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Her4 and Her2/neu Tyrosine Kinase Domains Dimerize and Activate in a Reconstituted in Vitro System*

  • John Monsey
    Footnotes
    Affiliations
    Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Wei Shen
    Footnotes
    Affiliations
    Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Paul Schlesinger
    Affiliations
    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Ron Bose
    Correspondence
    To whom correspondence should be addressed: 660 S. Euclid Ave., Campus Box 8069, St. Louis, MO 63110. Tel.: 314-747-9308; Fax: 314-747-9320;
    Affiliations
    Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Institutes of Health Grant K22CA128951 (NCI Transition Career Development Award, K22) (to R. B.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.
    1 Both authors contributed equally to this work.
Open AccessPublished:December 18, 2009DOI:https://doi.org/10.1074/jbc.M109.096032
      Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues 952ENI954 to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

      Introduction

      Her4 (ErbB-4) and Her2/neu (ErbB-2) are members of the ErbB family of receptor-tyrosine kinases, with the founding member of this family being epidermal growth factor receptor (EGFR)
      The abbreviations used are: EGFR
      epidermal growth factor receptor
      DOPC
      dioleoylphosphatidylcholine
      Ni-NTA-DOGS
      nickel-1,2-dioleoyl-sn-glycero-3-([N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl)-nickel salt
      SUV
      small unilamellar vesicles
      DTT
      dithiothreitol
      WT
      wild type.
      (
      • Carpenter G.
      ). The ErbB receptor-tyrosine kinases have an extracellular domain that is involved in ligand binding and receptor homo- or heterodimerization. Their intracellular domain consists of the juxtamembrane region, tyrosine kinase domain, and C-terminal tail. EGFR, Her2/neu, and Her4 contain catalytically active kinase domains, whereas Her3 (ErbB-3) has an inactive kinase domain and must heterodimerize with another ErbB receptor to signal (
      • Citri A.
      • Skaria K.B.
      • Yarden Y.
      ). Several studies on ErbB heterodimerization have shown that Her2/neu is the preferred heterodimerization partner for the other three ErbB receptors (
      • Tzahar E.
      • Waterman H.
      • Chen X.
      • Levkowitz G.
      • Karunagaran D.
      • Lavi S.
      • Ratzkin B.J.
      • Yarden Y.
      ,
      • Graus-Porta D.
      • Beerli R.R.
      • Daly J.M.
      • Hynes N.E.
      ,
      • Karunagaran D.
      • Tzahar E.
      • Beerli R.R.
      • Chen X.
      • Graus-Porta D.
      • Ratzkin B.J.
      • Seger R.
      • Hynes N.E.
      • Yarden Y.
      ). These studies also provide evidence for formation of EGFR-Her3 and EGFR-Her4 heterodimers in response to ligands that bind to Her3 or Her4 (
      • Tzahar E.
      • Waterman H.
      • Chen X.
      • Levkowitz G.
      • Karunagaran D.
      • Lavi S.
      • Ratzkin B.J.
      • Yarden Y.
      ,
      • Olayioye M.A.
      • Graus-Porta D.
      • Beerli R.R.
      • Rohrer J.
      • Gay B.
      • Hynes N.E.
      ).
      Both Her4 and Her2/neu play important roles in the development and the normal physiology of the cardiovascular and nervous systems (
      • Birchmeier C.
      ,
      • Pentassuglia L.
      • Sawyer D.B.
      ). Her4 and Her2/neu knock-out mice both die around embryonic day 10.5 because of malformation of the cardiac trabeculae, and additionally, they are found to have abnormalities in the hindbrain or in sensory ganglia and motor nerves (
      • Gassmann M.
      • Casagranda F.
      • Orioli D.
      • Simon H.
      • Lai C.
      • Klein R.
      • Lemke G.
      ,
      • Lee K.F.
      • Simon H.
      • Chen H.
      • Bates B.
      • Hung M.C.
      • Hauser C.
      ). In human breast cancers, Her2/neu gene amplification is found in 20–30% of patients, resulting in unregulated tyrosine kinase signaling that increases cancer cell proliferation, migration, and propensity to metastasize (
      • Hynes N.E.
      • MacDonald G.
      ). Treatment of Her2/neu-amplified breast cancers with the monoclonal antibody, trastuzumab (Herceptin), forms an essential part of state of the art, breast cancer treatment (
      • Romond E.H.
      • Perez E.A.
      • Bryant J.
      • Suman V.J.
      • Geyer Jr., C.E.
      • Davidson N.E.
      • Tan-Chiu E.
      • Martino S.
      • Paik S.
      • Kaufman P.A.
      • Swain S.M.
      • Pisansky T.M.
      • Fehrenbacher L.
      • Kutteh L.A.
      • Vogel V.G.
      • Visscher D.W.
      • Yothers G.
      • Jenkins R.B.
      • Brown A.M.
      • Dakhil S.R.
      • Mamounas E.P.
      • Lingle W.L.
      • Klein P.M.
      • Ingle J.N.
      • Wolmark N.
      ).
      Multiple crystal structures of the extracellular domain of all four ErbB receptor-tyrosine kinases have been solved and provide a detailed understanding of ligand binding and receptor dimerization (reviewed in Refs.
