IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell (1535-6108) 2006 imex curation Wang SE., Narasanna A., Perez-Torres M., Xiang B., Wu FY., Yang S., Carpenter G., Gazdar AF., Muthuswamy SK., Arteaga CL. Synapse - Interactions of proteins with an established role in the presynapse. Cancer - Interactions investigated in the context of cancer HER2WT or HER2YVMA receptors expressed in MCF10A cells were precipitated with a Myc antibody and an in vitro kinase assay was performed. Only protein-protein interactions 6 in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Wang SE., Narasanna A., Perez-Torres M., Xiang B., Wu FY., Yang S., Carpenter G., Gazdar AF., Muthuswamy SK., Arteaga CL. Synapse - Interactions of proteins with an established role in the presynapse. HER2WT or HER2YVMA receptors expressed in MCF10A cells were precipitated with a Myc antibody and an in vitro kinase assay was performed. Accepted 2006-OCT-10 by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 6 imex curation HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell (1535-6108) 2006 imex curation Wang SE., Narasanna A., Perez-Torres M., Xiang B., Wu FY., Yang S., Carpenter G., Gazdar AF., Muthuswamy SK., Arteaga CL. Synapse - Interactions of proteins with an established role in the presynapse. Cancer - Interactions investigated in the context of cancer HER2WT or HER2YVMA receptors expressed in MCF10A cells were precipitated with a Myc antibody and an in vitro kinase assay was performed. Only protein-protein interactions 6 human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line pull down pull down antibody detection identification by antibody antibody detection Wang SE., Narasanna A., Perez-Torres M., Xiang B., Wu FY., Yang S., Carpenter G., Gazdar AF., Muthuswamy SK., Arteaga CL. Synapse - Interactions of proteins with an established role in the presynapse. HER2WT or HER2YVMA receptors expressed in MCF10A cells were precipitated with a Myc antibody. An in vitro kinase assay was performed, and the products were analyzed by 7.5% SDS-PAGE followed by P-Tyr and Myc immunoblots. Accepted 2006-OCT-9 by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 6 imex curation HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell (1535-6108) 2006 imex curation Wang SE., Narasanna A., Perez-Torres M., Xiang B., Wu FY., Yang S., Carpenter G., Gazdar AF., Muthuswamy SK., Arteaga CL. Synapse - Interactions of proteins with an established role in the presynapse. Cancer - Interactions investigated in the context of cancer HER2WT or HER2YVMA receptors expressed in MCF10A cells were precipitated with a Myc antibody and an in vitro kinase assay was performed. Only protein-protein interactions 6 human-beas_2b Human bronchial epithelial cell line BEAS-2B beas_2b Human bronchial epithelial cell line BEAS-2B http://cancerres.aacrjournals.org/cgi/content/abstract/48/7/1904?ijkey=330aa4101c1c77d6fbc2b1fc3d60b4ead4af2511&keytype2=tf_ipsecsha pull down pull down antibody detection identification by antibody antibody detection Wang SE., Narasanna A., Perez-Torres M., Xiang B., Wu FY., Yang S., Carpenter G., Gazdar AF., Muthuswamy SK., Arteaga CL. Synapse - Interactions of proteins with an established role in the presynapse. 4C is covered in wang-2006c-1. Have removed the exp-modification Accepted 2006-OCT-9 by JYOTI. Cancer Cell (1535-6108) 2006 Only protein-protein interactions 6 imex curation erbb3_human Receptor tyrosine-protein kinase erbB-3 Tyrosine kinase-type cell surface receptor HER3 ERBB3 HER3 Proto-oncogene-like protein c-ErbB-3 protein protein human Homo sapiens Human MRANDALQVLGLLFSLARGSEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLTMALTVIAGLVVIFMMLGGTFLYWRGRRIQNKRAMRRYLERGESIEPLDPSEKANKVLARIFKETELRKLKVLGSGVFGTVHKGVWIPEGESIKIPVCIKVIEDKSGRQSFQAVTDHMLAIGSLDHAHIVRLLGLCPGSSLQLVTQYLPLGSLLDHVRQHRGALGPQLLLNWGVQIAKGMYYLEEHGMVHRNLAARNVLLKSPSQVQVADFGVADLLPPDDKQLLYSEAKTPIKWMALESIHFGKYTHQSDVWSYGVTVWELMTFGAEPYAGLRLAEVPDLLEKGERLAQPQICTIDVYMVMVKCWMIDENIRPTFKELANEFTRMARDPPRYLVIKRESGPGIAPGPEPHGLTNKKLEEVELEPELDLDLDLEAEEDNLATTTLGSALSLPVGTLNRPRGSQSLLSPSSGYMPMNQGNLGESCQESAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVNGYVMPDTHLKGTPSSREGTLSSVGLSSVLGTEEEDEDEEYEYMNRRRRHSPPHPPRPSSLEELGYEYMDVGSDLSASLGSTQSCPLHPVPIMPTAGTTPDEDYEYMNRQRDGGGPGGDYAAMGACPASEQGYEEMRAFQGPGHQAPHVHYARLKTLRSLEATDSAFDNPDYWHSRLFPKANAQRT DIP protein P21860 original sequence version: 175 DIP protein P21860 original sequence version: 125 DIP protein P21860 original sequence version: 172 DIP protein P21860 original sequence version: 130 DIP protein P21860 original sequence version: 157 DIP protein P21860 original sequence version: 137 7201E7F66CA374BD p85a_human Phosphatidylinositol 3-kinase regulatory subunit alpha PIK3R1 GRB1 Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha protein protein human Homo sapiens Human MSAEGYQYRALYDYKKEREEDIDLHLGDILTVNKGSLVALGFSDGQEARPEEIGWLNGYNETTGERGDFPGTYVEYIGRKKISPPTPKPRPPRPLPVAPGSSKTEADVEQQALTLPDLAEQFAPPDIAPPLLIKLVEAIEKKGLECSTLYRTQSSSNLAELRQLLDCDTPSVDLEMIDVHVLADAFKRYLLDLPNPVIPAAVYSEMISLAPEVQSSEEYIQLLKKLIRSPSIPHQYWLTLQYLLKHFFKLSQTSSKNLLNARVLSEIFSPMLFRFSAASSDNTENLIKVIEILISTEWNERQPAPALPPKPPKPTTVANNGMNNNMSLQDAEWYWGDISREEVNEKLRDTADGTFLVRDASTKMHGDYTLTLRKGGNNKLIKIFHRDGKYGFSDPLTFSSVVELINHYRNESLAQYNPKLDVKLLYPVSKYQQDQVVKEDNIEAVGKKLHEYNTQFQEKSREYDRLYEEYTRTSQEIQMKRTAIEAFNETIKIFEEQCQTQERYSKEYIEKFKREGNEKEIQRIMHNYDKLKSRISEIIDSRRRLEEDLKKQAAEYREIDKRMNSIKPDLIQLRKTRDQYLMWLTQKGVRQKKLNEWLGNENTEDQYSLVEDDEDLPHHDEKTWNVGSSNRNKAENLLRGKRDGTFLVRESSKQGCYACSVVVDGEVKHCVINKTATGYGFAEPYNLYSSLKELVLHYQHTSLVQHNDSLNVTLAYPVYAQQRR DIP protein P27986 original sequence version: 181 DIP protein P27986 original sequence version: 184 DIP protein P27986-1 original sequence version: 135 DIP protein P27986 original sequence version: 135 DIP protein P27986-1 original sequence version: 145 DIP protein P27986-1 original sequence version: 147 DIP protein P27986 original sequence version: 191 B9DAD8416C33140F erbb2_human Receptor tyrosine-protein kinase erbB-2 p185erbB2 MLN19 Proto-oncogene c-ErbB-2 Tyrosine kinase-type cell surface receptor HER2 Proto-oncogene Neu ERBB2 HER2 NEU NGL Metastatic lymph node gene 19 protein protein protein human Homo sapiens Human MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV DIP protein P04626 original sequence version: 198 DIP protein P04626 original sequence version: 122 DIP protein P04626 original sequence version: 206 DIP protein P04626 original sequence version: 195 DIP protein P04626 original sequence version: 150 DIP protein P04626 original sequence version: 179 DIP protein P04626 original sequence version: 146 DIP protein P04626 original sequence version: 147 39E9DFDA04DCF962 shc1_human SHC-transforming protein 1 SHC1 SHC SHCA Src homology 2 domain-containing-transforming protein C1 SHC-transforming protein A SHC-transforming protein 3 protein protein human Homo sapiens Human MDLLPPKPKYNPLRNESLSSLEEGASGSTPPEELPSPSASSLGPILPPLPGDDSPTTLCSFFPRMSNLRLANPAGGRPGSKGEPGRAADDGEGIVGAAMPDSGPLPLLQDMNKLSGGGGRRTRVEGGQLGGEEWTRHGSFVNKPTRGWLHPNDKVMGPGVSYLVRYMGCVEVLQSMRALDFNTRTQVTREAISLVCEAVPGAKGATRRRKPCSRPLSSILGRSNLKFAGMPITLTVSTSSLNLMAADCKQIIANHHMQSISFASGGDPDTAEYVAYVAKDPVNQRACHILECPEGLAQDVISTIGQAFELRFKQYLRNPPKLVTPHDRMAGFDGSAWDEEEEEPPDHQYYNDFPGKEPPLGGVVDMRLREGAAPGAARPTAPNAQTPSHLGATLPVGQPVGGDPEVRKQMPPPPPCPGRELFDDPSYVNVQNLDKARQAVGGAGPPNPAINGSAPRDLFDMKPFEDALRVPPPPQSVSMAEQLRGEPWFHGKLSRREAEALLQLNGDFLVRESTTTPGQYVLTGLQSGQPKHLLLVDPEGVVRTKDHRFESVSHLISYHMDNHLPIISAGSELCLQQPVERKL DIP protein P29353 original sequence version: 161 DIP protein P29353 original sequence version: 190 7EFA5CB185A548D1 egfr_human Epidermal growth factor receptor ERBB1 EGFR Receptor tyrosine-protein kinase erbB-1 Proto-oncogene c-ErbB-1 ERBB HER1 protein protein human Homo sapiens Human MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA DIP protein P00533 original sequence version: 213 DIP protein P00533 original sequence version: 214 DIP protein P00533 original sequence