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/ Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell (1535-6108) 2009 imex curation Junttila TT., Akita RW., Parsons K., Fields C., Lewis Phillips GD., Friedman LS., Sampath D., Sliwkowski MX. Cancer - Interactions investigated in the context of cancer Cells were treated with 100 nM antibody (Trastuzumab or Pertuzumab), followed by Heregulin when indicated. In the left panel, extracellular proteins were crosslinked with DTSSP. Lysates were then immunoprecipitated by using an anti-HER2 antibody, and HER3 was detected by Western Blot. marks@gene.com 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 3 human-mda_mb_361 Homo sapiens MDA MB 361 cell line. mda_mb_361 Human MDA MB 361 breast carcinoma cell line. MDA MB 361 cells are used as an in vitro model of breast carcinoma (adenocarcinoma) as they overexpress HER2 (erbB2 receptor tyrosine kinase) and contain constitutively tyrosine phosphorylated Shc, and and an activating PIK3CA mutation (E545K). MDA MB 361 cells are used as an in vitro model of breast carcinoma (adenocarcinoma)as they overexpress HER2 (erbB2 receptor tyrosine kinase) and contain constitutively tyrosine phosphorylated Shc, and and an activating PIK3CA mutation (E545K). anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Junttila TT., Akita RW., Parsons K., Fields C., Lewis Phillips GD., Friedman LS., Sampath D., Sliwkowski MX. Cancer - Interactions investigated in the context of cancer Erratum published in Cancer Cell December 2011:-molecular profiling of the MDA-MB-361.1 cell line revealed that it is actually a derivative of the MCF7 cell line that was stably transfected to overexpress HER2 (termed MCF7-neo/HER2). MCF7 cells also harbor the activating PIK3CA mutation (E545K). Although the conclusions from the study are unaffected, we apologize for the discrepancy. Cells were treated with 100 nM antibody (Trastuzumab or Pertuzumab), followed by Heregulin when indicated. In the left panel, extracellular proteins were crosslinked with DTSSP. Lysates were then immunoprecipitated by using an anti-HER2 antibody, and HER3 was detected by Western Blot. marks@gene.com Accepted 2010-MAR-23 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 3 imex curation Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell (1535-6108) 2009 imex curation Junttila TT., Akita RW., Parsons K., Fields C., Lewis Phillips GD., Friedman LS., Sampath D., Sliwkowski MX. Cancer - Interactions investigated in the context of cancer Cells were treated with 100 nM antibody (Trastuzumab or Pertuzumab), followed by Heregulin when indicated. In the left panel, extracellular proteins were crosslinked with DTSSP. Lysates were then immunoprecipitated by using an anti-HER2 antibody, and HER3 was detected by Western Blot. marks@gene.com 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 3 human-skbr3 Homo sapiens SKBR3 skbr3 SKBR3 Species: human; Tissue: breast; Tumor: adenocarcinoma; Derived from: metastatic site: pleural effusion crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Junttila TT., Akita RW., Parsons K., Fields C., Lewis Phillips GD., Friedman LS., Sampath D., Sliwkowski MX. Cancer - Interactions investigated in the context of cancer Cells were treated with 100 nM antibody (Trastuzumab or Pertuzumab), followed by Heregulin when indicated. In the left panel, extracellular proteins were crosslinked with DTSSP. Lysates were then immunoprecipitated by using an anti-HER2 antibody, and HER3 was detected by Western Blot. marks@gene.com Accepted 2010-MAR-23 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 3 imex curation Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell (1535-6108) 2009 imex curation Junttila TT., Akita RW., Parsons K., Fields C., Lewis Phillips GD., Friedman LS., Sampath D., Sliwkowski MX. Cancer - Interactions investigated in the context of cancer Cells were treated with 100 nM antibody (Trastuzumab or Pertuzumab), followed by Heregulin when indicated. In the left panel, extracellular proteins were crosslinked with DTSSP. Lysates were then immunoprecipitated by using an anti-HER2 antibody, and HER3 was detected by Western Blot. marks@gene.com 29-Mar-2010: Contacted by JYOTI. orchard Only protein-protein interactions 3 human-bt474 Homo sapiens breast ductal carcinoma cell line bt474 Human breast ductal carcinoma cell line BT474 Human breast ductal carcinoma cell line established from a solid, invasive ductal carcinoma of the breast obtained from a 60-year-old woman. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Junttila TT., Akita RW., Parsons K., Fields C., Lewis Phillips GD., Friedman LS., Sampath D., Sliwkowski MX. Cancer - Interactions investigated in the context of cancer Cells were treated with 100 nM antibody (Trastuzumab or Pertuzumab), followed by Heregulin when indicated. In the left panel, extracellular proteins were crosslinked with DTSSP. Lysates were then immunoprecipitated by using an anti-HER2 antibody, and HER3 was detected by Western Blot. marks@gene.com Accepted 2010-MAR-23 by ORCHARD. 29-Mar-2010: Contacted by JYOTI. Cancer Cell (1535-6108) 2009 orchard Only protein-protein interactions 3 imex curation 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 pk3ca_human Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Phosphoinositide-3-kinase catalytic alpha polypeptide Serine/threonine protein kinase PIK3CA PIK3CA Phosphatidylinositol 4,5-bisphosphate 3-kinase 110 kDa catalytic subunit alpha Phosphoinositide 3-kinase alpha protein protein human Homo sapiens Human MPPRPSSGELWGIHLMPPRILVECLLPNGMIVTLECLREATLITIKHELFKEARKYPLHQLLQDESSYIFVSVTQEAEREEFFDETRRLCDLRLFQPFLKVIEPVGNREEKILNREIGFAIGMPVCEFDMVKDPEVQDFRRNILNVCKEAVDLRDLNSPHSRAMYVYPPNVESSPELPKHIYNKLDKGQIIVVIWVIVSPNNDKQKYTLKINHDCVPEQVIAEAIRKKTRSMLLSSEQLKLCVLEYQGKYILKVCGCDEYFLEKYPLSQYKYIRSCIMLGRMPNLMLMAKESLYSQLPMDCFTMPSYSRRISTATPYMNGETSTKSLWVINSALRIKILCATYVNVNIRDIDKIYVRTGIYHGGEPLCDNVNTQRVPCSNPRWNEWLNYDIYIPDLPRAARLCLSICSVKGRKGAKEEHCPLAWGNINLFDYTDTLVSGKMALNLWPVPHGLEDLLNPIGVTGSNPNKETPCLELEFDWFSSVVKFPDMSVIEEHANWSVSREAGFSYSHAGLSNRLARDNELRENDKEQLKAISTRDPLSEITEQEKDFLWSHRHYCVTIPEILPKLLLSVKWNSRDEVAQMYCLVKDWPPIKPEQAMELLDCNYPDPMVRGFAVRCLEKYLTDDKLSQYLIQLVQVLKYEQYLDNLLVRFLLKKALTNQRIGHFFFWHLKSEMHNKTVSQRFGLLLESYCRACGMYLKHLNRQVEAMEKLINLTDILKQEKKDETQKVQMKFLVEQMRRPDFMDALQGFLSPLNPAHQLGNLRLEECRIMSSAKRPLWLNWENPDIMSELLFQNNEIIFKNGDDLRQDMLTLQIIRIMENIWQNQGLDLRMLPYGCLSIGDCVGLIEVVRNSHTIMQIQCKGGLKGALQFNSHTLHQWLKDKNKGEIYDAAIDLFTRSCAGYCVATFILGIGDRHNSNIMVKDDGQLFHIDFGHFLDHKKKKFGYKRERVPFVLTQDFLIVISKGAQECTKTREFERFQEMCYKAYLAIRQHANLFINLFSMMLGSGMPELQSFDDIAYIRKTLALDKTEQEALEYFMKQMNDAHHGGWTTKMDWIFHTIKQHALN DIP protein P42336 original sequence version: 110 DIP protein P42336 original sequence version: 119 DIP protein P42336 original sequence version: 166 DIP protein P42336 original sequence version: 139 041487231A9A1207 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 erbb3-erbb2 2 4 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association Fig 3A. The HER2/HER3 interaction was significantly increased when cells were pretreated with the HER3 ligand HRG. Trastuzumab Disrupts the Ligand-Independent HER2/HER3 Interaction. Heregulin. The HER2/HER3 interaction was significantly increased when cells were pretreated with the HER3 ligand HRG. Trastuzumab Disrupts the Ligand-Independent HER2/HER3 Interaction. erbb3-erbb2-4 3 4 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association Fig 3B, 3C. Trastuzumab disrupted the HER2/HER3 interaction in a dose-dependent manner. Trastuzumab erbb3-pik3ca 1 5 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait physical association physical association Fig 5B. Trastuzumab caused a similar decrease in pHER3 in MDA-MB-361 as was detected in trastuzumab-sensitive SKBR-3 cells. Dephosphorylation of HER3 also led to the dissociation of both p85 and p110 subunits of PI3K from HER3, again similar to the trastuzumab-sensitive SKBR-3. p85 subunit(s) were also identified. Trastuzumab caused a similar decrease in pHER3 in MDA-MB-361 as was detected in trastuzumab-sensitive SKBR-3 cells. Dephosphorylation of HER3 also led to the dissociation of both p85 and p110 subunits of PI3K from HER3, again similar to the trastuzumab-sensitive SKBR-3.