MOLECULAR AND CELLULAR BIOLOGY, Mar. 1988, P. 1019-1026 Vol. 8, No. 30270-7306/88/031019-08$02.00/0Copyright X 1988, American Society for Microbiology

Tumor Promoter and Epidermal Growth Factor StimulatePhosphorylation of the c-erbB-2 Gene Product in MKN-7

Human Adenocarcinoma CellsTETSU AKIYAMA,1 TOSHIYUKI SAITO,2 HIROSHI OGAWARA,3 KUMAO TOYOSHIMA,2

AND TADASHI YAMAMOTO2*Institute for Virus Research, Kyoto University, Kawaracho, Shogoin, Sakyo-ku, Kyoto 606,1 Institute of Medical Science,

University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108,2 and Department of Biochemistry, Meiji College ofPharmacy, 1-35-23 Nozawa, Setagaya-ku, Tokyo 154,3 Japan

Received 6 July 1987/Accepted 27 November 1987

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorboltetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapidincrease in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine.On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonineresidues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA inducedphosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGFincreased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulatedby long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by differentmechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylationof tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that wasactivated by EGF.

Growth factors exert their effects through their binding tospecific membrane-associated receptors (13). The receptorsfor epidermal growth factor (EGF), platelet-derived growthfactor, insulin, and insulinlike growth factor I are known topossess tyrosine kinase activity, and this activity is thoughtto be important in the regulation of cellular proliferation (9).Similar kinase activity is also associated with oncogeneproducts of the src family (2). These observations suggestthe possible link between growth factor receptors and onco-gene products. In fact, the oncogene of avian erythroblasto-sis virus, v-erbB, was found to correspond to the carboxylhalf of the EGF receptor (25, 29). Therefore, the transform-ing ability of the v-erbB gene product could be considered toresult from deregulated expression of the activity of the EGFreceptor. Overexpression of the EGF receptor in humansquamous carcinomas has also been observed (28).

Recently, another v-erbB-related cellular gene, c-erbB-2,was identified by analysis of the human genome (8, 22) andsuggested to encode a growth factor receptor similar to theEGF receptor; the product was predicted to consist of anexternal ligand-binding domain, a transmembrane anchordomain, and a cytoplasmic tyrosine kinase domain (27). Thec-erbB-2 gene product was recently identified as a 185-kilodalton glycoprotein associated with tyrosine kinase ac-tivity (3). Comparison of the sequence of c-erbB-2 with thatof neu oncogene (23) revealed that they are the same gene (4,27). The involvement of the c-erbB-2 gene in human cancerwas suggested by the observation that this gene is amplifiedin human adenocarcinomas (15, 22, 27, 30).The above observations suggest that subversion of the

normal signal pathway involving growth factor receptor mayresult in neoplastic transformation. Thus, elucidation of theroles of the normal receptor function, including receptor-associated tyrosine kinase activity, is important for under-

* Corresponding author.

standing the mechanism of cell transformation. In thepresent study, we found that phosphorylation of the c-erbB-2gene product was enhanced by treatment of MKN-7 cellswith EGF or phorbol tetradecanoate acetate (TPA).

MATERIALS AND METHODS

Materials. 32Pi and 1251 were purchased from AmershamCorp.; TPA, phosphoserine, phosphothreonine, and phos-photyrosine were from Sigma Chemical Co.; EGF was fromCollaborative Research; N-L-phenylalanyl chloromethyl ke-tone-trypsin was from Worthington Diagnostics. A src pep-tide, which is a tyrosine-containing peptide closely resem-bling the phosphorylation site present in pp6Osrc, wasobtained from Peninsula Laboratories.

