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/ Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Cell (0092-8674) 2007 imex curation Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions 12 in vitro In vitro nmr nuclear magnetic resonance NMR NMR nmr predetermined predetermined participant predetermined Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca Accepted 2007-Not a month!-12 by DAVET. Cell (0092-8674) 2007 davet Only protein-protein interactions 12 imex curation Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Cell (0092-8674) 2007 imex curation Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions 12 in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay western blot western blot Immuno blot Immuno blot Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca Accepted 2007-Not a month!-12 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 12 imex curation Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Cell (0092-8674) 2007 imex curation Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions 12 in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis The ranges for the Rsp5p, Nedd4-1, AIP4 and WWP2 C2 deletion mutants are based on the range of the C2 domain given in the UniProtKB entry for each protein. The RhoA used was determined to be human by examining the reference Zhang et al. (2000) JBC 275, 25299 referred to by reference Wang et al., 2006 referred to in the materials and methods. silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca Accepted 2007-Not a month!-12 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 12 imex curation Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. Cell (0092-8674) 2007 imex curation Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca 22-Jan-2008: Contacted by JYOTI. davet Only protein-protein interactions 12 in vitro In vitro pull down pull down predetermined predetermined participant predetermined Wiesner S., Ogunjimi AA., Wang HR., Rotin D., Sicheri F., Wrana JL., Forman-Kay JD. Apoptosis - Interactions involving proteins with a function related to apoptosis silke@pound.med.utoronto.ca silke@pound.med.utoronto.ca Accepted 2007-Not a month!-12 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 davet Only protein-protein interactions 12 imex curation nedd4_human E3 ubiquitin-protein ligase NEDD4 Neural precursor cell expressed developmentally down-regulated protein 4 NEDD4 KIAA0093 NEDD4-1 Cell proliferation-inducing gene 53 protein PIG53 HECT-type E3 ubiquitin transferase NEDD4 protein protein human Homo sapiens Human MAQSLRLHFAARRSNTYPLSETSGDDLDSHVHMCFKRPTRISTSNVVQMKLTPRQTALAPLIKENVQSQERSSVPSSENVNKKSSCLQISLQPTRYSGYLQSSNVLADSDDASFTCILKDGIYSSAVVDNELNAVNDGHLVSSPAICSGSLSNFSTSDNGSYSSNGSDFGSCASITSGGSYTNSVISDSSSYTFPPSDDTFLGGNLPSDSTSNRSVPNRNTTPCEIFSRSTSTDPFVQDDLEHGLEIMKLPVSRNTKIPLKRYSSLVIFPRSPSTTRPTSPTSLCTLLSKGSYQTSHQFIISPSEIAHNEDGTSAKGFLSTAVNGLRLSKTICTPGEVRDIRPLHRKGSLQKKIVLSNNTPRQTVCEKSSEGYSCVSVHFTQRKAATLDCETTNGDCKPEMSEIKLNSDSEYIKLMHRTSACLPSSQNVDCQININGELERPHSQMNKNHGILRRSISLGGAYPNISCLSSLKHNCSKGGPSQLLIKFASGNEGKVDNLSRDSNRDCTNELSNSCKTRDDFLGQVDVPLYPLPTENPRLERPYTFKDFVLHPRSHKSRVKGYLRLKMTYLPKTSGSEDDNAEQAEELEPGWVVLDQPDAACHLQQQQEPSPLPPGWEERQDILGRTYYVNHESRRTQWKRPTPQDNLTDAENGNIQLQAQRAFTTRRQISEETESVDNRESSENWEIIREDEATMYSNQAFPSPPPSSNLDVPTHLAEELNARLTIFGNSAVSQPASSSNHSSRRGSLQAYTFEEQPTLPVLLPTSSGLPPGWEEKQDERGRSYYVDHNSRTTTWTKPTVQATVETSQLTSSQSSAGPQSQASTSDSGQQVTQPSEIEQGFLPKGWEVRHAPNGRPFFIDHNTKTTTWEDPRLKIPAHLRGKTSLDTSNDLGPLPPGWEERTHTDGRIFYINHNIKRTQWEDPRLENVAITGPAVPYSRDYKRKYEFFRRKLKKQNDIPNKFEMKLRRATVLEDSYRRIMGVKRADFLKARLWIEFDGEKGLDYGGVAREWFFLISKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFKFIGRVAGMAVYHGKLLDGFFIRPFYKMMLHKPITLHDMESVDSEYYNSLRWILENDPTELDLRFIIDEELFGQTHQHELKNGGSEIVVTNKNKKEYIYLVIQWRFVNRIQKQMAAFKEGFFELIPQDLIKIFDENELELLMCGLGDVDVNDWREHTKYKNGYSANHQVIQWFWKAVLMMDSEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQSFTVEQWGTPEKLPRAHTCFNRLDLPPYESFEELWDKLQMAIENTQGFDGVD