer and by mass spectroscopy using a LCQ pension was mixed by end-over-end rotation for1hat4 °C.The beads Finnigan MAT ion trap mass spectrometer (Finnigan MAT, San Jose, were pelleted at 1000 3 g and 4 °C for 1 min, and the supernatant was CA) coupled with an on-line Hewlett-Packard 1090 high performance discarded. The beads were washed 4 3 10 min at 4 °C each time with 10 liquid chromatography. 1 mg of each peptide was dissolved in 1 ml of ml of buffer W1 (25 mM Tris-HCl, pH 7, 5, 150 mM NaCl, 10 mM water, and 1 ml was injected onto the high performance liquid chroma- b-mercaptoethanol, 1% Triton X-100) and 13 with buffer W2 (50 mM tograph and eluted with an isocratic gradient of 50% methyl alcohol, 1% Tris-HCl, pH 8, 150 mM NaCl, 10 mM b-mercaptoethanol). The bound formic acid at flow rate of 200 ml/min. EGF Receptor SHP-1 Interaction 24841 Phosphopeptide Activation Assay—Activation of SHP-1 by EGFR- related phosphopeptides was carried out in a 50-ml reaction mixture containing the reaction buffer (100 mM Hepes, pH 7, 4, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 10 mM p-nitrophenyl phosphate), 200 mM phosphopeptides, and 500 ng of SHP-1. After 30 min at 25 °C, the reactions were quenched with 100 mlof1N NaOH, and the absorbance at 405 nm was measured. Controls with unphosphorylated peptides were carried out in the same way. Competition of the EGFR-SHP-1 Interaction by Phosphopeptides— Solubilized A431 membranes were prepared as described earlier (15). For 15 binding reactions, the following mixture was prepared: 100 mlof solubilized membranes (25 mg of protein) in buffer S (50 mM Hepes, pH 7.4, 1% Triton X-100) were supplemented with 3 mM MnCl , 0.1 mM Na VO , and 2 mg/ml EGF (final concentrations). This reaction was 3 4 incubated on ice for 20 min. Autophosphorylation was started by adding 100 mM ATP and 38 mCi of [g- P]ATP. After a 5-min incubation on ice, the reaction mixture was applied onto a 0.5-ml WGA-agarose column (Amersham Pharmacia Biotech) equilibrated with WGA buffer (50 mM Hepes, pH 7.4, 1 mM Na VO , 0.1% Triton X-100). The column was 3 4 washed with 3 ml of WGA buffer. The radioactively labeled EGFR was eluted with 2 ml of WGA buffer containing 0.3 MN-acetylglucosamine, 100-ml fractions were collected, and aliquots were analyzed by scintil- lation counting. The peak fractions were pooled (500 ml). 50 mgof GST-fused SHP-1 SH2 domains (GST-SH2) were coupled to 30 mlof ml of the radiolabeled glutathione-Sepharose beads per reaction. 30 FIG.1. SHP-1 binds directly to EGFR. A431 cells were treated EGFR (;20, 000 cpm) supplemented with 200 mM phosphopeptides or with 100 ng/ml EGF (lanes 2 and 4) or a respective vehicle (lanes 1 and the unphosphorylated analogues were added to the immobilized GST- 3) for 5 min and subsequently lysed. Immunoprecipitation was carried SH2. The binding reactions were incubated with shaking for2hat4 °C. out using the anti-EGFR monoclonal antibody 425. To disrupt preex- Subsequently, the beads were washed three times with HNGT, 60 mlof isting protein complexes, the immunoprecipitates were denatured in 23 Laemmli buffer was added, and the samples were incubated for 5 the presence of SDS and b-mercaptoethanol. Subsequently, the dena- min at 95 °C. The extracted proteins were resolved by SDS-PAGE (7.5% tured protein solution was diluted with a Triton X-100-containing gels), and the gels were analyzed using a GS250 Molecular Imager buffer to a final SDS concentration of 0.1%. The partially renatured (Bio-Rad). proteins were incubated with GST-SHP-1 (lanes 3 and 4) or GST (lanes Binding Assay with EGFR YF Mutants, Cotransfections, and Dephos- 1 and 2) immobilized on glutathione-Sepharose. The beads were phorylation Assays—Point mutations were introduced into the EGFR washed, and the associated proteins were analyzed by SDS-PAGE and immunoblotting using anti-EGFR antibodies (A). Aliquots of the dena- cDNA using a M13 mutagenesis kit according to the manual of the tured first immunoprecipitates were analyzed to verify equal loading of supplier. 293 cells were transfected with the various EGFR mutants the second precipitation (B). using calcium phosphate coprecipitation (15). 20 mg of plasmid DNA was used for one 94-mm dish. After transfection, the cells were stimu- RESULTS lated with 100 ng/ml EGF for 5 min. Stimulation was stopped by ml lysis buffer. The lysates were removing the medium and lysis in 700- Mapping of an SHP-1 Binding Site on the EGFR—Signaling clarified by centrifugation at 25,000 3 g and 4 °C for 20 min. Protein molecules possessing SH2 domains can bind to autophospho- amounts were determined using the Bradford method, and the different rylated growth factor receptors in a direct or indirect manner. lysates were diluted to the same protein concentration with lysis buffer. An example of this is the adaptor protein Grb2, which binds to To monitor the binding of the EGFR mutants to SHP-1, 10 mgof the EGFR directly via phosphorylated Tyr-1086. In contrast, GST-SH2 or GST were coupled to 30 ml of glutathione-Sepharose. The lysates were incubated with the immobilized fusion proteins for2hat Grb2 binds indirectly to the phosphorylated PDGFb- receptor 4 °C with end-over-end rotation. The beads were washed three times Tyr-1009 site mediated by SHP-2 (37). We wished to know mlof23 Laemmli buffer was added. Noncovalently with HNGT, and 60 whether SHP-1 is capable of a direct binding to EGFR. To test bound proteins were extracted by boiling the beads for 5 min, and the this, EGFR immunoprecipitates from A431 cells were dena- supernatants were loaded onto a 7.5% SDS gel. Immunoblotting was tured by boiling in the presence of 1% SDS. Under these con- performed as described earlier (35). The bound EGFR was visualized ditions any possible adaptor protein should be inactivated; using anti-EGFR antibodies. Cotransfections of the EGFR mutants with SHP-1 were performed as however, the phosphotyrosine motifs in the autophosphoryl- described (35). The cells were stimulated with 100 ng/ml EGF for 5 min ated EGFR are expected to retain at least part of their binding and lysed in 200 ml of lysis buffer/well of a 6-well plate. Immunoblot affinity to corresponding SH2 domains (37). The denatured analysis was carried out as described (35). The difference of the tyrosine immunoprecipitates were diluted to reduce the SDS concentra- phosphorylation level of a given EGFR mutant in the absence or pres- tion to a nondenaturing level and were subjected to a binding ence of SHP-1 was used as measure of dephosphorylation activity reaction with a SHP-1 GST fusion protein. As demonstrated in toward the receptor. Fig. 1, EGFR can be recovered on GST-SHP-1 when the cells MAP Kinase Activation Assays—MAP kinase activity assay in vitro was performed as described (42). Briefly, 293 cells were cotransfected had been stimulated by EGF. From nonstimulated cells, much with 0.25 mg of pcDNA3 HA extracellular signal-regulated kinase 2, 0.5 less EGFR is bound to GST-SHP-1, and no EGFR is detectable mg of pRK5 EGFR wild type or mutants and 3.25 mg of pRK5 SHP-1 or on GST beads in either case. Although the amount of EGFR intact vector. The cells were stimulated with 100 ng/ml EGF for 5 min bound to GST-SHP-1 recovered from denatured immunopre- or left unstimulated. Thereafter, cells were lysed, and extracellular cipitates is only a fraction of what can be recovered with GST- signal-regulated kinase 2 was immunoprecipitated with an anti-HA SHP-1 from nondenatured lysates (not shown), this experiment antibody. The kinase reaction was started by the addition of 1.5 mg/ml indicates that SHP-1 is capable of a direct interaction with myelin basic protein, 75 mM ATP, 7.5 mM MgCl2 (final concentrations), and 1 mCi [g- P]ATP. The reaction was terminated with SDS-PAGE autophosphorylated EGFR. sample buffer, and the samples were boiled for 5 min. Proteins were We went on to test the possible involvement of the different separated by SDS-PAGE (12.5% gels), and the lower part of the gels autophosphorylation sites of EGFR in the SHP-1 binding. Ty- containing the phosphorylated myelin basic protein were dried and rosines 992, 1068, 1086, 1148, and 1173 have been reported to analyzed using a GS250 Molecular Imager (Bio-Rad). The amount of become tyrosine-phosphorylated by the autokinase activity of immunoprecipitated extracellular signal-regulated kinase 2 was deter- the EGFR (for a review see Ref. 43 and references therein). The mined by blotting the upper part of the gel and development with anti-HA antibodies. tyrosine 954 is a potential phosphorylation site and part of a 24842 EGF Receptor SHP-1 Interaction FIG.2. Effect of EGFR-related phosphopeptides on catalytic activity of recombinant SHP-1 and on binding of autophosphorylated EGFR to immobilized SHP-1 SH2 domains. Upper panel, activation assay. The PTPase activity of purified recombinant SHP-1 was measured using p-nitrophenyl phosphate as substrate in the presence of the EGFR-related phosphopeptides (200 mM) encompassing the sequences around the indicated EGFR tyrosine residues (filled bars) or their unphosphorylated analogues (striped bars). Lower panel, competition assay. The same peptides were used to compete binding of autophosphorylated EGFR to SHP-1 SH2 domains. For this, membranes from A431 cells were stimulated with EGF, and the EGFR was allowed to autophosphorylate in the presence of [g- P]ATP. The autophosphorylated EGFR was subsequently partially purified by WGA-agarose affinity chromatography. The semipurified, radiolabeled EGFR was incubated with immobilized SHP-1 SH2 domain GST fusion protein (GST-SH2) or immobilized GST alone (GST) in the presence of phosphopeptides or their unphosphorylated analogues, as indicated. The beads were washed, and the bound EGFR was visualized using SDS-PAGE and autoradiography. sequence that resembles the consensus sequence for binding of vitro, the Y954F point mutation had little effect on SHP-1 the N-terminal SH2 domain of SHP-1, hXY(P)XXh(h 5 hydro- binding, reducing binding in five independent experiments to phobic, X 5 any amino acid) (44 – 46). We synthesized a panel of only 80 6 10% of the wild type receptor (100%). This finding phosphopeptides encompassing EGFR sequences around ty- indicates that tyrosine 954 is most likely only poorly phospho- rosines 954, 992, 1068, 1086, 1148, and 1173 and the corre- rylated in vivo and therefore is of little importance for SHP-1 sponding unphosphorylated peptides. Phosphopeptides with binding. In contrast and in agreement with the phosphopeptide high affinity to the N-terminal SH2 domain of SHP-1 are data, mutation of Tyr-1173 led to a strong decrease of SHP-1 known to activate the recombinant enzyme in vitro (47, 48). We binding (Fig. 3, mean remaining binding 9 6 5%). Thus, Tyr- therefore tested the phosphopeptides for their capacity to acti- 1173 in its phosphorylated form is clearly the main SHP-1 vate purified recombinant SHP-1 (Fig. 2, upper panel). The binding site on the EGFR. We also observed some reduction in phosphopeptides corresponding to Tyr-954 and Tyr-1173 ex- binding by YF mutation of Tyr-1068 and Tyr-1148 (85 6 21% erted a strong SHP-1 activation, and the one corresponding to and 65 6 25%, respectively). Although the small effect of the