Antibodies. Antibodies to the c-erbB-2 gene product wereprepared by immunizing rabbits with a synthetic peptidecorresponding to amino acid residues 1242 to 1255 (anti-c-erbB-2) (3). Anti-EGF receptor antibodies against aminoacid residues 1173 to 1186 were prepared by the samemethod. These antibodies were purified by affinity chroma-tography on a column to which the synthetic peptide hadbeen covalently linked as described previously (1). Antise-rum prepared by immunizing mice with NIH 3T3 cells thathad been transfected with a human c-erbB-2 cDNA clonewith the mouse mammary tumor virus (MMTV) promoter(anti-MMTV-c-erbB-2) were also used for immunoprecipita-tion of the c-erbB-2 gene product. A monoclonal anti-EGFreceptor antibody (528IgG) was a gift from T. Kawamoto ofOkayama University (14).

Cells. Human adenocarcinoma MKN-7 cells were culturedin Dulbecco modified Eagle medium supplemented with 8%fetal calf serum.

Cell labeling. MKN-7 cells were labeled for 7 h in phos-phate-free Dulbecco modified Eagle medium supplementedwith 2% fetal calf serum and 700 FCi of 32p; per ml. TPA orEGF was added during the last 15 min of labeling.

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1020 AKIYAMA ET AL.

A TPA B EGF

1 2 3 4 5 1 2 3 4 5

185K-*p

-185K

0 0.5 5 50 200

1 2 3 4 5 6 7 8 9

-185K-170K

0 0.5 5 50 200 ng/mI

1 2

_- -185K c erbB 2

-170K EGF R

FIG. 1. Effects of TPA and EGF on phosphorylation of the c-erbB-2 gene product. MKN-7 cells were labeled with 32p, for 7 h and thentreated with TPA (A) or EGF (B) for 15 min. The cells were lysed, and the c-erbB-2 gene product was immunoprecipitated with anti-c-erbB-2antibodies and then analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Lane 1, Immunoprecipitates fromcontrol cells; lanes 2 to 5, immunoprecipitates from cells treated with TPA or EGF at 0.5, 5, 50, and 200 ng/ml. In panel C, the EGF receptorwas first immunoprecipitated with anti-EGF receptor antibody (lanes 1 to 3) from 32P-labeled MKN-7 cell lysates. Then the lysates, preclearedwith anti-EGF receptor antibody, were immunoprecipitated with anti-EGF receptor antibody again (lanes 4 to 6) or with anti-c-erbB-2antibodies (lanes 7 to 9). The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.Lanes 1, 4, and 7, Immunoprecipitates from control cells; lanes 2, 5, and 8, immunoprecipitates from cells treated with 50 ng of TPA per ml;lanes 3, 7, and 9, immunoprecipitates from cells treated with 50 ng of EGF per ml. In panel D, specificity of antitumor antisera(anti-MMTV-c-erbB-2 antibodies) is demonstrated. 35S-labeled MKN-7 cell lysates were immunoprecipitated with anti-MMTV-c-erbB-2antibodies (lane 1) or anti-EGF receptor antibody (lane 2).

Immunoprecipitation. The labeled cells were solubilized inmodified RIPA buffer (50 mM Tris hydrochloride [pH 7.4],1% Nonidet P-40, 0.1% sodium deoxycholate, 0.15 M NaCl,1 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyro-phosphate, 10 mM sodium fluoride, 4 mM EDTA, 2 mMsodium vanadate [26]), and the lysates were incubated withantibodies for 1 h. The immunocomplexes were adsorbed toprotein A-Sepharose 4B (Pharmacia) and washed exten-sively with modified RIPA buffer as described previously (3).The immunoprecipitates were analyzed by sodium dodecylsulfate (SDS)-polyacrylamide (7.5%) gel electrophoresis (16)followed by autoradiography.

Covalent cross-linking of 125I-labeled EGF to MKN-7 recep-tor. EGF was labeled with 1251I by the chloramine-T method

(6, 19). Confluent MKN-7 cells (6-cm dishes) were incubatedwith 50 ng of 125I-labeled EGF per ml in 2 ml of bindingmedium (Dulbecco modified Eagle medium, 5 mM HEPES[N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid]-NaOH [pH 7.5], 0.1% bovine serum albumin) for 60 min at37°C as described before (6, 19). The cells were washed withbinding medium and solubilized with the RIPA buffer; thenimmunoprecipitation was performed as described above.The immunoprecipitates were subjected to SDS-polyacryl-amide gel electrophoresis and autoradiography.Phosphoamino acid analysis and two-dimensional peptide

mapping. The phosphorylated proteins were eluted from thegel and subjected to acid hydrolysis in 6 N HCI for 1.5 h at110°C. The phosphoamino acids were resolved in two dimen-