DIP protein P46934 original sequence version: 76 D56EBBC50A34F13B myo-inositol 1,3,5-trisphosphate 1D-myo-inositol 1,3,5-trisphosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) bul1_yeast Ubiquitin ligase-binding protein BUL1 BUL1 DAG1 RDS1 YMR275C YM8021.01C YM8156.17C SMM2 Respiration deficiency suppressor 1 protein protein yeast Saccharomyces cerevisiae Baker's yeast MAKDLNDSGFPPKRKPLLRPQRSDFTANSSTTMNVNANTRGRGRQKQEGGKGSSRSPSLHSPKSWIRSASATGILGLRRPELAHSHSHAPSTGTPAGGNRSPLRRSTANATPVETGRSLTDGDINNVVDVLPSFEMYNTLHRHIPQGNVDPDRHDFPPSYQEANNSTATGAAGSSADLSHQSLSTDALGATRSSSTSNLENLIPLRTEHHSIAAHQSTAVDEDSLDIPPILDDLNDTDNIFIDKLYTLPKMSTPIEITIKTTKHAPIPHVKPEEESILKEYTSGDLIHGFITIENKSQANLKFEMFYVTLESYISIIDKVKSKRTIKRFLRMVDLSASWSYSKIALGSGVDFIPADVDYDGSVFGLNNSRVLEPGVKYKKFFIFKLPLQLLDVTCKQEHFSHCLLPPSFGIDKYRNNCKYSGIKVNRVLGCGHLGTKGSPILTNDMSDDNLSINYTIDARIVGKDQKASKLYIMKEREYNLRVIPFGFDANVVGERTTMSQLNDITKLVQERLDALRKIFQRLEKKEPITNRDIHGADLSGTIDDSIESDSQEILQRKLDQLHIKNRNNYLVNYNDLKLGHDLDNGRSGNSGHNTDTSRAWGPFVESELKYKLKNKSNSSSFLNFSHFLNSSSSSMSSSSNAGKNNHDLTGNKERTGLILVKAKIPKQGLPYWAPSLLRKTNVFESKSKHDQENWVRLSELIPEDVKKPLEKLDLQLTCIESDNSLPHDPPEIQSITTELICITAKSDNSIPIKLNSELLMNKEKLTSIKALYDDFHSKICEYETKFNKNFLELNELYNMNRGDRRPKELKFTDFITSQLFNDIESICNLKVSVHNLSNIFKKQVSTLKQHSKHALSEDSISHTGNGSSSSPSSASLTPVTSSSKSSLFLPSGSSSTSLKFTDQIVHKWVRIAPLQYKRDINVNLEFNKDIKETLIPSFESCLCCRFYCVRVMIKFENHLGVAKIDIPISVRQVTK 9253D207B894CE44 9253D207B894CE44 itch_human E3 ubiquitin-protein ligase Itchy homolog HECT-type E3 ubiquitin transferase Itchy homolog Atrophin-1-interacting protein 4 NFE2-associated polypeptide 1 ITCH protein protein human Homo sapiens Human MSDSGSQLGSMGSLTMKSQLQITVISAKLKENKKNWFGPSPYVEVTVDGQSKKTEKCNNTNSPKWKQPLTVIVTPVSKLHFRVWSHQTLKSDVLLGTAALDIYETLKSNNMKLEEVVVTLQLGGDKEPTETIGDLSICLDGLQLESEVVTNGETTCSENGVSLCLPRLECNSAISAHCNLCLPGLSDSPISASRVAGFTGASQNDDGSRSKDETRVSTNGSDDPEDAGAGENRRVSGNNSPSLSNGGFKPSRPPRPSRPPPPTPRRPASVNGSPSATSESDGSSTGSLPPTNTNTNTSEGATSGLIIPLTISGGSGPRPLNPVTQAPLPPGWEQRVDQHGRVYYVDHVEKRTTWDRPEPLPPGWERRVDNMGRIYYVDHFTRTTTWQRPTLESVRNYEQWQLQRSQLQGAMQQFNQRFIYGNQDLFATSQSKEFDPLGPLPPGWEKRTDSNGRVYFVNHNTRITQWEDPRSQGQLNEKPLPEGWEMRFTVDGIPYFVDHNRRTTTYIDPRTGKSALDNGPQIAYVRDFKAKVQYFRFWCQQLAMPQHIKITVTRKTLFEDSFQQIMSFSPQDLRRRLWVIFPGEEGLDYGGVAREWFFLLSHEVLNPMYCLFEYAGKDNYCLQINPASYINPDHLKYFRFIGRFIAMALFHGKFIDTGFSLPFYKRILNKPVGLKDLESIDPEFYNSLIWVKENNIEECDLEMYFSVDKEILGEIKSHDLKPNGGNILVTEENKEEYIRMVAEWRLSRGVEEQTQAFFEGFNEILPQQYLQYFDAKELEVLLCGMQEIDLNDWQRHAIYRHYARTSKQIMWFWQFVKEIDNEKRMRLLQFVTGTCRLPVGGFADLMGSNGPQKFCIEKVGKENWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGFGQE DIP protein Q96J02 original sequence version: 130 DIP protein Q96J02-1 original sequence version: 125 6777A2043C7B67BC 1-phosphatidyl-1d-myo-inositol 3,4,5-trisphosphate 1-phosphatidyl-1D-myo-inositol 3,4,5-trisphosphates small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) phosphatidic acids phosphatidic acids small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) wwp2_human NEDD4-like E3 ubiquitin-protein ligase WWP2 WW domain-containing protein 2 Atrophin-1-interacting protein 2 WWP2 HECT-type E3 ubiquitin transferase WWP2 protein protein human Homo sapiens Human