      • Burgess A.W.
      • Cho H.S.
      • Eigenbrot C.
      • Ferguson K.M.
      • Garrett T.P.
      • Leahy D.J.
      • Lemmon M.A.
      • Sliwkowski M.X.
      • Ward C.W.
      • Yokoyama S.
      ,
      • Lemmon M.A.
      ). However, our structural understanding of the intracellular domain of the ErbB receptors is less complete. Multiple crystal structures of the EGFR tyrosine kinase domain have been solved (reviewed in Ref.
      • Bose R.
      • Zhang X.
      ), and in 2008, two structures of the Her4 tyrosine kinase domain were published (
      • Qiu C.
      • Tarrant M.K.
      • Choi S.H.
      • Sathyamurthy A.
      • Bose R.
      • Banjade S.
      • Pal A.
      • Bornmann W.G.
      • Lemmon M.A.
      • Cole P.A.
      • Leahy D.J.
      ,
      • Wood E.R.
      • Shewchuk L.M.
      • Ellis B.
      • Brignola P.
      • Brashear R.L.
      • Caferro T.R.
      • Dickerson S.H.
      • Dickson H.D.
      • Donaldson K.H.
      • Gaul M.
      • Griffin R.J.
      • Hassell A.M.
      • Keith B.
      • Mullin R.
      • Petrov K.G.
      • Reno M.J.
      • Rusnak D.W.
      • Tadepalli S.M.
      • Ulrich J.C.
      • Wagner C.D.
      • Vanderwall D.E.
      • Waterson A.G.
      • Williams J.D.
      • White W.L.
      • Uehling D.E.
      ). An important advance in the understanding of how ErbB receptor dimerization activates the tyrosine kinase domain came from the crystal structure of an EGFR kinase domain dimer (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      ). The contact surfaces of the observed dimer are located on the C-lobe of one monomer (called the “donor” monomer) and the N-lobe of the other monomer (the “acceptor” monomer), and therefore, this dimer was named the asymmetric dimer. Comparison of this EGFR kinase domain dimer to the structure of the cyclin-cyclin-dependent kinase complex showed similarities in that the “donor” EGFR kinase domain monomer acted as a “cyclin-like” activator for the “acceptor” EGFR monomer (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      ). This asymmetric dimer model was supported by biochemical studies on recombinant EGFR kinase domain protein and by mutational studies on the full-length EGFR protein. The crystal structure of Her4 kinase domain shows that it also adopts a very similar, asymmetric dimer structure (
      • Qiu C.
      • Tarrant M.K.
      • Choi S.H.
      • Sathyamurthy A.
      • Bose R.
      • Banjade S.
      • Pal A.
      • Bornmann W.G.
      • Lemmon M.A.
      • Cole P.A.
      • Leahy D.J.
      ).
      The biochemical studies on EGFR kinase domain which supported the asymmetric dimer model involved the use of a reconstituted in vitro system. In this system, the EGFR kinase domain was anchored to the surface of a liposome and brought to a high local concentration, thereby mimicking what happens in vivo on the cell membrane when the full-length EGFR protein binds its ligand and dimerizes (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      ). The anchoring of the EGFR kinase domain to liposome was achieved by incorporating a polyhistidine tag on the kinase and a nickel-chelating lipid into the liposome. This in vitro system was first developed by Weis and co-workers (
      • Shrout A.L.
      • Montefusco D.J.
      • Weis R.M.
      ,
      • Montefusco D.J.
      • Asinas A.E.
      • Weis R.M.
      ) in their studies of a prokaryotic signal transduction system that mediates bacterial chemotaxis. Small unilamellar vesicles (SUVs or liposomes) containing this nickel-chelating lipid were used to anchor and cluster the polyhistidine-tagged Tar chemotaxis protein and form a 3-protein complex containing Tar, an adaptor protein, and the CheA protein kinase. This complex resulted in a 180-fold increase in CheA protein kinase activity. Application of this system to EGFR allowed for in vitro studies of EGFR kinase domain dimerization and discovery of the mechanism of action of Mig6/RALT, an endogenous inhibitory protein of EGFR (
      • Zhang X.
      • Pickin K.A.
      • Bose R.
      • Jura N.
      • Cole P.A.
      • Kuriyan J.
      ).
      In this report, we show that this nickel-chelating lipid-liposome system is broadly applicable to the ErbB receptor-tyrosine kinases. Her4 binds to and dimerizes on the surface of these liposomes, and this results in a marked enhancement of its tyrosine kinase activity. Likewise, Her2/neu is also activated by addition of these liposomes. This system offers the unique advantage that ErbB kinase domain heterodimerization can now be studied in vitro, and we utilize this to test predictions made by the EGFR asymmetric dimer model. Specifically, we observe that the ability of the Her3 kinase domain to activate Her4 does not follow the model predictions and that this difference is due to specific residues on the Her3 kinase domain C-lobe surface.

