MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. EMBO J. (0261-4189) 2011 imex curation Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease zhangyin@mail.nih.gov Only protein-protein interactions mouse-embryonic_fibroblast Mus musculus embryonic fibroblasts embryonic_fibroblast Embryonic fibroblast Fibroblasts taken from an embryonic as opposed to a foetal or adult source. Foetus is defined as the stage at which tissue diferentiation is initiated. Tissue taken from an embryonic as opposed to a foetal or adult source. Embryo is defined as the development stage prior to organ formation. anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease vog.hin.liam@niygnahz zhangyin@mail.nih.gov EMBO J. (0261-4189) 2011 Only protein-protein interactions imex curation Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. EMBO J. (0261-4189) 2011 imex curation Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease zhangyin@mail.nih.gov 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 tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease vog.hin.liam@niygnahz zhangyin@mail.nih.gov EMBO J. (0261-4189) 2011 Only protein-protein interactions imex curation Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. EMBO J. (0261-4189) 2011 imex curation Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease zhangyin@mail.nih.gov Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease The Flag-tagged Smad3 construct contains ser to ala mutations (described in PMID:9679060), however positions of the mutations are not described. This mutant behaves as activated Smad3. The GST-tagged Smad3 was phosphorylated in vitro by purified TbetaRI kinase domain prior to the binding experiment. vog.hin.liam@niygnahz zhangyin@mail.nih.gov EMBO J. (0261-4189) 2011 Only protein-protein interactions imex curation Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. EMBO J. (0261-4189) 2011 imex curation Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease zhangyin@mail.nih.gov Only protein-protein interactions in vitro In vitro peptide array peptide array western blot western blot Immuno blot Immuno blot Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease vog.hin.liam@niygnahz zhangyin@mail.nih.gov EMBO J. (0261-4189) 2011 Only protein-protein interactions imex curation Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. EMBO J. (0261-4189) 2011 imex curation Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease zhangyin@mail.nih.gov Only protein-protein interactions in vitro In vitro itc isothermal titration calorimetry ITC ITC itc predetermined predetermined participant predetermined Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease vog.hin.liam@niygnahz zhangyin@mail.nih.gov EMBO J. (0261-4189) 2011 Only protein-protein interactions imex curation Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3. EMBO J. (0261-4189) 2011 imex curation Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease zhangyin@mail.nih.gov Only protein-protein interactions mouse-embryonic_fibroblast Mus musculus embryonic fibroblasts embryonic_fibroblast Embryonic fibroblast Fibroblasts taken from an embryonic as opposed to a foetal or adult source. Foetus is defined as the stage at which tissue diferentiation is initiated. Tissue taken from an embryonic as opposed to a foetal or adult source. Embryo is defined as the development stage prior to organ formation. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Tang LY., Yamashita M., Coussens NP., Tang Y., Wang X., Li C., Deng CX., Cheng SY., Zhang YE. IBD - Inflammatory bowel disease vog.hin.liam@niygnahz zhangyin@mail.nih.gov EMBO J. (0261-4189) 2011 Only protein-protein interactions imex curation smad3_mouse Mothers against decapentaplegic homolog 3 Smad3 Madh3 SMAD family member 3 protein protein mouse Mus musculus Mouse MSSILPFTPPIVKRLLGWKKGEQNGQEEKWCEKAVKSLVKKLKKTGQLDELEKAITTQNVNTKCITIPRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELRAMELCEFAFNMKKDEVCVNPYHYQRVETPVLPPVLVPRHTEIPAEFPPLDDYSHSIPENTNFPAGIEPQSNIPETPPPGYLSEDGETSDHQMNHSMDAGSPNLSPNPMSPAHNNLDLQPVTYCEPAFWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS DIP protein Q8BUN5 original sequence version: 57 46DF5E8B371321AC ubc_human Polyubiquitin-C UBC protein protein human Homo sapiens Human MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGV B6E7BC06FEE77196 smad2_human Mothers against decapentaplegic homolog 2 SMAD2 MADH2 MADR2 Mad-related protein 2 