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A Discrete Three-amino Acid Segment (LVI) at the C-terminal End of Kinase-impaired ErbB3 Is Required for Transactivation of ErbB2

A Discrete Three-amino Acid Segment (LVI) at the C-terminal End of Kinase-impaired ErbB3 Is... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 2, Issue of January 8, pp. 859 –866, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Discrete Three-amino Acid Segment (LVI) at the C-terminal End of Kinase-impaired ErbB3 Is Required for Transactivation of ErbB2* (Received for publication, September 2, 1998, and in revised form, October 7, 1998) Gabriele Schaefer‡§, Robert W. Akita‡, and Mark X. Sliwkowski‡¶ From ‡Genentech, Inc., South San Francisco, California 94080 and the §Institut fu ¨ r Biologie III, University of Freiburg, D-79104 Freiburg im Breisgau, Germany ErbB3 is unique among other members of the receptor important role in signaling. ErbB2 is transactivated by het- tyrosine kinase family of growth factor receptors in that erodimerization with ligand-occupied EGFR, ErbB3, or ErbB4 its kinase domain is enzymatically impaired. This ren- (6 –9). ders it incapable of transducing a signal in response to The extensive interreceptor associations that occur in the ligand binding. However, in conjunction with ErbB2, ErbB family serve to increase the repertoire of cellular re- ErbB3 is a potent mediator of signaling by the growth sponses to growth factor stimulation and to fine-tune growth factor heregulin. Heregulin binding to ErbB3 induces factor signaling. At least 10 different homo- and heteromeric formation of a heterodimeric complex with ErbB2, and combinations of ErbB proteins have been reported (10, 11). this results in transactivation of the ErbB2 kinase. Al- However, these combinations are not equally favorable. The though interaction between the extracellular domains interreceptor interactions are hierarchically organized, where of these receptors is an essential part of this process, it ErbB2 is the preferred heteromeric partner, and it favors in- was not clear whether interaction between the cytoplas- teraction with ErbB3 (12, 13). mic domains is also necessary for transactivation. By Cross-talk between ErbB2 and ErbB3 is especially important examining the abilities of a series of cytoplasmic domain as the kinase of ErbB3 is dysfunctional. The impaired kinase mutants of ErbB3 to activate ErbB2, we have found a activity has been demonstrated in several systems. It was discrete sequence of three amino acid residues (LVI), initially reported in insect cells expressing ErbB3 (14), later by located at the carboxyl-terminal end of the impaired ErbB3 kinase region, that is obligatory for transactiva- demonstrating the lack of any biological activity in cells ex- tion. We conclude that formation of a functional ErbB2- pressing ErbB3 alone (10, 11), and recently by biochemical ErbB3 signaling complex requires the presence of a spe- analysis of the purified kinase domain (15). Alterations of four cific structural feature within the ErbB3 cytoplasmic amino acid residues in the kinase region that are otherwise domain and suggest that ErbB2 transactivation results conserved among all protein tyrosine kinases (16) may account from a physical interaction between the cytoplasmic for the lack of catalytic activity. ErbB2, however, is character- domains of these receptors. ized by a constitutively active kinase (17). The physiological significance of this heteromeric complex is emphasized by the fact that the presence of ErbB2 in ErbB3-expressing cells sig- Receptor tyrosine kinases play a pivotal role in the transduc- nificantly enhances the transformation ability (18, 19). Inhibi- tion of extracellular signals into the cells. The binding of cog- tion of ErbB2 and ErbB3 complex formation abolishes HRG- nate growth factors to these cell-surface receptors results in mediated signaling (9, 13, 20). Additionally, active ErbB2- receptor oligomerization and activation of the intrinsic kinase ErbB3 receptor complexes have been seen in several mammary activity (1, 2). This leads to receptor phosphorylation and trig- tumor cell lines, indicating the relevance of this heteromeric gers a cascade of intracellular signaling events that ultimately receptor aggregate in human neoplasia (18, 21). elicit a variety of cellular responses such as proliferation, dif- HRG binds with low affinity to kinase-inactive ErbB3. Re- ferentiation, survival, or migration. cruitment of ErbB2 into the HRG-ErbB3 complex leads to the An extensively characterized subgroup of this receptor su- formation of a high affinity HRG-binding receptor, which is perfamily is the ErbB group of receptors, also known as the capable of generating a tyrosine phosphorylation signal due to class I receptor tyrosine kinases. Members of this group include 1 the kinase activity of ErbB2 (7). Studies on the isolated extra- the epidermal growth factor receptor (EGFR or ErbB1), ErbB2 cellular domain of ErbB3 show that ligand binding is exclu- (also termed HER2 or Neu), ErbB3 (HER3), and ErbB4 sively mediated by the extracellular region of ErbB3 (22). Fur- (HER4). EGFR binds several distinct ligands including EGF thermore, the interaction between ErbB2 and ErbB3 upon and transforming growth factor-a (3). ErbB3 and ErbB4 bind HRG stimulation is seen with a modified version of the extra- isoforms of the heregulin family (also designated neuregulin or cellular domain of ErbB3 containing a glycosylphosphatidyli- Neu differentiation factor) (4, 5). A ligand that directly binds to nositol moiety anchoring it to the plasma membrane (23). Also, ErbB2 has not been identified. Nevertheless, ErbB2 plays an receptor IgGs consisting of the extracellular domains of ErbB3 and ErbB2 fused to an immunoglobulin Fc domain show in- * The costs of publication of this article were defrayed in part by the creased HRG binding affinity compared with ErbB3 binding payment of page charges. This article must therefore be hereby marked alone (24). Taken together, it appears that HRG binding and “advertisement” in accordance with 18 U.S.C. Section 1734 solely to the affinity shift for HRG binding in the presence of ErbB2 indicate this fact. To whom correspondence should be addressed: Genentech, Inc., require only the extracellular domains of these receptors. Mail Stop 63, South San Francisco, CA 94080. Tel.: 650-225-1247; Fax: Details of the molecular mechanism that leads to the activa- 650-225-5945; E-mail: [email protected]. tion of ErbB2 kinase are unknown, and in particular, the role The abbreviations used are: EGFR, epidermal growth factor recep- of the intracellular domain of ErbB3 is uncertain. We ques- tor; EGF, epidermal growth factor; HRG, heregulin; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. tioned whether structural elements in the intracellular domain This paper is available on line at http://www.jbc.org 859 This is an Open Access article under the CC BY license. 860 ErbB3 Transactivation of ErbB2 was carried out as recommended by the supplier. Samples were counted of ErbB3 were needed for the transactivation of ErbB2. In this using a 100 Series Iso Data g-counter. study, we designed a series of C-terminal deletion and substi- Tyrosine Phosphorylation Assay—COS-7 cells were transfected in tution mutants of ErbB3 and assessed the phosphorylation 12-well plates. After 24 –36 h, cells were washed with serum-free Ham’s status of the receptors in the complex. We report here that a F-12 medium/Dulbecco’s modified Eagle’s medium and serum-starved distinct three-amino acid segment (LVI) in the intracellular for 2– 4 h. Cells were stimulated with the indicated concentrations of domain of ErbB3 is required for transactivation of ErbB2 and HRG. After incubation for 8 min at room temperature, the medium was carefully aspirated, and reactions were stopped by adding 250 mlof propose that this is an intermediate step between ligand-in- sample buffer (5% SDS, 1% dithiothreitol, and 25 mM Tris-HCl, pH 6.8). duced receptor dimerization and kinase activation. Each sample (20 ml) was subjected to SDS-polyacrylamide electrophore- sis using a 4 –12% gradient gel (Novex) and then electrophoretically EXPERIMENTAL PROCEDURES transferred onto a nitrocellulose membrane. Blots were probed with Materials—Monoclonal anti-ErbB2 antibody 3E8 has been described monoclonal anti-phosphotyrosine antibody conjugated to horseradish previously (25). Polyclonal rabbit anti-ErbB2 antibody was acquired peroxidase (125 ng/ml), and immunoreactive bands were visual- from Dako Corp. (Carpinteria, CA). Polyclonal rabbit anti-ErbB3 (C-17) ized with chemiluminescence detection reagent (ECL, Amersham and rabbit anti-ErbB4 antibodies were purchased from Santa Cruz Pharmacia Biotech). Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody conju- Chemical Cross-linking and Immunoprecipitation—Transfected gated to horseradish peroxidase was obtained from Transduction Lab- COS-7 cells were resuspended in Hanks’ balanced salts containing 20 oratories (Lexington, KY). Monoclonal antibody 5B6 raised against the 125 mM HEPES, pH 7.4, and incubated with radiolabeled I-rHRGb1 (0.5 herpes simplex glycoprotein D signal sequence has been described else- nM) in the presence or absence of 200 nM unlabeled rHRGb1. Incubation where (26). The EGF-like domain of HRGb1-(177–244) was expressed was carried out at room temperature for 20 min. Chemical cross-linking in Escherichia coli, purified, and radioiodinated as described previously was performed by adding bis(sulfosuccinimidyl) suberate (Pierce) to a (7, 27). The EGF-like domain of HRGb1-(177–244) was used in all final concentration of 1 mM and allowing it to react for 20 min at room experiments and designated rHRGb1. temperature. Samples were run on 5% SDS-polyacrylamide gels, and Cell Culture and Transient Transfections—COS-7 cells (American cross-linked complexes were visualized by autoradiography. Immuno- Type Culture Collection, CRL1651) and K562 cells (CCL243) were precipitation experiments were performed on transfected COS-7 cells cultured in a 50:50 mixture of Ham’s F-12 medium and Dulbecco’s erbB3 after chemical cross-linking or on K562 cells transfected with vec- modified Eagle’s medium supplemented with 10% heat-inactivated fe- 753 tor alone, erbB2, or erbB2M cDNA. K562 cells were serum-starved tal bovine serum, 2 mM glutamine, and 10% penicillin/streptomycin. for 2 h and treated with rHRGb1 (10 nM) or buffer alone for 15 min at TM COS-7 cells were transfected using the LipofectAMINE protocol ob- 37 °C. K562 or COS-7 cells were lysed in lysis buffer (25 mM Tris, pH tained from Life Technologies, Inc. Transfections were carried out for 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% CHAPS, 0.5 mM 24 –36 h, and experiments were initiated immediately. A stable K562 Na VO , 0.2 mM phenylmethylsulfonyl fluoride, 50 trypsin inhibitory 3 4 cell line that expressed human ErbB3 was generated by electroporation units/liter aprotinin, and 10 mM leupeptin). Immunoprecipitations were using a derivative of pcDNA3 (Invitrogen) containing the open reading performed with anti-ErbB2 (3E8), anti-ErbB2, or anti-ErbB3 antibody. frame of erbB3. This cell line was transiently transfected with the Immune complexes were purified by absorption on immobilized protein appropriate ErbB2 expression plasmids using SuperFect transfection A/G (Ultralink immobilized protein A/G, Pierce), and samples were reagent purchased from QIAGEN, Inc. Cells (1 3 10 ) were transfected subjected to SDS-polyacrylamide gel electrophoresis. In the case of with 30 mg of DNA and 120 ml of SuperFect reagent according to the K562 samples, Western blot analysis with anti-phosphotyrosine anti- supplier’s standard protocol and were subjected to further analysis body was accomplished as described above. after 48 h. Construction of Receptor Mutants—Site-directed mutagenesis was RESULTS performed to obtain C-terminal ErbB3 truncation or internal deletion/ The Intracellular Domain of ErbB3 Is Necessary for Trans- substitution mutants (28). All receptor constructs were derived from a pRK7-based vector (29). The endogenous signal sequences of ErbB2 and activation of the ErbB2 Kinase—To determine whether acti- ErbB3 were replaced with the herpes simplex signal sequence of glyco- vation of the ErbB2 kinase by ErbB3 requires a specific protein D for quantitative analysis (30). The cDNA sequence of erbB2 interaction with the cytoplasmic domain of ErbB3, we con- was subcloned into the XbaI and ClaI sites of pBluescript SK (Strat- structed a truncated version of ErbB3. This mutant, designated agene). Using oligonucleotide 59-CTCTTGCCCCTCGAGGCCGCGAGC- 1– 665 ErbB3 , essentially lacks the entire cytoplasmic domain of 39,an XhoI site at the end of the erbB2 signal sequence was inserted by 1– 665 site-directed mutagenesis. A derivative of pRK7 (pChad) was used as an the receptor. The functional characteristics of ErbB3 were expression plasmid containing the coding sequence for amino acids examined by transiently expressing the truncated receptor in 1–51 of herpes simplex glycoprotein D. The XhoI-ClaI fragment encod- COS-7 cells. ing mature ErbB2 was subcloned into the XhoI and ClaI sites of the 1– 665 Expression of ErbB3 at the cell surface was confirmed pChad vector, fusing the glycoprotein sequence upstream of the erbB2 by competitive binding analysis using I-rHRGb1. Untrans- sequence. The open reading frame of erbB3 was subcloned into the KpnI fected COS-7 cells do not bind I-rHRGb1 because they lack site of pBluescript SK .A SalI site was engineered using oligonucleo- 1– 665 tide 59-GGAGCCCCGGTCGACGCTGAAAAG-39 at the end of the erbB3 endogenous ErbB3 and ErbB4. As expected, ErbB3 -ex- signal sequence. The erbB3 sequence was excised with SalI and sub- pressing cells displayed an rHRGb1-binding site (4.3 3 10 6 cloned in frame into the XhoI site of the pChad vector. C-terminal 3 7.0 3 10 sites/cell) with a dissociation constant of 1.56 6 0.22 truncation mutants were obtained by inserting a stop codon down- nM. This was comparable to cells that were transfected with stream of the last desired amino acid codon. Internal deletion and 4 3 full-length erbB3 (2.7 3 10 6 2.8 3 10 sites/cell) and dis- substitution mutants were engineered using oligonucleotides that re- played a binding affinity of 1.1 6 0.34 nM (Fig. 1A) (7). moved the amino acid codons or replaced the designated amino acid To determine whether deletion of the cytoplasmic domain codons with alanine codons, respectively. Confirmation of receptor con- structs was obtained by DNA sequencing using a dideoxy method mod- interfered with the ability of the receptor to interact with TM ified for double-stranded DNA (T7 Sequenase , Version 2.0, Amer- ErbB2, COS-7 cells were co-transfected with erbB2 and either sham Pharmacia Biotech). 1– 665 erbB3 or erbB3 cDNA, and chemical cross-linking experi- I-rHRG Binding Assay—COS-7 cells were transfected in 15-cm ments were performed using I-rHRGb1 (Fig. 1B). A cross- plates. After 30 h, cells were removed from dishes using 2 mM EDTA in linked product of 100 kDa was observed in cells expressing phosphate-buffered saline. Cells were transferred onto 96-well plates at 1– 665 a density of 1.8 3 10 cells/well in final volume of 250 ml of binding ErbB3 (Fig. 1B, lane 3). The mobility of this band was buffer (Dulbecco’s modified Eagle’s medium, 10% penicillin/streptomy- consistent with the predicted size of the mutated receptor. In cin, and 0.2% bovine serum albumin). Cells were incubated with varied addition, a higher molecular mass complex was also present. concentrations of rHRGb1 and a constant amount (75 pM)of I-labeled The mobility of this higher molecular mass complex was rHRGb1. Binding was carried out on ice for 14 h. To separate cell-bound 125 slightly faster than that observed when wild-type ErbB3 was I-rHRGb1 from free, cells were transferred onto a 96-well filtration co-expressed with ErbB2 (Fig. 1B, lane 1). plate assembly (Multiscreen assay system, Millipore Corp.) and placed 1– 665 on a vacuum manifold to wash and remove unbound label. Separation To confirm the association of ErbB3 with ErbB2, cross- ErbB3 Transactivation of ErbB2 861 FIG.1. Binding analysis of I- rHRGb1 to COS-7 cells expressing 1– 665 125 ErbB3 . A, displacement of I- rHRGb1 binding to COS-7 cell transfec- tants by unlabeled rHRGb1. Transiently 1– 665 transfected cells expressing ErbB3 (l) or ErbB3 (M) were incubated with radiolabeled rHRGb1 and the indicated amounts of unlabeled rHRGb1. Incuba- tion was carried out on ice overnight, and cell-bound radiolabeled ligand was sepa- rated from unbound. The results are shown as displacement curves or Scat- chard plot (inset). B, chemical cross-link- ing of I-HRGb1 to cell-surface recep- tors on COS-7 cell transfectants and subsequent immunoprecipitation with anti-ErbB2 antibody. Cells were co-trans- fected with full-length erbB2 and full- length erbB3 or with full-length erbB2 1– 665 and erbB3 expression plasmids, re- spectively. Cells were detached and, prior to cross-linking, were incubated with 0.5 nM I-rHRGb1 in the presence (1)or absence (2) of 200 nM unlabeled rHRGb1. Bis(sulfosuccinimidyl) suberate was added to the cell suspension, and incubations were continued. Cells were washed and resuspended in SDS sample buffer. For lanes 5 and 6, following cross-linking, cell lysates were prepared, and immunopre- cipitation (IP) was performed with anti- ErbB2 antibody 3E8. Samples were run on a 5% SDS-polyacrylamide gel, and radioac- tive cross-linked complexes were visualized by autoradiography. xsHRG, cross-linked HRG; IP, immunoprecipitation. 1– 665 linked proteins were immunoprecipitated using a monoclonal ErbB3 with ErbB2 was similar to that of the full-length antibody (3E8) directed against ErbB2. As shown in lanes 5 receptor. The results presented in Fig. 1 show that deletion of and 6 in Fig. 1B, a cross-linking pattern similar to that seen in the entire cytoplasmic region of ErbB3 does not interfere with lanes 1 and 3 was observed. Since I-rHRGb1 does not be- either its ligand binding characteristics or its ability to form come directly cross-linked to ErbB2, these data indicated that heteromeric complexes with ErbB2. 