Somatic Mutations of ErbB4
2008; Elsevier BV; Volume: 284; Issue: 9 Linguagem: Inglês
10.1074/jbc.m805438200
ISSN1083-351X
AutoresDenis Tvorogov, Maria Sundvall, Kari J. Kurppa, Maija Hollmén, Susanna Repo, Mark S. Johnson, Klaus Elenius,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoCancer drugs targeting ErbB receptors, such as epidermal growth factor receptor and ErbB2, are currently in clinical use. However, the role of ErbB4 as a potential cancer drug target has remained controversial. Recently, somatic mutations altering the coding region of ErbB4 were described in patients with breast, gastric, colorectal, or non-small cell lung cancer, but the functional significance of these mutations is unknown. Here we demonstrate that 2 of 10 of the cancer-associated mutations of ErbB4 lead to loss of ErbB4 kinase activity due to disruption of functionally important structural features. Interestingly, the kinase-dead ErbB4 mutants were as efficient as wild-type ErbB4 in forming a heterodimeric neuregulin receptor with ErbB2 and promoting phosphorylation of Erk1/2 and Akt in an ErbB2 kinase-dependent manner. However, the mutant ErbB4 receptors failed to phosphorylate STAT5 and suppressed differentiation of MDA-MB-468 mammary carcinoma cells. These findings suggest that the somatic ErbB4 mutations have functional consequences and lead to selective changes in ErbB4 signaling. Cancer drugs targeting ErbB receptors, such as epidermal growth factor receptor and ErbB2, are currently in clinical use. However, the role of ErbB4 as a potential cancer drug target has remained controversial. Recently, somatic mutations altering the coding region of ErbB4 were described in patients with breast, gastric, colorectal, or non-small cell lung cancer, but the functional significance of these mutations is unknown. Here we demonstrate that 2 of 10 of the cancer-associated mutations of ErbB4 lead to loss of ErbB4 kinase activity due to disruption of functionally important structural features. Interestingly, the kinase-dead ErbB4 mutants were as efficient as wild-type ErbB4 in forming a heterodimeric neuregulin receptor with ErbB2 and promoting phosphorylation of Erk1/2 and Akt in an ErbB2 kinase-dependent manner. However, the mutant ErbB4 receptors failed to phosphorylate STAT5 and suppressed differentiation of MDA-MB-468 mammary carcinoma cells. These findings suggest that the somatic ErbB4 mutations have functional consequences and lead to selective changes in ErbB4 signaling. The ErbB/epidermal growth factor receptor (EGFR) 3The abbreviations used are: EGFR, epidermal growth factor receptor; NRG, neuregulins; STAT5, signal transducer and activator of transcription 5; TKI, tyrosine kinase inhibitor; Erk, extracellular signal-regulated kinase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.3The abbreviations used are: EGFR, epidermal growth factor receptor; NRG, neuregulins; STAT5, signal transducer and activator of transcription 5; TKI, tyrosine kinase inhibitor; Erk, extracellular signal-regulated kinase; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide./HER receptor-tyrosine kinase subfamily includes EGFR, ErbB2, ErbB3, and ErbB4. ErbB receptors bind several EGF-like growth factors including the neuregulins (NRG). Ligand-induced extracellular homo- or heterodimerization of ErbB receptors is followed by autophosphorylation at intracellular tyrosine residues by juxtaposed tyrosine kinase domains. The phosphorylated tyrosines in the cytoplasmic receptor tail serve as binding sites for various intracellular signal transduction molecules that mediate the cellular responses to ErbB stimulation (1Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3456) Google Scholar, 2Hynes N.E. Lane H.A. Nat. Rev. Cancer. 2005; 5: 341-354Crossref PubMed Scopus (2622) Google Scholar). Recent crystallographic and biochemical analyses have indicated that intracellular tyrosine kinases of EGFR and ErbB4 are activated allosterically in an asymmetrical fashion (3Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar, 4Qiu 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. Structure. 