Identification of Autophosphorylation Sites of HER2/Neu

Cell Growth & Differentiation 3

Identification of Autophosphorylation Sites of HER2/neu

R. Hazan, B. Margolis,’ M. Dombalagian, A. Ullrich, consensus tyrosine in the kinase domain is phosphory- A. Zilberstein, and J. Schlessinger2 lated. Site-directed mutagenesis of this site impairs the Rorer Biotechnology, Inc., King of Prussia, Pennsylvania 19406 [R. H., B. ability of autophosphorylation to activate the kinase (7, M., M. D., A. Z., J. 5.], and Max-Planck-Institut f#{252}rBiochemie, 8033 8). In the EGFR, this consensus tyrosine has not been Martinsreid bei MOnchen, Federal Republic of Germany [A. U.] found to be a site of autophosphorylation.4 It has been found that all autophosphorylation sites of the EGFR are concentrated in the carboxyl-terminal domain of the Abstrad receptor outside of the consensus kinase domain (9, 10). HER2 or c-erbB-2 is a putative growth fador receptor In the EGFR, it has been postulated that the autophos- with sequence homology to the epidermal growth fac- phorylation sites act as competitive inhibitors of exoge- tor receptor. It is the human homologue of the rat nous substrate phosphorylation (6, 1 1) and it has been protooncogene neu and may have an important role in demonstrated that mutagenesis of the individual auto- human malignancies such as breast and ovarian can- phosphorylation sites increases the receptor affinity for cers. Like other growth fador receptors, HER2 has in- exogenous substrates in vitro (6). Additionally, cells which trinsic protein tyrosine kinase adivity and undergoes express these mutated receptors demonstrate an in- autophosphorylation. Recently, we have demonstrated creased mitogenic sensitivity to EGF (12). Thus, intrinsic that, similar to the epidermal growth fador receptor, autophosphorylation sites may compete with exogenous all autophosphorylation sites of HER2 are localized in substrate phosphorylation at low levels of receptor acti- the carboxyl terminus of this protein. In the present vation. study, immunopurified HER2 was allowed to autophos- HER2 or c-erbB-2 is the human homologue of the rat phorylate, and tryptic phosphopeptides were gener- protooncogene neu (13-15), which has great sequence ated. After purification of these phosphopeptides by identity with the EGFR but for which the natural ligand high performance liquid chromatography, microsequen- is not known. HER2 is overexpressed in many breast and cing was performed. Utilizing this approach, two au- ovarian cancers and thus may play an important role in tophosphorylation sites were unequivocally identified human carcinogenesis (16). Recently, we have demon- at Y1023 and Y1248. The sequences of two other tyro- strated that like EGFR, all of the autophosphorylation sine phosphorylated tryptic peptides were determined, sites of the HER2 are located in the carboxyl terminus of but the exad site of autophosphorylation could not be the receptor (10). This was demonstrated utilizing a chi- determined because multiple tyrosines were located on meric receptor where the carboxyl terminus of the EGFR each peptide. However, each of these peptides contains was replaced by the carboxyl terminus of the HER2 tyrosines that correspond to major autophosphorylation receptor. It was found that the autophosphorylation sites sites of the epidermal growth fador receptor, suggest- of this EGFR/HER2 chimera were identical to the auto- ing that, in addition to Y1023 and Y1248, Yl 139 and phosphorylation sites of the wild-type HER2 receptor, Yl 222 also serve as autophosphorylation sites of HER2. indicating that the carboxyl terminus contains all of the autophosphorylation sites. In the present study, we ex- Introdudion tend this work by purifying and sequencing the tryptic peptides that contain the autophosphorylation sites of Many growth factors bind to cell surface receptors with the HER2 receptor. We have unequivocally identified intrinsic protein tyrosine kinase activity (reviewed in Refs. two autophosphorylation sites by direct sequencing and 1-3). Upon ligand binding, the intracellular tyrosine ki- demonstrated that two phosphopeptides contain tyro- nase is activated, resulting in tyrosine phosphorylation of sine residues which correspond to two major autophos- substrates. One of the first substrates to be phosphory- phorylation sites of EGFR. It appears, therefore, that, lated is tyrosine residues in the carboxyl terminus of the except for one site, autophosphorylation sites of the receptor, resulting in receptor autophosphorylation (re- HER2 receptor are similar to those found in the EGFR. viewed in Refs. 1 -3). The exact role of receptor autophosphorylation in growth factor signal transduction is still being investigated and appears to vary for different Results receptors. For example, autophosphorylation has been The HER2 receptor was immunoprecipitated and auto- demonstrated to increase the Vmax of the insulin receptor phosphorylated in vitro and then purified by SDS-poly- kinase (4), but not the EGFR3 kinase (5, 6). In the insulin acrylamide gel electrophoresis. The gel slice with the receptor (and several nonreceptor tyrosine kinases) a HER2 receptor contained approximately 1 nmol of phos- phate, as determined by Cerenkov counting. Fig. 1 demonstrates the elution pattern of the phosphorylated tryptic peptides prepared and separated by reverse phase Received 9/25/89. HPLC at pH 2.0. Pools of fractions 7-9 (peptide A), 1 1 1 Fellow of the Medical Research Council of Canada. 2 To whom requests for reprints should be addressed, at Rorer Bio- and 12 (peptide B), 25 and 26 (peptide C), and 27-29 technology, Inc., 680 Allendale Rd., King of Prussia, PA 19406.