      DISCUSSION

      Use of nickel-chelating lipids and liposomes is potentially applicable to the study of many membrane-associated proteins and protein domains. In this study, we demonstrate that binding of Her4 and Her2/neu to nickel-liposomes increases their kinase-specific activity 40-fold and 3.5-fold, respectively. Her2/neu can be further activated by heterodimerizing with either Her3, which is intrinsically kinase-dead, or with a kinase-dead mutant of Her4. This activation of the ErbB kinase domains by nickel-liposomes can be disrupted through competition of the nickel-polyhistidine interaction with imidazole. Further, while the binding of ErbB kinase domains to the nickel-liposomes follows a single-site binding model, kinase activation is found to be a cooperative process, with a Hill coefficient of close to 2, consistent with formation of a kinase dimer. Mutation of the asymmetric dimer interface, either Her4 mutation I712Q or V954R, eliminates Her4 kinase activation. Mixing of these two mutant kinases allows for reconstitution of a functional asymmetric dimer interface and restoration of activation, providing additional evidence for the formation of an activated kinase domain dimer. We observe that this activation on the surface of the nickel-liposomes is greatest in the presence of Mg2+, rather than with Mn2+, and this difference is likely due to direct effects of Mn2+ in the kinase catalytic site rather than an effect on kinase dimerization.
      We used this nickel-liposome-based system to probe predictions made by the asymmetric dimer model. Based on the crystal structure of the EGFR kinase domain asymmetric dimer and multisequence alignment of the four ErbB kinase domains, it was proposed that all ErbB kinase domains should be effective donor monomers (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      ). Her3 is intrinsically kinase dead, and one of reasons for this effect is the catalytic base Asp residue, which is conserved among kinases, is an Asn residue in Her3. By making the same amino acid change in Her4 (Her4 D843N), we render Her4 kinase dead and generate a protein that we can directly compare with Her3. We observe that both Her3 and kinase dead Her4 are equally effective in their ability to activate Her2/neu. In contrast, there is a striking difference in the ability of Her3 and kinase-dead Her4 to activate Her4. Mutation of Her3 residues 952–954 to the corresponding sequence in Her4 (mutation B) increased the ability of Her3 to activate Her4. This demonstrates that differences in the predicted C-lobe surface amino acids influence the degree of activation between different ErbB kinase heterodimers. We note that Her3 mutation B does not reproduce the full activation seen with kinase-dead Her4, and it is likely that additional amino acid residues distant from the C-lobe contact surface also influence how the Her3 kinase domain heterodimerizes.
      We observe that the Her2/neu kinase domain showed a smaller degree of activation upon binding to nickel-liposomes than the Her4 kinase domain. While Her2/neu kinase specific activity is further increased upon heterodimerization with Her3 or Her4 (up to 5.9-fold), it still did not achieve the 40-fold activation observed with Her4 bound to nickel-liposomes. Prior reports have suggested that the recombinant Her2/neu kinase domain is an intrinsically less active kinase than EGFR or Her4 kinase domains (
      • Brignola P.S.
      • Lackey K.
      • Kadwell S.H.
      • Hoffman C.
      • Horne E.
      • Carter H.L.
      • Stuart J.D.
      • Blackburn K.
      • Moyer M.B.
      • Alligood K.J.
      • Knight W.B.
      • Wood E.R.
      ,
      • Fan Y.X.
      • Wong L.
      • Ding J.
      • Spiridonov N.A.
      • Johnson R.C.
      • Johnson G.R.
      ). Her2/neu residues in the loop between the αC helix and the β4 sheet were found to play a major role in controlling the kinase activity of Her2/neu (
      • Fan Y.X.
      • Wong L.
      • Ding J.
      • Spiridonov N.A.
      • Johnson R.C.
      • Johnson G.R.
      ). Mutating residues in this loop to match EGFR (Her2/neu G776S, G778D) or a cancer-associated insertion in this loop (Her2/neu G776YVMA) significantly increased Her2/neu kinase activity (
      • Fan Y.X.
      • Wong L.
      • Ding J.