2008; 16: 460-467Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In the activated dimer the C-terminal lobe of one kinase domain contacts with the N-terminal lobe of another kinase domain, thereby breaking its intrinsic autoinhibited conformation and facilitating catalysis (3Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar, 4Qiu 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. Structure. 2008; 16: 460-467Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Activation mechanisms of protein kinases have shared features, and the relative spatial orientation of certain residues that are highly conserved within the eukaryotic protein kinome is essential for successful catalysis (5Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). These include residues that participate in nucleotide binding and transfer of the γ-phosphate group of adenosine triphosphate (ATP) to the hydroxyl oxygen atom of a substrate. Conserved regulatory elements of protein kinases include the activation (A)-loop, αC-helix, phosphate binding (P)-loop, and catalytic (C)-loop. The A-loop is involved in stabilizing the inactive conformation, whereas the αC-helix, located in the N-terminal lobe, mediates conformational changes within the catalytic center that activate the kinase. The aspartate-phenylalanine-glycine (DFG) motif at the base of the A-loop and the P-loop participate in binding and coordination of ATP, whereas the C-loop contains the catalytic aspartate residue (Asp-843 in ErbB4), which processes the substrate tyrosine for catalysis (5Huse M. Kuriyan J. Cell. 2002; 109: 275-282Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). Although EGFR and ErbB2 are well characterized oncogenes and targets for cancer therapeutics (2Hynes N.E. Lane H.A. Nat. Rev. Cancer. 2005; 5: 341-354Crossref PubMed Scopus (2622) Google Scholar), the relevance of ErbB4 as a cancer drug target is poorly understood. Indeed, both roles as a human oncogene as well as a tumor suppressor gene have been proposed (6Gullick W.J. J. Pathol. 2003; 200: 279-281Crossref PubMed Scopus (51) Google Scholar, 7Sundvall M. Iljin K. Kilpinen S. Sara H. Kallioniemi O.P. Elenius K. J. Mammary Gland Biol. Neoplasia. 2008; 13: 259-268Crossref PubMed Scopus (100) Google Scholar). Lack of knowledge of tumor-associated genetic changes in ErbB4 has precluded addressing their potential loss-of-function or gain-of-function phenotypes. Recently, nine different somatic mutations targeting the ErbB4 kinase domain were reported in patients with either breast, gastric, colorectal, or non-small cell lung cancer (8Soung Y.H. Lee J.W. Kim S.Y. Wang Y.P. Jo K.H. Moon S.W. Park W.S. Nam S.W. Lee J.Y. Yoo N.J. Lee S.H. Int. J. Cancer. 2006; 118: 1426-1429Crossref PubMed Scopus (103) Google Scholar). In EGFR, similar mutations targeting the kinase domain sensitize patients to treatment with tyrosine kinase inhibitors (9Lynch 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. N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9823) Google Scholar, 10Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8337) Google Scholar). In addition, one somatic mutation targeting the cytoplasmic tail of ErbB4 has been reported in a colorectal cancer patient (11Parsons D.W. Wang T.L. Samuels Y. Bardelli A. Cummins J.M. DeLong L. Silliman N. Ptak J. Szabo S. Willson J.K. Markowitz S. Kinzler K.W. Vogelstein B. Lengauer C. Velculescu V.E. Nature. 2005; 436: 792Crossref PubMed Scopus (485) Google Scholar). However, the functional effect of the cancer-associated mutations on ErbB4 activity has not been addressed. Here, we exploited the recently reported crystal structure of ErbB4 kinase domain (4Qiu 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. Structure. 2008; 16: 460-467Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) to predict the functionality of 9 somatic ErbB4 kinase domain mutants (8Soung Y.H. Lee J.W. Kim S.Y. Wang Y.P. Jo K.H. Moon S.W. Park W.S. Nam S.W. Lee J.Y. Yoo N.J. Lee S.H. Int. J. Cancer. 