Tyr-1068 had a weaker activation effect. In all cases, the cor- Y1068F mutation would be in agreement with the phosphopep- responding unphosphorylated sequences had no effect on tide activation and competition data and may indicate that SHP-1 activity. We then tested the phosphopeptides for their Tyr(P)1068 is a minor binding site for SHP-1, the reason for the capacity to interfere with binding of SHP-1 SH2 domains to weak effect of the Y1148F mutation on SHP-1 binding is un- autophosphorylated EGFRs. The unphosphorylated analogs clear. The corresponding phosphopeptide neither activated served as internal controls (Fig. 2, lower panel). In these bind- SHP-1 nor showed detectable competition in the binding assay. ing assays, we observed competition by the phosphopeptides Possibly Tyr-1148 weakly mediates indirect binding of SHP-1. corresponding to Tyr-954, Tyr-1173, and Tyr-1068. In sum- YF mutation of Tyr-992 and Tyr-1086 had no effect on SHP-1 mary, the phosphopeptide activation and competition experi- binding (not shown). Interestingly, EGFR revealed 1171–1176EpoR ments suggested that the EGFR tyrosines 954, 1173, and 1068 a strongly elevated binding capacity for SHP-1 compared with have the capacity of SHP-1 binding in their phosphorylated wild type EGFR (Fig. 3). form. Next, a panel of mutated EGFRs was generated with We employed GST fusion proteins of SHP-1 with an inacti- replacement of all candidate tyrosines by phenylalanine, in- vated N-terminal or C-terminal SH2 domain to clarify the cluding single mutations and some combinations. We also re- importance of the two SH2 domains for recognition of the placed the sequence AEY LRV around the candidate bind- binding sites on the EGFR. As shown in Fig. 4, under the ing site Tyr-1173 by a known high affinity binding site for conditions of this assay, inactivation of either SH2 domain led SHP-1, namely the sequence LEY LYL of the EpoR (17) and to a reduction of EGFR binding to undetectable levels, confirm- thereby generated EGFR . Lysates of 293 cells ing the earlier finding (35) that both SH2 domains play a role 1171–1176EpoR overexpressing the different receptor mutants were incubated in receptor recognition. When EGFR was analyzed 1171–1176EpoR with a GST-SHP-1SH2 domain fusion protein, and the amount in the same assay, a reduced but substantial binding was of bound receptor was visualized by immunoblotting. This as- detectable employing the SHP-1 construct with inactivated say was applied instead of a previously used association assay C-terminal SH2 domain, whereas binding became undetectable (35), in particular because it was not hampered by the low level upon inactivation of the N-terminal SH2 domain (Fig. 4). Thus, of endogenous wild type EGFR in 293 cells; it was also more as expected, elevation of binding of SHP-1 to EGFR 1171– sensitive to changes in binding affinity. A representative result 1176EpoR seems mainly caused by increased affinity for the for selected mutants is shown in Fig. 3. Despite the potent N-terminal SH2 domain. Still, also in this setting, the C-ter- activating and competing effects of the phosphopeptide 954 in minal SH2 domain contributes to binding. EGF Receptor SHP-1 Interaction 24843 FIG.3. Differential binding of EGFR YF mutants to the SH2 domains of SHP-1. The indicated EGFR YF mutants were transiently expressed in 293 cells from a cytomegalovirus promoter-driven expression plasmid. After transfection, the cells were stimulated with EGF and lysed. Lysates were incubated with 10 mg of immobilized SHP-1 SH2 domain GST fusion protein (GST-SH2) or immobilized GST alone (GST). Beads were washed, and the bound EGFR was visualized by SDS-PAGE and immunoblotting using anti-EGFR antibodies (A). The numbers of the mutants correspond to the EGFR tyrosine residues, which were exchanged for phenylalanine. EGFR EPO corresponds to EGFR ,a 1171–1176EpoR mutant where the sequence AEY LRV was replaced by the known high affinity binding site for SHP-1 LEY LYL of the EpoR. The expression 1173 429 levels of the EGFR mutants used for the different binding reactions were monitored by immunoblot analyses using lysate aliquots (B). WT, wild type. of the Y1173F or Y954F,Y1173F mutants led to a reduction but not a complete loss in dephosphorylation activity. High affinity binding of SHP-1 to the EGFR resulted in elevated 1171–1176EpoR activity of SHP-1 toward the mutant receptor compared with the wild type; however, not in complete dephosphorylation (Fig. 5). Thus, the changes of binding strength modulated dephos- phorylation capacity of SHP-1; however, the changes of recep- tor-directed PTPase activity were less striking than the changes of SHP-1 binding. These data suggest that part of the receptor dephosphoryl- ation may be due to a fraction of SHP-1 that is not bound to the receptor. We considered the possibility that another protein that is tyrosine-phosphorylated in response to EGF may serve as a docking protein, bind, and activate a fraction of SHP-1, which in turn would participate in EGFR dephosphorylation. We reasoned that co-overexpression of such a protein may enhance the activity of SHP-1 toward the EGFR. We tested a number of known substrates of activated EGFR for a respective activity, including p85, SHC (66-kDa isoform), phospholipase C g, c-Cbl, and SHP-2. In all cases, these proteins were tyrosine- FIG.4. Effect of inactivating point mutations in the individual phosphorylated in an EGF-dependent manner, and coexpres- SH2 domains of SHP-1 on binding to wild type EGFR or sion of SHP-1 led to a reduced phosphorylation level. However, EGFR . Wild type EGFR (EGFR WT) and EGFR 1171–1176EpoR 1171– although the tyrosine phosphorylation level of the EGFR was 1176EpoR (EGFR EPO) were transiently expressed in 293 cells. The cells were stimulated with EGF and lysed, and the lysates were incubated reduced upon coexpression of phospholipase C g in the absence with 50 mg of immobilized GST fusion proteins of wild type SHP-1 (GST of SHP-1, none of the tested proteins enhanced receptor de- SHP-1), SHP-1 with inactivated N-terminal SH2-domain (RK32)or phosphorylation by SHP-1 (not shown), making it unlikely that SHP-1 with inactivated C-terminal SH2-domain (RK138). The beads any of these proteins plays the role of an auxiliary docking were washed, and bound EGFR was visualized by SDS-PAGE and immunoblotting (A). Lysate aliquots were analyzed for expression lev- protein. els by immunoblotting (B). We also analyzed the effects of SHP-1 coexpression on the downstream signaling of the different EGFR variants by eval- Attenuation of EGFR Signaling Activity by SHP-1 Depends uating EGF-dependent MAP kinase activation. For this we Partially on Binding—We investigated the effect of impaired or employed antibodies that specifically recognize activated MAP enhanced binding of SHP-1 to the EGFR by analyzing the kinase species (Fig. 5, panel B) or measured the activity of receptor phosphorylation level in EGF-stimulated cells ex- cotransfected, HA epitope-tagged MAP kinase (extracellular pressing wild type EGFR or mutant receptors in the absence or signal-regulated kinase 2) after immunoprecipitation in vitro presence of SHP-1. As shown in Fig. 5 and demonstrated ear- (Fig. 5, panel G) and quantitated the signal by phosphorimag- lier (13, 15, 35), coexpression of SHP-1 leads to a clear decrease ing. SHP-1 coexpression led to a reduced MAP kinase activity in receptor phosphorylation. Loss of SHP-1 binding in the case in EGF-stimulated cells overexpressing wild type EGFR (59 6 24844 EGF Receptor SHP-1 Interaction FIG.5. Effect of mutations in EGFR SHP-1 binding sites on receptor signaling activity in the presence of coexpressed SHP-1. A–E: EGFR mutants (designations as in Fig. 3) were transiently expressed in 293 cells in the absence or presence of SHP-1 as indicated. The cells were stimulated with EGF and lysed, and receptor tyrosine phosphorylation was monitored by immunoblot analyses using anti-phosphotyrosine antibodies (A). The figures above lanes 2, 4, 6, 8, and 14 represent mean values of receptor phosphorylation signal in the presence of SHP-1 compared with receptor phosphorylation in the absence of SHP-1 (100%) as derived from densitometric analysis of two independent experiments with a similar extent of dephosphorylation in the wild type (WT) setting. The same relation of the different mutants was observed in at least three further experiments. The phosphorylation and, therefore, activation of endogenous p42/44 MAP kinases was analyzed in cell lysates of the same experiment by immunoblotting using an antibody, which specifically recognizes the phosphorylated forms of p42/p44 MAP kinases (anti-active EGF Receptor SHP-1 Interaction 24845 16% in presence versus 100% in absence of SHP-1, p 5 0.019). possibility would be the interaction of SHP-1 with two EGFR The SHP-1-dependent reduction of MAP kinase activity was molecules in a receptor dimer, allowing binding of both SH2 similar in cells expressing the Y1148F mutant receptor (48 6 domains to Tyr(P)-1173. Such a model has been proposed for 20%), whereas the effect of SHP-1 was less pronounced in cells interaction of SHP-2 with the PDGFb receptor via phosphoty- expressing the Y1173F mutant (77 6 13) or the Y1173F,Y954F rosine 1009 (49). Further work is required to identify the bind- double mutant (72 6 17). In contrast, the reduction of MAP ing partner on the EGFR for the C-terminal SH2 domain of kinase activity upon SHP-1 coexpression was clearly more pro- SHP-1. nounced in case of the EGFR mutant (27 6 5%, p 5 1171–1176EpoR A phosphopeptide corresponding to the EGFR sequence 0.033 versus wild type). In summary, the effects of SHP-1 around Tyr-954 was found to potently activate SHP-1 and to expression on MAP kinase activity in cells expressing the dif- compete with EGFR binding. However, the Y954F mutant re- ferent receptor mutants mirrored the effects on the receptor ceptor was not significantly impaired with respect to SHP-1 phosphorylation level. binding. Most likely Tyr-954 is not effectively autophosphory- lated. Similarly, an EGFR Tyr-954 phosphopeptide was found DISCUSSION to bind with high affinity to SHP-2 SH2 domains and to block The SH2-domain PTPase SHP-1 binds to and dephosphoryl- SHP-2 EGFR interaction; however, the significance of this find- ates autophosphorylated EGFR and may participate in modu- ing for the in vivo situation is questionable (53). Another bind- lation of EGFR signaling in epithelial cells. Here, we describe ing site of much lower affinity than the one around Tyr-1173 mapping of the binding site for SHP-1 on the EGFR. Most may present Tyr-1068 in its phosphorylated from. As observed important for SHP-1 binding is phosphorylation of Tyr-1173, a for other SH2-domain proteins (54), SHP-1 binding may occur prominent autophosphorylation site at the extreme C terminus through alternative sites, albeit with very different strength. of the cytoplasmic tail of the receptor. The binding site was Two findings suggest that binding of SHP-1 to EGFR is at assigned based on analysis of YF receptor mutants, competi- least in part direct. First, SHP-1 binding could be observed tion of binding by a synthetic phosphopeptide corresponding to using recombinant GST SHP-1 and autophosphorylated