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/ E-cadherin regulates the association between beta-catenin and actinin-4. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. (0008-5472) 2005 imex curation Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-colon human colon colon anti bait coip anti bait coimmunoprecipitation anti bait coip ms participant Identification by mass spectrometry ms participant Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin regulates the association between beta-catenin and actinin-4. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. (0008-5472) 2005 imex curation Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-colon human colon colon anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp Accepted 2007-NOV-8 by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin regulates the association between beta-catenin and actinin-4. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. (0008-5472) 2005 imex curation Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp 13-Nov-2007: Contacted by JYOTI. 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 predetermined predetermined participant predetermined Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin regulates the association between beta-catenin and actinin-4. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. (0008-5472) 2005 imex curation Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-dld1 Human colorectal adenocarcinoma dld1 Human colon adenocarcinoma confocal microscopy confocal microscopy immunostaining immunostaining Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin regulates the association between beta-catenin and actinin-4. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. (0008-5472) 2005 imex curation Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-colon human colon colon confocal microscopy confocal microscopy immunostaining immunostaining Hayashida Y., Honda K., Idogawa M., Ino Y., Ono M., Tsuchida A., Aoki T., Hirohashi S., Yamada T. Cancer - Interactions investigated in the context of cancer tyamada@ncc.go.jp Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation actn4_human Alpha-actinin-4 ACTN4 Non-muscle alpha-actinin 4 protein protein human Homo sapiens Human MVDYHAANQSYQYGPSSAGNGAGGGGSMGDYMAQEDDWDRDLLLDPAWEKQQRKTFTAWCNSHLRKAGTQIENIDEDFRDGLKLMLLLEVISGERLPKPERGKMRVHKINNVNKALDFIASKGVKLVSIGAEEIVDGNAKMTLGMIWTIILRFAIQDISVEETSAKEGLLLWCQRKTAPYKNVNVQNFHISWKDGLAFNALIHRHRPELIEYDKLRKDDPVTNLNNAFEVAEKYLDIPKMLDAEDIVNTARPDEKAIMTYVSSFYHAFSGAQKAETAANRICKVLAVNQENEHLMEDYEKLASDLLEWIRRTIPWLEDRVPQKTIQEMQQKLEDFRDYRRVHKPPKVQEKCQLEINFNTLQTKLRLSNRPAFMPSEGKMVSDINNGWQHLEQAEKGYEEWLLNEIRRLERLDHLAEKFRQKASIHEAWTDGKEAMLKHRDYETATLSDIKALIRKHEAFESDLAAHQDRVEQIAAIAQELNELDYYDSHNVNTRCQKICDQWDALGSLTHSRREALEKTEKQLEAIDQLHLEYAKRAAPFNNWMESAMEDLQDMFIVHTIEEIEGLISAHDQFKSTLPDADREREAILAIHKEAQRIAESNHIKLSGSNPYTTVTPQIINSKWEKVQQLVPKRDHALLEEQSKQQSNEHLRRQFASQANVVGPWIQTKMEEIGRISIEMNGTLEDQLSHLKQYERSIVDYKPNLDLLEQQHQLIQEALIFDNKHTNYTMEHIRVGWEQLLTTIARTINEVENQILTRDAKGISQEQMQEFRASFNHFDKDHGGALGPEEFKACLISLGYDVENDRQGEAEFNRIMSLVDPNHSGLVTFQAFIDFMSRETTDTDTADQVIASFKVLAGDKNFITAEELRRELPPDQAEYCIARMAPYQGPDAVPGALDYKSFSTALYGESDL DIP protein O43707 original sequence version: 184 DIP protein O43707 original sequence version: 139 461580C3F22937D1 ctnb1_human Catenin beta-1 Beta-catenin OK/SW-cl.35 PRO2286 CTNNB1 CTNNB protein protein human Homo sapiens Human MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEEDVDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQFDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVVVNKAAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHREGLLAIFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMVALLNKTNVKFLAITTDCLQILAYGNQESKLIILASGGPQALVNIMRTYTYEKLLWTTSRVLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAATKQEGMEGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAGDREDITEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGLIRNLALCPANHAPLREQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIVEGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAEAIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEPMAWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGHHPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDL DIP protein P35222 original sequence version: 187 DIP protein P35222 original sequence version: 188 DIP protein P35222 original sequence version: 180 DIP protein P35222-1 original sequence version: 181 DIP protein P35222 original sequence version: 181 DIP protein P35222 original sequence version: 107 DIP protein P35222-1 original sequence version: 107 DIP protein P35222 original sequence version: 103 DIP protein P35222-1 original sequence version: 103 DIP protein P35222 original sequence version: 175 DIP protein P35222 original sequence version: 136 DIP protein P35222 original sequence version: 135 DIP protein P35222 original sequence version: 131 DIP protein P35222-1 original sequence version: 133 DIP protein P35222 original sequence version: 133 DIP protein P35222 original sequence version: 148 DIP protein P35222-1 original sequence version: 139 DIP protein P35222 original sequence version: 139 DIP protein P35222 original sequence version: 146 DIP protein P35222 original sequence version: 196 DIP protein P35222 original sequence version: 192 DIP protein P35222 original sequence version: 185 DIP protein P35222 original sequence version: 151 DIP protein P35222-1 original