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/ NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. (0008-5472) 2007 imex curation Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu davet Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu Accepted 2007-SEP-28 by DAVET. Cancer Res. (0008-5472) 2007 Only protein-protein interactions imex curation NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. (0008-5472) 2007 imex curation Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu davet Only protein-protein interactions in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa predetermined predetermined participant predetermined Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer Oligonucleotides were 5 end labeled with T4 polynucleotide kinase in the presence of gamma-[32P]ATP. gelmanne@georgetown.edu Accepted 2007-OCT-14 by DAVET. Cancer Res. (0008-5472) 2007 Only protein-protein interactions imex curation NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. (0008-5472) 2007 imex curation Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu davet Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma confocal microscopy confocal microscopy predetermined predetermined participant predetermined Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu Accepted 2007-SEP-28 by DAVET. Cancer Res. (0008-5472) 2007 Only protein-protein interactions imex curation NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. (0008-5472) 2007 imex curation Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu davet Only protein-protein interactions in vitro In vitro pull down pull down ms participant Identification by mass spectrometry ms participant Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu Accepted 2007-SEP-28 by DAVET. Cancer Res. (0008-5472) 2007 davet Only protein-protein interactions imex curation NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. NKX3.1 homeodomain protein binds to topoisomerase I and enhances its activity. Cancer Res. (0008-5472) 2007 imex curation Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer gelmanne@georgetown.edu davet Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Bowen C., Stuart A., Ju JH., Tuan J., Blonder J., Conrads TP., Veenstra TD., Gelmann EP. Cancer - Interactions investigated in the context of cancer left Fig 2 A as also shows interaction and shows camptothecin does not affect interaction. the range of TOPO I is 320-765 which I think is the shortest interacting construct gelmanne@georgetown.edu Accepted 2007-OCT-14 by DAVET. Cancer Res. (0008-5472) 2007 Only protein-protein interactions imex curation nkx31_human Homeobox protein Nkx-3.1 Homeobox protein NK-3 homolog A NKX3-1 NKX3.1 NKX3A protein protein human Homo sapiens Human MLRVPEPRPGEAKAEGAAPPTPSKPLTSFLIQDILRDGAQRQGGRTSSQRQRDPEPEPEPEPEGGRSRAGAQNDQLSTGPRAAPEEAETLAETEPERHLGSYLLDSENTSGALPRLPQTPKQPQKRSRAAFSHTQVIELERKFSHQKYLSAPERAHLAKNLKLTETQVKIWFQNRRYKTKRKQLSSELGDLEKHSSLPALKEEAFSRASLVSVYNSYPYYPYLYCVGSWSPAFW C99A0943E15B2A55 nkx3_1_recog_seq NKX3.1 recognition sequence ds dna double stranded deoxyribonucleic acid ds DNA ds dna chemical synthesis Chemical synthesis (Chemical synthesis) GTATATAAGTAGTTG CD24E5ED2984FBE6 topo1_bd_tetrahy_dna Tetrahymena 52-mer containing the Topoisomerase I binding site ds dna double stranded deoxyribonucleic acid ds DNA ds dna chemical synthesis Chemical synthesis (Chemical synthesis) TCTAGAGGATTTCGAAGACTTAGAGAAATTTCGAAGATCCCCGGGCGAGCTC 1B995B81F82E0DEA top1_human DNA topoisomerase 1 DNA topoisomerase I TOP1 protein protein human Homo sapiens Human MSGDHLHNDSQIEADFRLNDSHKHKDKHKDREHRHKEHKKEKDREKSKHSNSEHKDSEKKHKEKEKTKHKDGSSEKHKDKHKDRDKEKRKEEKVRASGDAKIKKEKENGFSSPPQIKDEPEDDGYFVPPKEDIKPLKRPRDEDDADYKPKKIKTEDTKKEKKRKLEEEEDGKLKKPKNKDKDKKVPEPDNKKKKPKKEEEQKWKWWEEERYPEGIKWKFLEHKGPVFAPPYEPLPENVKFYYDGKVMKLSPKAEEVATFFAKMLDHEYTTKEIFRKNFFKDWRKEMTNEEKNIITNLSKCDFTQMSQYFKAQTEARKQMSKEEKLKIKEENEKLLKEYGFCIMDNHKERIANFKIEPPGLFRGRGNHPKMGMLKRRIMPEDIIINCSKDAKVPSPPPGHKWKEVRHDNKVTWLVSWTENIQGSIKYIMLNPSSRIKGEKDWQKYETARRLKKCVDKIRNQYREDWKSKEMKVRQRAVALYFIDKLALRAGNEKEEGETADTVGCCSLRVEHINLHPELDGQEYVVEFDFLGKDSIRYYNKVPVEKRVFKNLQLFMENKQPEDDLFDRLNTGILNKHLQDLMEGLTAKVFRTYNASITLQQQLKELTAPDENIPAKILSYNRANRAVAILCNHQRAPPKTFEKSMMNLQTKIDAKKEQLADARRDLKSAKADAKVMKDAKTKKVVESKKKAVQRLEEQLMKLEVQATDREENKQIALGTSKLNYLDPRITVAWCKKWGVPIEKIYNKTQREKFAWAIDMADEDYEF