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/ The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. Oncogene (0950-9232) 2002 imex curation Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu 22-Jan-2008: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu Accepted 2007-DEC-7 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Oncogene (0950-9232) 2002 orchard Only protein-protein interactions imex curation The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. Oncogene (0950-9232) 2002 imex curation Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu 22-Jan-2008: Contacted by JYOTI. orchard Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu Accepted 2007-DEC-7 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Oncogene (0950-9232) 2002 orchard Only protein-protein interactions imex curation The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. Oncogene (0950-9232) 2002 imex curation Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu 22-Jan-2008: Contacted by JYOTI. orchard Only protein-protein interactions human-786_o Homo sapiens renal cell adenocarcinoma 786_o Human renal cell adenocarcinoma cell line CRL-1932 is the ATCC identifier. ATCC identifier is CRL-1932. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer The cell line was stably transfected with a wild-type VHL expression construct (WT8). robert.gemmill@uchsc.edu Accepted 2007-DEC-15 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Oncogene (0950-9232) 2002 orchard Only protein-protein interactions imex curation The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. Oncogene (0950-9232) 2002 imex curation Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu 22-Jan-2008: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu Accepted 2007-DEC-7 by ORCHARD. Oncogene (0950-9232) 2002 orchard Only protein-protein interactions imex curation The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway. Oncogene (0950-9232) 2002 imex curation Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer robert.gemmill@uchsc.edu 22-Jan-2008: Contacted by JYOTI. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Gemmill RM., Bemis LT., Lee JP., Sozen MA., Baron A., Zeng C., Erickson PF., Hooper JE., Drabkin HA. Cancer - Interactions investigated in the context of cancer The cell line was stably transfected with a wild-type VHL expression construct (WT8). robert.gemmill@uchsc.edu Accepted 2007-DEC-15 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. A Drosophila embryo library (Clontech) was used. Oncogene (0950-9232) 2002 orchard Only protein-protein interactions imex curation rn139_human E3 ubiquitin-protein ligase RNF139 Translocation in renal carcinoma on chromosome 8 protein RING-type E3 ubiquitin transferase RNF139 RING finger protein 139 RNF139 TRC8 protein protein human Homo sapiens Human MAAVGPPQQQVRMAHQQVWAALEVALRVPCLYIIDAIFNSYPDSSQSRFCIVLQIFLRLFGVFASSIVLILSQRSLFKFYTYSSAFLLAATSVLVNYYASLHIDFYGAYNTSAFGIELLPRKGPSLWMALIVLQLTFGIGYVTLLQIHSIYSQLIILDLLVPVIGLITELPLHIRETLLFTSSLILTLNTVFVLAVKLKWFYYSTRYVYLLVRHMYRIYGLQLLMEDTWKRIRFPDILRVFWLTRVTAQATVLMYILRMANETDSFFISWDDFWDLICNLIISGCDSTLTVLGMSAVISSVAHYLGLGILAFIGSTEEDDRRLGFVAPVLFFILALQTGLSGLRPEERLIRLSRNMCLLLTAVLHFIHGMTDPVLMSLSASHVSSFRRHFPVLFVSACLFILPVLLSYVLWHHYALNTWLFAVTAFCVELCLKVIVSLTVYTLFMIDGYYNVLWEKLDDYVYYVRSTGSIIEFIFGVVMFGNGAYTMMFESGSKIRAFMMCLHAYFNIYLQAKNGWKTFMNRRTAVKKINSLPEIKGSRLQEINDVCAICYHEFTTSARITPCNHYFHALCLRKWLYIQDTCPMCHQKVYIEDDIKDNSNVSNNNGFIPPNETPEEAVREAAAESDRELNEDDSTDCDDDVQRERNGVIQHTGAAAEEFNDDTD 