MASASSSRAGVALPFEKSQLTLKVVSAKPKVHNRQPRINSYVEVAVDGLPSETKKTGKRIGSSELLWNEIIILNVTAQSHLDLKVWSCHTLRNELLGTASVNLSNVLKNNGGKMENMQLTLNLQTENKGSVVSGGELTIFLDGPTVDLGNVPNGSALTDGSQLPSRDSSGTAVAPENRHQPPSTNCFGGRSRTHRHSGASARTTPATGEQSPGARSRHRQPVKNSGHSGLANGTVNDEPTTATDPEEPSVVGVTSPPAAPLSVTPNPNTTSLPAPATPAEGEEPSTSGTQQLPAAAQAPDALPAGWEQRELPNGRVYYVDHNTKTTTWERPLPPGWEKRTDPRGRFYYVDHNTRTTTWQRPTAEYVRNYEQWQSQRNQLQGAMQHFSQRFLYQSSSASTDHDPLGPLPPGWEKRQDNGRVYYVNHNTRTTQWEDPRTQGMIQEPALPPGWEMKYTSEGVRYFVDHNTRTTTFKDPRPGFESGTKQGSPGAYDRSFRWKYHQFRFLCHSNALPSHVKISVSRQTLFEDSFQQIMNMKPYDLRRRLYIIMRGEEGLDYGGIAREWFFLLSHEVLNPMYCLFEYAGKNNYCLQINPASSINPDHLTYFRFIGRFIAMALYHGKFIDTGFTLPFYKRMLNKRPTLKDLESIDPEFYNSIVWIKENNLEECGLELYFIQDMEILGKVTTHELKEGGESIRVTEENKEEYIMLLTDWRFTRGVEEQTKAFLDGFNEVAPLEWLRYFDEKELELMLCGMQEIDMSDWQKSTIYRHYTKNSKQIQWFWQVVKEMDNEKRIRLLQFVTGTCRLPVGGFAELIGSNGPQKFCIDKVGKETWLPRSHTCFNRLDLPPYKSYEQLREKLLYAIEETEGFGQE FCCD75CBA61F2204 1-phosphatidyl-1d-myo-inositol 3,5-bisphosphate 1-phosphatidyl-1D-myo-inositol 3,5-bisphosphates small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) pi5p 1-phosphatidyl-1D-myo-inositol 5-phosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) pi3p 1-phosphatidyl-1D-myo-inositol 3-phosphates small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) rhoa_human Transforming protein RhoA Rho cDNA clone 12 ARHA RHO12 RHOA ARH12 protein protein human Homo sapiens Human MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL DIP protein P61586 original sequence version: 86 DIP protein P61586 original sequence version: 123 DIP protein P61586 original sequence version: 57 DIP protein P61586 original sequence version: 88 DIP protein P61586 original sequence version: 87 DIP protein P61586 original sequence version: 85 DIP protein P61586 original sequence version: 125 DIP protein P61586 original sequence version: 132 C4DA2DC31FF858BC 1-phosphatidyl-1d-myo-inositol 4,5-bisphosphate 1-phosphatidyl-1D-myo-inositol 4,5-bisphosphates small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ubb_human Polyubiquitin-B UBB protein protein human Homo sapiens Human MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGC 33011162F1C48BB1 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 smurf2-smurf2 1 18 unspecified role unspecified role neutral component neutral component c-terminal C2 domain 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) 18 unspecified role unspecified role neutral component neutral component region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal HECT domain 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) direct interaction direct interaction addition of Smurf2 C2 domain to 15N-labeled HECT domain resulted in numerous chemical shift changes in the 1H,15N-HSQC spectrum of the HECT domain 1 B smurf2-smurf2-1 1 18 unspecified role unspecified role neutral component neutral component c-terminal C2 domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) 18 unspecified role unspecified role neutral component neutral component n-terminal HECT domain 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) direct interaction direct interaction 1 C, S2 A titration of the HECT domain to the 15N-labeled C2 domain also resulted in significant chemical shift changes in the 1H,15N-HSQC spectrum of the C2 domain smurf2-smurf2-2 1 18 unspecified role unspecified role neutral component neutral component n-terminal HECT domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region 13c label 13c label C13 13C C13 13C undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) 18 unspecified role unspecified role neutral component neutral component c-terminal C2 domain 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) direct interaction direct interaction 3 C we obtained high-quality 1H,13C-correlation (HMQC) methyl spectra that could be used for chemical shift mapping studies (Figure 3C). This approach is thus a useful alternative to 1H,15N-based chemical shift perturbation studies for proteins with limited stability and/or solubility. As in the 1H,15N-based chemical shift titration (Figure 1B), a subset of resonances of the Smurf2 HECT domain changed in the methyl HMQC