C

185K-170K-

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EGF-INDUCED PHOSPHORYLATION OF c-erbB-2 GENE PRODUCT

B TPA

P-Ser

P-Thr

P Tyr

D EGF 15 MIN

P Ser

PT hrP-Ser

P Thr

P-Tyr

P-Tyr

FIG. 2. Phosphoamino acid analysis of the c-erbB-2 gene prod-uct prepared from MKN-7 cells that had been treated with EGF (50ng/ml) for 0 (A), 1 (C), and 15 (D) min or with TPA (50 ng/ml) for 15min (B). Immunoprecipitation was performed with anti-MMTV-c-erbB-2 antibodies. The same results were obtained with anti-c-erbB-2 antibodies.

phosphothreonine but also phosphotyrosine of the c-erbB-2protein (Fig. 2). Figure 3 shows the time course of theincrease of EGF-induced phosphorylation of the c-erbB-2protein; phosphorylation increased gradually for 15 min.This progressive increase was largely due to the phosphor-ylation of serine and threonine residues of the c-erbB-2protein. These results are not accounted for by the copre-cipitation of the EGF receptor, since the same results wereobtained even after preclearing the cell lysates with anti-EGF receptor antibody (Fig. 1C). In addition, anti-c-erbB-2antibodies used in these experiments were specific to thec-erbB-2 gene product and did not precipitate the EGFreceptor (3) (Fig. 1D). Other growth factors such as platelet-derived growth factor, insulin, insulinlike growth factor I,transforming growth factor-P, fibroblast growth factor, bom-besin, IL-1, or gastrin did not stimulate phosphorylation ofthe c-erbB-2 gene product (data not shown).

Since the amino acid sequence of the extracellular domainof the c-erbB-2 gene product shows 44% homology with thatof the ligand-binding domain of the EGF receptor (27), weexamined whether EGF-mediated stimulation of the phos-phorylation is induced by direct binding of EGF to thec-erbB-2 gene product. When 125I-labeled EGF was cova-lently cross-linked to its receptor, the cross-linked EGFreceptor complex was precipitated by anti-EGF receptorantibodies, whereas no precipitation was observed withanti-c-erbB-2 antibodies (Fig. 4). The immunoprecipitatedc-erbB-2 gene product did not bind to EGF, while theimmunoprecipitated EGF receptor exhibited binding activity(data not shown). Moreover, when MKN-7 cells were pre-incubated with increasing concentrations of 528IgG, ananti-EGF receptor antibody that specifically inhibits EGFbinding to its receptor (14), binding of 1251I-labeled EGF toMKN-7 cells was blocked almost completely (Fig. 5). Thesedata imply that the c-erbB-2 protein does not possess EGF-binding activity. In a parallel experiment, blocking of theEGF receptor by 528IgG inhibited EGF-stimulated phos-

sions on thin-layer plates by chromatography at pH 1.9followed by electrophoresis at pH 3.5 (2). For tryptic phos-phopeptide mapping analysis, phosphorylated proteins wereexcised from gels and digested with trypsin (50 ,ug/ml).32P-labeled peptides were separated in two dimensions oncellulose thin-layer plates by electrophoresis at pH 8.9followed by chromatography (2).