SMAD family member 2 JV18-1 protein protein human Homo sapiens Human MSSILPFTPPVVKRLLGWKKSAGGSGGAGGGEQNGQEEKWCEKAVKSLVKKLKKTGRLDELEKAITTQNCNTKCVTIPSTCSEIWGLSTPNTIDQWDTTGLYSFSEQTRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEVCVNPYHYQRVETPVLPPVLVPRHTEILTELPPLDDYTHSIPENTNFPAGIEPQSNYIPETPPPGYISEDGETSDQQLNQSMDTGSPAELSPTTLSPVNHSLDLQPVTYSEPAFWCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSVRCSSMS DIP protein Q15796 original sequence version: 174 DIP protein Q15796 original sequence version: 100 DIP protein Q15796-1 original sequence version: 141 95406DB5FC0AA4C9 tgfr1_rat TGF-beta receptor type-1 Tgfbr1 Transforming growth factor-beta receptor type I TGF-beta type I receptor Serine/threonine-protein kinase receptor R4 protein protein rat Rattus norvegicus (Rat) Rat MEAASAALRRCLLLIVLVAAATLLPGAKALQCFCHLCTKDNFTCETDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGAVTYCCNQDHCNKIELPTTGPFSEKQSAGLGPVELAAVIAGPVCFVCIALMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM EAD7F8D37E456102 smad4_mouse Mothers against decapentaplegic homolog 4 Smad4 Dpc4 Madh4 Deletion target in pancreatic carcinoma 4 homolog SMAD family member 4 protein protein mouse Mus musculus Mouse MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITAITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFDLKCDSVCVNPYHYERVVSPGIDLSGLTLQSNAPSMLVKDEYVHDFEGQPSLPTEGHSIQTIQHPPSNRASTETYSAPALLAPAESNATSTTNFPNIPVASTSQPASILAGSHSEGLLQIASGPQPGQQQNGFTAQPATYHHNSTTTWTGSRTAPYTPNLPHHQNGHLQHHPPMPPHPGHYWPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPVVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD DIP protein P97471 original sequence version: 79 DIP protein P97471 original sequence version: 150 4FBDF5DED4442F86 smad7_mouse Mothers against decapentaplegic homolog 7 Smad7 Madh7 Madh8 Mothers against decapentaplegic homolog 8 SMAD family member 7 protein protein mouse Mus musculus Mouse MFRTKRSALVRRLWRSRAPGGEDEEEGVGGGGGGGELRGEGATDGRAYGAGGGGAGRAGCCLGKAVRGAKGHHHPHPPTSGAGAAGGAEADLKALTHSVLKKLKERQLELLLQAVESRGGTRTACLLLPGRLDCRLGPGAPASAQPAQPPSSYSLPLLLCKVFRWPDLRHSSEVKRLCCCESYGKINPELVCCNPHHLSRLCELESPPPPYSRYPMDFLKPTAGCPDAVPSSAETGGTNYLAPGGLSDSQLLLEPGDRSHWCVVAYWEEKTRVGRLYCVQEPSLDIFYDLPQGNGFCLGQLNSDNKSQLVQKVRSKIGCGIQLTREVDGVWVYNRSSYPIFIKSATLDNPDSRTLLVHKVFPGFSIKAFDYEKAYSLQRPNDHEFMQQPWTGFTVQISFVKGWGQCYTRQFISSCPCWLEVIFNSR BEEE751371C0E0CF smad4_human Mothers against decapentaplegic homolog 4 SMAD4 DPC4 MADH4 SMAD family member 4 Deletion target in pancreatic carcinoma 4 protein protein human Homo sapiens Human MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITAITTNGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFDLKCDSVCVNPYHYERVVSPGIDLSGLTLQSNAPSSMMVKDEYVHDFEGQPSLSTEGHSIQTIQHPPSNRASTETYSTPALLAPSESNATSTANFPNIPVASTSQPASILGGSHSEGLLQIASGPQPGQQQNGFTGQPATYHHNSTTTWTGSRTAPYTPNLPHHQNGHLQHHPPMPPHPGHYWPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD DIP protein Q13485 original sequence version: 149 DIP protein Q13485 original sequence version: 177 7EE3C4647712DA90 smuf2_human E3 ubiquitin-protein ligase SMURF2 SMURF2 SMAD ubiquitination regulatory factor 2 SMAD-specific E3 ubiquitin-protein ligase 2 HECT-type E3 ubiquitin transferase SMURF2 protein protein human Homo sapiens Human MSNPGGRRNGPVKLRLTVLCAKNLVKKDFFRLPDPFAKVVVDGSGQCHSTDTVKNTLDPKWNQHYDLYIGKSDSVTISVWNHKKIHKKQGAGFLGCVRLLSNAINRLKDTGYQRLDLCKLGPNDNDTVRGQIVVSLQSRDRIGTGGQVVDCSRLFDNDLPDGWEERRTASGRIQYLNHITRTTQWERPTRPASEYSSPGRPLSCFVDENTPISGTNGATCGQSSDPRLAERRVRSQRHRNYMSRTHLHTPPDLPEGYEQRTTQQGQVYFLHTQTGVSTWHDPRVPRDLSNINCEELGPLPPGWEIRNTATGRVYFVDHNNRTTQFTDPRLSANLHLVLNRQNQLKDQQQQQVVSLCPDDTECLTVPRYKRDLVQKLKILRQELSQQQPQAGHCRIEVSREEIFEESYRQVMKMRPKDLWKRLMIKFRGEEGLDYGGVAREWLYLLSHEMLNPYYGLFQYSRDDIYTLQINPDSAVNPEHLSYFHFVGRIMGMAVFHGHYIDGGFTLPFYKQLLGKSITLDDMELVDPDLHNSLVWILENDITGVLDHTFCVEHNAYGEIIQHELKPNGKSIPVNEENKKEYVRLYVNWRFLRGIEAQFLALQKGFNEVIPQHLLKTFDEKELELIICGLGKIDVNDWKVNTRLKHCTPDSNIVKWFWKAVEFFDEERRARLLQFVTGSSRVPLQGFKALQGAAGPRLFTIHQIDACTNNLPKAHTCFNRIDIPPYESYEKLYEKLLTAIEETCGFAVE DIP protein Q9HAU4 original sequence version: 112 DIP protein Q9HAU4 original sequence version: 123 DIP protein Q9HAU4 original sequence version: 144 3042B443A3755762 smad3_human Mothers against decapentaplegic homolog 3 SMAD3 MADH3 SMAD family member 3 JV15-2 protein protein human Homo sapiens Human MSSILPFTPPIVKRLLGWKKGEQNGQEEKWCEKAVKSLVKKLKKTGQLDELEKAITTQNVNTKCITIPRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELRAMELCEFAFNMKKDEVCVNPYHYQRVETPVLPPVLVPRHTEIPAEFPPLDDYSHSIPENTNFPAGIEPQSNIPETPPPGYLSEDGETSDHQMNHSMDAGSPNLSPNPMSPAHNNLDLQPVTYCEPAFWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS DIP protein P84022 original sequence version: 57 DIP protein P84022 original sequence version: 112 DIP protein P84022-1 original sequence version: 146 DIP protein P84022-1 original sequence version: 111 46DF5E8B371321AC smurf2-smad3 2 SMURF2 14 anti tag western anti tag western blot anti tag western anti tag western anti tag western blot anti tag western unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined SMAD3 15 anti tag western anti tag western blot anti tag western anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P84022:p.Thr179Val mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain T V Wed May 02 15:22:15 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr179val' Tue Jun 30 18:47:34 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr179Val' Thr179Val P84022:p.[Thr179Val;Ser204Ala;Ser208Ala;Ser213Ala] mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain T V certain certain sequence position certain certain certain sequence position certain S A Fri Jul 22 10:52:26 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'ser204ala,ser208ala,thr179val,ser213ala' Wed May 02 17:49:31 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr179val,ser204ala,ser208ala,ser213ala' Tue Jun 30 15:44:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Thr179Val;Ser204Ala;Ser208Ala;Ser213Ala]' Ser204Ala physical association physical association Fig. 5a homomint mint smad3-smurf2 4 SMAD3 15 peptide synthesis peptide synthesis western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait purified purified required phosphothr-179 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) SMURF2 14 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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. physical association physical association Fig. 5b homomint mint smad2-smurf2-1 4 SMURF2 14 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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. SMAD2 9 peptide synthesis peptide synthesis western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait purified purified required phosphothr-220 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association Fig. 5b homomint mint smad3-smurf2-1 5 SMURF2 14 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ww_dom 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) SMAD3 15 peptide synthesis peptide synthesis predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified phosphothr-179 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain prerequisite-ptm region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 37 40 direct interaction direct interaction homomint Fig. 5c domino mint Temp = 25C Smad3 peptide (173-189) phosphorylated at Thr179 binds to Smurf2 with Kd = 281 +/- 6 x 10E-9 molar. Unphosphorylated peptide binds much weaker, with Kd = 2971 +/- 158 x 10E-9 molar. Q9HAU4 decreases activity of P84022 Q9HAU4 is required for ubiquitination of P84022 Q9HAU4 decreases binding of P84022 to Q13485 Q9HAU4 decreases binding of P84022 to P84022 smad2-smurf2-2 5 SMURF2 14 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified ww_dom 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) SMAD2 9 peptide synthesis peptide synthesis predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain phosphothr-220 opthr O-phospho-L-threonine (2S,3R)-2-amino-3-(phosphonooxy)butanoic acid 2-amino-3-hydroxybutanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)butanoic acid MOD_RES Phosphothreonine O-phospho-L-threonine O-phosphorylated L-threonine O3-phosphothreonine threonine phosphate ester OPThr Phospho Phospho Threonyl Phosphorylation phosphorylationt certain certain sequence position certain certain certain sequence position certain prerequisite-ptm chemical synthesis Chemical synthesis (Chemical synthesis) 43 45 direct interaction direct interaction homomint Fig. 5c domino mint Temp = 25C smad3-smad4-7 6 Smad4 11 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level Smad3 7 western blot western blot Immuno blot Immuno blot western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association Fig. 7a homomint mint transforming growth factor beta (TGFbeta) smad4-smad3-2 3 Smad4 13 anti tag western anti tag western blot anti tag western anti tag western anti tag western blot anti tag western unspecified role unspecified role bait bait purified purified 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) Smad3 15 anti tag western anti tag western blot anti tag western anti tag western anti tag western blot anti tag western unspecified role unspecified role prey prey partially purified partially purified region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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. physical association physical association homomint mint Fig. 7d GST-Smad4 did not pull down ubiquitin-modified Flag-Smad3. smad3-smad4-8 1 Smad3 15 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined P84022:p.