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/ Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Oncogene (0950-9232) 2007 imex curation Kehn K., Berro R., Alhaj A., Bottazzi ME., Yeh WI., Klase Z., Van Duyne R., Fu S., Kashanchi F. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the recombinant cyclin D1 and recombinant BRCA1 since the organism of origin of these interactors could not be ascertained. Thus the pull down experiments and in vitro kinase assays in Fig. 4 could not be curated. The in vitro kinase assays in Fig. 5 could not be curated as it is not clear which protein(s) is carrying out the phosphorylations. The ChIP assays were carried out on cell extracts and thus could not be curated, as it is not clear which proteins were involved in nucleic acid binding. bcmfxk@gwumc.edu 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions human-t47d human breast cancer tumour cell line-epithelial cells t47d Epithelial cells derived from human breast tumour anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Kehn K., Berro R., Alhaj A., Bottazzi ME., Yeh WI., Klase Z., Van Duyne R., Fu S., Kashanchi F. Cancer - Interactions investigated in the context of cancer bcmfxk@gwumc.edu Accepted 2007-Not a month!-19 by DAVET. 22-Jan-2008: Contacted by JYOTI. Oncogene (0950-9232) 2007 davet Only protein-protein interactions imex curation Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Oncogene (0950-9232) 2007 imex curation Kehn K., Berro R., Alhaj A., Bottazzi ME., Yeh WI., Klase Z., Van Duyne R., Fu S., Kashanchi F. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the recombinant cyclin D1 and recombinant BRCA1 since the organism of origin of these interactors could not be ascertained. Thus the pull down experiments and in vitro kinase assays in Fig. 4 could not be curated. The in vitro kinase assays in Fig. 5 could not be curated as it is not clear which protein(s) is carrying out the phosphorylations. The ChIP assays were carried out on cell extracts and thus could not be curated, as it is not clear which proteins were involved in nucleic acid binding. bcmfxk@gwumc.edu 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells confocal microscopy confocal microscopy predetermined predetermined participant predetermined Kehn K., Berro R., Alhaj A., Bottazzi ME., Yeh WI., Klase Z., Van Duyne R., Fu S., Kashanchi F. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the recombinant cyclin D1 and recombinant BRCA1 since the organism of origin of these interactors could not be ascertained. Thus the pull down experiments and in vitro kinase assays in Fig. 4 could not be curated. The in vitro kinase assays in Fig. 5 could not be curated as it is not clear which protein(s) is carrying out the phosphorylations. The ChIP assays were carried out on cell extracts and thus could not be curated, as it is not clear which proteins were involved in nucleic acid binding. bcmfxk@gwumc.edu Accepted 2007-Not a month!-19 by DAVET. Oncogene (0950-9232) 2007 davet Only protein-protein interactions imex curation Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells. Oncogene (0950-9232) 2007 imex curation Kehn K., Berro R., Alhaj A., Bottazzi ME., Yeh WI., Klase Z., Van Duyne R., Fu S., Kashanchi F. Cancer - Interactions investigated in the context of cancer A unique UniProtKB entry could not be associated with the recombinant cyclin D1 and recombinant BRCA1 since the organism of origin of these interactors could not be ascertained. Thus the pull down experiments and in vitro kinase assays in Fig. 4 could not be curated. The in vitro kinase assays in Fig. 5 could not be curated as it is not clear which protein(s) is carrying out the phosphorylations. The ChIP assays were carried out on cell extracts and thus could not be curated, as it is not clear which proteins were involved in nucleic acid binding. bcmfxk@gwumc.edu 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Kehn K., Berro R., Alhaj A., Bottazzi ME., Yeh WI., Klase Z., Van Duyne R., Fu S., Kashanchi F. Cancer - Interactions investigated in the context of cancer bcmfxk@gwumc.edu Accepted 2007-Not a month!-19 by DAVET. 