9885F5915F019EF5 vhl_human von Hippel-Lindau disease tumor suppressor VHL pVHL Protein G7 protein protein human Homo sapiens Human MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD DIP protein P40337 original sequence version: 176 DIP protein P40337 original sequence version: 103 DIP protein P40337-1 original sequence version: 177 DIP protein P40337 original sequence version: 160 DIP protein P40337 original sequence version: 175 BA5D6765FBC16EA7 csn5_human COP9 signalosome complex subunit 5 Jun activation domain-binding protein 1 COPS5 CSN5 JAB1 protein protein human Homo sapiens Human MAASGSGMAQKTWELANNMQEAQSIDEIYKYDKKQQQEILAAKPWTKDHHYFKYCKISALALLKMVMHARSGGNLEVMGLMLGKVDGETMIIMDSFALPVEGTETRVNAQAAAYEYMAAYIENAKQVGRLENAIGWYHSHPGYGCWLSGIDVSTQMLNQQFQEPFVAVVIDPTRTISAGKVNLGAFRTYPKGYKPPDEGPSEYQTIPLNKIEDFGVHCKQYYALEVSYFKSSLDRKLLELLWNKYWVNTLSSSSLLTNADYTTGQVFDLSEKLEQSEAQLGRGSFMLGLETHDRKSEDKLAKATRDSCKTTIEAIHGLMSQVIKDKLFNQINIS DIP protein Q92905 original sequence version: 161 DIP protein Q92905 original sequence version: 87 DIP protein Q92905 original sequence version: 132 DIP protein Q92905 original sequence version: 149 DIP protein Q92905 original sequence version: 136 DIP protein Q92905 original sequence version: 101 B5742F4AAD03A1CF trc8_drome Protein TRC8 homolog Trc8 CG2304 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MSVRTKVLGLVDVMMRVPPVMVIDEILKMDMGMQSWLYPDKDKASGALATPTEAPAVPSGQDPFTSIKELFSHMTTSAQSVLEQSIEKASEAAKTHGMLSSGFNSLMNELSKDQTLADVLSTTTVKFVLCVFAFLSAACIFMLWTRHLVMVYMFLTSLGLTFLSYWSNVSALALTERSPSMVEDLMSLNTTRLLDSGGVVMSLAPHLMAQWFMGMLFAYIHLGPRFEHVQRSMPIIFASPILLAMLPLPAKVVQHLPVVAVFTPIILTKITLMQSAMEASRTVYNGYQYAMNFVSNFGLSALIENEWQRLNVPNVLRVFWTIRLIQGGYALATTESDEPLDLMTATQKLLVDGCETMTAVLGMTGVISMFCHYIGRGFQWYLLTYDDEEKSLGTVSAVLFYILALQTGLTSLSPDKRFIRLCRNLCLLMTALLHFLHNIVSPILMSLSAARNPSRKRHVRALSVCAFLVVLSVSLLYHLWSQQSISTWLLAVTAFSVEVVVKVLVSLATYTLFLLDARRQFFWEKLDDYLYYVRAFGNSVEFCFGILLFINGAWILIFESAQNATGGGIRAIMMCIHAYFNIWCEARAGWSVFMKRRSAVHKISALPEATPAQLQAFDDVCAICYQEMYSAKITRCRHFFHGVCLRKWLYVQDRCPLCHEIMMYTDKADENAPEAEPAPAAQAEQPMRIYPRDDANNAAAQRRSPERAPVEASEQAPATSSSSAAATIGAEAVSAIVESAAAVGEARSLVSVASSSSATHRISASGSSDSSYMTASAQSPPPTATSAAAVATAAASNTTHMFRMSQDQQ B61E172E80D05317 csn5_drome COP9 signalosome complex subunit 5 CSN5 CG14884 quo JAB1 homolog protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MDSDAAQKTWELENNIQTLPSCDEIFRYDAEQQRQIIDAKPWEKDPHFFKDIKISALALLKMVMHARSGGTLEVMGLMLGKVEDNTMIVMDAFALPVEGTETRVNAQAQAYEYMTAYMEAAKEVGRMEHAVGWYHSHPGYGCWLSGIDVSTQMLNQTYQEPFVAIVVDPVRTVSAGKVCLGAFRTYPKGYKPPNEEPSEYQTIPLNKIEDFGVHCKQYYPLEISYFKSALDRRLLDSLWNKYWVNTLGSSGLLTNTEYTTGQIMDLSEKLEQSENFLGRGTDVNEKRSEDKLSKATRDCSRSTIELIHGLMAQIVKDKLFNKVGLGK DIP protein Q9XZ58 original sequence version: 103 D0E94ED7ECC0ECF2 vhl_drome Protein Vhl Vhl CG13221 protein protein drome Drosophila melanogaster (Fruit fly) Fruit fly MALQIAQNNRDGQQLVGADQGKVEVYVLFANTTYRTLDLYWVCERERENMYLTLKPFEEVRVNTFTTHSWLFRDYYTGERMHVRSQRIFQPIRVRVPKSQQSPDQLVDVRSEVLIHFPMRSLRENCLWLVARWLIRTSNAPRRIIHGYHIPSTLKQQLLSLLTCIESYSRVAGTRRRR EF0AE70597F9F279 vhl-trc8 4 9 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-teminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain in vitro In vitro 11 