1– 665 the receptor complexes in both the ErbB3- and ErbB3 - We next looked at the ability of truncated ErbB3 to induce transfected cells contained ErbB2. Therefore, the high molec- tyrosine phosphorylation upon HRG stimulation. Since COS-7 ular mass complex represents cross-linked receptors and cross- cells naturally express low levels of ErbB2, which form hetero- linked radiolabeled HRG. Interestingly, these complexes meric complexes with ectopically expressed ErbB3, co-transfec- migrated slower than expected for an ErbB2-ErbB3 receptor tion with exogenous erbB2 was not necessary in these experi- dimer, suggesting that a higher order complex was formed ments. Cells that expressed full-length ErbB3 showed a dose- instead. The bands at 190 and 100 kDa in lanes 5 and 6 dependent increase in tyrosine phosphorylation after HRG 1– 665 represented ErbB3 and ErbB3 , respectively, which were stimulation (Fig. 2). The double band visible at ;185 kDa associated with ErbB2, but not covalently cross-linked to it. represented phosphorylated ErbB2 and ErbB3. In contrast, 1– 665 Furthermore, these data suggested that the interaction of although ErbB3 binds HRG and associates with ErbB2, it 862 ErbB3 Transactivation of ErbB2 that were designed to fine map the region responsible for ErbB2 transactivation. ErbB3D955–962, which lacked the eight amino acids that span the junction between the kinase domain and the C-terminal tail, was devoid of receptor phos- phorylation activity. Additional constructs with smaller inter- nal deletions were engineered and tested for transactivation 957 958 activity. Deletion of three amino acid residues, Leu , Val , and Ile (ErbB3DLVI), was sufficient to abolish phosphoryl- ation following HRG stimulation (Fig. 4B). In contrast, deletion 957 958 of two amino acid residues, Leu and Val (ErbB3DLV) and 958 959 Val and Ile (ErbB3DVI), did not affect the transactivation potential. FIG.2. HRG-stimulated receptor autophosphorylation. COS-7 1– 665 cells were transfected in 12-well plates with erbB3 or erbB3 ex- It is possible that deletion of three amino acids at positions pression plasmids. Cells were treated 28 –32 h post-transfection with 957–959 altered the spatial alignment of important structural the indicated amounts of rHRGb1. Cell lysates were subjected to SDS features on ErbB3 relative to corresponding sites on ErbB2. gel electrophoresis and then transferred to nitrocellulose. For the upper The inability of ErbB3DLVI to activate the ErbB2 kinase might panels, blots were probed with anti-phosphotyrosine antibody (aP-Tyr). The lower panels demonstrate ErbB3 expression. Blots were probed have resulted from a gross positional change in the ErbB3 with monoclonal antibody 5B6, which recognizes glycoprotein D (gD)- polypeptide backbone relative to ErbB2, rather than deletion of 1– 665 tagged ErbB3 and ErbB3 . a specific activation motif. To address this possibility, we re- 957 958 959 placed Leu , Val , and Ile with alanine residues. This was not able to activate the ErbB2 kinase. The results shown in removes hydrophobic side chains available for potential inter- Fig. 2 suggested that a direct or indirect interaction between molecular interactions, but maintains the length of the the intracellular domains of ErbB3 and ErbB2 was necessary polypeptide chain. A phosphorylation signal was not observed for HRG-mediated activation of the intrinsic kinase activity of in cells expressing L957A/V958A/I959A (Fig. 4B), indicating ErbB2. that the sequence LVI is required for transactivation. Mapping the Intracellular Transactivation Domain of To further characterize this area, a series of double and ErbB3—We questioned whether the entire intracellular do- single substitution mutants were designed. These mutants are main of ErbB3 or only a segment of it was required for ErbB2 outlined in schematic form in Fig. 4A along with results of the transactivation. To address this issue, a series of ErbB3 trun- phosphorylation analysis for each. Interestingly, in contrast to cation mutants containing smaller deletions from the C termi- ErbB3DLV, cells expressing L957A/V958A did not transacti- nus were constructed. These are shown in schematic form in vate ErbB2 upon HRG stimulation. One explanation for this Fig. 3A. The receptor mutants were transiently expressed and finding is that, in ErbB3DLV, isoleucine mimics leucine at subjected to HRG-induced receptor activation analysis as de- position 957. But in L957A/V958A, the substituted alanine scribed above. In these mutants, the phosphorylation signals residue was not able to replace Leu . This possibility was observed were exclusively due to tyrosine phosphorylation of further explored with mutant L957A. The replacement of endogenous ErbB2 because the truncation mutants lacked the leucine with an alanine residue resulted in a strong decrease in tyrosine phosphorylation sites located on the C terminus of the the phosphorylation signal, further indicating that Leu full-length receptor. Phosphorylation results are also indicated 1–952 played a major role in the transactivation. From these data, we in Fig. 3A. Surprisingly, ErbB3 did not show HRG-induced 1–963 conclude that the minimal region necessary for full transacti- phosphorylation, whereas ErbB3 showed a dose-depend- vation of the ErbB2 kinase upon HRG stimulation is the LVI ent increase in ErbB2 phosphorylation upon HRG stimulation. sequence beginning at position 957. Moreover, Leu appears To further map this area, we engineered additional truncation to play a key role in this hydrophobic motif. However, we mutants to identify regions that may contribute to ErbB2 1–959 cannot rule out the possibility that areas N-terminal to this transactivation. ErbB3 was able to stimulate receptor motif may also contribute to transactivation of ErbB2. phosphorylation, yet additional deletion of Ile resulted in a The LVI Segment Is Conserved in ErbB4 and Is Necessary for decreased phosphorylation signal, and deletion of Val in 1–957 ErbB2 Transactivation—Sequence alignment of the tyrosine ErbB3 completely abolished stimulation of tyrosine phos- kinase domains of members of the ErbB receptor family re- phorylation (Fig. 3B). Thus, we were able to localize the region vealed that the LVI segment is conserved in ErbB4 as well as responsible for the transactivation activity to an area N-termi- ErbB3 and EGFR (Fig. 5). We questioned whether a similar nal to amino acid 959. This critical region is found at the transactivation mechanism also occurred between ErbB2 and C-terminal end of the impaired kinase domain, as shown in Fig. ErbB4. To assess ErbB2 transactivation in an ErbB2-ErbB4 3C. heterodimer, we eliminated the intrinsic kinase activity of Fine Mapping of the Transactivation Area in the Context of ErbB4 by constructing an ErbB4 mutant in which Lys at the the Full-length Receptor by Internal Deletion and Substitution ATP-binding site was replaced with methionine (ErbB4M ) Mutants—Further characterization of the ErbB3 transactiva- (Fig. 6A). The lack of kinase activity in this mutant was verified tion region was carried out by constructing internal deletion by phosphorylation analysis using a human hematopoietic cell and substitution mutants in the context of the full-length re- line (K562) that is devoid of all ErbB family members (data not ceptor. Although Tyr on ErbB3 has not been described as a shown). Receptor activation analysis in COS-7 cells expressing potential phosphorylation site, we questioned whether Tyr might play a role in the regulation of transactivation, possibly ErbB4M showed a dose-dependent phosphorylation signal upon HRG treatment (Fig. 6B). These data confirmed that a by serving as a phosphorylation site for another kinase. We therefore replaced Tyr with phenylalanine in the full-length similar transactivation of ErbB2 also occurred in ErbB2-ErbB4 heterodimers. To investigate the importance of the LVI seg- ErbB3 receptor. Analysis of the substitution mutant Y956F showed a strong phosphorylation signal, indicating that Tyr ment in ErbB4, a three-amino acid deletion mutant lacking 960 961 962 does not play a direct role in the transactivation process (Fig. residues Leu , Val , and Ile was constructed in the con- 726 726 4A). text of ErbB4M . COS-7 cells expressing ErbB4M DLVI Fig. 4A summarizes a series of internal deletion mutants revealed no increase in tyrosine phosphorylation signal upon ErbB3 Transactivation of ErbB2 863 FIG.3. Receptor phosphorylation analysis of ErbB3 truncation mu- tants. A, schematic representation of C- terminal deletion mutants and their abil- ity to induce phosphorylation in COS-7 cells. The portion of the extracellular do- main is indicated as ECD; the transmem- brane domains (TM) are represented as hatched boxes; and the tyrosine kinase domains (TK) or portions of them are rep- resented as black boxes. The mature re- ceptor is indicated as ErbB3, whereas the various C-terminal truncation mutants are designated according to their remain- ing amino acid residues. C-terminal mu- tants were analyzed in phosphorylation assays as described in the legend to Fig. 2, and their ability (1) or inability (2)to induce receptor phosphorylation is indi- cated on the right. B, phosphorylation 1–959 1–958 analysis of ErbB3 , ErbB3 , and 1–957 ErbB3 . COS-7 cells were transfected with the corresponding expression plas- mids, and tyrosine phosphorylation assay was performed as described in the legend to Fig. 2. C, partial amino acid sequence of ErbB3 that spans the junction between the kinase domain and the C-terminal tail. Amino acid residues of the kinase domain are represented in boldface. The number beneath the amino acid isoleucine indicates its position in the mature receptor. aP-Tyr, anti-phosphotyrosine antibody; agD, anti-glycoprotein D antibody. HRG treatment (Fig. 6B). Thus, analogous