2006; 118: 1426-1429Crossref PubMed Scopus (103) Google Scholar). In addition, we analyzed the basal and ligand-induced phosphotyrosine content of the 9-kinase domain and 1-cytoplasmic domain mutations (8Soung Y.H. Lee J.W. Kim S.Y. Wang Y.P. Jo K.H. Moon S.W. Park W.S. Nam S.W. Lee J.Y. Yoo N.J. Lee S.H. Int. J. Cancer. 2006; 118: 1426-1429Crossref PubMed Scopus (103) Google Scholar, 11Parsons D.W. Wang T.L. Samuels Y. Bardelli A. Cummins J.M. DeLong L. Silliman N. Ptak J. Szabo S. Willson J.K. Markowitz S. Kinzler K.W. Vogelstein B. Lengauer C. Velculescu V.E. Nature. 2005; 436: 792Crossref PubMed Scopus (485) Google Scholar) in different cell backgrounds. Our data demonstrate that 2 of the 10 mutations disrupt the catalytic activity of the ErbB4 tyrosine kinase, and there are clear structural reasons for the observed effects. Intriguingly, despite loss of kinase activity, the two mutant receptors were able to form a functional heterodimer with ErbB2 and activate mitogen-activated protein kinase Erk and phosphoinositide 3-kinase/Akt pathways. However, kinase activity of ErbB4 was required for NRG-induced activation of signal transducer and activator of transcription 5 (STAT5), resulting in a failure of ErbB4 mutants to activate STAT5. Interestingly, when overexpressed in a breast cancer cell line, the two kinase-dead mutants gained an ability to suppress the formation of differentiated acinar structures, unlike the wild-type ErbB4, which promoted differentiation. Thus, although most of the ErbB4 mutations are surface mutations and are not located either near the binding/active site or near the dimerization surface and, hence, appear to be innocuous, for two of the mutants that occur within the binding/active site, the structural and experimental data suggest that the mutations affect kinase activity but not dimerization. These alterations also associate with a selective loss-of-function phenotype affecting specific signal transduction pathways and cellular responses in cancer cells. Structural Analysis and Molecular Modeling of ErbB4—The crystal structure of the ErbB4 kinase domain reported by Qiu et al. (4Qiu 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. Structure. 2008; 16: 460-467Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) (Protein Data Bank (PDB) (12Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (26227) Google Scholar) ID code: 3BCE) was used as a template for the structural models of ErbB4 with the mutations G802dup and D861Y. The model of ErbB4 with the point mutation D861Y was created with the Bodil modeling environment (13Lehtonen J.V. Still D.J. Rantanen V.V. Ekholm J. Bjorklund D. Iftikhar Z. Huhtala M. Repo S. Jussila A. Jaakkola J. Pentikainen O. Nyronen T. Salminen T. Gyllenberg M. Johnson M.S. J. Comput. Aided Mol. Des. 2004; 18: 401-419Crossref PubMed Scopus (187) Google Scholar). To investigate the structural consequences of the D861Y mutation on the function and ATP binding properties of ErbB4, the crystal structure of the EGFR kinase domain in complex with the non-hydrolyzable ATP analog AMP-PNP (PDB ID 2ITX) (14Yun C.H. Boggon T.J. Li Y. Woo M.S. Greulich H. Meyerson M. Eck M.J. Cancer Cell. 2007; 11: 217-227Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar) was used to position the ligand in the structure of ErbB4. The two kinase structures were first superimposed with Vertaa (15Johnson M. Lehtonen J. Bioinformatics. Oxford University Press, Oxford, UK2000: 15-50Google Scholar) implemented in Bodil, and then AMP-PNP and a conserved water molecule important for ATP binding were copied into the "mutated" model structure of ErbB4. Where necessary, new rotamers for side chains within the ATP-binding site were selected from the rotamer library (16Lovell S.C. Word J.M. Richardson J.S. Richardson D.C. Proteins. 