Insulin-EGF Receptor Chimerae Mediate Tyrosine

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VOl. 266. No. 15, Issue of May 25, pp. 9900-9906,1991

THEJOURNAL OF BIOLOGICAL CHEMISTRY CC?

Printed in U.S.A.

1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Insulin-EGF Receptor Chimerae Mediate Tyrosine Transphosphorylation andSerine/Threonine Phosphorylation of Kinase-deficient EGF Receptors* (Received for publication, October 31,

1990)

Sophie Tartare*, RobertBallotti*, Reiner LammersQ, Frangoise AlengrinS, Thomas Dull§, Joseph Schlessingerll, Axel Ullrichs, and Emmanuel Van ObberghenSIl From the Slnstitut National de la Sante et de la Recherche Medicale U145, Faculte de Medecine, Nice, CCdex 06034, France, §Max-Planck Institut fur Biochemie, 8033 Martinsriedbei Munchen, Federal Republic of Germany, and the lDepartmentof Pharmacology, New York University, New York, New York 10016

To study cross-talk between unoccupied epidermal membrane glycoprotein possessing an EGF-stimulable progrowthfactor (EGF) receptorsandactivated EGF tein tyrosine kinase activity. Binding of EGF stabilizes the receptorkinases,wehave used double-transfected receptor in an oligomeric state and causes stimulation of the cells, IHEP cells, expressingboth an enzymatically receptor tyrosine kinase activity leading to tyrosine phosactive insulin-EGF chimeric receptor and an inactive phorylation of cellular proteins, including the receptor itself kinase EGF receptor mutant. Using immunoaffinity- (Schlessinger, 1988a, 1988b; Ullrich and Schlessinger, 1990). purifiedreceptors, we show thatinsulin increased The tyrosines identified as the major autophosphorylation phosphorylation of the insulin-EGF chimericfl subunit and of the kinase-deficient EGF receptor. Stimulation sites are located at the carboxyl-terminal end of the receptor of intact IHEP cells with insulin leads to a rapid tyro- at positions 1068,1086,1148, and 1173 (Downward et al., sine autophosphorylation of the insulin-EGF chimeric 1984; Margolis et al., 1989). Analysis of an EGF receptor fl subunit and to tyrosine phosphorylation of the unoc- mutated on the ATP binding site have indicated that the intrinsic protein tyrosine kinase activity of the receptor is cupied kinase-deficient EGF receptor. Insulin-stimurequired for EGF-induced Ca2+ influx (Chen et al., 1987; latedtransphosphorylation of thekinase-deficient EGF receptor yields the same pattern of tryptic phos- Moolenaar et al., 1988), inositol phosphate formation (Moolphopeptides as those in EGF-induced autophosphory- enaar et al., 1988), Na+/H+exchange (Moolenaar et al., 1988), lation of the wild-type human EGF receptor. We con- glucose and amino acid transport (Scimeca et al., 1989), DNA clude that insulin, through activation of the insulin- synthesis (Honegger et al., 1987b; Chen et al., 1987), and cEGF chimeric receptor, mediates transphosphorylafos and c-myc expression and transformation (Honegger et al., tion of the kinase-deficient EGF receptor,further con- 1987b). The role of EGF receptor autophosphorylation is less firming that EGF receptor autophosphorylation may well defined. Studies of tyrosine kinases such as the insulin proceed by an intermolecular mechanism. In addition receptor (Rosen et al., 1983) and transforming proteins from to receptor tyrosine phosphorylation, we findthat exposure of cells to insulin results in enhanced phos- Rous sarcoma and Fujinami sarcoma viruses (Piwnica-Worms phorylation on serine and threonine residues of the et al., 1987; Meckling-Hansen et al., 1987) suggest that autounoccupied kinase-deficient EGF receptor. These re- phosphorylation of these enzymes activates their kinase acsults suggest that insulin-EGF chimeric receptor acti- tivity toward substrates. However, for the EGFreceptor convation stimulatesat least one serinelthreonine kinase, flicting data have been reported, indicating that autophoswhich inturn phosphorylates the kinase-deficient EGF phorylation enhances (Bertics andGill, 1985) or has no effect receptor. Finally, we show that transphosphorylation on kinase activity of the receptor (Downward et al., 1985). and coexpression of an active kinase cause a decrease Studies with EGF receptors mutated on the three individual in the number of cell surface kinase-deficient EGF autophosphorylation sites have shown that these receptors receptors without increasing their degradation rate. display a small increase in affinity of the EGF-stimulated tyrosine kinase activity toward a synthetic peptide substrate (Honegger et al., 1988b). Further, cells expressing these mutated receptors were found to bemore sensitive to EGFThe epidermal growth factor (EGF)’ receptor is a transinduced mitogenesis compared with cells expressing wild-type * This work was supported by the Institut National de la Sante et receptors (Honegger et al., 1988a). These data led the authors de la Recherche Medicale (INSERM), Association pour la Recherche to propose that autophosphorylation removes a competitive sur le Cancer (Grant 6432), Ligue FranGaise contre le Cancer, Uni- inhibition by phosphorylation of intramolecular sites leading versity of Nice, and Bayer Pharma, France. The costs of publication to a decrease in the K,,, toward substrates. It remains possible of this article were defrayed in part by the payment of page charges. that other phenomena are involved in the regulation of the This article must therefore be hereby marked “aduertisement” in affinity of substrates toward EGF receptor. In addition to accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed. Tel.: 33-93-85- receptor tyrosine autophosphorylation, binding of EGF also results in enhanced serine/threonine phosphorylation of the 16-54; Fax: 33-93-92-07-13. The abbreviations used are: EGF, epidermal growth factor; HER, receptor (Hunter and Cooper, 1981; Heisermann and Gill, human EGF receptor; HIR, human insulin receptor; IER, insulin1988).These serine/threoninephosphorylations are mediated, EGF receptor chimera; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; HEPES, 4-(2-hydroxyethyl)-l-piper- at least in part, by protein kinase C activation and seem to azineethanesulfonic acid; EGTA, [ethylene(oxyethylenenitrilo)]tet- regulate EGF receptor functions negatively (Schlessinger, 1988a; Ullrich and Schlessinger, 1990). raacetic acid. 9900

