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/ Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. 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 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. 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 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. 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 predetermined predetermined participant predetermined Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer The authors assigned confidence based on beta-galactosidase activity, the highest confidence given was (+++) and the lowest (-). fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. 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 bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. 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 predetermined predetermined participant predetermined Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. Mammalian tumor suppressor Int6 specifically targets hypoxia inducible factor 2 alpha for degradation by hypoxia- and pVHL-independent regulation. J. Biol. Chem. (0021-9258) 2007 Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Chen L., Uchida K., Endler A., Shibasaki F. Cancer - Interactions investigated in the context of cancer fshibasa@rinshoken.or.jp Accepted 2007-JUN-1 by JYOTI. 23-MAY-2007: Contacted by JYOTI. A pretransformed human heart MATCHMAKER cDNA library (Clonetech) was used. J. Biol. Chem. (0021-9258) 2007 eif3e_human Eukaryotic translation initiation factor 3 subunit E EIF3E INT6 EIF3S6 Eukaryotic translation initiation factor 3 subunit 6 eIF-3 p48 Viral integration site protein INT-6 homolog protein protein human Homo sapiens Human MAEYDLTTRIAHFLDRHLVFPLLEFLSVKEIYNEKELLQGKLDLLSDTNMVDFAMDVYKNLYSDDIPHALREKRTTVVAQLKQLQAETEPIVKMFEDPETTRQMQSTRDGRMLFDYLADKHGFRQEYLDTLYRYAKFQYECGNYSGAAEYLYFFRVLVPATDRNALSSLWGKLASEILMQNWDAAMEDLTRLKETIDNNSVSSPLQSLQQRTWLIHWSLFVFFNHPKGRDNIIDLFLYQPQYLNAIQTMCPHILRYLTTAVITNKDVRKRRQVLKDLVKVIQQESYTYKDPITEFVECLYVNFDFDGAQKKLRECESVLVNDFFLVACLEDFIENARLFIFETFCRIHQCISINMLADKLNMTPEEAERWIVNLIRNARLDAKIDSKLGHVVMGNNAVSPYQQVIEKTKSLSFRSQMLAMNIEKKLNQNSRSEAPNWATQDSGFY DIP protein P60228 original sequence version: 120 DIP protein P60228 original sequence version: 60 DIP protein P60228 original sequence version: 76 DIP protein P60228 original sequence version: 111 A5368651DD0DDD0C epas1_human Endothelial PAS domain-containing protein 1 EPAS1 HIF2A MOP2 Member of PAS protein 2 Basic-helix-loop-helix-PAS protein MOP2 BHLHE73 PASD2 Hypoxia-inducible factor 2-alpha HIF-1-alpha-like factor PAS domain-containing protein 2 Class E basic helix-loop-helix protein 73 protein protein human Homo sapiens Human MTADKEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSSHLDKASIMRLAISFLRTHKLLSSVCSENESEAEADQQMDNLYLKALEGFIAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENLSLKNGSGFGKKSKDMSTERDFFMRMKCTVTNRGRTVNLKSATWKVLHCTGQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQHPSHMDIPLDSKTFLSRHSMDMKFTYCDDRITELIGYHPEELLGRSAYEFYHALDSENMTKSHQNLCTKGQVVSGQYRMLAKHGGYVWLETQGTVIYNPRNLQPQCIMCVNYVLSEIEKNDVVFSMDQTESLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTKLKEEPEELAQLAPTPGDAIISLDFGNQNFEESSAYGKAILPPSQPWATELRSHSTQSEAGSLPAFTVPQAAAPGSTTPSATSSSSSCSTPNSPEDYYTSLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKFQQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNTQWPPDPPLHFGPTKWAVGDQRTEFLGAAPLGPPVSPPHVSTFKTRSAKGFGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQDLSGGDPPGGSTSHLMWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHLPLPQPPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGPSFESYLLPELTRYDCEVNVPVLGSSTLLQGGDLLRALDQAT DIP protein Q99814 original sequence version: 115 DIP protein Q99814 original sequence version: 155 DIP protein Q99814 original sequence version: 157 DIP protein Q99814 original sequence version: 137 DIP protein Q99814 original sequence version: 135 4838989598234FC1 epas1-eif3s6 2 9 unspecified role unspecified role prey prey 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 bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association yeast two-hybrid analysis was performed using the C-terminal region of HIF-2 (amino acids 571-828) as bait; this region represents the region of greatest sequence divergence between the HIF- proteins and is devoid of autoactivity (55). From a total of 1.2 106 cDNA clones in the initial screen of the human heart cDNA library, 326 were positive clones when assayed for -galactosidase expression for the first screen. An N-terminal Int6 fragment (amino acids 4 128, accession number U94175) was identified in 3 of the 28 clones in a high-stringency screening. PREY gene U94175 The region aa 571-828 of HIF-2 alpha is the region of greatest sequence divergence between the HIF-alpha proteins. epas1-eif3s6-1 7 10 unspecified role unspecified role bait bait region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association PREY gene U94175 interactions between the N-terminal of Int6 and truncated HIF-2 in yeast that were analyzed by a quantitative -galactosidase assay. The N-terminal of Int6 (Int6-N; amino acids 4-150) retained its -galactosidase activity in HIF-2 constructs that contained amino acids 571-828, 641-828, and 571-700. Constructs with amino acids 701-828 exhibited no activity. 