EGFR the sequence around Tyr-Y1173, and strong activation of re- from SDS-denatured cell lysate as partners. Second, as men- combinant SHP-1 by the Tyr-1173 phosphopeptide but not by tioned above, the sequence around Tyr-1173 matches a consen- its unphosphorylated analog. It is likely that Tyr-1173 in its sus sequence for the N-terminal SH2 domain of SHP-1. Our phosphorylated form permits binding of the N-terminal SH2 data do, however, not exclude the possibility that, additionally, domain of SHP-1, since the sequence AEY(P)LRV corresponds to direct binding an indirect binding of SHP-1 occurs. to the consensus motif hXY(P)XXh derived from phosphopep- Binding of SHP-1 to the EGFR was highly elevated upon tide library screens and known binding sites (17, 44 – 46). Also, changing the sequence around Tyr-1173 to the one of the activation of recombinant SHP-1 is expected for a binding known SHP-1 binding site around Tyr-429 in the EpoR. In this partner of the N-terminal SH2 domain (47, 48). setting the binding of SHP-1 is dominantly mediated via the Previous studies (35) and data presented in this paper (Fig. N-terminal SH2 domain. The observation supports assignment 4) clearly show that both SH2 domains are involved in SHP-1 of Tyr-1173 as the SHP-1 binding site; however, it indicates binding to the EGFR. Simultaneous occupation of the tandem that the binding of SHP-1 to wild type EGFR is weaker than SH2 domains with appropriate phosphotyrosine-containing in- binding to EpoR via Tyr-429 in its phosphorylated form. teraction partners is likely to confer much higher affinity bind- An interesting general question with respect to physiological ing (49) and more potent activation of the phosphatase than substrates of the SH2 domain PTPases is to what extent the occupation of only the N-terminal SH2 domain. Enhanced ac- SH2 domains may target the enzymes to substrates. We ob- tivation of SHP-1 with doubly phosphorylated peptides versus served that binding of SHP-1 to the EGFR to some extent monophosphorylated peptides has been reported (46). Simi- correlated with its capacity to dephosphorylate the receptor in larly, the simultaneous occupation of both SH2 domains of that the Y1173F mutant receptor was less effectively and the SHP-2 by a doubly phosphorylated peptide leads to an exclu- EGFR mutant was more readily dephosphoryl- sively strong activation (50). The crystal structure of the SH2 1171–1176EpoR ated, respectively. The changes observed in receptor dephos- domains of SHP-2 revealed sterical requirements for simulta- phorylation capacity for Y1173F and EGFR mu- neous ligand occupation of both domains with an optimal dis- 1171–1176EpoR tants were less striking than the changes in SHP-1 binding to tance of about 40Å (51). Based on the recent elucidation of the the receptor compared with the wild type. On the other hand, crystal structure of SHP-2, an activation mechanism for SHP-2 the capacity of SHP-1 to dephosphorylate the EGFR is critically has been proposed that involves sequential occupation of first dependent on intact SHP-1 SH2 domains (35). Two conclusions the C-terminal and then the N-terminal SH2 domain by a can be derived from these data. First, the fraction of receptor- doubly phosphorylated interaction partner (52). The structure bound SHP-1 has only moderate activity toward the receptor, of the SHP-1 SH2 domains may be very similar to the structure which may be due to the sterical position of the PTPase cata- of the SHP-2 SH2 domains (44, 45), and activation of SHP-1 is lytic site relative to the SH2-domains. Also, the catalytic site likely to occur similarly as activation of SHP-2. It is currently may have sterical access to only some of the autophosphoryla- not clear which EGFR phosphotyrosine binds to the C-terminal SH2 domain of SHP-1. One possible candidate may be Tyr(P)- tion sites when SHP-1 is immobilized at Tyr-1173. It is cur- rently unknown whether SHP-1 displays any selectivity with 1148, which could be in appropriate distance from Tyr(P)-1173 to allow simultaneous interaction of both sites with both SHP-1 respect to dephosphorylation of individual EGFR autophospho- SH2 domains. However, the effect of the Y1148F mutation on rylation sites. Second, a part of receptor dephosphorylating SHP-1 binding was only weak and does not compellingly sup- activity of SHP-1 is likely to be due to a fraction of PTPase that port a role of Tyr(P)-1148 for SHP-1 binding. An alternative is not bound to the receptor. To become activated, it should, p42/44 MAPK) (B). Equal expression levels for EGFR (C), MAP kinase (D), and SHP-1 (E) are revealed by immunoblot analysis using lysate aliquots. F and G, 293 cells were transfected with expression plasmids for EGFR mutants, HA epitope-tagged MAP kinase (extracellular signal-regulated kinase 2 (ERK)), and SHP-1 as indicated. The cells were left unstimulated or were stimulated with EGF as indicated. Extracellular signal-regulated kinase 2 was immunoprecipitated and subjected to the activity assay with myelin basic protein (MBP) as substrate (F) or to immunoblotting to reveal the amount of immunoprecipitated MAP kinase (G). 24846 EGF Receptor SHP-1 Interaction 1611–1614 however, be bound to a tyrosine-phosphorylated protein. We 14. Yi, T., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, 3350 –3358 propose that such an auxiliary docking protein may participate 15. Tomic, S., Greiser, U., Lammers, R., Kharitonenkov, A., Imyanitov, E., Ullrich, in regulation of EGFR-directed SHP-1 activity. Work is in A., and Bo ¨ hmer, F.-D. (1995) J. Biol. Chem. 270, 21277–21284 16. Yi, T. L., Mui, A. L. F., Krystal, G., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, progress to identify respective candidate proteins. A similar 7577–7586 dissociation of SHP-1 binding and dephosphorylation activity 17. Klingmu ¨ ller, U., Lorenz, U., Cantley, L. C., Neel, B. G., and Lodish, H. F. as described here has been reported for receptor tyrosine ki- (1995) Cell 80, 729 –738 18. David, M., Chen, H. E., Goelz, S., Larner, A. C., and Neel, B. G. (1995) Mol. nases in hematopoietic cells. SHP-1 dephosphorylates the Cell. Biol. 15, 7050 –7058 CSF-1 receptor; however, it does not bind to it. A 130-kDa 19. Cyster, J. G., and Goodnow, C. C. (1995) Immunity 2, 13–24 20. Pani, G., Kozlowski, M., Cambier, J. C., Mills, G. B., and Siminovitch, K. A. docking protein has been proposed to mediate activity of SHP-1 (1995) J. Exp. Med. 181, 2077–2084 toward the receptor (32). 21. Lorenz, U., Ravichandran, K. S., Burakoff, S. J., and Neel, B. G. (1996) Proc. Mitogenic signaling of the EGFR seems to critically depend Natl. Acad. Sci. U. S. A. 93, 9624 –9629 on activation of the MAP kinase cascade. We found that SHP-1 22. Frearson, J. A., and Alexander, D. R. (1997) Bioessays 19, 417– 427 23. Jackson, D. E., Ward, C. M., Wang, R. G., and Newman, P. J. (1997) J. Biol. coexpression attenuates MAP kinase activation via EGFR in Chem. 272, 6986 – 6993 the 293 cell system. Interference of SHP-1 with MAP kinase 24. Jackson, D. E., Kupcho, K. R., and Newman, P. J. (1997 J. Biol. Chem. 272, 24868 –24875 activation by Y1173F mutant EGFR was less pronounced, 25. Perkins, L. A., Larsen, I., and Perrimon, N. (1992) Cell 70, 225–236 whereas increased SHP-1 binding to the EGFR 1171–1176EpoR 26. Perkins, L. A., Johnson, M. R., Melnick, M. B., and Perrimon, N. (1996) Dev. receptor resulted in a more potent interference of SHP-1 with Biol. 180, 63– 81 27. Cossette, L. J., Hoglinger, O., Mou, L. J., and Shen, S. H. (1996) Exp. Cell Res. MAP kinase activation. These findings are in agreement with a 223, 459 – 466 reduced MAP kinase activation secondary to a reduced EGFR 28. Symes, A., Stahl, N., Reeves, S. A., Farruggella, T., Servidei, T., Gearan, T., autophosphorylation in the presence of SHP-1. A lower phos- Yancopoulos, G., and Fink, J. S. (1997) Curr. Biol. 7, 697–700 29. Banville, D., Stocco, R., and Shen, S. H. (1995) Genomics 27, 165–173 phorylation level of the EGFR is expected to lead to a reduction 30. Paulson, R. F., Vesely, S., Siminovitch, K. A., and Bernstein, A. (1996) Nat. in the recruitment of Grb2 and SHC, which are believed to be Genet. 13, 309 –315 the prime mediators of the MAP kinase response to EGF (55). 31. Lorenz, U., Bergemann, A. D., Steinberg, H. N., Flanagan, J. G., Li, X., Galli, S. J., and Neel, B. G. (1996) J. Exp. Med. 184, 1111–1126 Indeed, we observed reduced SHC phosphorylation in SHP-1- 32. Chen, H. E., Chang, S., Trub, T., and Neel, B. G. (1996) Mol. Cell. Biol. 16, overexpressing cells (not shown). Alternatively or in addition, 3685–3697 SHP-1 binding may compete for SHC binding to the receptor, 33. Yi, T. L., Zhang, J. L., Miura, O., and Ihle, J. N. (1995) Blood 85, 87–95 34. Jiao, H., Berrada, K., Yang, W., Tabrizi, M., Platanias, L. C., and Yi, T. (1996) which has been described to involve primarily phosphoty- Mol. Cell. Biol. 16, 6985– 6992 rosines 1173 and 1148 (55, 56). Finally, SHP-1 may also di- 35. Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, rectly dephosphorylate SHC and thereby reduce complex for- R., Krivtsov, A. V., Ullrich, A., and Bo ¨ hmer, F.-D. (1997) J. Biol. Chem. 272, 5966 –5973 mation with Grb2 and subsequent activation of the signaling 36. You, M., and Zhao, Z. Z. (1997) J. Biol. Chem. 272, 23376 –23381 cascade. An attenuation of nerve growth factor-dependent 37. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J., Cooper, J. A., MAP kinase activation in PC12 cells has likewise been ob- and Schlessinger, J. (1994) Mol. Cell. Biol. 14, 509 –517 38. Stewart, J. M., and Young, J. D. (1984) Solid Phase Peptide Synthesis, Pierce, served as a consequence of SHP-1 overexpression (57). In other Rockford, IL experimental systems a positive role of SHP-1 for MAP kinase 39. Atherton, E., and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis: A activation has been described (58, 59). Recently, SHP-1 has Practical Approach, Oxford University Press, Oxford 40. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., been demonstrated to have the capacity for activation of the and Cantley, L. C. (1994) Curr. Biol. 4, 973–982 cytoplasmic tyrosine kinase c-Src by dephosphorylation of the 41. Heinonen, J. K., and Lahti, R. J. (1981) Anal. Biochem. 113, 313–317 inhibitory phosphotyrosine at the C terminus (60), an event 42. Lopez-Ilasaca, M., Crespo, P., Pellici P. G., Gutkind J. S., and Wetzker, R. (1997) Science 275, 394 –397 that may be related to SHP-1-dependent activation of the MAP 43. Seedorf, K., and Ullrich, A. (1995) in The Protein Kinase Facts Book. Protein kinase cascade observed in the above-quoted studies. It is pos- Tyrosine Kinases (Hardie, G., and Hanks, S., eds) Vol. II, pp. 123–125, Academic Press, New York sible that, depending on the cellular background, i.e. levels and 44. Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, activation state of critical signaling proteins, a positive or neg- X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., ative net effect of SHP-1 activity on signaling cascades is ob- Ratnofsky, S., Feldman, R. A., and Cantley, L. C. (1994) Mol. Cell. Biol. 14, 2777–2785 servable. The availability of EGFR mutants with abrogated or 45. Cantley, L. C., and Songyang, Z. (1994) J. Cell Sci. 18, (suppl.) 