      • Spiridonov N.A.
      • Johnson R.C.
      • Johnson G.R.
      ,
      • Shigematsu H.
      • Takahashi T.
      • Nomura M.
      • Majmudar K.
      • Suzuki M.
      • Lee H.
      • Wistuba II,
      • Fong K.M.
      • Toyooka S.
      • Shimizu N.
      • Fujisawa T.
      • Minna J.D.
      • Gazdar A.F.
      ,
      • Wang S.E.
      • Narasanna A.
      • Perez-Torres M.
      • Xiang B.
      • Wu F.Y.
      • Yang S.
      • Carpenter G.
      • Gazdar A.F.
      • Muthuswamy S.K.
      • Arteaga C.L.
      ). We speculate that this may be an evolutionary adaptation as Her2/neu is the only member of the ErbB family that does not bind a ligand. The crystal structure of the Her2/neu extracellular domain has shown that it adopts an extended conformation with its dimerization surfaces exposed (
      • Cho H.S.
      • Mason K.
      • Ramyar K.X.
      • Stanley A.M.
      • Gabelli S.B.
      • Denney Jr., D.W.
      • Leahy D.J.
      ,
      • Garrett T.P.
      • McKern N.M.
      • Lou M.
      • Elleman T.C.
      • Adams T.E.
      • Lovrecz G.O.
      • Kofler M.
      • Jorissen R.N.
      • Nice E.C.
      • Burgess A.W.
      • Ward C.W.
      ). Given that the Her2/neu extracellular domain appears to be “poised” to interact with other EGFR family members, limiting the ability of the Her2/neu kinase domain to be activated by homo- or heterodimerization could represent an important physiologic regulatory mechanism.
      An important consideration in the interpretation of these experiments is that they measure the dimerization of the kinase domains only. The extracellular domain, transmembrane domain, and the intracellular juxtamembrane region also contribute significantly to homo- and heterodimerization of the ErbB receptor-tyrosine kinases (
      • Burgess A.W.
      • Cho H.S.
      • Eigenbrot C.
      • Ferguson K.M.
      • Garrett T.P.
      • Leahy D.J.
      • Lemmon M.A.
      • Sliwkowski M.X.
      • Ward C.W.
      • Yokoyama S.
      ,
      • Lemmon M.A.
      ,
      • Thiel K.W.
      • Carpenter G.
      ,
      • Macdonald-Obermann J.L.
      • Pike L.J.
      ), and they are not present in this reconstituted in vitro system. After the initial submission of this report, further biochemical and structural studies on the EGFR intracellular juxtamembrane region were published (
      • Red Brewer M.
      • Choi S.H.
      • Alvarado D.
      • Moravcevic K.
      • Pozzi A.
      • Lemmon M.A.
      • Carpenter G.
      ,
      • Jura N.
      • Endres N.F.
      • Engel K.
      • Deindl S.
      • Das R.
      • Lamers M.H.
      • Wemmer D.E.
      • Zhang X.
      • Kuriyan J.
      ). These studies clearly demonstrate that the juxtamembrane region contains an activation domain, also called the juxtamembrane latch. The crystal structure of this region demonstrates that the juxtamembrane activation domain of the acceptor monomer cradles the donor monomer (
      • Red Brewer M.
      • Choi S.H.
      • Alvarado D.
      • Moravcevic K.
      • Pozzi A.
      • Lemmon M.A.
      • Carpenter G.
      ). Kinase assays were performed comparing the activity of EGFR constructs containing the juxtamembrane region and the kinase domain in solution to kinase domain only constructs attached to nickel-liposomes (
      • Jura N.
      • Endres N.F.
      • Engel K.
      • Deindl S.
      • Das R.
      • Lamers M.H.
      • Wemmer D.E.
      • Zhang X.
      • Kuriyan J.
      ). We anticipate that these juxtamembrane containing protein constructs can also be tested with the nickel-liposome system. In vivo, the transmembrane domain and the lipids on the inner leaflet of the plasma membrane will affect how the juxtamembrane region interacts with the kinase domain, and this can be studied with this in vitro system.
      Assaying membrane-associated proteins, such as the kinase domains of receptor-tyrosine kinases, in the presence of nickel-liposome has multiple advantages. First, this assay method can be broadly applied to multiple receptor-tyrosine kinases. Zhang et al. (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      ) demonstrated its utility for studies on EGFR. We demonstrate its general applicability to the remainder of the ErbB family. Weis and co-workers (
      • Esposito E.A.