RESULTS

For determination of the effect ofTPA on phosphorylationof the human c-erbB-2 gene product, MKN-7 cells carryingan amplified c-erbB-2 gene were labeled with 32p for 7 h andthen treated with various concentrations of TPA for 15 min.The c-erbB-2 gene products from these cells were immuno-precipitated and resolved by SDS-polyacrylamide gal elec-trophoresis. Treatment with TPA increased the 32p contentof the c-erbB-2 gene product (Fig. 1A), the effect beinghalf-maximal at a TPA concentration of approximately 5ng/ml. Phosphorylation of the c-erbB-2 gene product in thepresence of TPA occurred on serine and threonine, but noton tyrosine (see Fig. 2B).We next examined the effect of EGF on phosphorylation

of the c-erbB-2 gene product in a similar way. Exposure ofcells to EGF also resulted in an increase in phosphorylationof the c-erbB-2 gene product (Fig. 1B), the effect beinghalf-maximal at an EGF concentration of between 5 and 50ng/ml. EGF induced increases of not only phosphoserine and

1 2 3 4 5

185K - * 44

0 1 3 8 15 MIN

FIG. 3. Time course of EGF-induced phosphorylation of thec-erbB-2 gene product. MKN-7 cells were labeled with 32p for 7 hand then incubated with 50 ng of EGF per ml for various times (lane1, 0 min; lane 2, 1 min; lane 3, 3 min; lane 4, 8 min; lane 5, 15 min).For immunoprecipitation of the c-erbB-2 gene product, anti-MMTV-c-erbB-2 antibodies were used. Immunoprecipitates were analyzedby SDS-polyacrylamide gel electrophoresis. The same results wereobtained by using anti-c-erbB-2 antibodies.

A CONTR()t

P SerP Thr

P Tyr

C FGF 1 MIN

- .._

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1022 AKIYAMA ET AL.

1 2 3 4 5 6 7 8

FIG. 4. Immunoprecipitation of covalently cross-linked '251-la-beled EGF receptor complexes. MKN-7 cells were incubated with125I-labeled EGF under covalent cross-linking conditions in theabsence (lanes 1 to 4) or presence (lanes 5 to 8) of excess unlabeledEGF (50 ,ug/ml). The cells were lysed and incubated with controlserum (lanes 1 and 5), anti-EGF receptor antibodies (lanes 2 and 6),anti-c-erbB-2 antibodies (lanes 3 and 7), or anti-c-erbB-2 antibodiesthat had been preincubated with excess synthetic peptide (lanes 4and 8). Immunoprecipitates were analyzed by SDS-polyacrylamidegel electrophoresis followed by autoradiography as described inMaterials and Methods.

phorylation of the c-erbB-2 gene product (Fig. 5). Thus,EGF-induced phosphorylation of the c-erbB-2 gene producton both serine/threonine and tyrosine residues was sug-gested to be mediated by the binding of EGF to the EGF

receptor and not by its direct binding to the c-erbB-2 geneproduct.To determine the site specificities of the phosphorylations

induced by EGF and TPA, we compared the two-dimen-sional tryptic phosphopeptide maps of the c-erbB-2 geneproducts immunoprecipitated from EGF- or TPA-treatedMKN-7 cells (Fig. 6). The c-erbB-2 gene products fromEGF- and TPA-treated cells gave similar patterns. In addi-tion to the many peptides constitutively phosphorylated inuntreated cells, cells after both treatments showed signifi-cantly enhanced phosphorylation of peptide X and alsophosphorylation of several unique peptides (e.g., peptide Yin Fig. 6). Phosphoamino acid analysis showed that phos-phoserine was the only phosphorylated component of pep-tide X, whereas phosphoserine, phosphothreonine, andphosphotyrosine were present in peptide Y. Except forpeptide Z, in which only threonine was phosphorylated,other major peptides were mainly phosphorylated on serineresidues (data not shown).Prolonged exposure of cells to TPA is known to result in

down-regulation of protein kinase C, the cellular effector ofTPA (20). As expected, TPA did not enhance phosphoryla-tion of the c-erbB-2 gene product in cells that had beenexposed to TPA (100 ng/ml) for 24 h (Fig. 7, lane 5). Incontrast, EGF was still able to induce phosphorylation of thec-erbB-2 protein in the TPA-pretreated cells (Fig. 7A, lane 6,and 7B). These observations indicated that a cellular serine/threonine kinase different from protein kinase C is appar-ently involved in EGF-induced phosphorylation of the c-erbB-2 protein.