[Lys333Arg;Lys341Arg;Lys378Arg;Lys409Arg] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R Wed May 02 16:31:42 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys333arg,lys341arg,lys378arg,lys409arg' Tue Jun 30 18:04:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Lys333Arg;Lys341Arg;Lys378Arg;Lys409Arg]' P84022:p.[Lys333Arg;Lys378Arg;Lys409Arg] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R certain certain sequence position certain certain certain sequence position certain K R Wed May 02 14:53:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys333arg,lys378arg,lys409arg' Tue Jun 30 19:54:47 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Lys333Arg;Lys378Arg;Lys409Arg]' Smad4 13 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell 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 Fig. 7b homomint mint Smad4 did not co-immunoprecipitate with Flag-Smad3 from MEF cells transfected with Myc-Smurf2 and HA-ubiquitin. smad3-ubc 1 Smad3 15 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell P84022:p.Lys341Arg mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R Tue Jul 12 10:58:55 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 17:09:00 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys341arg' Tue Jun 30 18:56:31 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys341Arg' P84022:p.Lys333Arg mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R Tue Jul 12 10:23:51 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 15:56:21 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys333arg' Tue Jun 30 19:11:09 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys333Arg' P84022:p.Lys378Arg mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R Tue Jul 12 10:05:52 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 20:04:13 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys378arg' Tue Jun 30 17:12:00 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys378Arg' P84022:p.Lys409Arg mutation decreasing interaction mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K R Tue Jul 12 10:07:03 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation decreasing' Wed May 02 16:48:56 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys409arg' Tue Jun 30 19:54:21 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys409Arg' flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Ubiquitin 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association homomint mint f4a f4c f6b sf6 On the Flag Smad3 western blot, however, we detected only the 72-kDa modified band after prolonged exposure (Figure 4A), implying that the 72-kDa band is the ubiquitin-modified Smad3 rather than a ubiquitinated protein associated with Smad3. A similar pattern of ubiquitinated Smad2 was also detected in WT MEFs, but its level decreased only slightly in the absence of Smurf2 (Figure 4B). The concerted attachment of two ubiquitin moieties to Smad3 could be accomplished either through adding two branched ubiquitins on a single lysine residue (di-ubiquitination) or attaching two separate ubiquitins to two lysine residues (dual mono-ubiquitination) to produce the 72-kDa ubiquitin-modified Smad3. To distinguish between these two possibilities, we reintroduced Smurf2 along with Flag Smad3 and a mutant ubiquitin, HA Ub(KO), that lacks all lysine residues and is unable to support the growth of any ubiquitin chain into Smurf2 / MEFs. If Smurf2 catalyses addition of two ubiquitin moieties onto one substrate lysine site, the above experiment would produce a single ubiquitin-modified Smad3 with a normal molecular weight of 63.5 kDa. Following immunoprecipitation of Flag Smad3 and western blot analysis for HA ubiquitin and Flag Smad3, we detected only the 72-kDa ubiquitinated Smad3 in the presence of Myc Smurf2 (Figure 4C, left panel), thus, indicating