121–126 elevated SHP-1 binding will help to clarify the role of EGFR 46. Burshtyn, D. N., Yang, W., Yi, T., and Long, E. O. (1997) J. Biol. Chem. 272, SHP-1 interaction for generation and modulation of receptor 13066 –13072 47. Pei, D., Lorenz, U., Klingmu ¨ ller, U., Neel, B. G., and Walsh, C. T. (1994) signals in appropriate stable cell lines. Biochemistry 33, 15483–15493 48. Pei, D. H., Wang, J., and Walsh, C. T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, Acknowledgments—We greatly acknowledge the generous provision 1141–1145 of various reagents and the critical discussion of the data by A. Ullrich 49. Ottinger, E. A., Botfield, M. C., and Shoelson, S. E. (1998) J. Biol. Chem. 273, and R. Lammers. 729 –735 50. Pluskey, S., Wandless, T. J., Walsh, C. T., and Shoelson, S. E. (1995) J. Biol. REFERENCES Chem. 270, 2897–2900 1. Swarup, G., Cohen, S., and Garbers, D. L. (1982) Biochem. Biophys. Res. 51. Eck, M. J., Pluskey, S., Trub, T., Harrison, S. C., and Shoelson, S. E. (1996) Commun. 107, 1104 –1109 Nature 379, 277–280 2. Mooney, R. A., and Anderson, D. L. (1990) J. Biol. Chem. 264, 6850 – 6857 52. Hof, P., Pluskey, S., Dhe-Paganon, S., Eck, M. J., and Shoelson, S. E. (1998) 3. Faure, R., Baquiran, G., Bergeron, J. J. M., and Posner, B. I. (1992) J. Biol. Cell 92, 441– 450 Chem. 267, 11215–11221 53. Case, R. D., Piccione, E., Wolf, G., Benett, A. M., Lechleider, R. J., Neel, B. G., 4. Bo ¨ hmer, F. D., Bo ¨ hmer, S. A., and Heldin, C. H. (1993) FEBS Lett. 331, and Shoelson, S. E. (1994) J. Biol. Chem. 269, 10467–10474 276 –280 54. Soler, C., Beguinot, L., and Carpenter, G. (1994) J. Biol. Chem. 269, 5. Lammers, R., Bossenmaier, B., Cool, D. E., Tonks, N. K., Schlessinger, J., 12320 –12324 Fischer, E. H., and Ullrich, A. (1993) J. Biol. Chem. 268, 22456 –22462 55. Batzer, A. G., Rotin, D., Urena, J. M., Skolnik, E. Y., and Schlessinger, J. 6. Bo ¨ hmer, F. D., Bo ¨ hmer, A., Obermeier, A., and Ullrich, A. (1995) Anal. Bio- (1994) Mol. Cell. Biol. 14, 5192–5201 chem. 228, 267–273 56. Batzer, A. G., Blaikie, P., Nelson, K., Schlessinger, J., and Margolis, B. (1995) 7. Kulas, D. T., Goldstein, B. J., and Mooney, R. A. (1996) J. Biol. Chem. 271, Mol. Cell. Biol. 15, 4403– 4409 748 –754 57. Wright, J. H., Drueckes, P., Bartoe, J., Zhao, Z. H., Shen, S. H., and Krebs, 8. Neel, B. G. (1993) Semin. Cell Biol. 4, 419 – 432 E. G. (1997) Mol. Biol. Cell 8, 1575–1585 9. Feng, G. S., and Pawson, T. (1994) Trends Genet. 10, 54 –58 58. Su, L., Zhao, Z., Bouchard, P., Banville, D., Fischer, E. H., Krebs, E. G., and 10. Adachi, M., Fischer, E. H., Ihle, J., Imai, K., Jirik, F., Neel, B., Pawson, T., Shen, S.-H. (1996) J. Biol. Chem. 271, 10385–10390 Shen, S. H., Thomas, M., Ullrich, A., and Zhao, Z. Z. (1996) Cell 85, 15 59. Krautwald, S., Buscher, D., Kummer, V., Buder, S., and Baccarini, M. (1996) 11. Shen, S. H., Bastien, L., Posner, B. I., and Chretien, P. (1991) Nature 352, Mol. Cell. Biol. 16, 5955–5963 736 –739 60. Somani, A.-K., Bignon, J. S., Mills, G. B., Siminovitch, K. A., and Branch, D. R. 12. Feng, G. S., Hui, C. C., and Pawson, T. (1993) Science 259, 1607–1611 13. Vogel, W., Lammers, R., Huang, J., and Ullrich, A. (1993) Science 259, (1997) J. Biol. Chem. 272, 21113–21119 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Phosphotyrosine 1173 Mediates Binding of the Protein-tyrosine Phosphatase SHP-1 to the Epidermal Growth Factor Receptor and Attenuation of Receptor Signaling

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 38, Issue of September 18, pp. 24839 –24846, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Phosphotyrosine 1173 Mediates Binding of the Protein-tyrosine Phosphatase SHP-1 to the Epidermal Growth Factor Receptor and Attenuation of Receptor Signaling* (Received for publication, April 7, 1997, and in revised form, June 14, 1998) Heike Keilhack‡, Tencho Tenev‡, Elke Nyakatura§, Jasminka Godovac-Zimmermann§, Lene Nielsen¶, Klaus Seedorf¶, and Frank-D. Bo ¨ hmer‡i From the ‡Research Unit “Molecular Cell Biology,” Medical Faculty, Friedrich Schiller University, D-07747 Jena, Germany, §Institute of Molecular Biotechnology e.V., Beutenbergstrasse 11, D-07745 Jena, Germany, and ¶Hagedorn Research Institute, Niels Steensen Vej 6, DK-2820 Gentofte, Denmark The protein-tyrosine phosphatase SHP-1 binds to and depending on the particular cell type or the activation state of cells. The PTPases with SH2 domains, SHP-1 and SHP-2 (8 – dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important 10), are prone for interaction with tyrosine-phosphorylated pro- for this interaction (Tenev, T., Keilhack, H., Tomic, S., teins and have been shown to bind to multiple receptor species Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, including transmembrane tyrosine kinases (11–15), cytokine A. V., Ullrich, A., and Bo ¨ hmer, F. D. (1997) J. Biol. Chem. receptors (16 –18), antigen receptors (19 –22), and adhesion 272, 5966 –5973). We mapped the EGFR phosphotyrosine molecules (23, 24). SHP-2 is a ubiquitously expressed enzyme 1173 as the major binding site for SHP-1 by a combina- and, as largely derived from genetic evidence for the Drosoph- tion of phosphopeptide activation, phosphopeptide ila SHP-2 homologue Csw, seems to present an essential com- competition, and receptor YF mutant analysis. Muta- ponent of a positive signaling cassette of multiple receptors (25, tional conversion of the EGFR sequence 1171–1176 AEY- 26), although negative modulation of receptor signaling by LRV into the high affinity SHP-1 binding sequence LEY- SHP-2 has also been reported (27, 28). SHP-1 is highly ex- LYL of the erythropoietin receptor (EpoR) led to a pressed in hematopoietic cells, and another closely related iso- highly elevated SHP-1 binding to the mutant EGFR form of SHP-1 is expressed from an alternative promotor in ) and in turn to an enhanced dephos- (EGFR 1171–1176EpoR epithelial cells (29). SHP-1 has been convincingly shown to phorylation of the receptor. SHP-1 expression inter- negatively regulate signaling of multiple receptors in hemato- fered with EGF-dependent mitogen-activated protein poietic cells, including Kit/SCF-receptor (30, 31), interleukin 3 kinase stimulation, and this effect was more pro- receptor (14), CSF-1 receptor (32), and the EpoR (17, 33). . Reduced SHP-1 nounced in case of EGFR 1171–1176EpoR SHP-1 binds to Kit/SCF receptor (14) but not to the CSF-1 binding to the EGFR Y1173F mutant resulted in a re- receptor (32) and may thus intercept with receptor signaling by duced receptor dephosphorylation by coexpressed different mechanisms. In case of the EpoR, SHP-1 is recruited SHP-1 and less interference with EGF-dependent mito- via its N-terminal SH2 domain to the receptor subsequent to gen-activated protein kinase stimulation. The effects of receptor mutations on SHP-1 binding were, however, EpoR phosphorylation by Jak2 kinase (17). A high affinity stronger than those on receptor dephosphorylation by SHP-1 binding site at Tyr-429 of EpoR has been characterized, SHP-1. Therefore, receptor dephosphorylation may be and its lack in respective YF mutants of EpoR or truncated the result of the combined activity of receptor-bound receptors leads to an enhanced EpoR signaling activity (17). SHP-1 and SHP-1 bound to an auxiliary docking protein. Negative modulation of EpoR signaling by SHP-1 appears to involve Jak2 dephosphorylation (17). In addition, direct bind- ing of SHP-1 to Jak2 and dephosphorylation of Jak2 by SHP-1 Activated and subsequently tyrosine-phosphorylated growth has also been demonstrated (34). factor receptors are rapidly dephosphorylated, a process that is The functional role of SHP-1 in epithelial cells is much less believed to negatively modulate signaling activity (1–7). The understood. SHP-1 can bind to the EGF receptor (11, 15, 35) PTPases involved in receptor dephosphorylation are fre- and to HER2 (13) and can dephosphorylate both in transient quently unknown. It is possible that multiple PTPases act in coexpression systems (13, 15) and in stably transfected epithe- concert on a given receptor, the identity of which may vary lial cells (36) . Thus, SHP-1 may negatively regulate HER family receptor signaling in epithelial cells. Both SH2 domains of SHP-1 are important for binding of SHP-1 to the EGFR and * This work was supported by grants from the Max-Planck Society (to for receptor dephosphorylation, although the N-terminal SH2 F. D. B.) and Deutsche Forschungsgemeinschaft (Bo 1043/3-1 to F. D. B. and J. G.-Z.; SFB197/A9). The costs of publication of this article domain contributes to a larger extent to the interaction (35). were defrayed in part by the payment of page charges. This article must The catalytic domain of SHP-1 provides the specificity for therefore be hereby marked “advertisement” in accordance with 18 EGFR dephosphorylation and cannot be replaced by the cata- U.S.C. Section 1734 solely to indicate this fact. lytic domain of SHP-2 in SHP-1/SHP-2 chimeras (35). To fur- To whom correspondence should be addressed: Research Unit “Mo- lecular Cell Biology,” Medical Faculty, Friedrich Schiller University, ther clarify the mechanism of SHP-1 EGFR interaction, we Drackendorfer Str. 1, D-07747 Jena, Germany. Tel.: 149 (3641) 304468; investigated the importance of different EGFR autophospho- Fax: 149 (3641) 304462; E-mail: [email protected]. rylation sites for the interaction with SHP-1. Activation of The abbreviations used are: PTPase, protein tyrosine phosphatase; purified recombinant SHP-1 by EGFR sequence-derived phos- EGF, epidermal growth factor; EGFR, EGF receptor; EpoR, erythropoi- etin receptor; HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; Fmoc, N-(9-fluorenyl) methoxycarbonyl; WGA, wheat germ T. Tenev, T. Bittorf, T. Beckers, and F.-D. Bo ¨ hmer, unpublished agglutinin. data. This paper is available on line at http://www.jbc.org 24839 This is an Open Access article under the CC BY license. 