Lastly, kinase activity of the c-erbB-2 protein immunopre-cipitated from TPA- or EGF-treated cells was examined byincubation in the presence of [-y-32P]ATP in vitro. Levels of

A B

-

0

z0ULPLL.0

C:zD0m

(.4

t -64 -3 -6210 10- 102ANTIBODY - ANTIBODY CONCENTRATION

(DILUTION)

11 2 3 4

__ -*

5

-185K

_- _._

FIG. 5. Effect of anti-EGF receptor antibody (528IgG) on 125I-labeled EGF binding to MKN-7 cells and phosphorylation of the c-erbB-2gene product. (A) MKN-7 cells were preincubated with the indicated concentrations of 528IgG at 37°C for 30 min. Then the cell monolayerswere washed, and their binding of "25I-labeled EGF was determined by incubating them with 50 ng of labeled EGF at 4°C for 1 h. (B) MKN-7cells labeled with 32p for 7 h were preincubated in the absence (lanes 1 and 2) or presence (lane 3, 10,000-fold diluted; lane 4, 1,000-fold diluted;lane 5, 100-fold diluted) of 528IgG at 37°C for 30 min and then incubated for an additional 15 min with no additions (lane 1) or with 50 ng ofEGF per ml (lanes 2 to 5). The cells were solubilized, and the c-erbB-2 gene product was immunoprecipitated with anti-c-erbB-2 antibodies.Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.

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EGF-INDUCED PHOSPHORYLATION OF c-erbB-2 GENE PRODUCT

A CONTROL B EGF

-YA

t

C TPA D MIX

tFIG. 6. Phosphopeptide mapping of the c-erbB-2 gene products of TPA- and EOF-treated MKN-7 cells. MKN-7 cell monolayers were

labeled with 32p for 7 h and then incubated for an additional 15 min in the absence (A) or presence of (B) 50 ng of EGF per ml or 50 ng ofTPA (C) per ml. The c-erbB-2 gene product was immunoprecipitated with anti-c-erbB-2 antibodies and subjected to phosphopeptide mappinganalysis as described in Materials and Methods. A mixture of the c-erbB-2 gene products from control cells and cells treated with TPA andEGF, respectively, was also-analyzed (D). The origin is marked by an arrow.

autophosphorylation of the c-erbB-2 protein precipitatedfrom TPA- or EGF-treated cells were not altered as com-pared with the control (Fig. 8). Significant alteration of thetyrosine kinase activity against exogenous substrate, the srcpeptide corresponding to the autophosphorylation site ofPP6src was not induced by either TPA or EGF treatment(data not shown).

DISCUSSION

In the present study, treatment of MKN-7 cells with EGFwas found to stimulate phosphorylation of the c-erbB-2 geneproduct on serine, threonine, and tyrosine residues. We didnot detect phosphotyrosine in our previous study (3), but wecould readily detect its appearance by using RIPA buffer

containing phosphatase inhibitors for immunoprecipitation(26). We have observed a similar rapid increase in thephosphotyrosine content of the c-erbB-2 gene product onEGF treatment of human epidermal carcinoma KB cells(13a). Moreover, Stern et al. mentioned that EGF stimulatedtyrosine phosphorylation of the neu (rat c-erbB-2) geneproduct in Rat-1 cells, but they did not show data (24). Thus,EGF-inducted tyrosine phosphorylation of the c-erbB-2 pro-tein is not confined to MKN-7, the c-erbB-2 overproducercells. The c-erbB-2 gene product exhibits extensive se-quence homology with the EGF receptor (27), but it did notshow detectable EGF-binding activity under conditions thatinduced increased phosphorylation. Thus, EGF-mediatedstimulation of the phosphorylation is not caused by directbinding of EGF to the c-erbB-2 gene product, but is induced

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1024 AKIYAMA ET AL.