spectra upon stepwise addition of the C2 domain smurf2-pi35p2 2 12 unspecified role unspecified role bait bait 18 unspecified role unspecified role prey prey 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-pi34p2 2 16 unspecified role unspecified role bait bait 18 unspecified role unspecified role prey prey 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-pi45p2 2 18 unspecified role unspecified role prey prey 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 16 unspecified role unspecified role bait bait physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-pi345p3 2 18 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role bait bait physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-pi3p 2 14 unspecified role unspecified role bait bait 18 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-pi5p 2 18 unspecified role unspecified role prey prey 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 13 unspecified role unspecified role bait bait physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-pi4p 2 13 unspecified role unspecified role bait bait 18 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-phosphatidic 2 10 unspecified role unspecified role bait bait 18 unspecified role unspecified role prey prey n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) physical association physical association the Smurf2 C2 domain interacted with phosphatidyl mono-, bi-, and triphosphates (PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3) and with phosphatidic acid (PA) in a Ca2+-independent manner S2 B smurf2-smurf2-3 3 18 unspecified role unspecified role neutral component neutral component region DeltaC2 truncated by 4 residues from the C terminus (-4aa) sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain Q9HAU4:p.Cys716Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C A Wed May 02 19:05:45 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys716ala' Tue Jun 30 17:38:59 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys716Ala' ecoli Escherichia coli (strain K12) 17 unspecified role unspecified role ancillary ancillary n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 3 E, 4 A, B, S6, S7, S8 A Deletion of the Smurf2 C2 domain enables Smurf2 Ub thioester formation. The DeltaC2 -4 aa mutant readily formed thioester-, but not isopeptide-linked Ub adducts. Together with chemical shift mapping studies, these results suggest that the C2 domain inhibits Smurf2 activity by obstructing accessibility of the catalytic cysteine of the HECT domain, thus blucking Smurf2~Ub thioester formation. When lysine-less (K0) ubiquitin, (cannot form polyUb chains), was used, Smurf2 DeltaC2 formed monoubiquitinated species conjugated with up to three K0 moieties, suggesting it contains three major ubiquitination sites. Smurf2 C2 domain - levels of autoubiquitinated DeltaC2 mutant decreased with increasing concentrations of isolated C2 domain. Smurf2 undergoes autoubiquitination. It is not clear whether the autoubiquitination is intra-, inter-molecular or both. smurf2-pi35p3 1 6 unspecified role unspecified role neutral component neutral component 18 unspecified role unspecified role neutral component neutral component 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction S2 C Using NMR titrations with unlabeled Ins(1,3,5)P3 and 15N-labeled Smurf2 C2 domain (Figure S2C), we mapped the phospholipid binding surface to hydrophobic and positively charged C2 domain residues in the b1-b2 loop, the b5-b6 loop including helix aI, and the b7-b8 loop smurf2-smurf2-4 3 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 18 unspecified role unspecified role neutral component neutral component c-terminal HECT domain sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 4 A, S6 Deletion of the C2 domain enhances Smurf2 auto-ubiquitination in vitro. It is not proven whether the autoubiquitination is intra-, inter-molecular or both. Smurf2 undergoes autoubiquitination. smurf2-smurf2-5 3 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 18 enzyme target enzyme target substrate substrate unspecified role unspecified role Q9HAU4:p.