24840 EGF Receptor SHP-1 Interaction GST fusion proteins were eluted with 1 ml of buffer E (50 mM Tris-HCl, phopeptides, competition of phosphopeptides with binding of pH 8, 5 mM reduced glutathione) with end-over-end rotation for 30 min autophosphorylated EGFR to immobilized SHP-1 SH2 do- at 4 °C. The beads were pelleted by centrifugation at 1000 3 g and 4 °C mains, and binding of SHP-1 to a panel of EGFR YF mutants for 1 min, and the supernatant was collected. This elution step was was employed to identify Tyr(P)1173 as the main binding site repeated twice, and the supernatants were combined (3 ml). The eluate for SHP-1 on the EGFR. Introduction of the EpoR high affinity was dialyzed against Xa buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 2 SHP-1 binding site into the EGFR strongly increases SHP-1 mM CaCl ,1mM dithiothreitol). The protein concentration was deter- mined using the Bradford method. To remove the GST tag, GST-SHP-1 binding and also increases receptor dephosphorylation by was digested with Factor Xa. For this the protein concentration of the SHP-1. Introduction of a Y1173F mutation reduces but does not fusion protein was adjusted to 1 mg/ml using Xa buffer, and Factor Xa abrogate receptor dephosphorylation by coexpressed SHP-1. was added with a ratio of protease:fusion protein of 1:200. The mixture Thus, SHP-1 activity toward EGFR is modulated but not ex- was incubated for 16 h at 4 °C. The proteolytic reaction was stopped by clusively dependent on receptor binding. adding 5 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mM dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone (final MATERIALS AND METHODS concentrations). The mixture was applied to a 1-ml glutathione-Sepha- Chemicals and Reagents—EGF was purchased from Pepro Tech, Inc., rose 4B column equilibrated with buffer A (50 mM Tris-HCl, pH 8, 5 mM (Rocky Hill, NJ). Polyclonal anti-phosphotyrosine antibodies and mono- dithiothreitol) to remove free GST and uncleaved fusion protein. The clonal anti-pan-extracellular signal-regulated kinase antibodies were unbound proteins were eluted with buffer A by collecting 250 ml frac- obtained from Transduction Laboratories (Lexington, KY). Monoclonal tions. The fractions were analyzed by SDS-PAGE and Coomassie stain- anti-EGFR antibody 425 was a kind gift from Dr. Luckenbach (Merck ing. The positive fractions were combined and loaded on a MonoQ AG, Darmstadt, Germany). Polyclonal anti-EGFR and anti-SHP-1 an- HR5/5 column (Amersham Pharmacia Biotech) connected to a fast tibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Acti- protein liquid chromatography system. The bound proteins were eluted vated MAP kinase was detected with a polyclonal anti-active MAP with a linear NaCl gradient (80 –200 mM within 20 ml). The fractions kinase antibody (Promega Corporation, Madison, WI). Anti-HA 12CA5 were analyzed by SDS-PAGE and silver staining. Positive fractions monoclonal antibody was purchased from Babco (Berkeley, CA), myelin were pooled and concentrated using 10-kDa centrifugal filters (Milli- basic protein was from Sigma. For introduction of point mutations in pore Corporation, Bedford, MA). 20% glycerol was added, and the pro- the EGFR cDNA, an M13 mutagenesis kit (Bio-Rad) was used. tein solution was stored at 270 °C. The purity of the preparation was [g- P]ATP was purchased from NEN Life Science Products. Factor Xa 95%; 300 mg of SHP-1 could be obtained from a 500-ml bacterial culture. was obtained from Boehringer Mannheim. Dansyl-L-glutamyl-glycyl-L- A431 Cell Treatment and Binding of SHP-1 to the EGFR—A431 cells arginine chloromethyl ketone was from Calbiochem-Novabiochem. were grown in 94-mm dishes to about 70% confluency in Dulbecco’s Cloning of SHP-1 and SH2 Domain Mutants to pGEX Expression modified Eagle’s medium supplemented with 10% fetal calf serum. The Vector—The SHP-1 construct in pRK5RS-SHP-1 (35), which contains cells were starved for 16 h using serum-free Dulbecco’s modified Eagle’s the originally cloned cDNA, was sequentially treated with XhoI, Pfu, medium and subsequently stimulated with 100 ng/ml EGF for 5 min and EcoRI, and the obtained SHP-1 DNA was cloned into SmaI/EcoRI- and lysed in 700 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM digested pBluescriptKS. The 39-untranslated sequences were removed NaCl, 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 1 mM phenylmeth- by replacing the poly(A)-containing Eco47III-XbaI fragment in this ylsulfonyl fluoride, 10 mg/ml leupeptin, 1 mg/ml pepstatin A, 1 mM construct with an analogous fragment without poly(A) from the earlier sodium orthovanadate). The lysates were centrifuged at 25,000 3 g for 2 2 1 described chimera SH2 hg CAT (35). 59-Untranslated sequences were 20 min. To investigate whether the interaction of SHP-1 and EGFR is removed by replacing the EcoRI-BamHI fragment of original SHP-1 direct or not, a sequential immuno- and affinity precipitation approach cDNA with an analogous fragment produced by polymerase chain re- was used (37). A431 cell lysates were prepared as described above. action with SHP-1 cDNA in pBluescriptKS as template using primers EGFR was immunoprecipitated using the monoclonal anti-EGFR anti- 1C-E, 59AAGAATTCCCCTACAGAGAGATGCTGTCC-39, and 2C, 59- body 425. The precipitates were denatured in 100 ml of SI buffer (50 mM CCGGAATTCGGCGCCTGAGGCCTCCTCAAT-39. The modified SHP-1 Hepes, pH 7.5, 1% SDS, 1% b-mercaptoethanol) for 5 min at 95 °C. 100 DNA was removed from pBKS using EcoRI/NotI and cloned in pGEX- ml of denatured protein solution was mixed with 900 ml of lysis buffer. 5X-1. To clone the SH2 domains only, an EcoRI/SmaI fragment of The partially renatured proteins were incubated with 50 mg of immo- modified SHP-1 was cloned into pGEX-5X-1. The SHP-1 SH2 domain bilized GST-SHP-1 or GST for2hat4 °C.The beads were washed three point mutants with inactivated N-terminal SH2 domain (R32K), with times with HNGT (20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton inactivated C-terminal SH2 domain (R138K), or both inactivated SH2 X-100, 10% glycerol, 1 mM Na VO ), 60 ml23 Laemmli buffer was 3 4 domains (R32K, R138K) were subcloned from the corresponding full- added, and the beads were boiled for 5 min. Proteins were separated by length DNAs in pRK5 (35) to pGEX-5X-1 using EcoRI/XhoI. SDS-PAGE, and bound proteins were visualized by immunoblotting. Preparation of GST Fusion Proteins and of Recombinant SHP-1— Peptide Synthesis and Purification—Six peptides encompassing the pGEX-5X-1 SHP-1, pGEX-5X-1 SH2, and the SH2 domain point mu- following EGFR sequences, PQRY LVIQGD, DADEY LIPQQGFF, 954 992 tants in pGEX-5X-1 were used to transform Escherichia coli strain VPEY INQSVPK, NPVY HNQPLN, NPDY QQDFFPK, 1068 1086 1148 BL21(DE3). A single colony was inoculated into 50 ml of LB medium NAEY LRVAPQS, were synthesized using Fmoc chemistry on an containing 50 mg/ml ampicillin and cultured overnight. 25 ml of this ABI 433 synthesizer (Applied Biosystems, Foster City, CA) and esta- overnight culture were added to 500 ml of LB/ampicillin and grown at blished procedures in solid phase peptide chemistry (38, 39). We syn- 37 °C to A 5 0.8. Isopropyl-1-thio-b-D-galactopyranoside was thesized each peptide with and without phosphorylation of tyrosine 600nm added to a final concentration of 0.1 mM, and the bacteria were kept for using either Fmoc-Tyr(PO3H2)-OH (Novabiochem) or Fmoc-Tyr(t-bu- 16 h at 25 °C under shaking. Thereafter, the bacteria were pelleted at tyl)-OH (Applied Biosystems, Foster City, CA). The peptides were ini- 6,000 3 g and 4 °C for 10 min. The pellet was resuspended in 10 ml of tially purified on a SMART chromatographic system (Amersham Phar- a buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM macia Biotech) using an reverse phase column C2/C18 Sephacil 2.1/10 b-mercaptoethanol, 2 mM EGTA, 1 mM EDTA, 5 mM b-glycerophos- column in 0.1% trifluoroacetic acid, 5% acetonitrile with a gradient of phate, 2 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mg/ml bestatin, 1 mg/ml 0 –100% acetonitrile in 60 min at a flow rate of 100 ml/min. The antipain, 1 mg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. phosphopeptides were further purified by chelating chromatography The suspension was three times frozen and thawed, lysozyme was (40) and subsequently desalted using C18 Sep-Pak Cartridges (Waters added (0.5 mg/ml final concentration), and the mixture was incubated Corporation, Milford, MA). The eluted phosphopeptides were dried in a at 25 °C for 20 min. Subsequently EDTA and Triton X-100 were added vacuum concentrator and redissolved in water. The phosphopeptide in final concentrations of 20 mM and 0.5%, respectively, and the mixture concentrations were determined by complete dephosphorylation using was incubated again for 20 min at 25 °C. The bacterial lysate was GST-SHP-1 and determination of released inorganic phosphate (41). centrifuged at 50,000 3 g and 4 °C for 30 min, and the supernatant was The sequences and the purity of the peptides were verified by complete transferred to a fresh 15-ml polypropylene tube containing 1 ml of N-terminal protein sequencing using an Applied Biosystems Procise glutathione-Sepharose 4B (Amersham Pharmacia Biotech). This sus- 494 4-cartridge sequencer and by mass spectroscopy using a LCQ pension was mixed by end-over-end rotation for1hat4 °C.The beads Finnigan MAT ion trap mass spectrometer (Finnigan MAT, San Jose, were pelleted at 1000 3 g and 4 °C for 1 min, and the supernatant was CA) coupled with an on-line Hewlett-Packard 1090 high performance discarded. The beads were washed 4 3 10 min at 4 °C each time with 10 liquid chromatography. 1 mg of each peptide was dissolved in 1 ml of ml of buffer W1 (25 mM Tris-HCl, pH 7, 5, 150 mM NaCl, 10 mM water, and 1 ml was injected onto the high performance liquid chroma- b-mercaptoethanol, 1% Triton X-100) and 13 with buffer W2 (50 mM tograph and eluted with an isocratic gradient of 50% methyl alcohol, 1% Tris-HCl, pH 8, 150 mM NaCl, 10 mM b-mercaptoethanol). The bound formic acid at flow rate of 200 ml/min. EGF Receptor SHP-1 Interaction 24841 Phosphopeptide Activation Assay—Activation of SHP-1 by EGFR- related phosphopeptides was carried out in a 50-ml reaction mixture containing the reaction buffer (100 mM Hepes, pH 7, 4, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 10 mM p-nitrophenyl phosphate), 200 mM phosphopeptides, and 500 ng of SHP-1. After 30 min at 25 °C, the reactions were quenched with 100 mlof1N NaOH, and the absorbance at 405 nm was measured. Controls with unphosphorylated peptides were carried out in the same way. Competition of the EGFR-SHP-1 Interaction by Phosphopeptides— Solubilized A431 membranes were prepared as described earlier (15). For 15 binding reactions, the following mixture was prepared: 100 mlof solubilized membranes (25 mg of protein) in buffer S (50 mM Hepes, pH 7.4, 1% Triton X-100) were supplemented with 3 mM MnCl , 0.1 mM Na VO , and 2 mg/ml EGF (final concentrations). This reaction was 3 4 incubated on ice for 20 min. Autophosphorylation was started by adding 100 mM ATP and 38 mCi of [g- P]ATP. After a 5-min incubation on ice, the reaction mixture was applied onto a 0.5-ml WGA-agarose column (Amersham Pharmacia Biotech) equilibrated with WGA buffer (50 mM Hepes, pH 7.4, 1 mM Na VO , 0.1% Triton X-100). The column was 3 4 washed with 3 ml of WGA buffer. The radioactively labeled EGFR was eluted with 2 ml of WGA buffer containing 0.3 MN-acetylglucosamine, 100-ml fractions were collected, and aliquots were analyzed by scintil- lation counting. The peak fractions were pooled (500 ml). 50 mgof GST-fused SHP-1 SH2 domains (GST-SH2) were coupled to 30 mlof ml of the radiolabeled glutathione-Sepharose beads per reaction. 30 FIG.1. SHP-1 binds directly to EGFR. A431 cells were treated EGFR (;20, 000 cpm) supplemented with 200 mM phosphopeptides or with 100 ng/ml EGF (lanes 2 and 4) or a respective vehicle (lanes 1 and the unphosphorylated analogues were added to the immobilized GST- 3) for 5 min and subsequently lysed. Immunoprecipitation was carried SH2. The binding reactions were incubated with shaking for2hat4 °C. out using the anti-EGFR monoclonal antibody 425. To disrupt preex- Subsequently, the beads were washed three times with HNGT, 60 mlof isting protein complexes, the immunoprecipitates were denatured in 23 Laemmli buffer was added, and the samples were incubated for 5 the presence of SDS and b-mercaptoethanol. Subsequently, the dena- min at 95 °C. The extracted proteins were resolved by SDS-PAGE (7.5% tured protein solution was diluted with a Triton X-100-containing gels), and the gels were analyzed using a GS250 Molecular Imager buffer to a final SDS concentration of 0.1%. The partially renatured (Bio-Rad). proteins were incubated with GST-SHP-1 (lanes 3 and 4) or GST (lanes Binding Assay with EGFR YF Mutants, Cotransfections, and Dephos- 1 and 2) immobilized on glutathione-Sepharose. The beads were phorylation Assays—Point mutations were introduced into the EGFR washed, and the associated proteins were analyzed by SDS-PAGE and immunoblotting using anti-EGFR antibodies (A). Aliquots of the dena- cDNA using a M13 mutagenesis kit according to the manual of the tured first immunoprecipitates were analyzed to verify equal loading of supplier. 