version: 119 DIP protein P00533 original sequence version: 207 DIP protein P00533 original sequence version: 132 DIP protein P00533-1 original sequence version: 197 DIP protein P00533 original sequence version: 163 DIP protein P00533-1 original sequence version: 159 DIP protein P00533 original sequence version: 184 DIP protein P00533-1 original sequence version: 174 DIP protein P00533-1 original sequence version: 191 DIP protein P00533 original sequence version: 218 DIP protein P00533 original sequence version: 221 DIP protein P00533-1 original sequence version: 221 DIP protein P00533 original sequence version: 226 DIP protein P00533 original sequence version: 166 DIP protein P00533-1 original sequence version: 166 DIP protein P00533 original sequence version: 194 DIP protein P00533-1 original sequence version: 194 DIP protein P00533-1 original sequence version: 224 DIP protein P00533 original sequence version: 189 DIP protein P00533-1 original sequence version: 207 DIP protein P00533 original sequence version: 177 DIP protein P00533 original sequence version: 168 D8A2A50B4EFB6ED2 erbb2-erbb2 1 6 enzyme target enzyme target substrate substrate unspecified role unspecified role insertion aa 776 Insertion TyrValMetAla at residue 776 biological feature biological feature certain certain sequence position certain certain certain sequence position certain n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range region optyr O4'-phospho-L-tyrosine (2S)-2-amino-3-(4-phosphonooxyphenyl)propanoic acid 2-azanyl-3-(4-phosphonooxyphenyl)propanoic acid MOD_RES Phosphotyrosine O4-phosphotyrosine tyrosine phosphate 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-phosphate O4'-phospho-L-tyrosine O4'-phosphorylated L-tyrosine OPTyr Phospho Phospho Tyrosinyl Phosphorylation phosphorylationy undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line 6 enzyme enzyme unspecified role unspecified role region optyr O4'-phospho-L-tyrosine (2S)-2-amino-3-(4-phosphonooxyphenyl)propanoic acid 2-azanyl-3-(4-phosphonooxyphenyl)propanoic acid MOD_RES Phosphotyrosine O4-phosphotyrosine tyrosine phosphate 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-phosphate O4'-phospho-L-tyrosine O4'-phosphorylated L-tyrosine OPTyr Phospho Phospho Tyrosinyl Phosphorylation phosphorylationy undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined insertion aa 776 insertion TyrValMetAla at residue 776 biological feature biological feature certain certain sequence position certain certain certain sequence position certain n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line physical association physical association Myc-tagged wild-type and mutant HER2 were precipitated from MCF10A/HER2WT and MCF10A/HER2YVMA cells, respectively, and the pull-downs were tested in an in vitro kinase reaction. When ATP was added, HER2YVMA showed markedly higher tyrosine phosphorylation than HER2WT 3 C HER2YVMA showed markedly higher tyrosine autophosphorylation than HER2WT. HER2 autophosphorylates. erbb2-egfr 3 8 unspecified role unspecified role prey prey 5 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 4 unspecified role unspecified role prey prey 1289 optyr O4'-phospho-L-tyrosine (2S)-2-amino-3-(4-phosphonooxyphenyl)propanoic acid 2-azanyl-3-(4-phosphonooxyphenyl)propanoic acid MOD_RES Phosphotyrosine O4-phosphotyrosine tyrosine phosphate 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-phosphate O4'-phospho-L-tyrosine O4'-phosphorylated L-tyrosine OPTyr Phospho Phospho Tyrosinyl Phosphorylation phosphorylationy certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range association association 4 A higher levels of EGFR, Y1068 P-EGFR, Shc, and p85 coprecipitated with Myc in cells expressing the insertion mutant compared to HER2WT cells (Figure 4A, lanes 7�12). Higher levels of Y1289 P-HER3 and increased HER3:HER2 association were observed in HER2YVMA-expressing MCF10A cells but not in BEAS2B cells, probably as a result