ATPAPRE TREATMENT

C T E C T E

185K- _ _ _ _ _ _

1 2 3 4 5

B6

-P-Ser

-P Thr

-P Tyr

FIG. 7. Effect of long-term TPA treatment on stimulations ofphosphorylation of the c-erbB-2 gene product by TPA and EGF. (A)MKN-7 cells were cultured in the absence (lanes 1 to 3) or presence(lanes 4 to 6) of 100 ng ofTPA per ml for 24 h and then labeled with32p for 5 h in the presence of 100 ng of TPA per ml. They were thenincubated for 15 min with no additions (lanes 1 and 4) or with 100 ngof TPA (lanes 2 and 5) or 50 ng of EGF (lanes 3 and 6) per ml. Thecells were then lysed, and the c-erbB-2 gene product was immuno-precipitated with anti-c-erbB-2 antibodies and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.(B) Phosphoamino acid analysis of the c-erbB-2 gene productprepared from 32P-labeled MKN-7 cells that had been pretreatedwith TPA and then stimulated with EGF as in lane 6 in panel A.

indirectly by an unknown mechanism which involves theEGF receptor. A very interesting possibility is that phos-phorylation of tyrosine in the c-erbB-2 gene product iscatalyzed directly by the EGF receptor. We observed pre-liminarily that the EGF receptor could phosphorylate thepartially purified c-erbB-2 protein in the presence of EGF invitro (data not shown).There is accumulating evidence that tyrosine phosphory-

lation may be closely involved in the regulation of cellgrowth or transformation (9). Several substrate proteins fortyrosine kinases have been identified, including vinculin,p81, p50, p42, p36, enolase, lactate dehydrogenase, andphosphoglycerate mutase (9). However, most of these sub-strate proteins are rather abundant cellular proteins, andnone of them has been found to play a critical role in cellular

proliferation or transformation. In this regard, the EGF-induced tyrosine phosphorylation of the c-erbB-2 gene prod-uct observed in this study is of particular interest, since thec-erbB-2 gene product itself is presumed to be the growthfactor receptor associated with tyrosine kinase activity.Studies on the autophosphorylation of tyrosine kinases havesuggested that phosphorylation of tyrosine in these enzymesactivates their kinase activity (9). Moreover, treatment ofcells with platelet-derived growth factor was recently shownto induce phosphorylation of tyrosine in pp60Orc and increaseits kinase activity (18). Accordingly, we have examined thetyrosine kinase activity of the c-erbB-2 gene product precip-itated from EGF-stimulated cells (Fig. 8). Although we didnot observe any alteration of tyrosine kinase activity, itshould be noted that we have measured only the basalactivity of the c-erbB-2 protein without ligand stimulation.Tyrosine phosphorylation may induce functional alterations(for example, inhibition of kinase activity) of the ligand-stimulated c-erbB-2 protein. This possibility could be testedwhen the ligand for the c-erbB-2 protein becomes available.Tumor promoters are known to enhance the phosphory-

lation of serine and threonine residues in the receptors forEGF, insulin, and insulinlike growth factor I (5, 7, 11, 12). Ofthe many TPA-stimulated phosphorylation sites of the EGFreceptor, one unique phosphorylation site detectable bytryptic peptide mapping was identified as threonine 654, andprotein kinase C is believed to catalyze the phosphorylationof this site (10). Corresponding to threonine 654 of the EGFreceptor, the c-erbB-2 gene product possesses a threonineresidue at position 686, which is surrounded by basic aminoacid residues (27). Therefore, TPA-induced phosphorylation

1 2 3

185K-

FIG. 8. Effect ofTPA or EGF treatment on autophosphorylatingactivity of the c-erbB-2 gene product. The c-erbB-2 gene productwas immunoprecipitated from MKN-7 cells (lane 1) and fromMKN-7 cells treated with TPA for 15 min (lane 2) or EGF for 3 min(lane 3) and then assayed for autophosphorylating activity byincubating with 20 mM HEPES-NaOH (pH 7.2-10 mM MgCl2-3mM MnCl2-10 ,uM [y-32P]ATP for 7 min at 25°C. The reactionproduct was analyzed by SDS-polyacrylamide gel electrophoresisfollowed by autoradiography.