[Lys87Glu;Lys88Glu] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K E certain certain sequence position certain certain certain sequence position certain K E Fri Jul 22 11:10:54 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'lys88glu,lys87glu' Wed May 02 14:41:29 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys87glu,lys88glu' Tue Jun 30 19:35:28 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Lys87Glu;Lys88Glu]' Q9HAU4:p.His86Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain H A Wed May 02 19:40:42 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'his86ala' Tue Jun 30 17:43:51 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.His86Ala' Q9HAU4:p.[Phe29Ala;Phe30Ala] mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain F A Wed May 02 17:31:20 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe29ala,phe30ala' Tue Jun 30 17:37:40 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe29Ala;Phe30Ala]' Q9HAU4:p.[Thr56Ala;Leu57Ala] mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 18:49:20 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr56ala,leu57ala' Tue Jun 30 20:11:11 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Thr56Ala;Leu57Ala]' ecoli Escherichia coli (strain K12) 18 enzyme enzyme unspecified role unspecified role Q9HAU4:p.[Lys87Glu;Lys88Glu] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain K E certain certain sequence position certain certain certain sequence position certain K E Wed May 02 19:53:09 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys87glu,lys88glu' Tue Jun 30 18:16:08 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Lys87Glu;Lys88Glu]' Q9HAU4:p.His86Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain H A Wed May 02 19:42:05 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'his86ala' Tue Jun 30 17:15:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.His86Ala' Q9HAU4:p.[Phe29Ala;Phe30Ala] mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain F A Wed May 02 19:23:36 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe29ala,phe30ala' Tue Jun 30 15:38:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe29Ala;Phe30Ala]' Q9HAU4:p.[Thr56Ala;Leu57Ala] mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain T A certain certain sequence position certain certain certain sequence position certain L A Wed May 02 18:08:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr56ala,leu57ala' Tue Jun 30 18:07:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Thr56Ala;Leu57Ala]' ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 4 B In total, we generated six mutants and tested their autoubiquitination efficiency by comparing protein levels of unmodified and ubiquitinated Smurf2 (Figure 4B). Smurf2 variants in which the C2 domain hydrophobic residues involved in HECT domain binding were mutated (F29A/F30A and T56A/L57A) displayed the highest ubiquitination levels. When lysine-less (K0) ubiquitin, (cannot form polyUb chains), was used, Smurf2 F29/30A formed monoubiquitinated species conjugated with up to three K0 moieties, suggesting it contains three major ubiquitination sites. Smurf2 C2 domain - levels of autoubiquitinated Smurf2 F29/30A mutant decreased with increasing concentrations of isolated C2 domain. The presence of increasing amounts of the CA mutant had no effect on the activity of F29/30A Smurf2. The results indicate that the interaction betweem the C2 and HECT domains serves an auto-inhibitory function, interacting in an intramolecular manner. Smurf2 undergoes autoubiquitination. It is not proven whether autoubiquitination is inter-, intra-molecular or both. smurf2-rhoa 3 15 enzyme target enzyme target substrate substrate unspecified role unspecified role 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 18 enzyme enzyme unspecified role unspecified role Q9HAU4:p.