293 cells were transfected with the various EGFR mutants the second precipitation (B). using calcium phosphate coprecipitation (15). 20 mg of plasmid DNA was used for one 94-mm dish. After transfection, the cells were stimu- RESULTS lated with 100 ng/ml EGF for 5 min. Stimulation was stopped by ml lysis buffer. The lysates were removing the medium and lysis in 700- Mapping of an SHP-1 Binding Site on the EGFR—Signaling clarified by centrifugation at 25,000 3 g and 4 °C for 20 min. Protein molecules possessing SH2 domains can bind to autophospho- amounts were determined using the Bradford method, and the different rylated growth factor receptors in a direct or indirect manner. lysates were diluted to the same protein concentration with lysis buffer. An example of this is the adaptor protein Grb2, which binds to To monitor the binding of the EGFR mutants to SHP-1, 10 mgof the EGFR directly via phosphorylated Tyr-1086. In contrast, GST-SH2 or GST were coupled to 30 ml of glutathione-Sepharose. The lysates were incubated with the immobilized fusion proteins for2hat Grb2 binds indirectly to the phosphorylated PDGFb- receptor 4 °C with end-over-end rotation. The beads were washed three times Tyr-1009 site mediated by SHP-2 (37). We wished to know mlof23 Laemmli buffer was added. Noncovalently with HNGT, and 60 whether SHP-1 is capable of a direct binding to EGFR. To test bound proteins were extracted by boiling the beads for 5 min, and the this, EGFR immunoprecipitates from A431 cells were dena- supernatants were loaded onto a 7.5% SDS gel. Immunoblotting was tured by boiling in the presence of 1% SDS. Under these con- performed as described earlier (35). The bound EGFR was visualized ditions any possible adaptor protein should be inactivated; using anti-EGFR antibodies. Cotransfections of the EGFR mutants with SHP-1 were performed as however, the phosphotyrosine motifs in the autophosphoryl- described (35). The cells were stimulated with 100 ng/ml EGF for 5 min ated EGFR are expected to retain at least part of their binding and lysed in 200 ml of lysis buffer/well of a 6-well plate. Immunoblot affinity to corresponding SH2 domains (37). The denatured analysis was carried out as described (35). The difference of the tyrosine immunoprecipitates were diluted to reduce the SDS concentra- phosphorylation level of a given EGFR mutant in the absence or pres- tion to a nondenaturing level and were subjected to a binding ence of SHP-1 was used as measure of dephosphorylation activity reaction with a SHP-1 GST fusion protein. As demonstrated in toward the receptor. Fig. 1, EGFR can be recovered on GST-SHP-1 when the cells MAP Kinase Activation Assays—MAP kinase activity assay in vitro was performed as described (42). Briefly, 293 cells were cotransfected had been stimulated by EGF. From nonstimulated cells, much with 0.25 mg of pcDNA3 HA extracellular signal-regulated kinase 2, 0.5 less EGFR is bound to GST-SHP-1, and no EGFR is detectable mg of pRK5 EGFR wild type or mutants and 3.25 mg of pRK5 SHP-1 or on GST beads in either case. Although the amount of EGFR intact vector. The cells were stimulated with 100 ng/ml EGF for 5 min bound to GST-SHP-1 recovered from denatured immunopre- or left unstimulated. Thereafter, cells were lysed, and extracellular cipitates is only a fraction of what can be recovered with GST- signal-regulated kinase 2 was immunoprecipitated with an anti-HA SHP-1 from nondenatured lysates (not shown), this experiment antibody. The kinase reaction was started by the addition of 1.5 mg/ml indicates that SHP-1 is capable of a direct interaction with myelin basic protein, 75 mM ATP, 7.5 mM MgCl2 (final concentrations), and 1 mCi [g- P]ATP. The reaction was terminated with SDS-PAGE autophosphorylated EGFR. sample buffer, and the samples were boiled for 5 min. Proteins were We went on to test the possible involvement of the different separated by SDS-PAGE (12.5% gels), and the lower part of the gels autophosphorylation sites of EGFR in the SHP-1 binding. Ty- containing the phosphorylated myelin basic protein were dried and rosines 992, 1068, 1086, 1148, and 1173 have been reported to analyzed using a GS250 Molecular Imager (Bio-Rad). The amount of become tyrosine-phosphorylated by the autokinase activity of immunoprecipitated extracellular signal-regulated kinase 2 was deter- the EGFR (for a review see Ref. 43 and references therein). The mined by blotting the upper part of the gel and development with anti-HA antibodies. tyrosine 954 is a potential phosphorylation site and part of a 24842 EGF Receptor SHP-1 Interaction FIG.2. Effect of EGFR-related phosphopeptides on catalytic activity of recombinant SHP-1 and on binding of autophosphorylated EGFR to immobilized SHP-1 SH2 domains. Upper panel, activation assay. The PTPase activity of purified recombinant SHP-1 was measured using p-nitrophenyl phosphate as substrate in the presence of the EGFR-related phosphopeptides (200 mM) encompassing the sequences around the indicated EGFR tyrosine residues (filled bars) or their unphosphorylated analogues (striped bars). Lower panel, competition assay. The same peptides were used to compete binding of autophosphorylated EGFR to SHP-1 SH2 domains. For this, membranes from A431 cells were stimulated with EGF, and the EGFR was allowed to autophosphorylate in the presence of [g- P]ATP. The autophosphorylated EGFR was subsequently partially purified by WGA-agarose affinity chromatography. The semipurified, radiolabeled EGFR was incubated with immobilized SHP-1 SH2 domain GST fusion protein (GST-SH2) or immobilized GST alone (GST) in the presence of phosphopeptides or their unphosphorylated analogues, as indicated. The beads were washed, and the bound EGFR was visualized using SDS-PAGE and autoradiography. sequence that resembles the consensus sequence for binding of vitro, the Y954F point mutation had little effect on SHP-1 the N-terminal SH2 domain of SHP-1, hXY(P)XXh(h 5 hydro- binding, reducing binding in five independent experiments to phobic, X 5 any amino acid) (44 – 46). We synthesized a panel of only 80 6 10% of the wild type receptor (100%). This finding phosphopeptides encompassing EGFR sequences around ty- indicates that tyrosine 954 is most likely only poorly phospho- rosines 954, 992, 1068, 1086, 1148, and 1173 and the corre- rylated in vivo and therefore is of little importance for SHP-1 sponding unphosphorylated peptides. Phosphopeptides with binding. In contrast and in agreement with the phosphopeptide high affinity to the N-terminal SH2 domain of SHP-1 are data, mutation of Tyr-1173 led to a strong decrease of SHP-1 known to activate the recombinant enzyme in vitro (47, 48). We binding (Fig. 3, mean remaining binding 9 6 5%). Thus, Tyr- therefore tested the phosphopeptides for their capacity to acti- 1173 in its phosphorylated form is clearly the main SHP-1 vate purified recombinant SHP-1 (Fig. 2, upper panel). The binding site on the EGFR. We also observed some reduction in phosphopeptides corresponding to Tyr-954 and Tyr-1173 ex- binding by YF mutation of Tyr-1068 and Tyr-1148 (85 6 21% erted a strong SHP-1 activation, and the one corresponding to and 65 6 25%, respectively). Although the small effect of the Tyr-1068 had a weaker activation effect. In all cases, the cor- Y1068F mutation would be in agreement with the phosphopep- responding unphosphorylated sequences had no effect on tide activation and competition data and may indicate that SHP-1 activity. We then tested the phosphopeptides for their Tyr(P)1068 is a minor binding site for SHP-1, the reason for the capacity to interfere with binding of SHP-1 SH2 domains to weak effect of the Y1148F mutation on SHP-1 binding is un- autophosphorylated EGFRs. The unphosphorylated analogs clear. The corresponding phosphopeptide neither activated served as internal controls (Fig. 2, lower panel). In these bind- SHP-1 nor showed detectable competition in the binding assay. ing assays, we observed competition by the phosphopeptides Possibly Tyr-1148 weakly mediates indirect binding of SHP-1. corresponding to Tyr-954, Tyr-1173, and Tyr-1068. In sum- YF mutation of Tyr-992 and Tyr-1086 had no effect on SHP-1 mary, the phosphopeptide activation and competition experi- binding (not shown). Interestingly, EGFR revealed 1171–1176EpoR ments suggested that the EGFR tyrosines 954, 1173, and 1068 a strongly elevated binding capacity for SHP-1 compared with have the capacity of SHP-1 binding in their phosphorylated wild type EGFR (Fig. 3). form. Next, a panel of mutated EGFRs was generated with We employed GST fusion proteins of SHP-1 with an inacti- replacement of all candidate tyrosines by phenylalanine, in- vated N-terminal or C-terminal SH2 domain to clarify the cluding single mutations and some combinations. We also re- importance of the two SH2 domains for recognition of the placed the sequence AEY LRV around the candidate bind- binding sites on the EGFR. As shown in Fig. 4, under the ing site Tyr-1173 by a known high affinity binding site for conditions of this assay, inactivation of either SH2 domain led SHP-1, namely the sequence LEY LYL of the EpoR (17) and to a reduction of EGFR binding to undetectable levels, confirm- thereby generated EGFR . Lysates of 293 cells ing the earlier finding (35) that both SH2 domains play a role 1171–1176EpoR overexpressing the different receptor mutants were incubated in receptor recognition. When EGFR was analyzed 1171–1176EpoR with a GST-SHP-1SH2 domain fusion protein, and the amount in the same assay, a reduced but substantial binding was of bound receptor was visualized by immunoblotting. This as- detectable employing the SHP-1 construct with inactivated say was applied instead of a previously used association assay C-terminal SH2 domain, whereas binding became undetectable (35), in particular because it was not hampered by the low level upon inactivation of the N-terminal SH2 domain (Fig. 4). Thus, of endogenous wild type EGFR in 293 cells; it was also more as expected, elevation of binding of SHP-1 to EGFR 1171– sensitive to changes in binding affinity. A representative result 1176EpoR seems mainly caused by increased affinity for the for selected mutants is shown in Fig. 3. Despite the potent N-terminal SH2 domain. Still, also in this setting, the C-ter- activating and competing effects of the phosphopeptide 954 in minal SH2 domain contributes to binding. EGF Receptor SHP-1 Interaction 24843 FIG.3. Differential binding of EGFR YF mutants to the SH2 domains of SHP-1. The indicated EGFR YF mutants were transiently expressed