to the ErbB3 situ- either erbB2M or control expression plasmids, HRG stimu- ation, activation of ErbB2 kinase also requires the LVI motif in lation caused no autophosphorylation on ErbB2 or ErbB3. The ErbB2-ErbB4 heterodimers. expression of ErbB2 and ErbB2M was verified by Western An Associated Cytoplasmic Kinase Is Not Involved in Recep- blot analysis (Fig. 7B). These data showed that deactivation of tor Autophosphorylation upon HRG Stimulation—It is conceiv- the ErbB2 kinase completely abolished the phosphorylation able that ErbB2 transactivation is not the result of a direct signal and confirmed that ErbB3 has no intrinsic kinase activ- interaction that occurs solely between ErbB3 and ErbB2. One ity. Autophosphorylation of the receptors was therefore not due scenario is that a third protein with tyrosine kinase activity is to a cytoplasmic kinase activated by binding to the transacti- bound to the cytoplasmic domain of ErbB3 and that this kinase vation segment of ErbB3, but was the result of the ErbB2 phosphorylates ErbB2. This would be similar to the transacti- kinase. vation mechanism observed in cytokine receptor and T-cell DISCUSSION antigen receptor signaling (31, 32). If the activity of a third kinase was directly regulated by ErbB3, the kinase activity of The allosteric oligomerization model proposed for EGFR by ErbB2 would not be required for receptor phosphorylation. To Schlessinger (33, 34) predicts that ligand binding induces for- test this possibility, we constructed a kinase-inactive ErbB2 mation of receptor dimers, which brings the intracellular do- mutant in which Lys at the ATP-binding site was replaced mains into close proximity, and causes them to phosphorylate with methionine. Ectopic expression of this mutant was then one another in trans. Because ErbB3 lacks intrinsic kinase performed in a hematopoietic cell line that expressed only activity and ErbB2 does not bind HRG, this model does not erbB3 ErbB3 (K562 ) (Fig. 7A). If a cytoplasmic kinase was re- fully explain the phosphorylation pattern observed in the sponsible for autophosphorylation, cells that co-express ErbB3 ErbB2-ErbB3 complex following HRG stimulation. It was also and kinase-inactive ErbB2 would still show a phosphorylation previously unclear whether activation of the intrinsic kinase signal upon HRG treatment. Cells transfected with wild-type required specific cytoplasmic domain interactions. Here, we erbB2, erbB2M , or control expression plasmids were incu- present data showing that transactivation of the ErbB2 kinase bated with HRG and then subjected to immunoprecipitation by ErbB3 requires the presence of a structural element within with antibodies directed against ErbB2 or ErbB3. Cells trans- the ErbB3 cytoplasmic domain. Using ErbB3 deletion and sub- fected with erbB2 cDNA demonstrated tyrosine phosphoryla- stitution mutants, we found a discrete sequence of three amino tion on ErbB2 and ErbB3 (Fig. 7B, lanes 1– 4). Interestingly, acids (LVI) at the carboxyl terminus of the inactive kinase ErbB2 was constitutively phosphorylated, whereas ErbB3 domain of ErbB3 that is necessary for ErbB2 transactivation. phosphorylation was HRG-dependent. In cells transfected with Deletion of segments distal to this region had no effect, indi- 864 ErbB3 Transactivation of ErbB2 FIG.4. Tyrosine phosphorylation analysis of internal and substitution mutants. A, schematic diagram of recep- tor mutants and their ability to induce receptor autophosphorylation. Receptors and receptor domains are depicted as de- scribed in the legend to Fig. 3. Internal deletion areas are shown as gaps. Substi- tuted amino acid residues are indicated beneath the receptors. Receptor mutants underwent phosphorylation analysis as described in the legend to Fig. 2, and their ability (1) or inability (2) to induce transphosphorylation is indicated on the right. B, phosphorylation analysis of ErbB3 mutants ErbB3DLVI and L957A/ V958A/I959A. COS-7 cells were trans- fected with corresponding expression plasmids and analyzed for receptor phos- phorylation as described in the legend to Fig. 2. aP-Tyr, anti-phosphotyrosine anti- body; agD, anti-glycoprotein D antibody. FIG.5. Sequence alignment of the C-terminal ends of the ErbB tyrosine kinase domains. Sequences are displayed in a single letter code; identical residues are denoted as dots. Amino acid sequences are numbered according to the mature proteins. The LVI segment is boxed. The C-terminal ends (C-term) of the tyrosine kinase (TK) domains were determined as described by Plowman et al. (5). cating that they are not required for this activity. At present, we cannot rule out the possibility that additional sequences N-terminal to this region may also contribute to the transacti- vation of ErbB2. The results of our study suggest at least two models whereby direct sequence-specific molecular interactions may result in receptor transactivation. These models extend the Schlessinger FIG.6. Receptor phosphorylation analysis of ErbB4 mutants. hypothesis (33, 34) to the ErbB2-ErbB3 system and are shown A, ErbB4 mutants shown as described in the legend to Fig. 3. The schematically in Fig. 8. In the first model, activation of ErbB2 ErbB4 receptor construct with Lys replaced by Met is designated results from a direct interaction between the intracellular do- 726 726 ErbB4M . ErbB4M DLVI lacked, in addition to the amino acid sub- 960 961 962 mains of the two receptors. In this model, HRG binding to stitution, Leu , Val , and Ile . B, phosphorylation analysis of ErbB4 mutants. COS-7 cells were transfected with the corresponding ErbB3 results in a conformational change in the extracellular expression plasmids, and cells were subjected to phosphorylation anal- domain of ErbB3 facilitating recruitment of ErbB2 and forma- ysis as described in the legend to Fig. 2. ErbB4 expression was verified tion of the heterodimeric complex. The interaction between the using a polyclonal anti-ErbB4 antibody. aP-Tyr, anti-phosphotyrosine extracellular domains of the receptors aligns their intracellular antibody. domains, bringing the LVI motif of ErbB3 into direct contact with an as yet undefined region of ErbB2. This, in turn, leads heterodimerization of ErbB3 with ErbB2 allows an adaptor to the activation of the ErbB2 kinase. An alternative model molecule with specific recognition sites for each receptor to assumes the participation of a third protein. Ligand-induced bridge their intracellular domains. The putative adaptor pro- ErbB3 Transactivation of ErbB2 865 erbB3 FIG.7. An associated cytoplasmic kinase is not involved in receptor autophosphorylation. A, ErbB3 expression in stable K562 transfectants. Anti-ErbB3 antibody was used to detect ErbB3 expression in the cell lysates of the transfectants. B, phosphorylation analysis in erbB3 732 K562 cells transiently transfected with erbB2 or kinase-inactive erbB2 (erbB2M ). Incubations with or without rHRGb1 (10 nM) were performed for 10 min at room temperature, 48 h post-transfection. Polyclonal antibodies to ErbB2 or ErbB3 were used for immunoprecipitations (IP). Immune complexes were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose. Receptor phosphorylation was determined using anti-phosphotyrosine antibody (aP-Tyr). ErbB2 expression was verified using monoclonal antibody 5B6, which recognizes glycoprotein D(gD)-tagged ErbB2 and ErbB2M . WB, Western blot. tive agent. To date, several approaches have been undertaken to intercept the signal generation (42). For example, antibodies that directly bind to the extracellular domain of ErbB2 or EGFR have been very efficient as antiproliferative reagents (43– 45). The selective inhibition of EGFR tyrosine kinase with various small molecules also results in antitumor activity (46). The identification of the transactivation sequence in ErbB3 suggests another way to block signaling in a heteromeric FIG.8. Transactivation models. The schematic depicts two models complex. A compound that directly interacts with the LVI of HRG-mediated transactivation of ErbB3. ErbB3 and ErbB2 are des- segment in ErbB3 could inhibit specific transactivation, re- ignated H3 and H2, respectively. The extracellular and intracellular ceptor phosphorylation, and consequently all downstream domains are shown as distinct barrels. The LVI segment in ErbB3 is symbolized as a black half-circle; a so far unidentified interaction region signaling pathways. in ErbB2 is shown as a hatched box. Acknowledgment— We thank the DNA synthesis/purification group at Genentech, Inc. for supplying oligonucleotides. tein binds to the LVI sequence in ErbB3 and to an unknown REFERENCES sequence in ErbB2. We postulate that the adaptor stabilizes 1. van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. 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A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 737–744 367–382 45. Baselga, J., and Mendelsohn, J. (1994) Breast Cancer Res. Treat. 29, 127–138 29. Gorman, C. M., Gies, D. R., and McCray, G. (1990) DNA Protein Eng. Tech. 2, 46. Klohs, W. D., Fry, D. W., and Kraker, A. J. (1997) Curr. Opin. Oncol. 9, 2–10 562–568 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

A Discrete Three-amino Acid Segment (LVI) at the C-terminal End of Kinase-impaired ErbB3 Is Required for Transactivation of ErbB2

Schaefer, Gabriele; Akita, Robert W.; Sliwkowski, Mark X.