EGF Receptor Transphosphorylation In the present report, we have investigated the cross-talk between an unoccupied EGF receptor and an activated EGF receptor kinase. Specifically, we have studied in thesame cells the phosphorylation of an unoccupied EGF receptor after activation of an EGF receptor kinase. We have used doubletransfected cells, IHE2, expressing both an insulin-EGF receptor chimera (IER) and akinase-deficient EGF receptor, K721A, mutated on the ATP binding site. First, we showed in cell lysates and in intact IHE2 cells that insulin activation of the chimeric receptor led to an increased phosphorylation of IER andof the kinase-deficient EGF receptor. This insulinstimulated transphosphorylation of the unoccupied kinasenegative EGF receptor occurred on tyrosine residues and yielded the same phosphopeptides as those in EGF-induced "autophosphorylation" of the wild-type human EGF receptor. Our data furtherconfirm that EGF receptor autophosphorylation may proceed by an intermolecular mechanism (Honegger et al., 1989, 1990b;Lammers et al., 1990). In addition, we have observed an increased serinelthreonine phosphorylation of the unoccupied HER K721A mutant following activation of the chimeric receptor. Finally, we have taken advantage of IHE2 cells to investigate the role of phosphorylation in the cellular fate of EGF receptors. Previous studies have indicated that EGF does not induce down-regulation and degradation of the kinase-deficient EGF receptor (Honegger et al., 1987a; Glenney et al., 1988; Felder et al., 1990). Here we have investigated whether, in IHE2 cells, insulin-induced transphosphorylation of HER K721A is able to restore itsdown-regulation. Our results show that activation of the chimeric receptor induces a loss of cell surface kinase-deficient EGF receptor, which cannot be explained by an increase in receptor degradation rate. MATERIALS ANDMETHODS

[3'P]Orthophosphate and [35S]methionine were obtained from Amersham Corp. EGF "receptor grade" was from Sigma and insulin from Novo BioLabs. Triton X-100, Nonidet P-40, protein A-Sepharose, and bovine serum albumin (A7030) were from Sigma. Phenylmethanesulfonyl fluoride was from Serva. All reagents for SDSPAGE were from Bio-Rad. The standards used, and their molecular weights, were: myosin, 200,000; &galactosidase, 116,000;phosphorylase b, 97,000; bovine serum albumin, 66,000; ovalbumin, 42,000; carbonic anhydrase, 31,000; trypsininhibitor, 21,000; lysozyme, 14,000. Anti-phosphotyrosine antibodies were obtained by immunization of a sheep with phosphotyrosine coupled to bovine IgG. Antiphosphotyrosine antibodies were affinity-purified on a phosphotyrosine-agarose column (Ballotti et al., 1989b). mAblO8 is a monoclonal antibody against the extracellular domain of the human EGF receptor, and anti-C is a rabbit antiserum generated against a synthetic peptide from the COOH terminus of the human EGF receptor (residues 1176-1186) (Honegger et al., 1989). mAbB6 is a monoclonal antibody directed against the extracellular domain of the human insulin receptor (Ballotti et al., 1989a). Cell Lines and Cell Culture-Mouse NIH3T3 fibroblasts were transfected with expression plasmids encoding wild-type human EGF receptor (DHER14 cells) (Honegger et al., 1987a) or both the kinasedeficient human EGF receptor, HER K721A, and a chimeric receptor IER composed of the extracellular domain of the human insulin receptor linked to the transmembrane and the cytosolic domain of the human EGF receptor (IHE2 cells) (Honegger et al., 1987a; Riedel et al., 1989). Cells were grown in Dulbecco's modified Eagle'smedium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin, and streptomycin. Cells were cultured at 37 "C in the presence of 5% CO,. The number of cell surface receptors was obtained by Scatchard analysis of binding experiments of '2511-insulin (McClain et al., 1987)or '""I-labeled EGF (Honegger et al., 1987a). IHE2 cells expressed 2 X 105IER receptors and 2 X IO5 HER K721A receptors per cell. MetabolicLabeling o j Cells"IHE2 cells in 100-mm dishes were washed twice with phosphate-buffered saline and incubated for 15 h a t 37"C in methionine-free Dulbecco'smodified Eagle's medium supplemented with ["S]methionine (100 pCi/ml) and 10% dialyzed