3 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; PLC-y, phospholipase C-y. 4 5. Bianchi and J. schlessinger, unpublished observation. 4 HER2 Autophosphorylation Sites

Discussion We have utilized standard tryptic peptide purification and sequencing to identify the in vitro autophosphory-

B. lation sites of the HER2 protein. Two sites were unequivocally assigned as Y1023 and Y1248. Two other sites 4, 4, were localized to specific tryptic peptides, but the exact residue could not be assigned due to multiple tyrosines

C in these peptides. However, tyrosine residues located in these later peptides correspond to major autophosphorylation sites of the EGFR, suggesting that they also serve i as autophosphorylation sites. It is noteworthy that the autophosphorylated HER2 protein was generated by an in vitro reaction, and it is possible that additional or other sites are phosphorylated in vivo. However, quantitative comparisons of phosphopeptide maps of EGFR and 30 40 50 HER2 after either in vitro or in vivo autophosphorylation

S ACETONITRILE indicated that similar sites were phosphorylated for each of the two receptors under both in vitro and in vivo Fig. 1. HPLC separation of tryptic phosphopeptides from HER2. Auto- conditions. It is likely, therefore, that the in vitro autophosphorylated HER2 protein from transfected NIH/3T3 cells (inset) and phosphorylation sites described in this study represent HPLC separation of phosphopeptides. HER2 receptors were immunopre- the same sites which are phosphorylated in living cells. cipitated from transfected NIH/3T3 cells and allowed to autophosphor- These results are consistent with our previous results ylate in the presence of [.‘2PJATP The phosphorylated receptor was purified by 5D5-polyacrylamide gel electrophoresis, and the gel slice demonstrating that all autophosphorylation sites of HER2 containing the receptor was excised. After washing with 10% methanol, are localized in the carboxyl terminus of the receptor. the gel slice was dried and treated three times with 5 ig trypsin. The EGFR and HER2 are highly homologous in the kinase tryptic peptides were applied to a C, reverse phase column and eluted with increasing acetonitrile concentration at pH 2.0 as described in domain but have only 35% sequence identity in the Materials and Methods. Fractions of 0.5 ml were collected, and Cer- carboxyl terminus (11, 12). Nonetheless, three ofthe four enkov counts were determined. Arrows, the major fractions that were tyrosine autophosphorylation sites of EGFR are also con- subjected to further purification. served. All of these HER2 tyrosine residues were located on phosphorylated tryptic peptides, strongly suggesting that these sites of autophosphorylation are highly con- (peptide D) were selected as representing the major served between these two receptor molecules. However, peaks and were further purified in preparation for se- one site (Y1086) is phosphorylated in EGFR but not in quencing. HER2, and one site (Y1023) is phosphorylated in HER2 Fig. 2 represents the elution pattern of radioactive but not in EGFR (Fig. 3). peptides in the second HPLC purification at pH 6.5. The The role of autophosphorylation in HER2 signal trans- largest radioactive peak from each fraction was directly duction is unknown. In EGFR, tyrosine autophosphory- applied to the gas phase sequencer, and the sequences lation sites have been shown to act as competitive inhib- obtained are shown in Fig. 2. Tryptic peptides B and C itors of exogenous substrate phosphorylation (6), but this contain only one tyrosine each, Y1248 and Y1023, re- may not be their only function. The exact location of the spectively, indicating that these must be sites of auto- autophosphorylation sites in the carboxyl terminus would phosphorylation. The tyrosine residues at these positions not be critical if these sites functioned only as competi- are not sequenced, as the gas phase sequencer cannot tive inhibitors. Yet, despite the variability in the carboxyl- identify phosphotyrosine. Amino acid Y1248 of HER2 terminal sequence of EGFR and HER2, the locations of corresponds to Vi 1 73 of the EGFR, which is a major the autophosphorylation sites appear highly conserved. autophosphorylation site of EGFR (9). Amino acid Y1023 Recently, our laboratory has been studying the ability corresponds to EGFR Y992, which does not appear to of EGFR to associate physically with its substrate, PLC-y undergo autophosphorylation. Both tryptic peptides A (1 7). The association of EGFR