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Synthetic Phosphopeptide Immunogens Yield Activation-Specific Antibodies to the C-Erbb-2 Receptor (Trosine Kinases/Oncogencs/Phophorylation/Breast Neopsims) RICHARD J

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Synthetic Phosphopeptide Immunogens Yield Activation-Specific Antibodies to the C-Erbb-2 Receptor (Trosine Kinases/Oncogencs/Phophorylation/Breast Neopsims) RICHARD J Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10435-10439, November 1992 Medical Sciences Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor (trosine kinases/oncogencs/phophorylation/breast neopsims) RICHARD J. EPSTEIN*t, BRIAN J. DRUKER*t, THOMAS M. ROBERTS*1, AND CHARLES D. STILES*§ *Division of Cell and Molecular Biology, Dana-Farber Cancer Institute, and Departments of tMedicine, VPathology, and §Microbiology and Molecular Genetics, Harvard Medical School, 44 Binney Street, Boston, MA 02115 Communicated by Robert A. Weinberg, July 29, 1992 ABSTRACT We inoculated rabbits with synthetic phos- 10% (vol/vol) bovine calf serum, glutamine (60 mg/liter), phopeptides, duplicating a major autophosphorylation site of penicillin (10 units/ml), streptomycin (0.1 ,ug/ml), and 0.3 the c-rbB-2 protooncogene product. The rabbits produced AuM methotrexate. All other cell lines were obtained from the antisera that, after reverse immunaffinity purfcation, selec- American Type Culture Collection. Cell manipulation, im- tively recognize the erbB-2 protein in its enzymaticafly active munoblot protocols, polyacrylamide gel electrophoresis, and configuration. These anti-phosphopeptide antisera identify a reagents were as described (11). Cells were lysed in lysis subset of erbB-2-positive human cell lines wherein the protein buffer (LB) consisting of10 mM Na2HPO4, 10 mM NaH2PO4, is constitutively active as a tyrosine kinase. Synthetic phos- 150 mM NaCI, 1% Nonidet P.40, 10%6 (vol/vol) glycerol, 50 phopeptides incorporating informative protein phosphoryla- mM sodium fluoride, 10 mM sodium pyrophosphate, prote- tion sites may prove useful for generating antibodies that ase inhibitors [40 indicate the activation state of additional tyrosine kinases and ,uM leupeptin, aprotinin (10 ,ug/ml), and 1 perhaps other proteins phosphorylated on serine and threonine mM phenylmethylsulfonyl fluoride], and tyrosine phospha- residues. tase inhibitors [1 mM sodium orthovanadate, benzamidine hydrochloride (50 ,g/ml), and sodium molybdate (50 tug/ The c-erbB-2 (neu or HER-2) gene product is a cell-surface ml)]. Approximately 250 ,ug oftotal protein lysate was added receptor tyrosine kinase structurally related to the epidermal to each well for immunoblot analysis. To provide c-erbB-2- growth factor (EGF) receptor. Clinical studies have estab- inactive controls, G8/DHFR cell samples were treated with lished a link between tumor-associated c-erbB-2 protein 10%o bovine calf serum for 15 min to transmodulate the overexpression and reduced survival of primary breast can- c-erbB-2 receptor (11) and then lysed in the absence of cer patients with metastatic involvement of axillary lymph tyrosine phosphatase inhibitors; the transmodulating effi- nodes (1-3). However, the association between c-erbB-2 ciency of bovine calf serum is batch-dependent, probably protein expression and human breast cancer is neither per- reflecting varying platelet-derived growth factor content vasive nor internally consistent. Overexpression of c-erbB-2 (R.J.E., unpublished observations). For immunoblot con- in human breast tumors occurs more frequently in preinva- trols, pAb-1 rabbit polyclonal antibody (pan-erbB-2), raised sive than invasive lesions (4-6) and is seldom if ever asso- against the unphosphorylated C-terminal (residues 1243- ciated with receptor-activating transmembrane domain mu- 1255) peptide sequence of the neu gene product (Triton tations (7). In lymph-node-negative disease-where the need Biosciences, Alameda, CA), was used at a 1:100 dilution in for a reliable prognostic indicator is especially pressing- TBST buffer (10 mM Tris'HCl, pH 8.0/150 mM NaCl/0.05% correlations between expression of c-erbB-2 protein and Tween 20), and monoclonal phosphotyrosine [Tyr(P)] anti- clinical outcome are doubtful (1, 3, 8-10). body [anti-Tyr(P)] was purified over a Staphylococcus au- Since only the activated form of a tyrosine kinase is likely reus protein A affinity column and used at a 1:1000 dilution. to influence cell growth and differentiation, these discrepan- Immunoprecipitation ofthe c-erbB-2 receptor was performed cies could reflect heterogeneity ofreceptor activation in vivo. using a 1:10 dilution of the monoclonal antibody mAb-1 Patients with tumors expressing activated c-erbB-2 (due to (Triton Biosciences), which recognizes an extracellular do- mutations or paracrine/autocrine ligand production, for ex- main receptor epitope. ample) could prove to have a natural history different from In Vitro Kinase Assays and in Vivo Phospholabeling. All that of patients whose tumors express predominantly inac- manipulations were carried out at 40C. Cell samples were tivated receptor. To explore this possibility, we have devel- lysed in LB and immunoprecipitated as described (11). The oped antibodies that distinguish the kinase-active form of immunoprecipitates were washed twice with LB, twice with