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fetal calf serum. Cells were solubilized in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mMMgC12, 1 mM EGTA, 10%glycerol, 1% Triton X-100, and protease inhibitors: 100 units/ml aprotinin and 1 mM phenylmethanesulfonyl fluoride), and receptors were obtained by sequential immunoprecipitation using domain-specific EGF receptor or insulin receptor antibodies prebound on protein A-Sepharose. The washed pellets were resuspended in Laemmli buffer (3% SDS) and subjected to SDS-PAGE in reducing or nonreducing conditions (Laemmli, 1970). The gel with 35S-labeledproteins was fixed for 30 min in 30% acetic acid, incubated for 30 min in Amplify (Amersham), then dried and exposed to film. Phosphorylation ina CellLysate-Cell lysates were incubated without or with ligand for 15 min at 37 "C, then transfected receptors were immunoprecipitated with their appropriate antibody prebound on protein A-Sepharose. The phosphorylation of precipitated proteins was initiated by adding 10 pCi of [Y-~'P]ATP(3000 Ci/mmol) and 5 mMMnC12. After 1 min on ice, the reaction was stopped by addition of 3% SDS. Finally, the receptors were analyzed by SDS-PAGE and visualized by autoradiography. 32PLabeling of Cells and Zmmunoblotting-Cell monolayers in 100mm dishes were incubated for 3h at 37 "C with 5 mCiof["PI orthophosphate (1 mCi/ml) as described (Ballotti et al., 1989a). After M) was added for different times. Cells were labeling, insulin solubilized for 15 min on ice in buffer A (buffer A: 50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 10 mM EDTA, 100 mM NaF, 2 mM vanadate, 10 mM Na4P207, and protease inhibitors: 100 units/ml aprotinin and 1 mM phenylmethanesulfonyl fluoride) containing 1% Triton X-100. The lysate obtained was subjected to immunoprecipitation (as described above) and analyzed by SDS-PAGE. Labeled proteins were visualized by autoradiography. In the experiments where the cells were not labeled with ["PI orthophosphate and where phosphotyrosine was revealed with antiphosphotyrosine antibodies (immunoblotting analysis), the cell extract was processed the same way as above. After protein transfer, the Immobilon P membrane (Millipore) was saturated for 2h at 40"C in 10 mM Tris (pH 7.5),140 mM NaCI, 4% bovine serum albumin, then incubated overnight at 4 "C with affinity-purified antiphosphotyrosine antibodies (10 pg/ml) in the same buffer. The membrane was washed four times in buffer containing 10 mM Tris (pH 7.5), 140 mM NaC1, 0.4% bovine serum albumin, 0.5% Nonidet P-40, and incubated for 2 h with a peroxidase-conjugated antiserum directed against sheep immunoglobulins. After the same washing procedure, the immunoblot was revealed by a colorimetric method with 4-chloro1-naphthol as substrate of the peroxidase. For phosphopeptides and phosphoamino acid analysis of the EGF receptor, the receptors were phosphorylated in intact cells as described. After SDS-PAGE, the 32P-labeledreceptors were localized by autoradiography. The gel pieces corresponding to the labeled bands were excised, and incubated in 50 mM NH4HC03 (pH8.0) for 12 h at 37 "C. For phosphopeptide analysis, diphenylcarbamyl chloridetreated trypsin (Sigma) was then added at a final concentration of 20 pg/ml and the incubation continued for 24 h at 37 "C. For each sample the eluted phosphopeptides were lyophilized and dissolved in 15 mM NH,OH. Phosphopeptides were separated by two-dimensional analysis on cellulose thin-layerplates as described (Beemon and Hunter, 1978; Ballottiet al., 1989a). The plates were dried and subjected to autoradiography on Kodak X-AR film. For phosphoamino acid analysis, after elution from the gel pieces the phosphorylated receptors were hydrolyzed in 6 N HC1 for 90 min at 110 "C.The phosphoamino acids obtained were separated on cellulose thin-layer plates by electrophoresis at pH 3.5 for 2 h at 800 V and analyzed by autoradiography as described (Cooper et al., 1983). Receptor Down-regulutionand Degradation-Cells in 24-well dishes were incubated with insulin or EGF for 3 h at 37 "C. After washing, plates werecooled on ice and incubated for 2 h with mAbl08 or mAbB6, monoclonal antibodies directed against the extracellular domains of the EGF receptor and of the insulin receptor, respectively. Specific antibody binding was determined by incubation with 500,000 cpm/ml of '251-proteinA for 1 h. Then cells were washed three times with phosphate-buffered saline and solubilized with 0.2 N NaOH and counted (Honegger et al., 1987a). To study receptor degradation IHE2 cells were metabolically labeled with [35S]methionineand then incubated in medium containing unlabeled methionine (10 mM) without or with EGF M) and insulin M). At the indicated times following the chase immunoprecipitation was performed using a mixture of antibodies to insulin and toEGF receptor. After separation of the proteins by SDS-PAGE the intensity of labeling of IER and

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HER K721A was quantitated by densitometric scanning of the autoradiograph.