[Phe29Ala;Phe30Ala] mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain F A certain certain sequence position certain certain certain sequence position certain F A Wed May 02 16:46:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe29ala,phe30ala' Tue Jun 30 17:42:49 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe29Ala;Phe30Ala]' ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 4 D RhoA was efficiently ubiquitinated by the F29/30A Smurf2 mutant Interfering with the Smurf2 C2 domain and HECT domain interface enhances Smurf2 ubiquitination of RhoA. RhoA becomes ubiquitinated. smurf2-smurf2-6 4 18 unspecified role unspecified role prey prey n-terminal HECT domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 ecobb Escherichia coli (strain B / BL21) 18 unspecified role unspecified role bait bait c-terminal C2 domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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) 112 115 direct interaction direct interaction 4 F the Smurf2 C2 domain interacted with the HECT domain Smad7 N-terminal domain (NTD) - Increasing concentrations of Smad7 NTD interfered with HECT domain binding to the C2 domain. bul1-bul1 3 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component region C2 deletion mutant sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain range range range range certain certain sequence position certain ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 5 we detected almost no difference in the autoubiquitination efficiency for Rsp5p and AIP4 in the absence or presence of their C2 domains Deletion of the C2 domain of Rsp5p had little effect on its autoubiquitination efficiency. Rsp5p undergoes autoubiquitination. It is not clear whether the autoubiquitination is intra- or inter-molecular or both. nedd4-nedd4 3 5 unspecified role unspecified role neutral component neutral component 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 5 Deletion of the C2 domain of Nedd4-1 significantly enhanced autoubiquitination. Nedd4-1 undergoes autoubiquitination. It is not clear whether the autoubiquitination is intra- or inter-molecular or both. As for Smurf2, wild-type Nedd4-1 and WWP2 enzymes showed low ubiquitination levels that were significantly enhanced upon deletion of their C2 domains itch-itch 3 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 5 Deletion of the C2 domain of AIP4 had little effect on its autoubiquitination. AIP4 undergoes autoubiquitination. It is not clear whether the autoubiquitination is intra- or inter-molecular or both. we detected almost no difference in the autoubiquitination efficiency for Rsp5p and AIP4 in the absence or presence of their C2 domains wwp2-wwp2 3 11 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 17 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction 5 Deletion of the C2 domain of WWP2 significantly enhanced autoubiquitination. WWP2 undergoes autoubiquitination. It is not clear whether the autoubiquitination is intra- or inter-molecular or both. As for Smurf2, wild-type Nedd4-1 and WWP2 enzymes showed low ubiquitination levels that were significantly enhanced upon deletion of their C2 domains