with PLC--y appears to be and D contain more than one tyrosine residue; conse- enhanced by receptor activation and autophosphoryla- quently, the autophosphorylation site(s) cannot be iden- tion, suggesting that autophosphorylation may be impor- tified precisely. Peptide A contains three tyrosine resi- tant for substrate receptor interactions (18). A preliminary dues, Y1112, Y1127, and Y1139. HER2 Y1139 corre- study ofa mutated EGFR with a 126-amino acid carboxyl- sponds to EGFR Y1068, an EGFR autophosphorylation terminal deletion demonstrates that this receptor has a site (Fig. 3). A tyrosine residue was detected by the gas reduced ability to phosphorylate and associate with PLC- phase sequencer at residue Yl 127, suggesting that this is ,y. Additionally, NIH/3T3 cells expressing this deletion not an autophosphorylation site. However, this cannot mutant receptor have a reduced mitogenic sensitivity to be determined with certainty, as it is possible that Yl 127 EGF.5 It has also been found that this mutated EGFR, was phosphorylated but then was dephosphorylated dur- unlike wild-type receptor, is unable to transform chicken ing peptide purification. Peptide D also contains three tyrosines, Y1196, Y1221, and Y1222. Y1222 of HER2 corresponds to EGFR Yl 148, another autophosphorylation site of EGFR (Fig. 3). Again, Y1196 was recognized S A. M. Honegger, A. 5chmidt, A. Ullrich, and I. schlessinger. Evidence as tyrosine by the sequencer, suggesting that this is not for EGF-induced intermolecular autophosphorylation of EGF-receptors the phosphorylated residue in this peptide. in living cells, Mol. Cell Biol., in press. Cell Growth & Differentiation S

A. B.

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Fig. 2. Second HPLC purification of HER2 tryptic phosphopeptides. Pools of fractions 7-9 (peptide A), 1 1 and 12 (peptide B), 25 and 26 (peptide C), and 27-29 (peptide D) from the initial HPLC separation were dried with 10 zg -lactoglobulin A as a carrier protein. These peptides were then separated by reverse phase HPLC at pH 6.5 as described in “Materials and Methods.” Fractions of 0.1 ml were collected, and the Cerenkov counts were determined. The major peak obtained from each peptide was then directly applied to the gas phase sequencer. The amino acid sequences of each peptide are indicated. x, no specific amino acid could be identified for this residue; end of the sequence, point at which no further amino acids could be determined; x*, position of tyrosine residues unequivocally identified as sites of autophosphorylation.

erythroblasts, although it did retain transforming activity Materials and Methods in chicken fibroblasts (19). Carboxyl-terminal deletions Cell Culture. NIH/3T3 2.2 cells devoid of endogenous of the insulin receptor result in the loss of two autophos- EGFR or HER2 receptors were transfected with the full- phorylation sites and blunt the ability of the receptor to length complementary DNA construct of HER2 as pre- enhance cellular glucose uptake (20). Although deletion viously described (21-23). The cells were grown in Dul- mutations may affect the tertiary structure of the recep- becco’s modified Eagle’s minimal essential medium with tor, the data suggest that there may be another function 10% bovine calf serum, penicillin, and streptomycin. for the carboxyl terminus, possibly involving substrate Immunoprecipitation and Autophosphorylation. Cells recognition, and it is possible that the function of the were lysed into a 1% Triton X-100-containing lysis buffer carboxyl terminus depends on the presence of intact with protease inhibitors as previously described (10). For autophosphorylation sites. Further information as to the protein microsequencing, HER2 receptors were immu- exact function of receptor autophosphorylation will de- noprecipitated from approximately 1O cells. Immuno- pend in part on the generation of point mutations of precipitation was performed with a monoclonal antibody individual autophosphorylation sites of HER2. The pres- directed against the extracellular domain of HER2 (mAb4) ent study represents a first step in identifying the auto- linked to Protein A-Sepharose. The immunoprecipitate phosphorylation sites of the HER2 receptor and provides was washed three times with HNTG (20 m 4-(2-hydrox- the foundation for the generation of these autophos- yethyl)-1-piperazineethanesulfonic acid, 150 mi NaCI, phorylation site mutants. 0.1% Triton X-100, and 10% glycerol), and then auto- 6 HER2 Autophosphorylation Sites