Journal of Biological Chemistry – Jan 1, 1999
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10.1074/jbc.274.2.859
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 2, Issue of January 8, pp. 859 –866, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Discrete Three-amino Acid Segment (LVI) at the C-terminal End of Kinase-impaired ErbB3 Is Required for Transactivation of ErbB2* (Received for publication, September 2, 1998, and in revised form, October 7, 1998) Gabriele Schaefer‡§, Robert W. Akita‡, and Mark X. Sliwkowski‡¶ From ‡Genentech, Inc., South San Francisco, California 94080 and the §Institut fu ¨ r Biologie III, University of Freiburg, D-79104 Freiburg im Breisgau, Germany ErbB3 is unique among other members of the receptor important role in signaling. ErbB2 is transactivated by het- tyrosine kinase family of growth factor receptors in that erodimerization with ligand-occupied EGFR, ErbB3, or ErbB4 its kinase domain is enzymatically impaired. This ren- (6 –9). ders it incapable of transducing a signal in response to The extensive interreceptor associations that occur in the ligand binding. However, in conjunction with ErbB2, ErbB family serve to increase the repertoire of cellular re- ErbB3 is a potent mediator of signaling by the growth sponses to growth factor stimulation and to fine-tune growth factor heregulin. Heregulin binding to ErbB3 induces factor signaling. At least 10 different homo- and heteromeric formation of a heterodimeric complex with ErbB2, and combinations of ErbB proteins have been reported (10, 11). this results in transactivation of the ErbB2 kinase. Al- However, these combinations are not equally favorable. The though interaction between the extracellular domains interreceptor interactions are hierarchically organized, where of these receptors is an essential part of this process, it ErbB2 is the preferred heteromeric partner, and it favors in- was not clear whether interaction between the cytoplas- teraction with ErbB3 (12, 13). mic domains is also necessary for transactivation. By Cross-talk between ErbB2 and ErbB3 is especially important examining the abilities of a series of cytoplasmic domain as the kinase of ErbB3 is dysfunctional. The impaired kinase mutants of ErbB3 to activate ErbB2, we have found a activity has been demonstrated in several systems. It was discrete sequence of three amino acid residues (LVI), initially reported in insect cells expressing ErbB3 (14), later by located at the carboxyl-terminal end of the impaired ErbB3 kinase region, that is obligatory for transactiva- demonstrating the lack of any biological activity in cells ex- tion. We conclude that formation of a functional ErbB2- pressing ErbB3 alone (10, 11), and recently by biochemical ErbB3 signaling complex requires the presence of a spe- analysis of the purified kinase domain (15). Alterations of four cific structural feature within the ErbB3 cytoplasmic amino acid residues in the kinase region that are otherwise domain and suggest that ErbB2 transactivation results conserved among all protein tyrosine kinases (16) may account from a physical interaction between the cytoplasmic for the lack of catalytic activity. ErbB2, however, is character- domains of these receptors. ized by a constitutively active kinase (17). The physiological significance of this heteromeric complex is emphasized by the fact that the presence of ErbB2 in ErbB3-expressing cells sig- Receptor tyrosine kinases play a pivotal role in the transduc- nificantly enhances the transformation ability (18, 19). Inhibi- tion of extracellular signals into the cells. The binding of cog- tion of ErbB2 and ErbB3 complex formation abolishes HRG- nate growth factors to these cell-surface receptors results in mediated signaling (9, 13, 20). Additionally, active ErbB2- receptor oligomerization and activation of the intrinsic kinase ErbB3 receptor complexes have been seen in several mammary activity (1, 2). This leads to receptor phosphorylation and trig- tumor cell lines, indicating the relevance of this heteromeric gers a cascade of intracellular signaling events that ultimately receptor aggregate in human neoplasia (18, 21). elicit a variety of cellular responses such as proliferation, dif- HRG binds with low affinity to kinase-inactive ErbB3. Re- ferentiation, survival, or migration. cruitment of ErbB2 into the HRG-ErbB3 complex leads to the An extensively characterized subgroup of this receptor su- formation of a high affinity HRG-binding receptor, which is perfamily is the ErbB group of receptors, also known as the capable of generating a tyrosine phosphorylation signal due to class I receptor tyrosine kinases. Members of this group include 1 the kinase activity of ErbB2 (7). Studies on the isolated extra- the epidermal growth factor receptor (EGFR or ErbB1), ErbB2 cellular domain of ErbB3 show that ligand binding is exclu- (also termed HER2 or Neu), ErbB3 (HER3), and ErbB4 sively mediated by the extracellular region of ErbB3 (22). Fur- (HER4). EGFR binds several distinct ligands including EGF thermore, the interaction between ErbB2 and ErbB3 upon and transforming growth factor-a (3). ErbB3 and ErbB4 bind HRG stimulation is seen with a modified version of the extra- isoforms of the heregulin family (also designated neuregulin or cellular domain of ErbB3 containing a glycosylphosphatidyli- Neu differentiation factor) (4, 5). A ligand that directly binds to nositol moiety anchoring it to the plasma membrane (23). Also, ErbB2 has not been identified. Nevertheless, ErbB2 plays an receptor IgGs consisting of the extracellular domains of ErbB3 and ErbB2 fused to an immunoglobulin Fc domain show in- * The costs of publication of this article were defrayed in part by the creased HRG binding affinity compared with ErbB3 binding payment of page charges. This article must therefore be hereby marked alone (24). Taken together, it appears that HRG binding and “advertisement” in accordance with 18 U.S.C. Section 1734 solely to the affinity shift for HRG binding in the presence of ErbB2 indicate this fact. To whom correspondence should be addressed: Genentech, Inc., require only the extracellular domains of these receptors. Mail Stop 63, South San Francisco, CA 94080. Tel.: 650-225-1247; Fax: Details of the molecular mechanism that leads to the activa- 650-225-5945; E-mail: [email protected]. tion of ErbB2 kinase are unknown, and in particular, the role The abbreviations used are: EGFR, epidermal growth factor recep- of the intracellular domain of ErbB3 is uncertain. We ques- tor; EGF, epidermal growth factor; HRG, heregulin; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. tioned whether structural elements in the intracellular domain This paper is available on line at http://www.jbc.org 859 This is an Open Access article under the CC BY license. 860 ErbB3 Transactivation of ErbB2 was carried out as recommended by the supplier. Samples were counted of ErbB3 were needed for the transactivation of ErbB2. In this using a 100 Series Iso Data g-counter. study, we designed a series of C-terminal deletion and substi- Tyrosine Phosphorylation Assay—COS-7 cells were transfected in tution mutants of ErbB3 and assessed the phosphorylation 12-well plates. After 24 –36 h, cells were washed with serum-free Ham’s status of the receptors in the complex. We report here that a F-12 medium/Dulbecco’s modified Eagle’s medium and serum-starved distinct three-amino acid segment (LVI) in the intracellular for 2– 4 h. Cells were stimulated with the indicated concentrations of domain of ErbB3 is required for transactivation of ErbB2 and HRG. After incubation for 8 min at room temperature, the medium was carefully aspirated, and reactions were stopped by adding 250 mlof propose that this is an intermediate step between ligand-in- sample buffer (5% SDS, 1% dithiothreitol, and 25 mM Tris-HCl, pH 6.8). duced receptor dimerization and kinase activation. Each sample (20 ml) was subjected to SDS-polyacrylamide electrophore- sis using a 4 –12% gradient gel (Novex) and then electrophoretically EXPERIMENTAL PROCEDURES transferred onto a nitrocellulose membrane. Blots were probed with Materials—Monoclonal anti-ErbB2 antibody 3E8 has been described monoclonal anti-phosphotyrosine antibody conjugated to horseradish previously (25). Polyclonal rabbit anti-ErbB2 antibody was acquired peroxidase (125 ng/ml), and immunoreactive bands were visual- from Dako Corp. (Carpinteria, CA). Polyclonal rabbit anti-ErbB3 (C-17) ized with chemiluminescence detection reagent (ECL, Amersham and rabbit anti-ErbB4 antibodies were purchased from Santa Cruz Pharmacia Biotech). Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibody conju- Chemical Cross-linking and Immunoprecipitation—Transfected gated to horseradish peroxidase was obtained from Transduction Lab- COS-7 cells were resuspended in Hanks’ balanced salts containing 20 oratories (Lexington, KY). Monoclonal antibody 5B6 raised against the 125 mM HEPES, pH 7.4, and incubated with radiolabeled I-rHRGb1 (0.5 herpes simplex glycoprotein D signal sequence has been described else- nM) in the presence or absence of 200 nM unlabeled rHRGb1. Incubation where (26). The EGF-like domain of HRGb1-(177–244) was expressed was carried out at room temperature for 20 min. Chemical cross-linking in Escherichia coli, purified, and radioiodinated as described previously was performed by adding bis(sulfosuccinimidyl) suberate (Pierce) to a (7, 27). The EGF-like domain of HRGb1-(177–244) was used in all final concentration of 1 mM and allowing it to react for 20 min at room experiments and designated rHRGb1. temperature. Samples were run on 5% SDS-polyacrylamide gels, and Cell Culture and Transient Transfections—COS-7 cells (American cross-linked complexes were visualized by autoradiography. Immuno- Type Culture Collection, CRL1651) and K562 cells (CCL243) were precipitation experiments were performed on transfected COS-7 cells cultured in a 50:50 mixture of Ham’s F-12 medium and Dulbecco’s erbB3 after chemical cross-linking or on K562 cells transfected with vec- modified Eagle’s medium supplemented with 10% heat-inactivated fe- 753 tor alone, erbB2, or erbB2M cDNA. K562 cells were serum-starved tal bovine serum, 2 mM glutamine, and 10% penicillin/streptomycin. for 2 h and treated with rHRGb1 (10 nM) or buffer alone for 15 min at TM COS-7 cells were transfected using the LipofectAMINE protocol ob- 37 °C. K562 or COS-7 cells were lysed in lysis buffer (25 mM Tris, pH tained from Life Technologies, Inc. Transfections were carried out for 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% CHAPS, 0.5 mM 24 –36 h, and experiments were initiated immediately. A stable K562 Na VO , 0.2 mM phenylmethylsulfonyl fluoride, 50 trypsin inhibitory 3 4 cell line that expressed human ErbB3 was generated by electroporation units/liter aprotinin, and 10 mM leupeptin). Immunoprecipitations were using a derivative of pcDNA3 (Invitrogen) containing the open reading performed with anti-ErbB2 (3E8), anti-ErbB2, or anti-ErbB3 antibody. frame of erbB3. This cell line was transiently transfected with the Immune complexes were purified by absorption on immobilized protein appropriate ErbB2 expression plasmids using SuperFect transfection A/G (Ultralink immobilized protein A/G, Pierce), and samples were reagent purchased from QIAGEN, Inc. Cells (1 3 10 ) were transfected subjected to SDS-polyacrylamide gel electrophoresis. In the case of with 30 mg of DNA and 120 ml of SuperFect reagent according to the K562 samples, Western blot analysis with anti-phosphotyrosine anti- supplier’s standard protocol and were subjected to further analysis body was accomplished as described above. after 48 h. Construction of Receptor Mutants—Site-directed mutagenesis was RESULTS performed to obtain C-terminal ErbB3 truncation or internal deletion/ The Intracellular Domain of ErbB3 Is Necessary for Trans- substitution mutants (28). All receptor constructs were derived from a pRK7-based vector (29). The endogenous signal sequences of ErbB2 and activation of the ErbB2 Kinase—To determine whether acti- ErbB3 were replaced with the herpes simplex signal sequence of glyco- vation of the ErbB2 kinase by ErbB3 requires a specific protein D for quantitative analysis (30). The cDNA sequence of erbB2 interaction with the cytoplasmic domain of ErbB3, we con- was subcloned into the XbaI and ClaI sites of pBluescript SK (Strat- structed a truncated version of ErbB3. This mutant, designated agene). Using oligonucleotide 59-CTCTTGCCCCTCGAGGCCGCGAGC- 1– 665 ErbB3 , essentially lacks the entire cytoplasmic domain of 39,an XhoI site at the end of the erbB2 signal sequence was inserted by 1– 665 site-directed mutagenesis. A derivative of pRK7 (pChad) was used as an the receptor. The functional characteristics of ErbB3 were expression plasmid containing the coding sequence for amino acids examined by transiently expressing the truncated receptor in 1–51 of herpes simplex glycoprotein D. The XhoI-ClaI fragment encod- COS-7 cells. ing mature ErbB2 was subcloned into the XhoI and ClaI sites of the 1– 665 Expression of ErbB3 at the cell surface was confirmed pChad vector, fusing the glycoprotein sequence upstream of the erbB2 by competitive binding analysis using I-rHRGb1. Untrans- sequence. The open reading frame of erbB3 was subcloned into the KpnI fected COS-7 cells do not bind I-rHRGb1 because they lack site of pBluescript SK .A SalI site was engineered using oligonucleo- 1– 665 tide 59-GGAGCCCCGGTCGACGCTGAAAAG-39 at the end of the erbB3 endogenous ErbB3 and ErbB4. As expected, ErbB3 -ex- signal sequence. The erbB3 sequence was excised with SalI and sub- pressing cells displayed an rHRGb1-binding site (4.3 3 10 6 cloned in frame into the XhoI site of the pChad vector. C-terminal 3 7.0 3 10 sites/cell) with a dissociation constant of 1.56 6 0.22 truncation mutants were obtained by inserting a stop codon down- nM. This was comparable to cells that were transfected with stream of the last desired amino acid codon. Internal deletion and 4 3 full-length erbB3 (2.7 3 10 6 2.8 3 10 sites/cell) and dis- substitution mutants were engineered using oligonucleotides that re- played a binding affinity of 1.1 6 0.34 nM (Fig. 1A) (7). moved the amino acid codons or replaced the designated amino acid To determine whether deletion of the cytoplasmic domain codons with alanine codons, respectively. Confirmation of receptor con- structs was obtained by DNA sequencing using a dideoxy method mod- interfered with the ability of the receptor to interact with TM ified for double-stranded DNA (T7 Sequenase , Version 2.0, Amer- ErbB2, COS-7 cells were co-transfected with erbB2 and either sham Pharmacia Biotech). 1– 665 erbB3 or erbB3 cDNA, and chemical cross-linking experi- I-rHRG Binding Assay—COS-7 cells were transfected in 15-cm ments were performed using I-rHRGb1 (Fig. 1B). A cross- plates. After 30 h, cells were removed from dishes using 2 mM EDTA in linked product of 100 kDa was observed in cells expressing phosphate-buffered saline. Cells were transferred onto 96-well plates at 1– 665 a density of 1.8 3 10 cells/well in final volume of 250 ml of binding ErbB3 (Fig. 1B, lane 3). The mobility of this band was buffer (Dulbecco’s modified Eagle’s medium, 10% penicillin/streptomy- consistent with the predicted size of the mutated receptor. In cin, and 0.2% bovine serum albumin). Cells were incubated with varied addition, a higher molecular mass complex was also present. concentrations of rHRGb1 and a constant amount (75 pM)of I-labeled The mobility of this higher molecular mass complex was rHRGb1. Binding was carried out on ice for 14 h. To separate cell-bound 125 slightly faster than that observed when wild-type ErbB3 was I-rHRGb1 from free, cells were transferred onto a 96-well filtration co-expressed with ErbB2 (Fig. 1B, lane 1). plate assembly (Multiscreen assay system, Millipore Corp.) and placed 1– 665 on a vacuum manifold to wash and remove unbound label. Separation To confirm the association of ErbB3 with ErbB2, cross- ErbB3 Transactivation of ErbB2 861 FIG.1. Binding analysis of I- rHRGb1 to COS-7 cells expressing 1– 665 125 ErbB3 . A, displacement of I- rHRGb1 binding to COS-7 cell transfec- tants by unlabeled rHRGb1. Transiently 1– 665 transfected cells expressing ErbB3 (l) or ErbB3 (M) were incubated with radiolabeled rHRGb1 and the indicated amounts of unlabeled rHRGb1. Incuba- tion was carried out on ice overnight, and cell-bound radiolabeled ligand was sepa- rated from unbound. The results are shown as displacement curves or Scat- chard plot (inset). B, chemical cross-link- ing of I-HRGb1 to cell-surface recep- tors on COS-7 cell transfectants and subsequent immunoprecipitation with anti-ErbB2 antibody. Cells were co-trans- fected with full-length erbB2 and full- length erbB3 or with full-length erbB2 1– 665 and erbB3 expression plasmids, re- spectively. Cells were detached and, prior to cross-linking, were incubated with 0.5 nM I-rHRGb1 in the presence (1)or absence (2) of 200 nM unlabeled rHRGb1. Bis(sulfosuccinimidyl) suberate was added to the cell suspension, and incubations were continued. Cells were washed and resuspended in SDS sample buffer. For lanes 5 and 6, following cross-linking, cell lysates were prepared, and immunopre- cipitation (IP) was performed with anti- ErbB2 antibody 3E8. Samples were run on a 5% SDS-polyacrylamide gel, and radioac- tive cross-linked complexes were visualized by autoradiography. xsHRG, cross-linked HRG; IP, immunoprecipitation. 1– 665 linked proteins were immunoprecipitated using a monoclonal ErbB3 with ErbB2 was similar to that of the full-length antibody (3E8) directed against ErbB2. As shown in lanes 5 receptor. The results presented in Fig. 1 show that deletion of and 6 in Fig. 1B, a cross-linking pattern similar to that seen in the entire cytoplasmic region of ErbB3 does not interfere with lanes 1 and 3 was observed. Since I-rHRGb1 does not be- either its ligand binding characteristics or its ability to form come directly cross-linked to ErbB2, these data indicated that heteromeric complexes with ErbB2. 