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction 4 B In vitro labeled MYC-tagged DTrc8578 - 809 and HA-tagged DVhl proteins were mixed and immunoprecipitated (Figure 4b). The precipitation of HA-DVhl with anti-HA antibodies co-precipitated MYC-DTrc8 trc8-csn5 1 10 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 9 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) 11 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro association association 4 C both GST-DTrc8562 - 809 and GST-DTrc8562 - 744 proteins strongly interacted with labeled DVhl JAB1 interacted with GST-DTrc8562 - 809 and GST-DTrc8562 - 744, both of which include the highly conserved segment (55% identity/73% similarity) of 109 amino acids encompassing the RING-H2 finger rnf139-vhl 2 6 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 4 F In vivo interaction was verified by co-immunoprecipitation from HEK 293 cells transfected with the human homologues of TRC8 and VHL. HA-tagged VHL was co-precipitated with the anti-TRC8 antibody, 645/646 P40337 transfection rnf139-vhl-1 2 7 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role bait bait region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-teminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association 4 F P40337; Q8WU17 transfection Transfection of a GFP fusion gene encoding the C-terminus and RING-H2 domain (C-ter, including aa491 - 664), although expressed at low levels, nevertheless co-precipitated more VHL (lane 3) than endogenous TRC8 GFP-TRC8 (aa 491-664) coimmunoprecipitated more VHL than endogenous TRC8 and GFP-TRC8 (aa547-584), encompassing the RING-H2 domain inhibited VHL coimmunoprecipitation. trc8-csn5-1 1 11 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 9 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-teminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 10 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region dansyl label dansyl label 5-dimethylaminonaphthalene-1-sulfonyl tag dansyl tag 5-dimethylaminonaphthalene-1-sulfonyl tag deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro association association 4 D JAB1 interacted with GST-DTrc8562 - 809 and GST-DTrc8562 - 744, both of which include the highly conserved segment (55% identity/73% similarity) of 109 amino acids encompassing the RING-H2 finger In vitro labeled fragments of DVhl corresponding to a minimal C-terminal truncation (aa 1 - 170) and to the b-domain (aa 1 - 107) both bound DTrc8 vhl-cops5 3 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 5 E When tested for possible physical interactions, we found JAB1 could be co-immunoprecipitated with HA-tagged wild type VHL from WT8 but not from PRC3 cells that lack HA-tagged VHL P40337 transfection MG132 - the amount of coprecipitated JAB1 was increased when cells were treated with the proteasome inhibitor MG132. trc8-csn5-2 5 9 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role prey prey physical association physical association we performed a yeast 2-hybrid screen of a Drosophila embryo library (Clontech) using the Gal4 DNA binding domain fused to DTrc8 amino acids 277 - 744 containing the Patched-like loop and RING-H2 finger but lacking the C-terminal PEST sequences (Figure 4a). Two independent isolates of Drosophila CSN-5/ JAB1 (Claret et al., 1996) were identified 4 A