mono-ubiquitination of two lysine residues. To investigate the structural basis for Smurf2-mediated ubiquitination of Smad3, we set out to identify the lysine residue(s) of Smad3 to which the ubiquitin moiety is attached. To this end, a series of Smad3 deletion mutants were constructed and their ubiquitination patterns were analysed in Smurf2 / MEFs with or without expression of Myc Smurf2. In the absence of Smurf2, both Smad3 NL and LC were ubiquinated as detected by HA (Ub)-blot (Figure 6B, top panel, lanes 8 and 9). Adding back Myc Smurf2 to Smurf2 / MEFs significantly enhanced the ubiquitination of Smad3LC (Figure 6B, lane 4 versus 9) and only slightly so that of Smad3NL (lane 3 versus 8). In contrast, Smurf2 had no effect on Smad3 mutants containing only the MH1 (N) or the MH2 (C) domain (Figure 6B, top panel, comparing lanes 2 and 5 to lanes 7 and 10), which is consistent with the role of the linker in mediating the Smad3 and Smurf2 interaction. Of note is the poly-ubiquitin modification of Smad3LC, which likely resulted from aggregation of other ubiquitinated proteins onto Smad3LC in the proteasomes. In the light of this, we carried out western analysis with Flag Smad3 and only detected mono- or oligo-ubiquitin modification of full-length Smad3 and Smad3LC, which was further enhanced in the presence of Smurf2 (Figure 6B, middle panel, comparing lanes 1 and 4 to lanes 6 and 9, respectively). Since there is no lysine residue in the linker region, the major ubiquitin attachment site(s) probably lies in the MH2 domain. Double replacement of K333 and K378, the two lysines that are directly involved in hydrogen bonding detectably reduced the level of ubiquitin modification of Smad3; however, simultaneously replacing three lysines, K333, K378, and K409 (ACD mutant) or all four lysines (Q mutant) abolished Smurf2-induced Smad3 mono-ubiquitination entirely (Figure 6C; Supplementary Figure S6). smad2-ubc 1 Ubiquitin 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Smad2 9 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association homomint mint f4b f4e A similar pattern of ubiquitinated Smad2 was also detected in WT MEFs, but its level decreased only slightly in the absence of Smurf2 (Figure 4B). In contrast, Smurf2 only caused slightly more ubiquitination of Smad2 following expression of Myc Smurf2 and Flag Smad2 in Smurf2 / MEFs (Figure 4E). ubc-smad3 1 Ubiquitin 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Smad3 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f4g homomint mint smurf2-smad3-2 1 SMAD3 15 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined SMURF2 14 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell Q9HAU4:p.Cys716Gly mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C G Wed May 02 18:17:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys716gly' Tue Jun 30 19:26:11 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys716Gly' 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 Suppl. Fig. S1c homomint mint smad4-ubc 1 Smad4 13 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Ubiquitin 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association sf4 homomint mint Smurf2 has no effect on ubiquitination of TβRI, Smad7 or Smad4. smad7-ubc 1 Smad7 12 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Ubiquitin 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association sf4 homomint mint Smurf2 has no effect on ubiquitination of TβRI, Smad7 or Smad4. tgfbr1-ubc 1 TbetaRI 10 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Ubiquitin 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell ha tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association sf4 homomint mint Smurf2 has no effect on ubiquitination of TβRI, Smad7 or Smad4. smad3-smad3 3 15 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 ecobb Escherichia coli (strain B / BL21) 15 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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. physical association physical association Fig. 7d GST-Smad3 did not pull down ubiquitin-modified Flag-Smad3. smad3-smad3-1 1 15 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 15 unspecified role unspecified role prey prey 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 physical association physical association Fig. 7c Less GFP-Smad3 was co-immunoprecipitated with Flag-Smad3 from MEF cells transfected with Myc-Smurf2 and HA-ubiquitin.