1– 665 the receptor complexes in both the ErbB3- and ErbB3 - We next looked at the ability of truncated ErbB3 to induce transfected cells contained ErbB2. Therefore, the high molec- tyrosine phosphorylation upon HRG stimulation. Since COS-7 ular mass complex represents cross-linked receptors and cross- cells naturally express low levels of ErbB2, which form hetero- linked radiolabeled HRG. Interestingly, these complexes meric complexes with ectopically expressed ErbB3, co-transfec- migrated slower than expected for an ErbB2-ErbB3 receptor tion with exogenous erbB2 was not necessary in these experi- dimer, suggesting that a higher order complex was formed ments. Cells that expressed full-length ErbB3 showed a dose- instead. The bands at 190 and 100 kDa in lanes 5 and 6 dependent increase in tyrosine phosphorylation after HRG 1– 665 represented ErbB3 and ErbB3 , respectively, which were stimulation (Fig. 2). The double band visible at ;185 kDa associated with ErbB2, but not covalently cross-linked to it. represented phosphorylated ErbB2 and ErbB3. In contrast, 1– 665 Furthermore, these data suggested that the interaction of although ErbB3 binds HRG and associates with ErbB2, it 862 ErbB3 Transactivation of ErbB2 that were designed to fine map the region responsible for ErbB2 transactivation. ErbB3D955–962, which lacked the eight amino acids that span the junction between the kinase domain and the C-terminal tail, was devoid of receptor phos- phorylation activity. Additional constructs with smaller inter- nal deletions were engineered and tested for transactivation 957 958 activity. Deletion of three amino acid residues, Leu , Val , and Ile (ErbB3DLVI), was sufficient to abolish phosphoryl- ation following HRG stimulation (Fig. 4B). In contrast, deletion 957 958 of two amino acid residues, Leu and Val (ErbB3DLV) and 958 959 Val and Ile (ErbB3DVI), did not affect the transactivation potential. FIG.2. HRG-stimulated receptor autophosphorylation. COS-7 1– 665 cells were transfected in 12-well plates with erbB3 or erbB3 ex- It is possible that deletion of three amino acids at positions pression plasmids. Cells were treated 28 –32 h post-transfection with 957–959 altered the spatial alignment of important structural the indicated amounts of rHRGb1. Cell lysates were subjected to SDS features on ErbB3 relative to corresponding sites on ErbB2. gel electrophoresis and then transferred to nitrocellulose. For the upper The inability of ErbB3DLVI to activate the ErbB2 kinase might panels, blots were probed with anti-phosphotyrosine antibody (aP-Tyr). The lower panels demonstrate ErbB3 expression. Blots were probed have resulted from a gross positional change in the ErbB3 with monoclonal antibody 5B6, which recognizes glycoprotein D (gD)- polypeptide backbone relative to ErbB2, rather than deletion of 1– 665 tagged ErbB3 and ErbB3 . a specific activation motif. To address this possibility, we re- 957 958 959 placed Leu , Val , and Ile with alanine residues. This was not able to activate the ErbB2 kinase. The results shown in removes hydrophobic side chains available for potential inter- Fig. 2 suggested that a direct or indirect interaction between molecular interactions, but maintains the length of the the intracellular domains of ErbB3 and ErbB2 was necessary polypeptide chain. A phosphorylation signal was not observed for HRG-mediated activation of the intrinsic kinase activity of in cells expressing L957A/V958A/I959A (Fig. 4B), indicating ErbB2. that the sequence LVI is required for transactivation. Mapping the Intracellular Transactivation Domain of To further characterize this area, a series of double and ErbB3—We questioned whether the entire intracellular do- single substitution mutants were designed. These mutants are main of ErbB3 or only a segment of it was required for ErbB2 outlined in schematic form in Fig. 4A along with results of the transactivation. To address this issue, a series of ErbB3 trun- phosphorylation analysis for each. Interestingly, in contrast to cation mutants containing smaller deletions from the C termi- ErbB3DLV, cells expressing L957A/V958A did not transacti- nus were constructed. These are shown in schematic form in vate ErbB2 upon HRG stimulation. One explanation for this Fig. 3A. The receptor mutants were transiently expressed and finding is that, in ErbB3DLV, isoleucine mimics leucine at subjected to HRG-induced receptor activation analysis as de- position 957. But in L957A/V958A, the substituted alanine scribed above. In these mutants, the phosphorylation signals residue was not able to replace Leu . This possibility was observed were exclusively due to tyrosine phosphorylation of further explored with mutant L957A. The replacement of endogenous ErbB2 because the truncation mutants lacked the leucine with an alanine residue resulted in a strong decrease in tyrosine phosphorylation sites located on the C terminus of the the phosphorylation signal, further indicating that Leu full-length receptor. Phosphorylation results are also indicated 1–952 played a major role in the transactivation. From these data, we in Fig. 3A. Surprisingly, ErbB3 did not show HRG-induced 1–963 conclude that the minimal region necessary for full transacti- phosphorylation, whereas ErbB3 showed a dose-depend- vation of the ErbB2 kinase upon HRG stimulation is the LVI ent increase in ErbB2 phosphorylation upon HRG stimulation. sequence beginning at position 957. Moreover, Leu appears To further map this area, we engineered additional truncation to play a key role in this hydrophobic motif. However, we mutants to identify regions that may contribute to ErbB2 1–959 cannot rule out the possibility that areas N-terminal to this transactivation. ErbB3 was able to stimulate receptor motif may also contribute to transactivation of ErbB2. phosphorylation, yet additional deletion of Ile resulted in a The LVI Segment Is Conserved in ErbB4 and Is Necessary for decreased phosphorylation signal, and deletion of Val in 1–957 ErbB2 Transactivation—Sequence alignment of the tyrosine ErbB3 completely abolished stimulation of tyrosine phos- kinase domains of members of the ErbB receptor family re- phorylation (Fig. 3B). Thus, we were able to localize the region vealed that the LVI segment is conserved in ErbB4 as well as responsible for the transactivation activity to an area N-termi- ErbB3 and EGFR (Fig. 5). We questioned whether a similar nal to amino acid 959. This critical region is found at the transactivation mechanism also occurred between ErbB2 and C-terminal end of the impaired kinase domain, as shown in Fig. ErbB4. To assess ErbB2 transactivation in an ErbB2-ErbB4 3C. heterodimer, we eliminated the intrinsic kinase activity of Fine Mapping of the Transactivation Area in the Context of ErbB4 by constructing an ErbB4 mutant in which Lys at the the Full-length Receptor by Internal Deletion and Substitution ATP-binding site was replaced with methionine (ErbB4M ) Mutants—Further characterization of the ErbB3 transactiva- (Fig. 6A). The lack of kinase activity in this mutant was verified tion region was carried out by constructing internal deletion by phosphorylation analysis using a human hematopoietic cell and substitution mutants in the context of the full-length re- line (K562) that is devoid of all ErbB family members (data not ceptor. Although Tyr on ErbB3 has not been described as a shown). Receptor activation analysis in COS-7 cells expressing potential phosphorylation site, we questioned whether Tyr might play a role in the regulation of transactivation, possibly ErbB4M showed a dose-dependent phosphorylation signal upon HRG treatment (Fig. 6B). These data confirmed that a by serving as a phosphorylation site for another kinase. We therefore replaced Tyr with phenylalanine in the full-length similar transactivation of ErbB2 also occurred in ErbB2-ErbB4 heterodimers. To investigate the importance of the LVI seg- ErbB3 receptor. Analysis of the substitution mutant Y956F showed a strong phosphorylation signal, indicating that Tyr ment in ErbB4, a three-amino acid deletion mutant lacking 960 961 962 does not play a direct role in the transactivation process (Fig. residues Leu , Val , and Ile was constructed in the con- 726 726 4A). text of ErbB4M . COS-7 cells expressing ErbB4M DLVI Fig. 4A summarizes a series of internal deletion mutants revealed no increase in tyrosine phosphorylation signal upon ErbB3 Transactivation of ErbB2 863 FIG.3. Receptor phosphorylation analysis of ErbB3 truncation mu- tants. A, schematic representation of C- terminal deletion mutants and their abil- ity to induce phosphorylation in COS-7 cells. The portion of the extracellular do- main is indicated as ECD; the transmem- brane domains (TM) are represented as hatched boxes; and the tyrosine kinase domains (TK) or portions of them are rep- resented as black boxes. The mature re- ceptor is indicated as ErbB3, whereas the various C-terminal truncation mutants are designated according to their remain- ing amino acid residues. C-terminal mu- tants were analyzed in phosphorylation assays as described in the legend to Fig. 2, and their ability (1) or inability (2)to induce receptor phosphorylation is indi- cated on the right. B, phosphorylation 1–959 1–958 analysis of ErbB3 , ErbB3 , and 1–957 ErbB3 . COS-7 cells were transfected with the corresponding expression plas- mids, and tyrosine phosphorylation assay was performed as described in the legend to Fig. 2. C, partial amino acid sequence of ErbB3 that spans the junction