of the lower levels of HER3 in the latter cells The band corresponding to HER3 is very faint and the authors state that BEAS2B cells have lower levels of HER3 than MCF10A cells. The authors refer to a protein they callp85 but give no further information. Uniprot there are severalproteins which fit this decription. However, one of the references refers to the P85alpha regulatory subunit of PI3K. It has been assumed that this is the protein the authors are referring to. Only one of the two bands corresponding to Shc when HER2YVMA is the bait is seen when HER2WT is the bait. erbb2-egfr-1 3 8 unspecified role unspecified role prey prey 5 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait insertion aa 776 insertion TyrValMetAla at residue 776 biological feature biological feature certain certain sequence position certain certain certain sequence position certain 1248 optyr O4'-phospho-L-tyrosine (2S)-2-amino-3-(4-phosphonooxyphenyl)propanoic acid 2-azanyl-3-(4-phosphonooxyphenyl)propanoic acid MOD_RES Phosphotyrosine O4-phosphotyrosine tyrosine phosphate 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-phosphate O4'-phospho-L-tyrosine O4'-phosphorylated L-tyrosine OPTyr Phospho Phospho Tyrosinyl Phosphorylation phosphorylationy certain certain sequence position certain certain certain sequence position certain n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range association association 4 A To determine the activation status of ectopic HER2, Myc pull-downs were tested by P-Tyr and Y1248 P-HER2 immunoblots. Y1248 P-HER2 was only detected in the Myc precipitate from HER2YVMA-expressing cells. A much stronger P-Tyr band coprecipitated with Myc in these cells compared with HER2WT-expressing cells. The fainter P-Tyr band in HER2WT cells, where Y1248 P-HER2 was not detected in the Myc precipitates, suggests that HER2 is phosphorylated in tyrosines other than Y1248 in MCF10A/HER2WT cells. Finally, higher levels of EGFR, Y1068 P-EGFR, Shc, and p85 coprecipitated with Myc in cells expressing the insertion mutant compared to HER2WT cells (Figure 4A, lanes 7�12). Higher levels of Y1289 P-HER3 and increased HER3:HER2 association were observed in HER2YVMA-expressing MCF10A cells but not in BEAS2B cells, probably as a result of the lower levels of HER3 in the latter cells The authors refer to a protein they callp85 but give no further information. Uniprot there are severalproteins which fit this decription. However, one of the references refers to the P85alpha regulatory subunit of PI3K. It has been assumed that this is the protein the authors are referring to. 2 bands correspond to Shc. Bands corresponding to P-EGFRY1068 and P-HER2Y1248 are seen which are not seen for HER2WT. erbb2-egfr-2 2 8 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range 5 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey association association 4 A To determine the activation status of ectopic HER2, Myc pull-downs were tested by P-Tyr and Y1248 P-HER2 immunoblots. Y1248 P-HER2 was only detected in the Myc precipitate from HER2YVMA-expressing cells. A much stronger P-Tyr band coprecipitated with Myc in these cells compared with HER2WT-expressing cells. The fainter P-Tyr band in HER2WT cells, where Y1248 P-HER2 was not detected in the Myc precipitates, suggests that HER2 is phosphorylated in tyrosines other than Y1248 in MCF10A/HER2WT cells. Finally, higher levels of EGFR, Y1068 P-EGFR, Shc, and p85 coprecipitated with Myc in cells expressing the insertion mutant compared to HER2WT cells (Figure 4A, lanes 7�12). Higher levels of Y1289 P-HER3 and increased HER3:HER2 association were observed in HER2YVMA-expressing MCF10A cells The authors refer to a protein they callp85 but give no further information. Uniprot there are severalproteins which fit this decription. However, one of the references refers to the P85alpha regulatory subunit of PI3K. It has been assumed that this is the protein the authors are referring