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/ http://www.ebi.ac.uk/ wiesner-2007-2 Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. 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 Cell (0092-8674) 2007 Only protein-protein interactions 12 imex curation Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca wiesner-2007-3 Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. 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 Cell (0092-8674) 2007 Only protein-protein interactions 12 imex curation Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca wiesner-2007-1 Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. 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 Cell (0092-8674) 2007 Only protein-protein interactions 12 imex curation Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca wiesner-2007-4 Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain. 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 Cell (0092-8674) 2007 Only protein-protein interactions 12 imex curation Cancer - Interactions investigated in the context of cancer silke@pound.med.utoronto.ca,wrana@mshri.on.ca,forman@sickkids.ca 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) 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 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) 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) pi3p 1-phosphatidyl-1D-myo-inositol 3-phosphates 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) phosphatidic acids phosphatidic acids small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ubb_human Polyubiquitin-B UBB protein protein human Homo sapiens Human MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGC rhoa_human Transforming protein RhoA Rho cDNA clone 12 ARHA RHO12 RHOA ARH12 protein protein human Homo sapiens Human MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL 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 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 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 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 myo-inositol 1,3,5-trisphosphate 1D-myo-inositol 1,3,5-trisphosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) smurf2-pi35p2 SMURF2 C2 domain interacts with PtdIns(3,5)P2 in a protein lipid overlay assay 667355 N/A 667359 unspecified role unspecified role bait bait N/A 667360 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 false n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false S2 B smurf2-pi34p2 SMURF2 C2 domain interacts with PtdIns(3,4)P2 in a protein lipid overlay assay 667355 n/a 667361 unspecified role unspecified role bait bait N/A 667360 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 false n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) physical association physical association false false false S2 B smurf2-pi45p2 SMURF2 C2 domain interacts with PtdIns(4,5)P2 in a protein lipid overlay assay 667355 N/A 667360 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 false n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 667361 unspecified role unspecified role bait bait physical association physical association false false false S2 B smurf2-pi345p3 SMURF2 C2 domain interacts with PtdIns(3,4,5)P3 in a protein lipid overlay assay 667355 N/A 667360 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 false 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 false ecoli Escherichia coli (strain K12) N/A 667362 unspecified role unspecified role bait bait physical association physical association false false false S2 B smurf2-pi3p SMURF2 C2 domain interacts with PtdIns(3)P in a protein lipid overlay assay 667355 N/A 667363 unspecified role unspecified role bait bait N/A 667360 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 false 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false S2 B smurf2-pi5p SMURF2 C2 domain interacts with PtdIns(5)P in a protein lipid overlay assay 667355 N/A 667360 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 false n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 667364 unspecified role unspecified role bait bait physical association physical association false false false S2 B smurf2-pi4p SMURF2 C2 domain interacts with PtdIns(4)P in a protein lipid overlay assay 667355 n/a 667364 unspecified role unspecified role bait bait N/A 667360 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 false 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false S2 B smurf2-phosphatidic SMURF2 C2 domain interacts with Phosphatidic acid in a protein lipid overlay assay 667355 N/A 667365 unspecified role unspecified role bait bait N/A 667360 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 false 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false S2 B smurf2-smurf2-3 Smurf2 DeltaC2 undergoes autoubiquitination in vitro 667356 N/A 667360 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 false Q9HAU4:p.Cys716Ala mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 667366 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 false 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 false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 3 E, 4 A, B, S6, S7, S8 A 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-smurf2-4 Smurf2 HECT domain undergoes autoubiquitination in vitro 667356 N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) N/A 667360 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 false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 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 Smurf2 C2 domain mutations enhances autoubiquitination in vitro 667356 N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) N/A 667360 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 false certain certain sequence position certain certain certain sequence position certain false 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 false 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 false certain certain sequence position certain certain certain sequence position certain false 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 false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 667360 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 false certain certain sequence position certain certain certain sequence position certain false 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 false 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 false certain certain sequence position certain certain certain sequence position certain false 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 false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 4 B 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 RhoA is ubiquitinated by the F29/30A Smurf2 mutant in vitro 667356 N/A 667367 enzyme target enzyme target substrate substrate unspecified role unspecified role N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) N/A 667360 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 false certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 4 D Interfering with the Smurf2 C2 domain and HECT domain interface enhances Smurf2 ubiquitination of RhoA. RhoA becomes ubiquitinated. bul1-bul1 Rsp5p undergoes autoubiquitination in vitro 667356 N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) N/A 667368 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 false range range certain certain sequence position certain false ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 5 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 Nedd4-1 undergoes autoubiquitination in vitro 667356 N/A 667369 unspecified role unspecified role neutral component neutral component N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 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. itch-itch AIP4 undergoes autoubiquitination in vitro 667356 N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) N/A 667370 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 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. wwp2-wwp2 WWP2 undergoes autoubiquitination in vitro 667356 N/A 667371 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) N/A 667366 unspecified role unspecified role ancillary ancillary ecoli Escherichia coli (strain K12) enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction false false false 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. smurf2-smurf2 15N-HECT domain of SMURF2 interacts with C2 domain of SMURF2 as shown by NMR titration 667357 N/A 667360 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 false ecobb Escherichia coli (strain B / BL21) N/A 667360 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 false n-terminal HECT domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false 1 B smurf2-smurf2-1 15N-C2 domain of SMURF2 interacts with HECT domain of SMURF2 as shown by NMR titration 667357 N/A 667360 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 false region 15n label 15n label 15N N15 15N N15 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecobb Escherichia coli (strain B / BL21) N/A 667360 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 false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false 1 C, S2 A smurf2-smurf2-2 13N-HECT domain of SMURF2 interacts with C2 domain of SMURF2 as shown by NMR titration 667357 N/A 667360 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 false region 13c label 13c label C13 13C C13 13C undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false ecobb Escherichia coli (strain B / BL21) N/A 667360 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 false ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction false false false 3 C smurf2-pi35p3 SMURF2 C2 domain interacts with Ins(1,3,5)P3 as shown by NMR titration 667357 N/A 667372 unspecified role unspecified role neutral component neutral component N/A 667360 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 false n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false S2 C smurf2-smurf2-6 GST-tagged C2 domain pulls down His-tagged HECT domain 667358 N/A 667360 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 false 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 false ecobb Escherichia coli (strain B / BL21) N/A 667360 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 false 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 false ecobb Escherichia coli (strain B / BL21) 667482 667479 direct interaction direct interaction false false false 4 F Smad7 N-terminal domain (NTD) - Increasing concentrations of Smad7 NTD interfered with HECT domain binding to the C2 domain.