2000; 40: 389-408Crossref PubMed Scopus (882) Google Scholar) implemented in Bodil. The structural model of ErbB4 with the insertion G802dup was produced with Modeler Version 9.4 (17Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10294) Google Scholar). The model was energy-minimized with Gromacs Version 3.3.3 (18van der Spoel D. Lindahl E. Hess B. Groenhof G. Mark A.E. Berendsen H.J. J. Comput. Chem. 2005; 26: 1701-1718Crossref PubMed Scopus (10500) Google Scholar) using the steepest descent algorithm and OPLS_AA/L all-atom force field until the potential energy change between two steps was less than 2000 kJ mol–1 nm–1. Thereafter, the model was visually analyzed, and if necessary for docking purposes, new rotamers for side chains within the ATP-binding site were introduced using Bodil. The ATP analog AMP-PNP was docked into the G802dup model with GOLD Version 3.2 (19Jones G. Willett P. Glen R.C. Leach A.R. Taylor R. J. Mol. Biol. 1997; 267: 727-748Crossref PubMed Scopus (5162) Google Scholar) by imposing a hydrogen-bonding constraint between the main-chain nitrogen atom of Met-799 and the N1 atom of AMP-PNP. The active site radius was set to 10 Å centered on the Hγ atom of Val-732. The most appropriate docking pose of AMP-PNP to the ErbB4 model was chosen based on visual analysis of the docked conformations. Although ATP binds to ErbB4 and EGFR complexed with a metal cation (20Brignola 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. J. Biol. Chem. 2002; 277: 1576-1585Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), we decided to construct the models without the addition of the cations because there were no Mg2+ atoms in the EGFR structure that was used as a template in the docking of AMP-PNP. Figures (Fig. 1 and Fig. 3) of the models were produced with Pymol Version 1.1 (21DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, Palo Alto, CA2007Google Scholar).FIGURE 3Structural models of ErbB4 D861Y and G802dup mutations. The structural models of the mutated proteins were generated as described under "Experimental Procedures." The docked ATP analog, AMP-PNP, is shown as stick representations with green carbon, blue nitrogen, red oxygen, and orange phosphate atoms. In A, the active site of the ErbB4 kinase domain is shown in detail with the αC-helix highlighted in a light brown color (right side, center). The amino acids important for the function of ErbB4 are labeled, and their side chains are shown as stick representations, putative hydrogen bonds with dashed yellow lines and the conserved water molecule with a red sphere (w1). At position 861, the side chains of both the aspartate of wild-type ErbB4 (centrally located and hydrogen-bonded to w1) and tyrosine of the mutant (oriented to the left; carbon atoms in magenta) are shown. AMP-PNP docked to the wild-type ErbB4 crystal structure (cyan; B) and to the G802dup model structure (pink; C); the Connolly surface, generated with the program Pymol, is shown. The location of important side chains is labeled and indicated with yellow surface color.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell Culture, Expression Plasmids, and Transfection—COS-7, MCF-7, and 32D cells were cultured as described (22Sundvall M. Korhonen A. Paatero I. Gaudio E. Melino G. Croce C.M. Aqeilan R.I. Elenius K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4162-4167Crossref PubMed Scopus (82) Google Scholar, 23Maatta J.A. Sundvall M. Junttila T.T. Peri L. Laine V.J. Isola J. Egeblad M. Elenius K. Mol. Biol. Cell. 2006; 17: 67-79Crossref PubMed Scopus (116) Google Scholar). MDA-MB-468 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum (Promocell), 50 μg/ml streptomycin (Sigma), and 100 IU/ml penicillin (Sigma). Mutations targeting the tyrosine kinase or cytoplasmic domains of ErbB4 reported by Soung et al. (8Soung Y.H. Lee J.W. Kim S.Y. Wang Y.P. Jo K.H. Moon S.W. Park W.S. Nam S.W. Lee J.Y. Yoo N.J. Lee S.H. Int. J. Cancer. 2006; 118: 1426-1429Crossref PubMed Scopus (103) Google Scholar) and Parsons et al. (11Parsons D.W. Wang T.L. Samuels Y. Bardelli A. Cummins J.M. DeLong L. Silliman N. Ptak J. Szabo S. Willson J.K. Markowitz S. Kinzler K.W. Vogelstein B. Lengauer C. Velculescu V.E. Nature. 