in 293 cells from a cytomegalovirus promoter-driven expression plasmid. After transfection, the cells were stimulated with EGF and lysed. Lysates were incubated with 10 mg of immobilized SHP-1 SH2 domain GST fusion protein (GST-SH2) or immobilized GST alone (GST). Beads were washed, and the bound EGFR was visualized by SDS-PAGE and immunoblotting using anti-EGFR antibodies (A). The numbers of the mutants correspond to the EGFR tyrosine residues, which were exchanged for phenylalanine. EGFR EPO corresponds to EGFR ,a 1171–1176EpoR mutant where the sequence AEY LRV was replaced by the known high affinity binding site for SHP-1 LEY LYL of the EpoR. The expression 1173 429 levels of the EGFR mutants used for the different binding reactions were monitored by immunoblot analyses using lysate aliquots (B). WT, wild type. of the Y1173F or Y954F,Y1173F mutants led to a reduction but not a complete loss in dephosphorylation activity. High affinity binding of SHP-1 to the EGFR resulted in elevated 1171–1176EpoR activity of SHP-1 toward the mutant receptor compared with the wild type; however, not in complete dephosphorylation (Fig. 5). Thus, the changes of binding strength modulated dephos- phorylation capacity of SHP-1; however, the changes of recep- tor-directed PTPase activity were less striking than the changes of SHP-1 binding. These data suggest that part of the receptor dephosphoryl- ation may be due to a fraction of SHP-1 that is not bound to the receptor. We considered the possibility that another protein that is tyrosine-phosphorylated in response to EGF may serve as a docking protein, bind, and activate a fraction of SHP-1, which in turn would participate in EGFR dephosphorylation. We reasoned that co-overexpression of such a protein may enhance the activity of SHP-1 toward the EGFR. We tested a number of known substrates of activated EGFR for a respective activity, including p85, SHC (66-kDa isoform), phospholipase C g, c-Cbl, and SHP-2. In all cases, these proteins were tyrosine- FIG.4. Effect of inactivating point mutations in the individual phosphorylated in an EGF-dependent manner, and coexpres- SH2 domains of SHP-1 on binding to wild type EGFR or sion of SHP-1 led to a reduced phosphorylation level. However, EGFR . Wild type EGFR (EGFR WT) and EGFR 1171–1176EpoR 1171– although the tyrosine phosphorylation level of the EGFR was 1176EpoR (EGFR EPO) were transiently expressed in 293 cells. The cells were stimulated with EGF and lysed, and the lysates were incubated reduced upon coexpression of phospholipase C g in the absence with 50 mg of immobilized GST fusion proteins of wild type SHP-1 (GST of SHP-1, none of the tested proteins enhanced receptor de- SHP-1), SHP-1 with inactivated N-terminal SH2-domain (RK32)or phosphorylation by SHP-1 (not shown), making it unlikely that SHP-1 with inactivated C-terminal SH2-domain (RK138). The beads any of these proteins plays the role of an auxiliary docking were washed, and bound EGFR was visualized by SDS-PAGE and immunoblotting (A). Lysate aliquots were analyzed for expression lev- protein. els by immunoblotting (B). We also analyzed the effects of SHP-1 coexpression on the downstream signaling of the different EGFR variants by eval- Attenuation of EGFR Signaling Activity by SHP-1 Depends uating EGF-dependent MAP kinase activation. For this we Partially on Binding—We investigated the effect of impaired or employed antibodies that specifically recognize activated MAP enhanced binding of SHP-1 to the EGFR by analyzing the kinase species (Fig. 5, panel B) or measured the activity of receptor phosphorylation level in EGF-stimulated cells ex- cotransfected, HA epitope-tagged MAP kinase (extracellular pressing wild type EGFR or mutant receptors in the absence or signal-regulated kinase 2) after immunoprecipitation in vitro presence of SHP-1. As shown in Fig. 5 and demonstrated ear- (Fig. 5, panel G) and quantitated the signal by phosphorimag- lier (13, 15, 35), coexpression of SHP-1 leads to a clear decrease ing. SHP-1 coexpression led to a reduced MAP kinase activity in receptor phosphorylation. Loss of SHP-1 binding in the case in EGF-stimulated cells overexpressing wild type EGFR (59 6 24844 EGF Receptor SHP-1 Interaction FIG.5. Effect of mutations in EGFR SHP-1 binding sites on receptor signaling activity in the presence of coexpressed SHP-1. A–E: EGFR mutants (designations as in Fig. 3) were transiently expressed in 293 cells in the absence or presence of SHP-1 as indicated. The cells were stimulated with EGF and lysed, and receptor tyrosine phosphorylation was monitored by immunoblot analyses using anti-phosphotyrosine antibodies (A). The figures above lanes 2, 4, 6, 8, and 14 represent mean values of receptor phosphorylation signal in the presence of SHP-1 compared with receptor phosphorylation in the absence of SHP-1 (100%) as derived from densitometric analysis of two independent experiments with a similar extent of dephosphorylation in the wild type (WT) setting. The same relation of the different mutants was observed in at least three further experiments. The phosphorylation and, therefore, activation of endogenous p42/44 MAP kinases was analyzed in cell lysates of the same experiment by immunoblotting using an antibody, which specifically recognizes the phosphorylated forms of p42/p44 MAP kinases (anti-active EGF Receptor SHP-1 Interaction 24845 16% in presence versus 100% in absence of SHP-1, p 5 0.019). possibility would be the interaction of SHP-1 with two EGFR The SHP-1-dependent reduction of MAP kinase activity was molecules in a receptor dimer, allowing binding of both SH2 similar in cells expressing the Y1148F mutant receptor (48 6 domains to Tyr(P)-1173. Such a model has been proposed for 20%), whereas the effect of SHP-1 was less pronounced in cells interaction of SHP-2 with the PDGFb receptor via phosphoty- expressing the Y1173F mutant (77 6 13) or the Y1173F,Y954F rosine 1009 (49). Further work is required to identify the bind- double mutant (72 6 17). In contrast, the reduction of MAP ing partner on the EGFR for the C-terminal SH2 domain of kinase activity upon SHP-1 coexpression was clearly more pro- SHP-1. nounced in case of the EGFR mutant (27 6 5%, p 5 1171–1176EpoR A phosphopeptide corresponding to the EGFR sequence 0.033 versus wild type). In summary, the effects of SHP-1 around Tyr-954 was found to potently activate SHP-1 and to expression on MAP kinase activity in cells expressing the dif- compete with EGFR binding. However, the Y954F mutant re- ferent receptor mutants mirrored the effects on the receptor ceptor was not significantly impaired with respect to SHP-1 phosphorylation level. binding. Most likely Tyr-954 is not effectively autophosphory- lated. Similarly, an EGFR Tyr-954 phosphopeptide was found DISCUSSION to bind with high affinity to SHP-2 SH2 domains and to block The SH2-domain PTPase SHP-1 binds to and dephosphoryl- SHP-2 EGFR interaction; however, the significance of this find- ates autophosphorylated EGFR and may participate in modu- ing for the in vivo situation is questionable (53). Another bind- lation of EGFR signaling in epithelial cells. Here, we describe ing site of much lower affinity than the one around Tyr-1173 mapping of the binding site for SHP-1 on the EGFR. Most may present Tyr-1068 in its phosphorylated from. As observed important for SHP-1 binding is phosphorylation of Tyr-1173, a for other SH2-domain proteins (54), SHP-1 binding may occur prominent autophosphorylation site at the extreme C terminus through alternative sites, albeit with very different strength. of the cytoplasmic tail of the receptor. The binding site was Two findings suggest that binding of SHP-1 to EGFR is at assigned based on analysis of YF receptor mutants, competi- least in part direct. First, SHP-1 binding could be observed tion of binding by a synthetic phosphopeptide corresponding to using recombinant GST SHP-1 and autophosphorylated EGFR the sequence around Tyr-Y1173, and strong activation of re- from SDS-denatured cell lysate as partners. Second, as men- combinant SHP-1 by the Tyr-1173 phosphopeptide but not by tioned above, the sequence around Tyr-1173 matches a consen- its unphosphorylated analog. It is likely that Tyr-1173 in its sus sequence for the N-terminal SH2 domain of SHP-1. Our phosphorylated form permits binding of the N-terminal SH2 data do, however, not exclude the possibility that, additionally, domain of SHP-1, since the sequence AEY(P)LRV corresponds to direct binding an indirect binding of SHP-1 occurs. to the consensus motif hXY(P)XXh derived from phosphopep- Binding of SHP-1 to the EGFR was highly elevated upon tide library screens and known binding sites (17, 44 – 46). Also, changing the sequence around Tyr-1173 to the one of the activation of recombinant SHP-1 is expected for a binding known SHP-1 binding site around Tyr-429 in the EpoR. In this partner of the N-terminal SH2 domain (47, 48). setting the binding of SHP-1 is dominantly mediated via the Previous studies (35) and data presented in this paper (Fig. N-terminal SH2 domain. The observation supports assignment 4) clearly show that both SH2 domains are involved in SHP-1 of Tyr-1173 as the SHP-1 binding site; however, it indicates binding to the EGFR. Simultaneous occupation of the tandem that the binding of SHP-1 to wild type EGFR is weaker than SH2 domains with appropriate phosphotyrosine-containing in- binding to EpoR via Tyr-429 in its phosphorylated form. teraction partners is likely to confer much higher affinity bind- An interesting general question with respect to physiological ing (49) and more potent activation of the phosphatase than substrates of the SH2 domain PTPases is to what extent the occupation of only the N-terminal SH2 domain. Enhanced ac- SH2 domains may target the enzymes to substrates. We ob- tivation of SHP-1 with doubly phosphorylated peptides versus served that binding of SHP-1 to the EGFR to some extent monophosphorylated peptides has been reported (46). Simi- correlated with its capacity to dephosphorylate the receptor in larly, the simultaneous occupation of both SH2 domains of that the Y1173F mutant receptor was less effectively and the SHP-2 by a doubly phosphorylated peptide leads to an exclu- EGFR mutant was more readily dephosphoryl- sively strong activation (50). The crystal structure of the SH2 1171–1176EpoR ated, respectively. The changes observed in receptor dephos- domains of SHP-2 revealed sterical requirements for simulta- phorylation capacity for Y1173F and EGFR mu- neous ligand occupation of both domains with an optimal dis- 1171–1176EpoR tants were less striking than the changes in SHP-1 binding to tance of about 40Å (51). Based on the recent elucidation of the the receptor compared with the wild type. On the other hand, crystal structure of SHP-2, an activation mechanism for SHP-2 the capacity of SHP-1 to dephosphorylate the EGFR is critically has been proposed that involves sequential occupation of first dependent on intact SHP-1 SH2 domains (35). Two conclusions the C-terminal and then the N-terminal SH2 domain by a can be derived from these data. First, the fraction of receptor- doubly phosphorylated interaction partner (52). The structure bound SHP-1 has only moderate activity toward