1 C The approx. beta-galactosidase activity for HIF-2alpha constructs: (571-700) 15, (571-828) 8, (641-828) 7, (701-828) 0. epas1-eif3s6-3 4 9 unspecified role unspecified role prey prey 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 bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association author score author-based confidence author score +++ (highest score given) PREY gene U94175 The region aa 571-828 of HIF-2 alpha is the region of greatest sequence divergence between the HIF-alpha proteins. 1 B The Int6 fragment bound to HIF-2, but neither to HIF-1 nor HIF-3; this indicated subtype specificity (Fig. 1B) Int6 interacted with HIF-2alpha, but not to HIF-1alpha nor HIF-3alpha. epas1-eif3s6-2 5 9 unspecified role unspecified role neutral component neutral component region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 10 unspecified role unspecified role neutral component neutral component region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization 2 A Colocalization of HA-Int6-wt with Myc-HIF-2-wt was detected in MCF-7 cells by immunofluorescence analysis under normoxia as described previously (55). Cytoplasmic localization of HA-Int6-wt changed to nuclear following coexpression with Myc-HIF-2; both proteins were expressed in the nucleus The author identifier for P60228 is U94175. Q99814; P60228 transfection When Myc-HIF-2 alpha was coexpressed, HA-Int6 localization changed from cytoplasmic to nuclear. eif3s6-epas1-2 8 9 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 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 10 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 B BAIT gene U94175. Q99814; P60228 transfection Lysates from cells cotransfected with pcDNA3-Myc-HIF-2; and pcDNA3-HA-Int6-wt (lane 1), pcDNA3-HA-Int6-;C (lane 2, amino acids 1-326), pcDNA3-HA-Int6-N (lane 3, amino acids 1-150) or pcDNA3-HA-Bax (lane 4) were immunoprecipitated (IP) with anti-HA-conjugated agarose (middle panel, input), and detected by anti-HA antibody on western blots. Coimmunoprecipitated (Co-IP) Myc-HIF-2; was detected by western blotting with anti-Myc polyclonal antibody This interaction occurs under normoxia. eif3s6-epas1-1 3 9 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association 2 C BAIT gene U94175. In support of our results in yeast, HIF-2 was coprecipitated with Int6 under normoxia (Fig. 2B). For further confirmation, the endogenous binding was examined and also observed in HeLa, MCF7, and 786-O cell lines, even under normoxia (Fig. 2C). The interaction occurs under normoxia. eif3s6-epas1-3 1 10 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association 2 C BAIT gene U94175. In support of our results in yeast, HIF-2 was coprecipitated with Int6 under normoxia (Fig. 2B). For further confirmation, the endogenous binding was examined and also observed in HeLa, MCF7, and 786-O cell lines, even under normoxia (Fig. 2C). The interaction occurs under normoxia. eif3s6-epas1-4 6 9 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey physical association physical association 2 C BAIT gene U94175. In support of our results in yeast, HIF-2 was coprecipitated with Int6 under normoxia (Fig. 2B). For further confirmation, the endogenous binding was examined and also observed in HeLa, MCF7, and 786-O cell lines, even under normoxia (Fig. 2C). The interaction occurs under normoxia. epas1-eif3s6-4 4 9 unspecified role unspecified role prey prey 10 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 physical association physical association author score author-based confidence author score +++ (highest score given) PREY gene U94175 The region aa 571-828 of HIF-2 alpha is the region of greatest sequence divergence between the HIF-alpha proteins. 1 B The Int6 fragment bound to HIF-2, but neither to HIF-1 nor HIF-3; this indicated subtype specificity (Fig. 1B). Int6 did not interact with HIF-1alpha nor HIF-3alpha. eif3s6-epas1-6 7 10 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role bait bait region required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association PREY gene U94175 in order to determine the region within Int6 that is essential for HIF-2; binding, we constructed a vector containing the activated domain (AD) fused to HIF-2; fragment (amino acids 571-828). (indicated in Fig. 1D) and introduced this into yeast along with a vector expressing the DBD fused to an Int6 mutant fragments. The C-terminal HIF-2; fragment also binds the N-terminal region (amino acids 4�128) of Int6 1 D The approx. beta-galactosidase activity for Int6 constructs: (4-128) 17, (wt) 6, (129-455) 2, (129-260) 2.