Receptor Phosphorylation in u Cell Lysate-A cell extract of IHE2 was incubated in lysis buffer with insulin or EGF and thereafter immunoprecipitated with antibodies mAbl08 RESULTS to human EGF receptor (Fig. 2, lanes A and B ) , antibodies Expression of Receptor Mutants-We first examined the mAbB6 to insulin receptor (Fig. 2, lunes C and D),or both expression of the chimeric IER and the kinase-deficient HER (Fig. 2, lunes E-H). The immunoprecipitates were subjected K721A receptors in the stably transfected IHE2 cell line by to phosphorylation with a[y3'P]ATP mixture, and thephosimmunoprecipitation and analysis of [35S]methionine-labeled phoproteins were analyzed by SDS-PAGEunder reducing cells. The receptors were obtained by sequential immunopre- conditions. As shown in lane B, HER K721A receptor was cipitation and analyzed by SDS-PAGE under nonreducing unable to undergo autophosphorylation. As previously shown conditions (Fig. 1, lunesA and B ) or under reducingconditions by Riedel et ul. (1989), insulin enhanced the autophosphory(Fig. 1, lanes C and D). In the first immunoprecipitation, the lation of the chimeric receptor p subunit (IER p ) and also the monoclonal antibody, mAbl08, directed againstthe extracel- phosphorylation of the truncated receptor form, which aplular domain of the human EGF receptor, recognized the HER peared here as a 100-kDa phosphoprotein (lune D). When K721A that appeared in both nonreducing and reducing con- IER and HERK721A were co-immunoprecipitated (lane G), ditions as a monomeric protein of 170 kDa (lunes A and C). we observed that insulin stimulated autophosphorylation of Thereafter, the supernatant was precipitated by anti-C, an IER as well as the phosphorylation of HER K721A. This antiserum generated against a peptide corresponding to the increase inHER K721A phosphorylation was more significant carboxyl-terminal sequence of the human EGF receptor, or when the receptors were exposed simultaneously to insulin by mAbB6, a monoclonal antibody to theextracellular domain and EGF(lune H ) . These data with immunoaffinity-purified of the human insulin receptor. Under reducing conditions, receptors used in a cell-free system indicate that the kinasewith anti-C (lune D) we found two major proteins of 130 and negative receptor HER K721A is a substrate of the kinase120 kDa corresponding, respectively, to the insulin receptor active IER receptor, demonstrating intermolecular phosa subunit (IER a),and the chimeric p subunit composed of phorylation between homologous cytoplasmic domains of the extracellular domain of the insulin receptor p subunit EGF receptors. linked to the transmembrane and the cytoplasmic domains of Receptor Phosphorylation in Intact Cells-IHE2 cells were the EGF receptor (IER p). In addition, we observed a faint labeled with [32P]orthophosphate during 3 h and thenexposed band of approximately 97 kDa corresponding to a truncated to insulin for 15 min. Labeled proteins were solubilized, form of the p IER subunit. Anti-C also precipitated a small immunoprecipitated by specific antibodies, and subjected to amount of HER K721A, which was not precipitated during SDS-PAGE under reducing conditions. Immunoprecipitation thefirst immunoprecipitation. With mAbB6 we observed bymAbB6 (anti-HIR Fig. 3, lunes A and B ) showed that (lune B ) , under nonreducing conditions, two high molecular insulin increased IER /3 phosphorylation. WhenmAbl08 weight species, which have been shown to be disulfide-linked (anti-HER Fig. 3, lanes C and D) was used, we observed basal forms of a and p IER subunits (Riedel et ul., 1989). phosphorylation of HER K721A, which was markedly inOur results show that IHE2 cells express both chimeric creased by insulin. Next, we wished to determine, by immuIER receptors and HER K721A receptors. Further, using noblottingwithanti-phosphotyrosine antibodies, whether mAbB6 and mAblO8 no coprecipitation of the two receptors this phosphorylation of HER K721A occurred on tyrosine was observed, indicating that neither covalent association nor residues. Cells were stimulated with insulin for increasing persistent noncovalent association exists between these two times up to 45 min, lysed, and afterimmunoprecipitation with receptors. a mixture of antibodies against IER or HER, analyzed by A

B

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FIG. 1. [35S]Methionine labeling of IHE2 cells. Cells were labeled overnight with ["S]methionine in methionine-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum. After cell lysis, sequential immunoprecipitation of cell extracts was performed using a first immunoprecipitation with antibody to HER (mAblO8, lanes A and C ) . Thereafter, the supernatantwas immunoprecipitated either with an antipeptide to the carboxyl-terminal sequence of HER (anti-C, lane D ) or with a monoclonal antibody to HIR (mAbB6, lane B ) . The precipitated proteins were analyzed by SDS-PAGE under nonreducing conditions (lanes A and B ) or under reducing conditions (lanes C and D ) . An autoradiograph of the gel is shown. D T T , dithiothreitol; IER a,insulin receptor a subunit; IER 8, chimeric p subunit.