2005; 436: 792Crossref PubMed Scopus (485) Google Scholar) were introduced into pcDNA3.1ErbB4JM-aCYT-1, pcDNA3.1ErbB4JM-aCYT-2, or pBABE-puroErbB4JM-aCYT-2 using the QuikChange site-directed mutagenesis kit (Stratagene). Similarly, a conserved lysine residue (Lys-751) within the ATP-binding site of ErbB4 was mutated to arginine in pcDNA3.1ErbB4JM-aCYT-2-HA to generate kinase-dead pcDNA3.1ErbB4JM-aCYT-2K751R-HA. All mutations were confirmed by sequencing. pcDNA3.1ErbB2 was generated by cloning a 4.2-kilobase insert of human ErbB2 (24Coussens L. Yang-Feng T.L. Liao Y.C. Chen E. Gray A. McGrath J. Seeburg P.H. Libermann T.A. Schlessinger J. Francke U. Levinson A. Ullrich A. Science. 1985; 230: 1132-1139Crossref PubMed Scopus (1546) Google Scholar) into the HindIII site of pcDNA3.1(–) vector (Invitrogen). pME18S-STAT5a has been described (25Pircher T.J. Flores-Morales A. Mui A.L. Saltiel A.R. Norstedt G. Gustafsson J.A. Haldosen L.A. Mol. Cell. Endocrinol. 1997; 133: 169-176Crossref PubMed Scopus (46) Google Scholar). COS-7 and MCF-7 cells were transfected with pcDNA3. 1ErbB4JM-aCYT-2 wild type and mutants using the FuGENE 6 Transfection Reagent (Roche Applied Science) according to the manufacturer's recommendations. To generate stable 32D or MDA-MB-468 cell lines expressing wild-type ErbB4 JM-a CYT-2 or its G802dup or D861Y mutants, Phoenix Ampho HEK293 cells were transfected with the corresponding pBABE-puroErbB4JM-aCYT-2 mutant constructs and used for retrovirus production. Subsequently, 32D and MDA-MB-468 cells were infected with retroviral supernatants, and stable pools were selected with puromycin (Sigma). Immunoprecipitation and Western Blotting—To analyze ErbB4 tyrosine phosphorylation in MCF-7 transfectants, cells were starved overnight, stimulated for 10 min with 25 ng/ml NRG-1 (R&D Systems, Inc.), and lysed. ErbB4 was immunoprecipitated from lysates with anti-ErbB4 antibodies (sc-283; Santa Cruz Biotechnology, Inc.) and analyzed for phosphotyrosine content by Western blotting with anti-phosphotyrosine antibodies (4G10; Upstate Biotechnology Inc.). ErbB4 loading was controlled by Western blotting with the sc-283 antibody. To analyze ErbB4 tyrosine phosphorylation in the MDA-MB-468 background, cells were starved overnight and stimulated for 15 min with 50 ng/ml NRG-1. Cell lysates were analyzed by Western blotting using a phospho-specific antibody against Tyr-1284 of ErbB4 (Cell Signaling). Loading was controlled by Western blotting with anti-ErbB4 (E-200; Abcam) and anti-actin (sc-1616; Santa Cruz) antibodies. COS-7 transfectants transiently expressing the indicated ErbB4 constructs with or without ErbB2 or STAT5a were starved overnight in the absence of serum and stimulated for 10 min with 25 ng/ml NRG-1. Cell lysates were analyzed for ErbB4, Erk1/2, Akt, and STAT5 phosphorylation by Western blotting using phospho-specific antibodies (Cell Signaling). Loading was controlled by Western blotting with antibodies recognizing total ErbB4 (sc-283), Erk1/2 (Cell Signaling), Akt (sc-1618; Santa Cruz), or STAT5 (sc-835; Santa Cruz). ErbB2 was immunoprecipitated with anti-ErbB2 antibodies trastuzumab (Roche Applied Science) or sc-284 (Santa Cruz) and analyzed for phosphorylation by Western blotting with an anti-phosphotyrosine antibody (4G10). ErbB2 loading was controlled by Western blotting with an anti-ErbB2 antibody (sc-284). To inhibit tyrosine kinase activity, cells were incubated for 1 h in the presence of 1 μm gefitinib (AstraZeneca) or M578440 (AZ10398863; AstraZeneca) before stimulation with or without NRG-1. In Vitro Kinase Assay—32D cells stably expressing wild-type ErbB4 JM-a CYT-2 or its engineered G802dup or D861Y mutants were analyzed for ErbB4 in vitro kinase activity in the presence and absence of 10 μm ATP (Roche Applied Science) as described (23Maatta J.A. Sundvall M. Junttila T.T. Peri L. Laine V.J. Isola J. Egeblad M. Elenius K. Mol. Biol. Cell. 2006; 17: 67-79Crossref PubMed Scopus (116) Google Scholar). ErbB4 was immunoprecipitated with anti-ErbB4 antibodies (sc-283) and analyzed for phosphotyrosine content by Western blotting with anti-phosphotyrosine antibodies (4G10). ErbB4 loading was controlled by Western blotting with the sc-283 antibody. Three-dimensional Cultures—2 × 104 MDA-MB-468 transfectants in 20 μl of Dulbecco's modified Eagle's medium + 