976 A I N 0 C ( 0 N 0 D V V D A 0 ( Y I I P Q Q G F F S S P S T - S ft - EGIR

1007 S I I ( 0 0 D N G 0 1 V 0 A E E I V P Q Q G F F C P D P A P G A G G N V H H R H ft S S S T N S G G H(R2

1023 Fig. 3. Localization of the auto- 1008 ---TPLL SSL--S--ATSMNSTVACID-RNGLQSCPIKID (GFR phosphorylation sites of HER2 1057 6 D I T I G L ( P S E ( ( A P R S P 1 A P S ( G A G S D V F 0 G 0 1 G N G A A K G I Q S I P 1 H D P H(R2 and EGFR. The amino acid se- 1068 quences of the carboxy-terminal regions of EGFR (top) and HER2 1040 S F L Q 8 V S S 0 P T G A I - T E 0 S I 0 D T F I - - - P V P ( tI N Q - S V P K R P A G S - V Q N (GIN (bottom) are compared. Tyrosine 1107 S P 1 Q I Y S ( 0 P T V P 1 P S ( T 0 G Y V A P L T C S P 0 P ( V V N Q P 0 V I P Q P P 5 P N ( C P H(12 residues of HER2 Y1023 and Y1248 are unequivocally identi- 106 1139 fied as autophosphorylation sites 1084 PVTHNQPLNPAPSR-_-OPHYQOPNST AVGNP(YLNTVQPTCVN (GIN (solid arrows). Tyrosine residues 1157 1 P A A N P A C A T I ( N A K T L S P G K N G V V K 0 V F A F G C A V ( N P ( V I - T P 0 C C A A P H(R2 Y1139 and Y1222 (thin arrows) hf8 reside in phosphorylated peptides which contain multiple ty- 1125 S T F 0 - - - - S P A H N A 0 K G S H 0 I S I 0 N P D ! - o Q D F F P K ( A K P N C I F K C S - T A (GIN rosine residues. The positions of 1206 Q P H P P P A F S P A F 0 N I V Y N D Q 0 - P P ( N G A P P S T F K C T P T A H(R2 the residues match the positions of Y1068 and Y1148 of EGFR, 11j3 1222 respectively. 1169 #{163}NA(TLIVAPQSSEFIGA 1244 ENPEYLGLDVPV 1248

phosphorylation was performed in HNTG with 10 mri umn (2.1 mm x 30 mm) in an Applied Biosystems 130A MnCl2, 200 M sodium orthovanadate, and 40 Ci [‘y- high performance liquid chromatograph. Buffer A was 10 32P]ATP (0.4 nmoI/aCi) for 20 mm at 4#{176}C.Sample buffer mM ammonium acetate, pH 6.5, and buffer B was 10 miii 3x was added, and the protein was concentrated in an ammonium acetate in 80% acetonitrile. The gradient run Amicon Centricon 30. The phosphorylated receptor was at 200 I/min was as follows: 0-5 mm, 100% buffer A; purified on a 7% SDS-gel, and the receptor band was and 5-45 mm, a linear gradient to 40% buffer B. The excised. The gel slice was washed for 4 h with 10% 100-Ll fractions were counted, and those with significant methanol, dried, and treated three times with 5 g trypsin radioactivity were sequenced on an Applied Biosystems (sequencing grade, Boehringer-Mannheim, Indianapolis, 470A gas phase sequencer. IN) in 50 mM ammonium bicarbonate. Generation of Monoclonal Antibodies against HER2. Acknowledgments Monoclonal antibodies against HER2 protein were gen- We thank Rosalie Ratkiewicz for typing this manuscript. erated by immunizing BALB/c mice with intact NIH/3T3 cells transfected with HER2 complementary DNA expression vector and expressing about 106 HER2 receptors/ References

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