ANTIBODY TO

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HIR

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FIG.2. Phosphorylation of immunoaffinity-purified insulin-EGF receptor chimerae (IER) and kinase-deficient EGF receptor (HER K721A) in a cell lysate. A lysate of IHE2 cells was incubated in the absence or presence of insulin ( I N S ) (lo-' M ) or EGF (lo-' M) for 15 min a t 37 "C and then subjected to immunoprecipitation with antibody to HER (mAbl08, lanes A and B ) , antibody to HIR (mAbB6, lanes C and D),or both (lanes E-H). The washed immunoprecipitates were incubated in the presence of a cell lysate phosphorylation assay mixture. Finally, the phosphorylated receptors were analyzed by SDS-PAGE under reducing conditions and subjected to autoradiography.

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EGF Receptor Transphosphorylation -

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FIG. 3. Receptor phosphorylation in intact IHEZ cells. Cells were incubated with ["'Plorthophosphate (1 mCi/ml) for 3 h a t 37 "C and subsequently for 15 min without or with insulin (lo-' M). The cells were solubilized and thereceptors precipitated with antibody to HIR (mAbB6, lunes A and B ) or with antibody to HER (mAbl08, lunes C and D).The precipitates were analyzed by SDS-PAGE under reducing conditions. An autoradiograph of the gel is shown. IER /3, chimeric 0 subunit. OR

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FIG. 5. Phosphoamino acid analysis of phosphorylated HER K721A after 15 min of insulin stimulation. "P-Labeled IHEZ cells were incubated without or with insulin for 15 min a t 37 "C.The receptors were analyzed by SDS-PAGE as described in the legend to Fig. 3. The phosphoprotein corresponding to the HER K721A was eluted from the gel pieces and hydrolyzed in 6 N HCI for 90 min a t 110 "C. The phosphoamino acids were separated on cellulose plate by electrophoresis, and theplate was autoradiographed. p , phospho. ELECTROPHORESIS

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0 0.5 5 15 45

FIG. 4. Time course of receptor phosphorylation in intact IHE2 cells: immunoblotting with anti-phosphotyrosine antibodies. Cells were incubated in the absence or presence of insulin M) for indicated times a t 37 "C. The cells were solubilized and the extracted proteins subjected to immunoprecipitation with a mixture of antibodies to HER (mAbl08) and HIR(mAbB6). The precipitated proteins were separated by SDS-PAGE under reducing conditions and transferred to an Immobilon P membrane. The tyrosinephosphorylated proteins were revealed by anti-phosphotyrosine antibodies (as described under "Materialsand Methods"). A photograph of the blot is shown. IER 0, chimeric 0 subunit.

SDS-PAGE. After transfer to an Immobilon membrane, the tyrosine-phosphorylated proteinswere revealed by anti-phosphotyrosine antibodies. Fig. 4 shows the time course of insulin stimulation. Exposure of cells to the hormone induced tyrosine phosphorylation of IER p as well as of HER K721A. On both receptors this effect was visible at the same time and appeared to be extremely rapid, occurring within 30 s of insulin stimulation. IER tyrosine phosphorylation persisted for at least 45 min after insulin addition, while within 5 min the stimulatory effect of insulinon HER K721A tyrosine phosphorylation started todisappear. Further, phosphoamino acid analysis of HER K721A showed that 15min of exposure to insulin resulted in anincrease in phosphotyrosine but also led to aneven more important increase in phosphoserine and phosphothreonine (Fig. 5). This serine and threonine phosphorylation of HER K721A appeared later than thereceptor tyrosine phosphorylation and remained constant for a t least 1h after insulin stimulation (data notshown). Next we compared phosphopeptide maps of wild-type EGF

I

HER K721A

FIG. 6. Phosphopeptide map analysis of phosphorylated HER K721A and wild-type HER. IHEZ cells and DHER14 cells were labeled with [32P]orthophosphate andexposed to insulin (lo" M) (IHE2 cells) or EGF (lo-' M) (DHER14 cells) for 1 min a t 37 "C. The receptors were analyzed by SDS-PAGE as described in the legend to Fig. 3. The phosphoproteins corresponding to theHER K721A and the wild-type ( W T )HER were eluted and digested with trypsin (20 gg/ml) in 50 mM NH4HC03 (pH 8.0) for 24 h at 37 "C. The eluted peptides were lyophilized and resuspended in 15mM NH40H. Radioactive peptides were spotted on cellulose plates and separated by electrophoresis followed by chromatography. An autoradiograph of the plates is shown.