between the kinase domain and the C-terminal tail. Amino acid residues of the kinase domain are represented in boldface. The number beneath the amino acid isoleucine indicates its position in the mature receptor. aP-Tyr, anti-phosphotyrosine antibody; agD, anti-glycoprotein D antibody. HRG treatment (Fig. 6B). Thus, analogous to the ErbB3 situ- either erbB2M or control expression plasmids, HRG stimu- ation, activation of ErbB2 kinase also requires the LVI motif in lation caused no autophosphorylation on ErbB2 or ErbB3. The ErbB2-ErbB4 heterodimers. expression of ErbB2 and ErbB2M was verified by Western An Associated Cytoplasmic Kinase Is Not Involved in Recep- blot analysis (Fig. 7B). These data showed that deactivation of tor Autophosphorylation upon HRG Stimulation—It is conceiv- the ErbB2 kinase completely abolished the phosphorylation able that ErbB2 transactivation is not the result of a direct signal and confirmed that ErbB3 has no intrinsic kinase activ- interaction that occurs solely between ErbB3 and ErbB2. One ity. Autophosphorylation of the receptors was therefore not due scenario is that a third protein with tyrosine kinase activity is to a cytoplasmic kinase activated by binding to the transacti- bound to the cytoplasmic domain of ErbB3 and that this kinase vation segment of ErbB3, but was the result of the ErbB2 phosphorylates ErbB2. This would be similar to the transacti- kinase. vation mechanism observed in cytokine receptor and T-cell DISCUSSION antigen receptor signaling (31, 32). If the activity of a third kinase was directly regulated by ErbB3, the kinase activity of The allosteric oligomerization model proposed for EGFR by ErbB2 would not be required for receptor phosphorylation. To Schlessinger (33, 34) predicts that ligand binding induces for- test this possibility, we constructed a kinase-inactive ErbB2 mation of receptor dimers, which brings the intracellular do- mutant in which Lys at the ATP-binding site was replaced mains into close proximity, and causes them to phosphorylate with methionine. Ectopic expression of this mutant was then one another in trans. Because ErbB3 lacks intrinsic kinase performed in a hematopoietic cell line that expressed only activity and ErbB2 does not bind HRG, this model does not erbB3 ErbB3 (K562 ) (Fig. 7A). If a cytoplasmic kinase was re- fully explain the phosphorylation pattern observed in the sponsible for autophosphorylation, cells that co-express ErbB3 ErbB2-ErbB3 complex following HRG stimulation. It was also and kinase-inactive ErbB2 would still show a phosphorylation previously unclear whether activation of the intrinsic kinase signal upon HRG treatment. Cells transfected with wild-type required specific cytoplasmic domain interactions. Here, we erbB2, erbB2M , or control expression plasmids were incu- present data showing that transactivation of the ErbB2 kinase bated with HRG and then subjected to immunoprecipitation by ErbB3 requires the presence of a structural element within with antibodies directed against ErbB2 or ErbB3. Cells trans- the ErbB3 cytoplasmic domain. Using ErbB3 deletion and sub- fected with erbB2 cDNA demonstrated tyrosine phosphoryla- stitution mutants, we found a discrete sequence of three amino tion on ErbB2 and ErbB3 (Fig. 7B, lanes 1– 4). Interestingly, acids (LVI) at the carboxyl terminus of the inactive kinase ErbB2 was constitutively phosphorylated, whereas ErbB3 domain of ErbB3 that is necessary for ErbB2 transactivation. phosphorylation was HRG-dependent. In cells transfected with Deletion of segments distal to this region had no effect, indi- 864 ErbB3 Transactivation of ErbB2 FIG.4. Tyrosine phosphorylation analysis of internal and substitution mutants. A, schematic diagram of recep- tor mutants and their ability to induce receptor autophosphorylation. Receptors and receptor domains are depicted as de- scribed in the legend to Fig. 3. Internal deletion areas are shown as gaps. Substi- tuted amino acid residues are indicated beneath the receptors. Receptor mutants underwent phosphorylation analysis as described in the legend to Fig. 2, and their ability (1) or inability (2) to induce transphosphorylation is indicated on the right. B, phosphorylation analysis of ErbB3 mutants ErbB3DLVI and L957A/ V958A/I959A. COS-7 cells were trans- fected with corresponding expression plasmids and analyzed for receptor phos- phorylation as described in the legend to Fig. 2. aP-Tyr, anti-phosphotyrosine anti- body; agD, anti-glycoprotein D antibody. FIG.5. Sequence alignment of the C-terminal ends of the ErbB tyrosine kinase domains. Sequences are displayed in a single letter code; identical residues are denoted as dots. Amino acid sequences are numbered according to the mature proteins. The LVI segment is boxed. The C-terminal ends (C-term) of the tyrosine kinase (TK) domains were determined as described by Plowman et al. (5). cating that they are not required for this activity. At present, we cannot rule out the possibility that additional sequences N-terminal to this region may also contribute to the transacti- vation of ErbB2. The results of our study suggest at least two models whereby direct sequence-specific molecular interactions may result in receptor transactivation. These models extend the Schlessinger FIG.6. Receptor phosphorylation analysis of ErbB4 mutants. hypothesis (33, 34) to the ErbB2-ErbB3 system and are shown A, ErbB4 mutants shown as described in the legend to Fig. 3. The schematically in Fig. 8. In the first model, activation of ErbB2 ErbB4 receptor construct with Lys replaced by Met is designated results from a direct interaction between the intracellular do- 726 726 ErbB4M . ErbB4M DLVI lacked, in addition to the amino acid sub- 960 961 962 mains of the two receptors. In this model, HRG binding to stitution, Leu , Val , and Ile . B, phosphorylation analysis of ErbB4 mutants. COS-7 cells were transfected with the corresponding ErbB3 results in a conformational change in the extracellular expression plasmids, and cells were subjected to phosphorylation anal- domain of ErbB3 facilitating recruitment of ErbB2 and forma- ysis as described in the legend to Fig. 2. ErbB4 expression was verified tion of the heterodimeric complex. The interaction between the using a polyclonal anti-ErbB4 antibody. aP-Tyr, anti-phosphotyrosine extracellular domains of the receptors aligns their intracellular antibody. domains, bringing the LVI motif of ErbB3 into direct contact with an as yet undefined region of ErbB2. This, in turn, leads heterodimerization of ErbB3 with ErbB2 allows an adaptor to the activation of the ErbB2 kinase. An alternative model molecule with specific recognition sites for each receptor to assumes the participation of a third protein. Ligand-induced bridge their intracellular domains. The putative adaptor pro- ErbB3 Transactivation of ErbB2 865 erbB3 FIG.7. An associated cytoplasmic kinase is not involved in receptor autophosphorylation. A, ErbB3 expression in stable K562 transfectants. Anti-ErbB3 antibody was used to detect ErbB3 expression in the cell lysates of the transfectants. B, phosphorylation analysis in erbB3 732 K562 cells transiently transfected with erbB2 or kinase-inactive erbB2 (erbB2M ). Incubations with or without rHRGb1 (10 nM) were performed for 10 min at room temperature, 48 h post-transfection. Polyclonal antibodies to ErbB2 or ErbB3 were used for immunoprecipitations (IP). Immune complexes were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose. Receptor phosphorylation was determined using anti-phosphotyrosine antibody (aP-Tyr). ErbB2 expression was verified using monoclonal antibody 5B6, which recognizes glycoprotein D(gD)-tagged ErbB2 and ErbB2M . WB, Western blot. tive agent. To date, several approaches have been undertaken to intercept the signal generation (42). For example, antibodies that directly bind to the extracellular domain of ErbB2 or EGFR have been very efficient as antiproliferative reagents (43– 45). The selective inhibition of EGFR tyrosine kinase with various small molecules also results in antitumor activity (46). The identification of the transactivation sequence in ErbB3 suggests another way to block signaling in a heteromeric FIG.8. Transactivation models. The schematic depicts two models complex. A compound that directly interacts with the LVI of HRG-mediated transactivation of ErbB3. ErbB3 and ErbB2 are des- segment in ErbB3 could inhibit specific transactivation, re- ignated H3 and H2, respectively. The extracellular and intracellular ceptor phosphorylation, and consequently all downstream domains are shown as distinct barrels. The LVI segment in ErbB3 is symbolized as a black half-circle; a so far unidentified interaction region signaling pathways. in ErbB2 is shown as a hatched box. Acknowledgment— We thank the DNA synthesis/purification group at Genentech, Inc. for supplying oligonucleotides. tein binds to the LVI sequence in ErbB3 and to an unknown REFERENCES sequence in ErbB2. We postulate that the adaptor stabilizes 1. van der Geer, P., Hunter, T., and Lindberg, R. A. (1994) Annu. 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Published: Jan 1, 1999

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