      • Shrout A.L.
      • Weis R.M.
      ) recently reported activation of insulin receptor, Tie2, and the Ephrin receptor, EphB2, kinase activity by a nickel-chelating lipid containing nanoparticle. Additionally, this method permits the generation of membrane associated protein complexes in vitro and opens up important research approaches and experimental designs that are not possible in intact cells. The ability to generate a four protein prokaryotic signaling complex on the nickel-liposomes has been demonstrated (
      • Shrout A.L.
      • Montefusco D.J.
      • Weis R.M.
      ). Given that this system permits rapid autophosphorylation of the Her4 C-terminal tail tyrosine sites, testing SH2-containing proteins for their ability to complex in vitro with Her4 and mediate subsequent signaling steps should be feasible.
      Nickel-liposomes can also be used to study disease associated mutations in vitro. An activating mutation in EGFR, L858R, has been found in lung cancer patients who are never-smokers, and this mutation is associated with a markedly enhanced response to EGFR-targeted tyrosine kinase inhibitor drugs (
      • Pao W.
      • Miller V.
      • Zakowski M.
      • Doherty J.
      • Politi K.
      • Sarkaria I.
      • Singh B.
      • Heelan R.
      • Rusch V.
      • Fulton L.
      • Mardis E.
      • Kupfer D.
      • Wilson R.
      • Kris M.
      • Varmus H.
      ,
      • Paez J.G.
      • Jänne P.A.
      • Lee J.C.
      • Tracy S.
      • Greulich H.
      • Gabriel S.
      • Herman P.
      • Kaye F.J.
      • Lindeman N.
      • Boggon T.J.
      • Naoki K.
      • Sasaki H.
      • Fujii Y.
      • Eck M.J.
      • Sellers W.R.
      • Johnson B.E.
      • Meyerson M.
      ,
      • Lynch T.J.
      • Bell D.W.
      • Sordella R.
      • Gurubhagavatula S.
      • Okimoto R.A.
      • Brannigan B.W.
      • Harris P.L.
      • Haserlat S.M.
      • Supko J.G.
      • Haluska F.G.
      • Louis D.N.
      • Christiani D.C.
      • Settleman J.
      • Haber D.A.
      ). Assaying this mutation using nickel-liposomes demonstrated that EGFR L858R kinase domain has an ∼20-fold increase in catalytic efficiency (kcat/Km) as compared with WT EGFR kinase (
      • Zhang X.
      • Gureasko J.
      • Shen K.
      • Cole P.A.
      • Kuriyan J.
      ). In addition, using the dimeric and activated form of tyrosine kinases in small molecule inhibitor screens opens up many important pharmacological and pharmaceutical possibilities. The affinity and detailed structure-activity relationship of inhibitors for the dimeric state of the kinases could be directly probed. Screens for inhibitors of kinase dimerization can also be constructed. Of course, adequate controls to evaluate for effects of lipophilic, small molecules partitioning into liposomes would need to be included.
      In conclusion, this nickel-chelating lipid-liposome system is potentially applicable to the study of many membrane-associated proteins and protein domains. We demonstrate that Her4 and Her2/neu kinase domains dimerize and become activated upon binding to the nickel-liposomes. This system is used to induce the formation of ErbB kinase domain heterodimers in vitro. The Her3 kinase domain is found to readily activate Her2/neu but to be a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model, and we observed that mutating Her3 residues 952–954 to the corresponding sequence in Her4 increased the ability of Her3 to activate Her4. This ability to study ErbB heterodimerization in vitro is a unique feature of the nickel-liposome system and offers significant future potential for analyzing the function of the entire intracellular half of the EGFR-ErbB family of receptor-tyrosine kinases.

      Acknowledgments

      We thank Linda Pike, Daniel J. Leahy, Philip A. Cole, and Aruna Sathyamurthy for critical reading of the manuscript.

      Note Added in Proof

      A crystal structure of the Her3 kinase domain has been recently published (Jura, N., Shan, Y., Cao, X., Shaw, D. E., and Kuriyan, J. (2009) Proc. Natl. Acad. Sci. U.S.A.106, 21608–21613). Review of this crystal structure (Protein Data Bank code 3KEX) shows that Her3 residues Asp-951, Asn-953, and Ile-954 are surface-exposed.

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