receptor stimulated by EGF in DHER14 cells with those of HER K721A phosphorylated inIHE2 cells exposed to insulin. As illustrated in Fig. 6, insulin produced HER K721A transphosphorylation on the same peptides as those obtained by EGF-stimulatedautophosphorylation of wild-type human EGF receptor. These data suggest that HER K721A transphosphorylation in intactIHEZ cells can be accounted for, at least in part, by a mechanism similar to that used in EGF receptor autophosphorylation in intactDHER14 cells. Receptor Down-regulation and Degradation-Previous

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Transphosphorylation EGF Receptor

studies have indicated that EGF-induced down-regulation is impaired incells expressing the kinase-deficient EGF receptor (Honegger et al., 1987a; Glenney et al., 1988; Felder et al., 1990). However, the respective role of receptor phosphorylation and kinase activity in this process was not clearly established. To approach this questionwe investigated whether, in IHE2 cells, insulin-inducedtransphosphorylation of HER K721A mutant was able to restore its down-regulation. Cell surface receptors were quantitated by binding experiments with monoclonal antibodies against HIR or HER (Fig.7). When IHE2 cells were exposed to insulin for3 h at 37 "C, we observeda 50% decrease in chimeric receptors and a 20% reduction of kinase-negative EGF receptors expressed on the 25 cell surface. Exposure of cells to EGF did not change the E number of IER receptors butreduced by approximately 30% 10 I I the number of HER K721A receptors. 2 3 0 1 HOURS To investigate whether this receptor loss was due to an increase in the degradation rate of IER and HER K721.4, FIG. 8. Receptor degradation in IHEB cells. Cells were labeled cells were labeled with [:'%]methionine and subjected to a with[%]methionine for 15 h andthenincubated for increasing chase with unlabeled methionine either in the absence or periods in medium containing unlabeled methionine (lo-' M ) without M ) or EGF M). After the different presence of insulin and EGF(Fig. 8). As previously shown by or with insulin ( I N S ) chase times, the cells were lysed, and IER and HER K721A were Honegger et al. (1987a), EGF did not increase the degradation immunoprecipitated and then analyzed by SDS-PAGE. Intensity of rate of the HER K721A receptor (tlh= 7.5 h). While insulin radioactive bands was quantitated by densitometric scanning of the induced an accelerated loss of IER receptors (tl12= 1 h com- autoradiograph. The relative intensities were plottedagainstthe pared with tnlz= 5 h without hormone), no detectable accel- chase time. IER degradation is shown in the absence (0)or in the presence of insulin (A)and EGF (W). HER K721A degradation is eration of HER K721A degradation was found. Taken together, our data suggest that inIHEB cells, insulin, shown in the absence (0)or in the presence of EGF (W) and insulin probably through IER activation, provoked a small decrease (A). in the number of cell surface HER K721A receptors, which cannot be accounted for by a n increase in receptor degrada- and intactcells the abilityof the chimeric insulin-EGF receptor to phosphorylate the kinase-deficient EGF receptor. In an tion. IHEB cell lysate insulin increased both IER and HER K721A phosphorylation. This effect was enhanced by EGF, which DISCUSSION transformed HER K721A into a better substrate for IERA murine cell line, IHE2, was generated, which expresses mediated phosphorylation byinducingafavorableconforboth an enzymatically active receptor (IER) and a substrate mational change in HER K721A and/or by increasing its receptor (HER K721A). These two receptors are structurally aggregation state.Sincethephosphorylationphenomenon and immunologically distinguishable. IER is an insulin-rewasobserved with immunoaffinity-purifiedreceptors, and sponsive tyrosine kinasecomposed of the extracellular domain since HER K721A is devoid of kinase activity, we conclude of the human insulin receptor and of the transmembrane and that insulin, acting through the IER,induced transphosphointracellular domain of the human EGF receptor. As previ- rylation of the kinase-deficient EGF receptor. These data are ously described by Riedel et al. (1989), metaboliclabeling with in agreement with studies by Honegger et al. (1989, 1990b) [:"S]methionine showed that this receptor displays the same showing in a cell-free system and in living cells intermolecular oligomeric structure as native insulin receptors. The substrate autophosphorylation between two mutated EGF receptors, a receptor corresponds to the kinase-deficient EGF receptor, kinase-deficient receptor anda carboxyl truncation mutant. HER K721A, mutated on the ATP-binding site, and it appears In intact IHE2 cells labeled with ["'Plorthophosphate, we as a monomeric protein of 170 kDa (Honegger et al., 1987a). found that insulin increased phosphorylation of IER and of In the present work we have studied in both cell lysates HER K721A. Maximal hormonaleffect on HERK721A phosphorylation was observed after 15 min of stimulation. Immunoblotting analysis with anti-phosphotyrosine antibodies showed thattyrosinephosphorylation of IERandHER K721A appeared within 1 min of exposure to insulin. While comparablekinetics of tyrosinephosphorylation were observed for both receptors, HER K721A tyrosine dephosphorylation tookplace more rapidly than IERdephosphorylation. Comparison of phosphopeptide maps indicated that in intact IHE2 cells exposedto insulin, the kinase-deficient EGF receptor mutant was phosphorylated on sites virtuallyidentical to O L those modified in phosphorylated wild-type EGF receptors after stimulation by EGF. On the basis of these results, we Additions 0 INS EGF 0 INS EGF " confirm that autophosphorylation and transphosphorylation occur by a similar or the same mechanism and that EGF anti-HIR anti-HER receptor tyrosine phosphorylation can proceed via an interFIG. 7. Receptor down-regulation in IHE2 cells. IHE2 cells molecular mechanism (Honegger et al., 1989,1990b; Lammers were incubated with insulin ( I N S ) (lo-' M ) or EGF (lo-' M) for 3 h at 37 "C. Thereafter, thecells plates were cooled on ice and incubated et al., 1990). Note that the phosphopeptidesobserved after 1 for 2 h withantibody to HIR (mAbB6) or to HER (mAbl08). Antibody min of stimulation contain mainly phosphotyrosine, but itis possible that some of these peptides are serine or threonine binding was determined by incubation with '""I-labeled protein A.