the receptor, of the SHP-1 SH2 domains may be very similar to the structure which may be due to the sterical position of the PTPase cata- of the SHP-2 SH2 domains (44, 45), and activation of SHP-1 is lytic site relative to the SH2-domains. Also, the catalytic site likely to occur similarly as activation of SHP-2. It is currently may have sterical access to only some of the autophosphoryla- not clear which EGFR phosphotyrosine binds to the C-terminal SH2 domain of SHP-1. One possible candidate may be Tyr(P)- tion sites when SHP-1 is immobilized at Tyr-1173. It is cur- rently unknown whether SHP-1 displays any selectivity with 1148, which could be in appropriate distance from Tyr(P)-1173 to allow simultaneous interaction of both sites with both SHP-1 respect to dephosphorylation of individual EGFR autophospho- SH2 domains. However, the effect of the Y1148F mutation on rylation sites. Second, a part of receptor dephosphorylating SHP-1 binding was only weak and does not compellingly sup- activity of SHP-1 is likely to be due to a fraction of PTPase that port a role of Tyr(P)-1148 for SHP-1 binding. An alternative is not bound to the receptor. To become activated, it should, p42/44 MAPK) (B). Equal expression levels for EGFR (C), MAP kinase (D), and SHP-1 (E) are revealed by immunoblot analysis using lysate aliquots. F and G, 293 cells were transfected with expression plasmids for EGFR mutants, HA epitope-tagged MAP kinase (extracellular signal-regulated kinase 2 (ERK)), and SHP-1 as indicated. The cells were left unstimulated or were stimulated with EGF as indicated. Extracellular signal-regulated kinase 2 was immunoprecipitated and subjected to the activity assay with myelin basic protein (MBP) as substrate (F) or to immunoblotting to reveal the amount of immunoprecipitated MAP kinase (G). 24846 EGF Receptor SHP-1 Interaction 1611–1614 however, be bound to a tyrosine-phosphorylated protein. We 14. Yi, T., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, 3350 –3358 propose that such an auxiliary docking protein may participate 15. Tomic, S., Greiser, U., Lammers, R., Kharitonenkov, A., Imyanitov, E., Ullrich, in regulation of EGFR-directed SHP-1 activity. Work is in A., and Bo ¨ hmer, F.-D. (1995) J. Biol. Chem. 270, 21277–21284 16. Yi, T. L., Mui, A. L. F., Krystal, G., and Ihle, J. N. (1993) Mol. Cell. Biol. 13, progress to identify respective candidate proteins. A similar 7577–7586 dissociation of SHP-1 binding and dephosphorylation activity 17. Klingmu ¨ ller, U., Lorenz, U., Cantley, L. C., Neel, B. G., and Lodish, H. F. as described here has been reported for receptor tyrosine ki- (1995) Cell 80, 729 –738 18. David, M., Chen, H. E., Goelz, S., Larner, A. C., and Neel, B. G. (1995) Mol. nases in hematopoietic cells. SHP-1 dephosphorylates the Cell. Biol. 15, 7050 –7058 CSF-1 receptor; however, it does not bind to it. A 130-kDa 19. Cyster, J. G., and Goodnow, C. C. (1995) Immunity 2, 13–24 20. Pani, G., Kozlowski, M., Cambier, J. C., Mills, G. B., and Siminovitch, K. A. docking protein has been proposed to mediate activity of SHP-1 (1995) J. Exp. Med. 181, 2077–2084 toward the receptor (32). 21. Lorenz, U., Ravichandran, K. S., Burakoff, S. J., and Neel, B. G. (1996) Proc. Mitogenic signaling of the EGFR seems to critically depend Natl. Acad. Sci. U. S. A. 93, 9624 –9629 on activation of the MAP kinase cascade. We found that SHP-1 22. Frearson, J. A., and Alexander, D. R. (1997) Bioessays 19, 417– 427 23. Jackson, D. E., Ward, C. M., Wang, R. G., and Newman, P. J. (1997) J. Biol. coexpression attenuates MAP kinase activation via EGFR in Chem. 272, 6986 – 6993 the 293 cell system. Interference of SHP-1 with MAP kinase 24. Jackson, D. E., Kupcho, K. R., and Newman, P. J. (1997 J. Biol. Chem. 272, 24868 –24875 activation by Y1173F mutant EGFR was less pronounced, 25. Perkins, L. A., Larsen, I., and Perrimon, N. (1992) Cell 70, 225–236 whereas increased SHP-1 binding to the EGFR 1171–1176EpoR 26. Perkins, L. A., Johnson, M. R., Melnick, M. B., and Perrimon, N. (1996) Dev. receptor resulted in a more potent interference of SHP-1 with Biol. 180, 63– 81 27. Cossette, L. J., Hoglinger, O., Mou, L. J., and Shen, S. H. (1996) Exp. Cell Res. MAP kinase activation. These findings are in agreement with a 223, 459 – 466 reduced MAP kinase activation secondary to a reduced EGFR 28. Symes, A., Stahl, N., Reeves, S. A., Farruggella, T., Servidei, T., Gearan, T., autophosphorylation in the presence of SHP-1. A lower phos- Yancopoulos, G., and Fink, J. S. (1997) Curr. Biol. 7, 697–700 29. Banville, D., Stocco, R., and Shen, S. H. (1995) Genomics 27, 165–173 phorylation level of the EGFR is expected to lead to a reduction 30. Paulson, R. F., Vesely, S., Siminovitch, K. A., and Bernstein, A. (1996) Nat. in the recruitment of Grb2 and SHC, which are believed to be Genet. 13, 309 –315 the prime mediators of the MAP kinase response to EGF (55). 31. Lorenz, U., Bergemann, A. D., Steinberg, H. N., Flanagan, J. G., Li, X., Galli, S. J., and Neel, B. G. (1996) J. Exp. Med. 184, 1111–1126 Indeed, we observed reduced SHC phosphorylation in SHP-1- 32. Chen, H. E., Chang, S., Trub, T., and Neel, B. G. (1996) Mol. Cell. Biol. 16, overexpressing cells (not shown). Alternatively or in addition, 3685–3697 SHP-1 binding may compete for SHC binding to the receptor, 33. Yi, T. L., Zhang, J. L., Miura, O., and Ihle, J. N. (1995) Blood 85, 87–95 34. Jiao, H., Berrada, K., Yang, W., Tabrizi, M., Platanias, L. C., and Yi, T. (1996) which has been described to involve primarily phosphoty- Mol. Cell. Biol. 16, 6985– 6992 rosines 1173 and 1148 (55, 56). Finally, SHP-1 may also di- 35. Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, rectly dephosphorylate SHC and thereby reduce complex for- R., Krivtsov, A. V., Ullrich, A., and Bo ¨ hmer, F.-D. (1997) J. Biol. Chem. 272, 5966 –5973 mation with Grb2 and subsequent activation of the signaling 36. You, M., and Zhao, Z. Z. (1997) J. Biol. Chem. 272, 23376 –23381 cascade. An attenuation of nerve growth factor-dependent 37. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J., Cooper, J. A., MAP kinase activation in PC12 cells has likewise been ob- and Schlessinger, J. (1994) Mol. Cell. Biol. 14, 509 –517 38. Stewart, J. M., and Young, J. D. (1984) Solid Phase Peptide Synthesis, Pierce, served as a consequence of SHP-1 overexpression (57). In other Rockford, IL experimental systems a positive role of SHP-1 for MAP kinase 39. Atherton, E., and Sheppard, R. C. (1989) Solid Phase Peptide Synthesis: A activation has been described (58, 59). Recently, SHP-1 has Practical Approach, Oxford University Press, Oxford 40. Songyang, Z., Blechner, S., Hoagland, N., Hoekstra, M. F., Piwnica-Worms, H., been demonstrated to have the capacity for activation of the and Cantley, L. C. (1994) Curr. Biol. 4, 973–982 cytoplasmic tyrosine kinase c-Src by dephosphorylation of the 41. Heinonen, J. K., and Lahti, R. J. (1981) Anal. Biochem. 113, 313–317 inhibitory phosphotyrosine at the C terminus (60), an event 42. Lopez-Ilasaca, M., Crespo, P., Pellici P. G., Gutkind J. S., and Wetzker, R. (1997) Science 275, 394 –397 that may be related to SHP-1-dependent activation of the MAP 43. Seedorf, K., and Ullrich, A. (1995) in The Protein Kinase Facts Book. Protein kinase cascade observed in the above-quoted studies. It is pos- Tyrosine Kinases (Hardie, G., and Hanks, S., eds) Vol. II, pp. 123–125, Academic Press, New York sible that, depending on the cellular background, i.e. levels and 44. Songyang, Z., Shoelson, S. E., McGlade, J., Olivier, P., Pawson, T., Bustelo, activation state of critical signaling proteins, a positive or neg- X. R., Barbacid, M., Sabe, H., Hanafusa, H., Yi, T., Ren, R., Baltimore, D., ative net effect of SHP-1 activity on signaling cascades is ob- Ratnofsky, S., Feldman, R. A., and Cantley, L. C. (1994) Mol. Cell. Biol. 14, 2777–2785 servable. The availability of EGFR mutants with abrogated or 45. Cantley, L. C., and Songyang, Z. (1994) J. Cell Sci. 18, (suppl.) 121–126 elevated SHP-1 binding will help to clarify the role of EGFR 46. Burshtyn, D. N., Yang, W., Yi, T., and Long, E. O. (1997) J. Biol. Chem. 272, SHP-1 interaction for generation and modulation of receptor 13066 –13072 47. Pei, D., Lorenz, U., Klingmu ¨ ller, U., Neel, B. G., and Walsh, C. T. (1994) signals in appropriate stable cell lines. Biochemistry 33, 15483–15493 48. Pei, D. H., Wang, J., and Walsh, C. T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, Acknowledgments—We greatly acknowledge the generous provision 1141–1145 of various reagents and the critical discussion of the data by A. Ullrich 49. Ottinger, E. A., Botfield, M. C., and Shoelson, S. E. (1998) J. Biol. Chem. 273, and R. Lammers. 729 –735 50. Pluskey, S., Wandless, T. J., Walsh, C. T., and Shoelson, S. E. (1995) J. Biol. REFERENCES Chem. 270, 2897–2900 1. Swarup, G., Cohen, S., and Garbers, D. L. (1982) Biochem. Biophys. Res. 51. Eck, M. J., Pluskey, S., Trub, T., Harrison, S. C., and Shoelson, S. E. (1996) Commun. 107, 1104 –1109 Nature 379, 277–280 2. Mooney, R. A., and Anderson, D. L. (1990) J. Biol. Chem. 264, 6850 – 6857 52. Hof, P., Pluskey, S., Dhe-Paganon, S., Eck, M. J., and Shoelson, S. E. (1998) 3. Faure, R., Baquiran, G., Bergeron, J. J. M., and Posner, B. I. (1992) J. Biol. Cell 92, 441– 450 Chem. 267, 11215–11221 53. Case, R. D., Piccione, E., Wolf, G., Benett, A. M., Lechleider, R. J., Neel, B. G., 4. Bo ¨ hmer, F. D., Bo ¨ hmer, S. A., and Heldin, C. H. (1993) FEBS Lett. 331, and Shoelson, S. E. (1994) J. Biol. Chem. 269, 10467–10474 276 –280 54. Soler, C., Beguinot, L., and Carpenter, G. (1994) J. Biol. Chem. 269, 5. Lammers, R., Bossenmaier, B., Cool, D. E., Tonks, N. K., Schlessinger, J., 12320 –12324 Fischer, E. H., and Ullrich, A. (1993) J. Biol. Chem. 268, 22456 –22462 55. Batzer, A. G., Rotin, D., Urena, J. M., Skolnik, E. Y., and Schlessinger, J. 6. Bo ¨ hmer, F. D., Bo ¨ hmer, A., Obermeier, A., and Ullrich, A. (1995) Anal. Bio- (1994) Mol. Cell. Biol. 14, 5192–5201 chem. 228, 267–273 56. Batzer, A. G., Blaikie, P., Nelson, K., Schlessinger, J., and Margolis, B. (1995) 7. Kulas, D. T., Goldstein, B. J., and Mooney, R. A. (1996) J. Biol. Chem. 271, Mol. Cell. Biol. 15, 4403– 4409 748 –754 57. Wright, J. H., Drueckes, P., Bartoe, J., Zhao, Z. H., Shen, S. H., and Krebs, 8. Neel, B. G. (1993) Semin. Cell Biol. 4, 419 – 432 E. G. (1997) Mol. Biol. Cell 8, 1575–1585 9. Feng, G. S., and Pawson, T. (1994) Trends Genet. 10, 54 –58 58. Su, L., Zhao, Z., Bouchard, P., Banville, D., Fischer, E. H., Krebs, E. G., and 10. Adachi, M., Fischer, E. H., Ihle, J., Imai, K., Jirik, F., Neel, B., Pawson, T., Shen, S.-H. (1996) J. Biol. Chem. 271, 10385–10390 Shen, S. H., Thomas, M., Ullrich, A., and Zhao, Z. Z. (1996) Cell 85, 15 59. Krautwald, S., Buscher, D., Kummer, V., Buder, S., and Baccarini, M. (1996) 11. Shen, S. H., Bastien, L., Posner, B. I., and Chretien, P. (1991) Nature 352, Mol. Cell. Biol. 16, 5955–5963 736 –739 60. Somani, A.-K., Bignon, J. S., Mills, G. B., Siminovitch, K. A., and Branch, D. R. 12. Feng, G. S., Hui, C. C., and Pawson, T. (1993) Science 259, 1607–1611 13. Vogel, W., Lammers, R., Huang, J., and Ullrich, A. (1993) Science 259, (1997) J. Biol. Chem. 272, 21113–21119

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