10% fetal calf serum were suspended into 180 μl of cold Matrigel (BD Biosciences) supplemented with or without 50 ng/ml NRG-1. The cell-Matrigel suspension was divided to duplicate 96-well plate wells, and the Matrigel was allowed to polymerize at 37 °C for 30 min. One hundred μl of Dulbecco's modified Eagle's medium + 10% fetal calf serum containing 0 or 50 ng/ml NRG-1 was added on top of the polymerized Matrigel. The cells were maintained in Matrigel at 37 °C for 6 days, after which formed colonies were counted using Olympus CK40 light microscope. From each well the colonies were counted from 4 individual views through the whole thickness of the Matrigel using 200× magnification. MTT Proliferation Assay—To analyze the proliferation of MDA-MB-468 transfectants, 3 × 103 cells were plated onto 96-well plate wells in triplicates in 100 μl of Dulbecco's modified Eagle's medium + 10% fetal calf serum containing 0 or 50 ng/ml NRG-1. At the indicated time points, the number of viable cells was estimated using a CellTiter 96 nonradioactive cell proliferation assay (MTT; Promega) following the manufacturer's recommendations. Nine of the recently reported somatic mutations of ErbB4 target the tyrosine kinase domain (8Soung Y.H. Lee J.W. Kim S.Y. Wang Y.P. Jo K.H. Moon S.W. Park W.S. Nam S.W. Lee J.Y. Yoo N.J. Lee S.H. Int. J. Cancer. 2006; 118: 1426-1429Crossref PubMed Scopus (103) Google Scholar), whereas a missense mutation, I1030M, targets the cytoplasmic tail of ErbB4 (11Parsons D.W. Wang T.L. Samuels Y. Bardelli A. Cummins J.M. DeLong L. Silliman N. Ptak J. Szabo S. Willson J.K. Markowitz S. Kinzler K.W. Vogelstein B. Lengauer C. Velculescu V.E. Nature. 2005; 436: 792Crossref PubMed Scopus (485) Google Scholar) (Fig. 1A). To systematically analyze the location and model significance of the nine kinase domain mutations, we exploited the recently reported structure of the ErbB4 kinase domain (4Qiu 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. Structure. 2008; 16: 460-467Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). In the case of four of the mutations in ErbB4, R782Q, G802dup, P854Q, and D861Y, the targeted amino acids are conserved within the human ErbB receptor family (26Plowman G.D. Culouscou J.M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.G. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (675) Google Scholar); three other mutated positions, Ala-773, Glu-810, and Glu-872, are conserved in all family members except for the weak kinase ErbB3, whereas Val-721 varies as leucine and isoleucine, and Thr-926 varies as alanine and leucine. In Fig. 1B the mutated amino acids are mapped to the structure of a monomer of the wild-type ErbB4 kinase domain, showing their positioning relative to important structural and functional features of the ErbB4 structure. The mutation V721I is located in close proximity to the asymmetrical dimer interface, but the equivalent position in the wild-type EGFR kinase domain is isoleucine, and in the structure of the asymmetric dimer this position is not involved in forming the dimer interface (3Zhang X. Gureasko J. Shen K. Cole P.A. Kuriyan J. Cell. 2006; 125: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (1124) Google Scholar). The mutations A773S and R782Q are located close to each other on the surface of the kinase domain at the C-terminal end of the αC-helix (A773S) and within the loop connecting the αC-helix to the β-sheet of the N-terminal lobe of the kinase domain (R782Q). G802dup is located on the surface of the kinase domain, in a loop connecting the N-terminal and C-terminal lobes, also called the hinge region, whereas D861Y is located at the base of the A-loop. E810K, P854Q, E872K, and T926M are located on the surface of the kinase domain. Glu-810 is at the C-terminal end of helix 3, Pro-854 is within a loop connecting the two strands within the small β-sheet of the C-terminal lobe, Glu-872 is within the A-loop, and Thr-926 is located at the beginning of helix 9. To address their function in vivo, the somatic mutations were engineered by site-directed mutagenesis to one of