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EGF-INDUCED PHOSPHORYLATION OF c-erbB-2 GENE PRODUCT

of the c-erbB-2 gene product described in this study may alsooccur on threonine 686 by the action of activated proteinkinase C. However, peptide mapping analysis showed thatmost of the increase in phosphorylation occurred at sitesconstitutively phosphorylated in untreated cells. We are nowtrying to identify the tryptic peptide containing threonine686.The c-erbB-2 gene product from EGF-treated cells gave

almost the same phosphopeptide mapping pattern as that ofTPA-treated cells. Addition of EGF to the cells has beenshown to increase phosphoinositide turnover and the Ca2+level (17, 21), which in turn activate protein kinase C. Thus,our results were consistent with the hypothesis that acti-vated protein kinase C may also be, at least in part, respon-sible for EGF-mediated phosphorylation of the c-erbB-2gene product. However, our finding that EGF could inducephosphorylation of the c-erbB-2 gene product in cells thathad became refractory to TPA indicate that the mechanismsby which EGF and TPA induce phosphorylation are dif-ferent. We propose here that a cellular serine/threoninekinase is activated by EGF receptor kinase to phosphorylatethe c-erbB-2 protein.We have shown here that the EGF receptor and the

c-erbB-2 gene product, which are similar to one another,interact by means of serine, threonine, and tyrosine phos-phorylation. Protein kinase C, a tumor promoter receptor,and yet unidentified serine/threonine kinase were also shownto participate in the receptor communication at the surfaceof MKN-7 cells. Experiments to determine the consequenceof these interactions are currently under way.

ACKNOWLEDGMENTS

We thank T. Kawamoto for providing 5281gG. We also thank C.Sudo, B. Ryu, and T. Jinbo for technical assistance.

This work was partly supported by grants-in-aid from the Ministryof Education, Science and Culture of Japan.

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2. Akiyama, T., T. Kadowaki, E. Nishida, T. Kadooka, H. Oga-wara, Y. Fukami, H. Sakai, F. Takaku, and M. Kasuga. 1986.Substrate specificities of tyrosine-specific protein kinasestoward cytoskeletal proteins in vitro. J. Biol. Chem. 261:14797-14803.

3. Akiyama, T., C. Sudo, H. Ogawara, K. Toyoshima, and T.Yamamoto. 1986. The product of the human c-erbB-2 gene: a185,000 dalton glycoprotein with tyrosine kinase activity. Sci-ence 232:1644-1646.

4. Bargmann, C. I., M.-C. Huang, and R. A. Weinberg. 1986. Theneu oncogene encodes an epidermal growth factor receptor-related protein. Nature (London) 319:226-230.

5. Cochet, C., G. N. Gill, J. Meisenhelder, J. A. Cooper, and T.Hunter. 1984. C kinase phosphorylated the epidermal growthfactor receptor and reduces its epidermal growth factor stimu-lated tyrosine-protein kinase activity. J. Biol. Chem. 259:2553-2558.

6. Comens, P. G., R. L. Simmer, and J. B. Baker. 1982. Directlinkage of 125I-EGF to cell surface receptors. J. Biol. Chem. 257:42-45.

7. Davis, R., and M. P. Czech. 1984. Tumor-promoting phorboldiesters mediate phosphorylation of the epidermal growth factorreceptor. J. Biol. Chem. 259:8545-8549.

8. Fukushige, S., K. Matsubara, M. Yoshida, M. Sasaki, T. Suzuki,K. Semba, K. Toyoshima, and T. Yamamoto. 1986. Localizationof a novel v-erbB-related gene, c-erbB-2, on human chromo-

some 17 and its amplification in a gastric cancer cell line. Mol.Cell. Biol. 6:955-958.

9. Hunter, T., and J. A. Cooper. 1985. Protein-tyrosine kinases.Annu. Rev. Biochem. 54:897-930.

10. Hunter, T., N. Ling, and J. A. Cooper. 1984. Protein kinase Cphosphorylation of the EGF receptor at a threonine residueclose to a cytoplasmic face of the plasma membrane. Nature(London) 311:480-483.

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