c-erbB-2 from the inert kinase-inactive configuration. Our distilled H20, and twice with tyrosine kinase buffer (KB = 10 approach is based on the use of synthetic phosphopeptide mM MnCl2/20 mM Tris-HCl, pH 7.4). The beads were then immunogens that duplicate a major c-erbB-2 receptor auto- drained, resuspended in 40 ,ul of kinase buffer containing 0.5 phosphorylation site. ."I of [y-32P]ATP (specific activity, 3000 Ci/mol; 1 Ci = 37 GBq), and incubated at 40C for 20 min. The reaction was MATERIALS AND METHODS stopped by addition of 1 ml of LB. The beads were washed once, resuspended in 50,ul ofsample buffer, boiled for 5 min, Cells and Cell Lysis. Stock cultures of G8/DHFR murine and loaded onto a 7.5% polyacrylamide gel. After fixation fibroblasts containing an amplified dicistronic rat c-neu- and drying, the gel was exposed and analyzed using Phos- dihydrofolate reductase construct (a gift ofRobert Weinberg, phorlmager model 400S; band quantification was carried out Whitehead Institute, Cambridge, MA) were maintained in using IMAGEQUANT Version 2.0 Dulbecco's modified Eagle's medium supplemented with software (Molecular Dynam- Abbreviations: EGF, epidermal growth factor; APHID, activation- The publication costs ofthis article were defrayed in part by page charge specific phosphoprotein immunodetection; Tyr(P), phosphoty- payment. This article must therefore be hereby marked "advertisement" rosine; RIP, reverse immunoaffinity purification; apt, anti-phospho- in accordance with 18 U.S.C. §1734 solely to indicate this fact. peptide. 10435 Downloaded by guest on October 1, 2021 10436 Medical Sciences: Epstein et al. Proc. NatL Acad Sci. USA 89 (1992) ics, Sunnyvale, CA). In vivo phospholabeling was carried out RESULTS as described (11). Synthesis of Tyrosine-Phosphorylated Peptides and Immu- Phosphopeptide humunogens Yield Antsera Regi nization Protocol. A major autophosphorylation site of the the Parent Io n. Our point of departure was Tyr- c-erbB-2 receptor expressed in NIH 3T3 cells is Tyr-1248 (12, 1248, which is a major autophosphorylation site of the 13). The C-terminal (residues 1243-1255) c-erbB-2 peptide c-erbB-2 receptor (12, 13). The tyrosine-phosphorylated sequence was synthesized in association with an N-terminal C-terminal (residues 1243-1255) c-erbB-2 peptide sequence cysteine. Instead of incorporating a tyrosine residue at po- was synthesized. This phosphopeptide was coupled to hemo- sition 1248, however, a phosphorylated peptide was synthe- cyanin, gel-purified, and inoculated into rabbits. To identify sized by standard Merrifield solid-phase synthesis proce- activation-specific antisera, we needed a cell culture system dures using the t-butoxycarbonyl strategy. Synthesis was in which the c-erbB-2 protein could be reversibly converted performed on an ABI model 430 peptide synthesizer using between the inert and activated configurations. Although small-scale (0.1 mmol) rapid cycles. Tyr(P) was incorporated initially hindered by the lack of an available receptor- as tert-butoxycarbonyl-O-(dibenzyl)phosphono-L-tyrosine activating ligand, we found that c-erbB-2 receptor activity (Peninsula Laboratories, Belmont, CA). The fully protected can be rapidly modified by heterologous transmodulating from the ligands. When G8/DHFR cells are cultured as density- peptide was deprotected and cleaved phenylaceta- arrested monolayers, they express a 175-kDa c-erbB-2 pro- midomethyl resin using trifluoromethanesulfonic acid. Re- tein (p175) that is active as a tyrosine kinase in vitro and in verse-phase analysis of the crude peptide was achieved on a vivo; within minutes of exposure to platelet-derived growth Vydac C18 5-,um column 250 mm x 2.1 mm. A linear gradient factor or protein kinase C agonists, however, the activated from 0 to 60 min was run with 0.1% trifluoroacetic acid and c-erbB-2 receptor is phosphorylated on serine/threonine 0.09% trifluoroacetic acid in acetonitrile. Amino acid analysis residues and converted to a 185-kDa isoform (p185) with was performed using standard Pico-Tag procedures (Waters). markedly reduced tyrosine kinase activity (11). Hydrolysis was carried out with 6 M HCl at 145°C for 2 h. As shown in Fig. 1A, commercially available polyclonal Composition analysis showed an almost complete conversion antibodies to the unphosphorylated (residues 1243-1255) of Tyr(P) to tyrosine due to the hydrolysis condition. To peptide recognized both the tyrosine-phosphorylated pl75/ show that Tyr(P) was indeed incorporated into the peptide, c-erbB-2 and the transmodulated p185/c-erbB-2. Crude anti- sequence analysis was performed using an ABI model 477 phosphopeptide (apt-i) antiserum preferentially recognized gas-phase sequencer. The presence of a Tyr(P) residue was the activated p175 isoform but did not bind as selectively to determined by the lack of signal at the expected cycle [since p175 as did Tyr(P) antibody (Fig. 1A). Continued immuni- Tyr(P) is not extracted by the solvents used] and the reemer- zations with the phosphorylated peptide increased antiserum gence of the expected sequence on subsequent cycles. Phos- photyramine was prepared as described (14) and linked to A Affi-Gel 10 beads (Bio-Rad). Amino acid analysis of Tyr(P)- Receptor 1248-containing peptides revealed minimal unphosphory- activity 200--.
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