the ErbB4 isoforms expressed in cancer, ErbB4 JM-a CYT-2 (27Junttila T.T. Sundvall M. Lundin M. Lundin J. Tanner M. Harkonen P. Joensuu H. Isola J. Elenius K. Cancer Res. 2005; 65: 1384-1393Crossref PubMed Scopus (151) Google Scholar). Unexpectedly, none of the 10 mutations demonstrated enhanced basal or ligand-induced tyrosine phosphorylation in MCF-7 breast cancer or COS-7 cells compared with wild-type ErbB4 (Fig. 2A; data not shown). Moreover, sensitivity of the ErbB4 mutants to inhibition by the tyrosine kinase inhibitor gefitinib was not different from wild-type ErbB4 in COS-7 cells (IC50 for ErbB4 ≈ 0.5 μm). In contrast, two mutants, G802dup and D861Y, showed clearly reduced tyrosine phosphorylation in vivo (Fig. 2A). Moreover, results of an in vitro kinase assay demonstrate that in 32D cells devoid of endogenous ErbB expression, both G802dup and D861Y mutants had markedly reduced tyrosine kinase activity (Fig. 2B). Even after a maximal exposure time, only a weak phosphotyrosine signal was detected in the G802dup mutant, whereas the D861Y mutant was completely kinase-dead (data not shown). The findings were reproduced by analyzing the mutants in the context of the other cancer-associated isoform, ErbB4 JM-a CYT-1 (27Junttila T.T. Sundvall M. Lundin M. Lundin J. Tanner M. Harkonen P. Joensuu H. Isola J. Elenius K. Cancer Res. 2005; 65: 1384-1393Crossref PubMed Scopus (151) Google Scholar) (supplemental Fig. 1), as well as in the context of stable transfections into NIH 3T3 cells (data not shown). These data indicate that 2 of 10 ErbB4 mutants, G802dup and D861Y, have lost their kinase activity. The D861Y mutation is located in the DFG motif, a highly conserved triplet at the base of the activation loop of eukaryotic protein kinases that is crucial for successful phosphotransfer (28Hanks S.K. Quinn A.M. Hunter T. Science. 1988; 241: 42-52Crossref PubMed Scopus (3775) Google Scholar). In ErbB4, the DFG motif consists of Asp-861, Phe-862, and Gly-863. To investigate the structural consequences of introducing a tyrosine residue at amino acid position 861, we generated a model as described under "Experimental Procedures," replacing the side chain of aspartate by tyrosine in the ErbB4 crystal structure. Based on our model of wild-type ErbB4 bound to the non-hydrolyzable ATP analog, AMP-PNP, it is likely that upon ATP binding, Asp-861 is hydrogen-bonded to a conserved water molecule (w1 in Fig. 3A). This water molecule would additionally be coordinated by two key residues conserved throughout the family; that is, the side chains of Asp-843 and Asn-848 from the C-loop (Fig. 3A). Furthermore, Asp-843 is thought to act as a catalytic base, removing the hydrogen atom from the hydroxyl group of the substrate tyrosine residue (29Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1156) Google Scholar). Thus, introduction of a bulky tyrosine residue into the DFG motif by mutation of Asp-861 not only disrupts the hydrogen bonding network that functions to properly position the ATP terminal phosphate and the side chain of the catalytic Asp-843 (Fig. 3A), but the mutation may also directly interfere with the binding of the substrate. Moreover, a compensatory hydrogen bond cannot be formed between the hydroxyl group of tyrosine at position 861 and the side chain of Asp-843 due to steric hindrance (replacing the Asp-861–w1–Asp-843 interactions by direct hydrogen bonding of D861Y to Asp-843). Furthermore, the size difference, tyrosine versus aspartate, is sufficient to expect some alterations of the local structure, too. These observations indicate that D861Y mutation disrupts the functionally important DFG motif, resulting in catalytic incompetence. To investigate the structural consequences of the insertion of an additional glycine residue adjacent to Gly-802 in the hinge region of the kinase domain, we generated a model of ErbB4 G802dup. Because Gly-802 forms part of the ATP-binding site and is located in close proximity to the adenine ring, the ATP analog AMP-PNP was docked into the model to ass
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