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phosphorylated. The fact that the phosphopeptides maps areal., 1989a) and transient expression (Lammers et al., 1990). virtually identical as discussed above indicates that a similar Thisreceptorcross-talk could account for directreceptor transregulation. According to this scenario tyrosine transmechanism is responsiblefor theserine/threoninephosphorylation of wild-type EGF receptor and of HER K721A. phosphorylation could lead to transactivation of unoccupied EGF receptors and amplificationof the EGF response at the 5 min of exposuretoinsulin,tyrosine Inaddition,after phosphorylation of HER K721A decreased significantly and receptor level. Additionally, we show here that activation of approached basal levels (Fig. 4). In contrast, after 15 min of IER leads to serine and threonine phosphorylation of unocexposure to insulin the stimulatingeffect of the hormone on cupied EGF receptors. This phosphorylation, in agreement "total" phosphorylation remained clearly above the basallevel with previous studies (Ullrich andSchlessinger, 1990; Schlesseen without insulin(Fig. 3). Thissuggested to us thatat this singer, 1988a), would be involved in the negative transregutime of stimulation HERK721A mutant phosphorylationwas lation of the EGF receptor function. due to an increase in phosphorylation on serine and threonine The existence of a complex network of transregulations residues. This was entirely confirmed by phosphoamino acid between homologous receptors or between heterologous but closely related structures (e.g. pl85"'"/EGF-R) (Kadowaki et analysis (Fig. 5 ) . We interpret these results to mean that al., 1987; King et al., 1988; Stern and Kamps,1988) could play through IER activation, insulin stimulates at least one serine/ inthecontrol of cellularfunctioning. Any threonine kinase, which in turn phosphorylates the unoccu- acrucialrole impairment in this network could lead to an abnormal and pied kinase-deficient EGFreceptor. We next addressed therole of transphosphorylation in the deregulated cell growth. cellular fateof the EGFreceptor. Previous studieshave shown Acknowledgments-We thank Drs. Y. Le Marchand-Brustel and that kinase-deficient EGF receptors were not down-regulated E. Van Ohberghen-Schilling for critical readingof the manuscript,C. or degraded in response to EGF (Honegger et al., 1987a; Chen Filloux for technical assistance, J. Duch for secretarial help, and A. et al., 1987; Glenney et al., 1988; Felder et al., 1990). This led Grima and G. Visciano for graphics. the authors to conclude that intrinsic receptor tyrosine kinase activity was necessaryfor thenormal cellular routing of REFERENCES internalized EGF receptors. The signal, culminating in the Ballotti, R., Lammers, R., Scimeca, J. C., Dull, T., Schlessinger, J., degradation of EGFreceptorsafter endocytosis,could be Ullrich, A,, and Van Obherghen, E. (198%) EMEO J. 8,3303-3309 either phosphorylation of the receptor itself or receptor-me- Ballotti, R., Scimeca, J. C., Kowalski, A,, and VanOhberghen, E. (1989h) Cell. Signalling 1, 195-204 diated phosphorylation of cellular structures (Glenney et al., 1988). Taking advantage of our model system we have here Beemon, K., and Hunter, T.(1978) J. Virol. 28, 551-566 investigated first, the abilityof insulin to causea decrease in Bertics, P. J., and Gill, G. N. (1985) J . Biol. Chem. 260,14642-14647 Chen, W. S., Lazar, C. S., Poenie, M., Tsien, R. Y., Gill, G. N., and the number of HER K721A at the cell surface, and second, Rosenfeld, M. G. (1987) Nature 328, 820-823 the effect of insulinonthedegradation of metabolically Cooper, J. A,, Sefton, B.M., and Hunter,T. (1983) Methods Enzymol. labeled HER K721A. In response to insulin, IER was down99,387-402 regulated and rapidly degraded. By contrast, EGF and to a Downward, J., Parker, P., and Waterfield, M. D.