DIP protein P11387 original sequence version: 189 6FBED540BCF7BE28 nkx3_1-top1 4 9 unspecified role unspecified role prey prey human-pc3 Human PC3 Caucasian prostate adenocarcinoma pc3 Human Caucasian prostate adenocarcinoma 6 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) physical association physical association 1 PC3 cells do not express endogenous NKX3-1. top1-nkx3_1 5 6 cofactor cofactor coenzyme 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 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-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association 2 A, 3 B Treatment with camptothecin, DNase or RNase did not affect this interaction. NKX3.1 bound to GST-Topo D26 and GST-Topo 70 but not to GST-Topo A2 nkx3_1-top1-1 5 9 cofactor cofactor coenzyme 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 6 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction 2 B binding in vitro transcribed and translated [35S]Topo I to a His-NKX3.1 fusion protein bound to a MagZ binding column and showed preferential binding to the fusion protein compared with a polyhistidine peptide alone topo1_bd-top1 2 8 unspecified role unspecified role bait bait 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 in vitro In vitro direct interaction direct interaction This interaction was suggested by an EMSA using the 50-mer Tetrahymena sequence as a probe and purified proteins. The amount of NKX3.1 used in this assay alone did not bind to the Tetrahymena 50-mer probe (data not shown). However, NKX3.1 enhanced Topo I binding as shown by the increasing intensity of the two species formed by Topo I and the DNA probe and the depletion of the probe-alone band. Moreover, a supershifted complex can be seen, suggesting the formation of a complex of probe, Topo I, and NKX3.1 2 C NKX3.1 - the affinity of Top1 for the oligonucleotide appeared to increase in the presence of NKX3-1. A supershifted complex can be seen, suggesting the formation of a complex of probe, Topo I, and NKX3.1. nkx3_1_recog-nkx3_1 2 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait direct interaction direct interaction 2 D Topoisomerase I - this interaction only occurred in the presence topoisomerase I but at higher concentrations Topo I competes NKX3.1 from its cognate DNA sequence. Binding of Topo to NKX3.1 is of sufficiently high affinity to compete NKX3.1 from its cognate DNA sequence at a Topo I/NKX3.1 molar ratio as low as 2:1 nkx3_1-top1-2 1 6 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association Endogenous NKX3.1 and Topo I from LNCaP cells could be coprecipitated with NKX3.1 antibody. 8 nkx3_1-top1-3 3 9 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component physical association physical association NKX3.1 and Topo I were localized by antibody staining to aggregates in the nucleus as shown on Fig. 9A Exposure to 20 Gy gamma-irradiation resulted in delocalization from the nuclear aggregates but continued colocalization of NKX3.1 and Topo I up to 45 min after irradiation. 9 A nkx3_1-top1-4 3 6 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component region EGFP tag 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 physical association physical association 9 B We also transfected LNCaP cells with a fusion construct of Topo I and EGFP. The EGFP-Topo I fusion protein localized similarly to Topo I. NKX3.1 was detected by antibody staining. The two proteins were seen to colocalize in the nuclei After exposure to mitomycin C, which generates double-stranded DNA breaks, both signals delocalized but remained nuclear and continued to colocalize to a substantial degree.