sequence version: 151 DIP protein P35222-1 original sequence version: 171 DIP protein P35222-1 original sequence version: 148 DIP protein P35222 original sequence version: 177 DIP protein P35222 original sequence version: 178 DIP protein P35222-1 original sequence version: 178 CB78F165A3EEF86E cadh1_mouse Cadherin-1 Epithelial cadherin Uvomorulin ARC-1 Cdh1 protein protein mouse Mus musculus Mouse MGARCRSFSALLLLLQVSSWLCQELEPESCSPGFSSEVYTFPVPERHLERGHVLGRVRFEGCTGRPRTAFFSEDSRFKVATDGTITVKRHLKLHKLETSFLVRARDSSHRELSTKVTLKSMGHHHHRHHHRDPASESNPELLMFPSVYPGLRRQKRDWVIPPISCPENEKGEFPKNLVQIKSNRDKETKVFYSITGQGADKPPVGVFIIERETGWLKVTQPLDREAIAKYILYSHAVSSNGEAVEDPMEIVITVTDQNDNRPEFTQEVFEGSVAEGAVPGTSVMKVSATDADDDVNTYNAAIAYTIVSQDPELPHKNMFTVNRDTGVISVLTSGLDRESYPTYTLVVQAADLQGEGLSTTAKAVITVKDINDNAPVFNPSTYQGQVPENEVNARIATLKVTDDDAPNTPAWKAVYTVVNDPDQQFVVVTDPTTNDGILKTAKGLDFEAKQQYILHVRVENEEPFEGSLVPSTATVTVDVVDVNEAPIFMPAERRVEVPEDFGVGQEITSYTAREPDTFMDQKITYRIWRDTANWLEINPETGAIFTRAEMDREDAEHVKNSTYVALIIATDDGSPIATGTGTLLLVLLDVNDNAPIPEPRNMQFCQRNPQPHIITILDPDLPPNTSPFTAELTHGASVNWTIEYNDAAQESLILQPRKDLEIGEYKIHLKLADNQNKDQVTTLDVHVCDCEGTVNNCMKAGIVAAGLQVPAILGILGGILALLILILLLLLFLRRRTVVKEPLLPPDDDTRDNVYYYDEEGGGEEDQDFDLSQLHRGLDARPEVTRNDVAPTLMSVPQYRPRPANPDEIGNFIDENLKAADSDPTAPPYDSLLVFDYEGSGSEAASLSSLNSSESDQDQDYDYLNEWGNRFKKLADMYGGGEDD DIP protein P09803 original sequence version: 92 DIP protein P09803 original sequence version: 116 DIP protein P09803 original sequence version: 124 DIP protein P09803 original sequence version: 128 DIP protein P09803 original sequence version: 141 DIP protein P09803 original sequence version: 174 7A6444148D3D5983 ctnnb1-actn4 2 8 inhibitor inhibitor prey prey 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait association association 4 C Although the overall expression level of actinin-4 was unaffected by E-cadherin transfection (Fig. 3C, left), the amount of actinin-4 protein present in the immunoprecipitants with anti-beta-catenin antibody was significantly reduced. E-cadherin - Transfection with E-cadherin significantly reduced the amount of actinin-4 coimmunoprecipitated with beta-catenin. This indicates that recovery of E-cadherin-mediated cell adhesion inhibits the aberrant interaction between actinin-4 and beta-catenin in SW480 cells. actn4-ctnnb1 3 7 unspecified role unspecified role prey prey n-terminal required to bind necessary binding region required to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 6 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 A two-hybrid assay revealed that the NH2-terminal amino acids 1 to 161 of actinin-4 and the NH2-terminal amino acids 1 to 249 of h-catenin were responsible for the interaction 2 H The UniProtKB entry O43707 contains an additional 27 aa at the N-terminus not found in the sequence referenced by the authors for actinin-4, BA24447. BAIT protein BA24447; PREY protein NP_001895 ctnnb1-actn4-1 1 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey physical association physical association 2 A, B An f100-kDa protein and a few other proteins were constantly coimmunoprecipitated with anti-h-catenin antibody but not with normal control IgG (Fig. 2A). Peptide mass fingerprinting (Fig. 2B) and a protein database search revealed that the protein was actinin-4 actn4-ctnnb1-1 5 7 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component colocalization colocalization Double immunofluorescence cytochemistry (Fig. 2E-G) showed that the h-catenin (Fig. 2F) and actinin-4 (Fig. 2E) proteins were colocalized in bleb-like protrusions formed on the free surfaces of SW480 cell clusters 2 E-G Beta-catenin and actinin-4 were colocalized in bleb-like protrusions formed on the free surfaces of SW480 cell clusters. SW480 cells lack expression of E-cadherin. The bleb-like protrusions disappeared when actinin-4 was knocked down and beta-catenin was diffusely distributed in the cytoplasm. actn4-ctnnb1-2 4 7 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component colocalization colocalization The expression level of h-catenin was significantly elevated by the inhibition of E-cadherin expression (Fig. 3I and L), and h-catenin and actinin-4 proteins jointly distributed in the bleb-like protrusions formed at the free cell surfaces (Fig. 3J, arrowheads), the same as in SW480 cells lacking expression of E-cadherin (Fig. 2G). In the control transfection, h-catenin and actinin-4 proteins overlapped mainly in the nucleus, and hardly any association between h-catenin and actinin-4 was detected in the cell membranes 3 H-M The expression level of beta-catenin was significantly elevated by the inhibition of E-cadherin expression with siRNA, and beta-catenin and actinin-4 proteins jointly distributed in the bleb-like protrusions formed at the free cell surfaces, as in SW480 cells lacking expression of E-cadherin. In control cells the colocalization mainly took place in the nucleus. actn4-ctnnb1-3 4 7 unspecified role unspecified role neutral component neutral component 6 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 colocalization colocalization 4 D-I Immunofluorescence microscopy revealed that the h-catenin and actinin-4 proteins were colocalized at the leading edges of these wavy protrusions This interaction occurred when expression of HA-actinin-4 was induced by removal of doxycycline from culture medium of DLD-1 Tet-off ACTN-4 cells. The proteins colocalized at the leading edge of wavy protrusions.