(1984) Nature 311, 483-485 lesser extent, insulin, caused a decrease in cell surface HER K721A without affecting its degradation rate. A similar effect Downward, J., Waterfield, M. D., and Parker, P. J. (1985) J. Riol. Chem. 260,14538-14546 of EGF on HERK721A was recently reportedby Felder et al. Felder, S., Miller, K., Moehren, G., Ullrich, A,, Schlessinger, J., and (1990) and Honegger et al. (1990a). It is possible that the Hopkins, C. R. (1990) Cell 61, 623-634 decrease in cell surface receptor without increased degradaGlenney, J. R., Chen, W. S., Lazar, C. S., Walton, G. M., Zokas, L. M., Rosenfeld, M. G., and Gill, G. N. (1988) Cell 52, 675-684 tion is the resultof receptor sequestration as has been shown t o occur for the insulin receptor (Krupp and Lane, 1981). At Heisermann, G. J., and Gill, G. N.(1988) J . Biol. Chem. 263,1315213158 anyrate,ourresults suggest thatinstablytransfected Heldin, C.-H., Ernlund, A., Rorsman, C., and Ronnstrand, L. (1989) NIH3T3 cells the loss of cell surface HER K721A receptors J. Biol. Chem. 264,8905-8912 induced by transphosphorylation or IER activation cannotbe Honegger, A. M., Dull, T. J., Felder, S., Van Ohherghen, E., Bellot, F., Szapary, D., Schmidt, A., Ullrich, A,, and Schlessinger, J. accounted for by anincreaseindegradation of the HER (1987a) Cell 51, 199-209 K721A mutant. Our observations are in agreement with studies showing that degradation of EGF receptors was not af- Honegger, A. M., Szapary, D., Schmidt, A,, Lyall, R., Van Obherghen, E., Dull, T. J., Ullrich, A.? and Schlessinger, J. (1987h) Mol. Cell. fected by replacements or eliminations of three major autoBiol. 7,4568-4571 phosphorylation sites (Honegger et al., 1988a; Livneh et al., Honegger, A. M., Dull, T. J., Bellot, F., Van Ohberghen, E., Szapary, 1986). Furthermore, our data also stress that the presence in D., Schmidt, A,, Ullrich, A., and Schlessinger, J. (1988a) EMEO J. IHE2 cells of an active EGF receptor tyrosine kinase did not 7,3045-3052 suffice to restore down-regulation of HER K721A as suggested Honegger, A. M., Dull, T. J., Szapary, D.,Komoriya, A., Kris, R., Ullrich, A,, and Schlessinger,J. (1988h) EMEO J . 7, 3053-3060 previously by Honegger et al. (1990a). Honegger, A. M., Kris, R. M., Ullrich, A., and Schlessinger, J. (1989) Autophosphorylation of various receptors with tyrosine kiProc. Natl. Acad. Sci. U. S. A. 86, 925-929 nase activity like the CSF-1 receptor (Ohtsuka et al., 1990), Honegger, A. M., Schmidt, A., Ullrich, A,, andSchlessinger, J. (1990a) et al., 1989), and the EGF receptor J. Cell Bid. 110, 1541-1548 the PDGF receptor (Heldin (Honegger et al., 1989; 1990b) appears, at least in part, tobe Honegger, A. M., Schmidt, A,,Ullrich, A,, and Schlessinger, J. (1990h) Mol. Cell. Eiol. 10, 4035-4044 mediated by an intermolecular reaction. For the above menHunter, T., and Cooper, J. A. (1981) Cell 24, 741-752 tioned receptors, transphosphorylation takes place between Kadowaki, T., Kasuga, M., Tobe, K., Takaku, F., Nishida, F., Sakai, receptorswithin adimeric structureand reflects receptor H., Koyaso, S., Yahara, T., Toyoshima,K., Yamamoto, T., and Akiyama, T. (1987) Biochem. Biophys. Res. Commun. 144, 699cross-talk involvingtwoligand-occupiedreceptors. In contrast, we describe in this study tyrosine transphosphorylation 704 of an unoccupied monomeric receptor (HER K721A), occur- King, C. R., Borrello, I., Bellot, F., Comoglio, P., and Schlessinger,J. (1988) EMBO J. 7, 1647-1651 ring after activationof a naturally oligomeric receptor (IER). Krupp, M., and Lane, M. D. (1981) J . Biol. Chem. 256, 1689-1694 A similar receptor cross-talk was previously described forthe Laemmli, U. K. (1970) Nature 227, 680-685 insulin receptor using stably transfectedcell lines (Ballotti et Lammers, R., Van Ohherghen, E., Ballotti, R., Schlessinger, J., and

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