22-Jan-2008: Contacted by JYOTI. Oncogene (0950-9232) 2007 davet Only protein-protein interactions imex curation brca1_human Breast cancer type 1 susceptibility protein BRCA1 RNF53 RING finger protein 53 RING-type E3 ubiquitin transferase BRCA1 protein protein human Homo sapiens Human MDLSALRVEEVQNVINAMQKILECPICLELIKEPVSTKCDHIFCKFCMLKLLNQKKGPSQCPLCKNDITKRSLQESTRFSQLVEELLKIICAFQLDTGLEYANSYNFAKKENNSPEHLKDEVSIIQSMGYRNRAKRLLQSEPENPSLQETSLSVQLSNLGTVRTLRTKQRIQPQKTSVYIELGSDSSEDTVNKATYCSVGDQELLQITPQGTRDEISLDSAKKAACEFSETDVTNTEHHQPSNNDLNTTEKRAAERHPEKYQGSSVSNLHVEPCGTNTHASSLQHENSSLLLTKDRMNVEKAEFCNKSKQPGLARSQHNRWAGSKETCNDRRTPSTEKKVDLNADPLCERKEWNKQKLPCSENPRDTEDVPWITLNSSIQKVNEWFSRSDELLGSDDSHDGESESNAKVADVLDVLNEVDEYSGSSEKIDLLASDPHEALICKSERVHSKSVESNIEDKIFGKTYRKKASLPNLSHVTENLIIGAFVTEPQIIQERPLTNKLKRKRRPTSGLHPEDFIKKADLAVQKTPEMINQGTNQTEQNGQVMNITNSGHENKTKGDSIQNEKNPNPIESLEKESAFKTKAEPISSSISNMELELNIHNSKAPKKNRLRRKSSTRHIHALELVVSRNLSPPNCTELQIDSCSSSEEIKKKKYNQMPVRHSRNLQLMEGKEPATGAKKSNKPNEQTSKRHDSDTFPELKLTNAPGSFTKCSNTSELKEFVNPSLPREEKEEKLETVKVSNNAEDPKDLMLSGERVLQTERSVESSSISLVPGTDYGTQESISLLEVSTLGKAKTEPNKCVSQCAAFENPKGLIHGCSKDNRNDTEGFKYPLGHEVNHSRETSIEMEESELDAQYLQNTFKVSKRQSFAPFSNPGNAEEECATFSAHSGSLKKQSPKVTFECEQKEENQGKNESNIKPVQTVNITAGFPVVGQKDKPVDNAKCSIKGGSRFCLSSQFRGNETGLITPNKHGLLQNPYRIPPLFPIKSFVKTKCKKNLLEENFEEHSMSPEREMGNENIPSTVSTISRNNIRENVFKEASSSNINEVGSSTNEVGSSINEIGSSDENIQAELGRNRGPKLNAMLRLGVLQPEVYKQSLPGSNCKHPEIKKQEYEEVVQTVNTDFSPYLISDNLEQPMGSSHASQVCSETPDDLLDDGEIKEDTSFAENDIKESSAVFSKSVQKGELSRSPSPFTHTHLAQGYRRGAKKLESSEENLSSEDEELPCFQHLLFGKVNNIPSQSTRHSTVATECLSKNTEENLLSLKNSLNDCSNQVILAKASQEHHLSEETKCSASLFSSQCSELEDLTANTNTQDPFLIGSSKQMRHQSESQGVGLSDKELVSDDEERGTGLEENNQEEQSMDSNLGEAASGCESETSVSEDCSGLSSQSDILTTQQRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEKAVLTSQKSSEYPISQNPEGLSADKFEVSADSSTSKNKEPGVERSSPSKCPSLDDRWYMHSCSGSLQNRNYPSQEELIKVVDVEEQQLEESGPHDLTETSYLPRQDLEGTPYLESGISLFSDDPESDPSEDRAPESARVGNIPSSTSALKVPQLKVAESAQSPAAAHTTDTAGYNAMEESVSREKPELTASTERVNKRMSMVVSGLTPEEFMLVYKFARKHHITLTNLITEETTHVVMKTDAEFVCERTLKYFLGIAGGKWVVSYFWVTQSIKERKMLNEHDFEVRGDVVNGRNHQGPKRARESQDRKIFRGLEICCYGPFTNMPTDQLEWMVQLCGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNGFHAIGQMCEAPVVTREWVLDSVALYQCQELDTYLIPQIPHSHY DIP protein P38398 original sequence version: 183 DIP protein P38398 original sequence version: 111 DIP protein P38398 original sequence version: 149 DIP protein P38398-1 original sequence version: 149 DIP protein P38398 original sequence version: 142 DIP protein P38398 original sequence version: 212 DIP protein P38398 original sequence version: 163 DIP protein P38398 original sequence version: 185 DIP protein P38398 original sequence version: 148 DIP protein P38398 original sequence version: 210 89C6D83FF56312AF ccnd1_human G1/S-specific cyclin-D1 CCND1 BCL1 PRAD1 PRAD1 oncogene BCL-1 oncogene B-cell lymphoma 1 protein protein protein human Homo sapiens Human MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDI DIP protein P24385 original sequence version: 163 DIP protein P24385 original sequence version: 165 DIP protein P24385 original sequence version: 160 DIP protein P24385 original sequence version: 121 DIP protein P24385 original sequence version: 122 DIP protein P24385 original sequence version: 143 DIP protein P24385 original sequence version: 127 3CC00C9905F58D3A brca1-ccnd1 2 4 unspecified role unspecified role neutral component neutral component 5 unspecified role unspecified role neutral component neutral component colocalization colocalization 2 Asynchronous MCF-7 cells, hydroxyurea (HU; early S phase blocker) and nocodazole (G2/M blocker)-treated cells were fixed and fluorescently stained for both cyclin D1 and BRCA1 proteins (Figure 2). BRCA1 was detected in both the nucleus and cytoplasm of untreated cells, previously reported for MCF-7 cells (Fabbro et al., 2002). Cyclin D1 staining was also found in both the cytoplasm and nucleus (Figure 2, untreated cells). However, both cyclin D1 and BRCA1 were present mostly in the nucleus after HU treatment, indicating that they most likely migrate between the cytoplasm and nucleus at various stages of cell cycle. When red and green channels were merged, colocalization could be observed These proteins colocalized in both cytoplasm and nucleus in asynchronous cells and mostly in the nucleus after hydroxyurea treatment (when blocked in early S phase). brca1-ccnd1-1 3 4 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association 1 B, 3 D We next determined if cyclin D1 and BRCA1 complexed at various stages of cell cycle. A prominent complex was observed at the early G1 and some at the early S phase (panel d). This complex was specific since both BRCA1 and cyclin D1 peptides competed out the antibodies in the immunoprecipitation reaction (panel d). The association between BRCA1 and cyclin D1 is prominent in early G1 and less so at early S phase. This interaction was specific since both BRCA1 and cyclin D1 peptides competed out the antibodies in the immunoprecipitation reaction. brca1-ccnd1-2 1 4 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey physical association physical association 1 B This interaction was specific since competing peptides competed out the antibodies in the immunoprecipitation reaction. Using immunoprecipitation assays, we find that cyclin D1 complexes with BRCA1 in MCF-7 and T47D cells