to. The band corresponding to EGFR is much fainter than for HER2YVMA. The top band corresponding to Shc is much fainter than for the HER2YVMA. erbb2-egfr-3 2 8 unspecified role unspecified role prey prey 5 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range insertion aa 776 insertion TyrValMetAla at residue 776 biological feature biological feature certain certain sequence position certain certain certain sequence position certain 1248 optyr O4'-phospho-L-tyrosine (2S)-2-amino-3-(4-phosphonooxyphenyl)propanoic acid 2-azanyl-3-(4-phosphonooxyphenyl)propanoic acid MOD_RES Phosphotyrosine O4-phosphotyrosine tyrosine phosphate 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-phosphate O4'-phospho-L-tyrosine O4'-phosphorylated L-tyrosine OPTyr Phospho Phospho Tyrosinyl Phosphorylation phosphorylationy certain certain sequence position certain certain certain sequence position certain association association 4 A To determine the activation status of ectopic HER2, Myc pull-downs were tested by P-Tyr and Y1248 P-HER2 immunoblots. Y1248 P-HER2 was only detected in the Myc precipitate from HER2YVMA-expressing cells. A much stronger P-Tyr band coprecipitated with Myc in these cells compared with HER2WT-expressing cells. The fainter P-Tyr band in HER2WT cells, where Y1248 P-HER2 was not detected in the Myc precipitates, suggests that HER2 is phosphorylated in tyrosines other than Y1248 in MCF10A/HER2WT cells. Finally, higher levels of EGFR, Y1068 P-EGFR, Shc, and p85 coprecipitated with Myc in cells expressing the insertion mutant compared to HER2WT cells (Figure 4A, lanes 7�12). Higher levels of Y1289 P-HER3 and increased HER3:HER2 association were observed in HER2YVMA-expressing MCF10A cells The authors refer to a protein they callp85 but give no further information. Uniprot there are severalproteins which fit this decription. However, one of the references refers to the P85alpha regulatory subunit of PI3K. It has been assumed that this is the protein the authors are referring to. 2 bands correspond to Shc. Bands corresponding to HER3, P-HER3Y1289 and P-Her2Y1248 are seen which are not seen for wild-type HER-2. erbb2-egfr-4 1 8 enzyme target enzyme target substrate substrate unspecified role unspecified role lys721arg kinase dead (KD) biological feature biological feature certain certain sequence position certain certain certain sequence position certain c-terminal gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range mouse-32d Mouse 32d bone marrow cells 32d established from long-term bone marrow cultures of C3H/HeJ mice infected with the Friend murine leukemia virus; cells are constitutively growth factor-dependent 6 enzyme enzyme unspecified role unspecified role undefined mutation insertion TyrValMetAla at residue 776 mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain Wed Jul 06 16:58:48 BST 2016 Sequence change details about this feature cannot be ascertained or do not fit with the current version of the referenced protein, so they have been deleted as a result of our quality control procedures. The original label was 'insertion aa 776' n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range human-mcf10a Human non-transformed breast epithelial cell line mcf10a non-transformed human breast epithelial cell line physical association physical association 4 C we performed an in vitro kinase assay using GFP-fused K721R (kinase-dead) EGFR (Ewald et al., 2003) (EGFRKD) as substrate. Myc-tagged wild-type and mutant HER2 receptors were precipitated from transfected MCF10A cells and incubated with EGFRKD-GFP in the presence or absence of ATP. In the absence of ATP, no phosphorylation of EGFRKD-GFP was detected. Both HER2WT and HER2YVMA phosphorylated EGFRKD-GFP in vitro, but this effect was at least 5-fold higher with the insertion mutant The phosphorylation of the kinase dead EGFR-GFP by the HER2YVMA was at least five times that by HER2WT. The kinase dead EGFR-GFP was phosphorylated.