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/ Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. J. Biol. Chem. (0021-9258) 2010 imex curation Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K., Scheffner M. Cancer - Interactions investigated in the context of cancer Only done earlier part of the paper as further images look to be variants on ptms only, or inappropriate (impossible?) to annotate. The authors confirm p53, E1(UBE1) are of human origin, and ubiquitin is of bovine origin. martin.scheffner@uni-konstanz.de 19-Jun-2012: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column anti tag western anti tag western blot anti tag western Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K., Scheffner M. Cancer - Interactions investigated in the context of cancer Only done earlier part of the paper as further images look to be variants on ptms only, or inappropriate (impossible?) to annotate. Superdex S75 column The authors confirm p53, E1(UBE1) are of human origin, and ubiquitin is of bovine origin. martin.scheffner@uni-konstanz.de Accepted 2012-FEB-27 by ORCHARD J. Biol. Chem. (0021-9258) 2010 Only protein-protein interactions imex curation Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. J. Biol. Chem. (0021-9258) 2010 imex curation Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K., Scheffner M. Cancer - Interactions investigated in the context of cancer Only done earlier part of the paper as further images look to be variants on ptms only, or inappropriate (impossible?) to annotate. The authors confirm p53, E1(UBE1) are of human origin, and ubiquitin is of bovine origin. martin.scheffner@uni-konstanz.de 19-Jun-2012: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro ubiquitinase assay ubiquitinase assay ubiquitinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K., Scheffner M. Cancer - Interactions investigated in the context of cancer Only done earlier part of the paper as further images look to be variants on ptms only, or inappropriate (impossible?) to annotate. The authors confirm p53, E1(UBE1) are of human origin, and ubiquitin is of bovine origin. martin.scheffner@uni-konstanz.de Accepted 2012-FEB-27 by ORCHARD 19-Jun-2012: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2010 Only protein-protein interactions imex curation Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. Turning the RING domain protein MdmX into an active ubiquitin-protein ligase. J. Biol. Chem. (0021-9258) 2010 imex curation Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K., Scheffner M. Cancer - Interactions investigated in the context of cancer Only done earlier part of the paper as further images look to be variants on ptms only, or inappropriate (impossible?) to annotate. The authors confirm p53, E1(UBE1) are of human origin, and ubiquitin is of bovine origin. martin.scheffner@uni-konstanz.de 19-Jun-2012: Contacted by JYOTI. Only protein-protein interactions human-h1299 Homo sapiens lung lymph node carcinoma h1299 Human lung lymph node carcinoma p53 null. These cells have also been reported to secrete the peptide hormone neuromedin B (NMB), but not gastrin releasing peptide (GRP). Also known as NCI H1299 or CRL-5803. http://www.lgcpromochem.com/atcc/ ATCC number: CRL-5803. ATCC number of the cell line is CRL-5803. pull down pull down western blot western blot Immuno blot Immuno blot Iyappan S., Wollscheid H.P., Rojas-Fernandez A., Marquardt A., Tang H.C., Singh R.K., Scheffner M. Cancer - Interactions investigated in the context of cancer Only done earlier part of the paper as further images look to be variants on ptms only, or inappropriate (impossible?) to annotate. The authors confirm p53, E1(UBE1) are of human origin, and ubiquitin is of bovine origin. martin.scheffner@uni-konstanz.de Accepted 2012-FEB-27 by ORCHARD 19-Jun-2012: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2010 Only protein-protein interactions imex curation p53_human Cellular tumor antigen p53 TP53 P53 Tumor suppressor p53 Phosphoprotein p53 Antigen NY-CO-13 protein protein human Homo sapiens Human MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD DIP protein P04637 original sequence version: 121 DIP protein P04637 original sequence version: 115 DIP protein P04637-1 original sequence version: 224 DIP protein P04637 original sequence version: 117 DIP protein P04637-1 original sequence version: 226 DIP protein P04637 original sequence version: 226 DIP protein P04637 original sequence version: 212 DIP protein P04637 original sequence version: 220 DIP protein P04637-1 original sequence version: 220 DIP protein P04637-1 original sequence version: 145 DIP protein P04637 original sequence version: 146 DIP protein P04637 original sequence version: 145 DIP protein P04637 original sequence version: 166 DIP protein P04637-1 original sequence version: 166 DIP protein P04637 original sequence version: 169 DIP protein P04637 original sequence version: 177 DIP protein P04637 original sequence version: 175 DIP protein P04637-1 original sequence version: 175 DIP protein P04637-1 original sequence version: 172 DIP protein P04637-1 original sequence version: 169 DIP protein P04637 original sequence version: 170 DIP protein P04637 original sequence version: 172 DIP protein P04637-1 original sequence version: 194 DIP protein P04637 original sequence version: 196 DIP protein P04637 original sequence version: 185 DIP protein P04637-1 original sequence version: 191 DIP protein P04637 original sequence version: 191 DIP protein P04637 original sequence version: 180 DIP protein P04637-1 original sequence version: 197 DIP protein P04637 original sequence version: 183 DIP protein P04637-1 original sequence version: 183 DIP protein P04637-1 original sequence version: 185 DIP protein P04637 original sequence version: 199 DIP protein P04637 original sequence version: 201 DIP protein P04637 original sequence version: 189 DIP protein P04637 original sequence version: 235 DIP protein P04637 original sequence version: 230 DIP protein P04637 original sequence version: 234 DIP protein P04637-1 original sequence version: 234 DIP protein P04637-1 original sequence version: 235 DIP protein P04637 original sequence version: 239 DIP protein P04637-1 original sequence version: 212 DIP protein P04637-1 original sequence version: 213 DIP protein P04637 original sequence version: 213 DIP protein P04637 original sequence version: 206 DIP protein P04637-1 original sequence version: 188 DIP protein P04637-1 original sequence version: 228 DIP protein P04637 original sequence version: 208 DIP protein P04637 original sequence version: 240 DIP protein P04637-1 original sequence version: 240 DIP protein P04637 original sequence version: 204 DIP protein P04637-1 original sequence version: 204 DIP protein P04637-1 original sequence version: 209 DIP protein P04637-1 original sequence version: 203 DIP protein P04637 original sequence version: 221 DIP protein P04637-1 original sequence version: 152 DIP protein P04637 original sequence version: 217 DIP protein P04637-1 original sequence version: 140 DIP protein P04637 original sequence version: 215 DIP protein P04637 original sequence version: 214 DIP protein P04637-1 original sequence version: 182 DIP protein P04637 original sequence version: 182 DIP protein P04637 original sequence version: 188 DIP protein P04637 original sequence version: 192 DIP protein P04637-1 original sequence version: 192 DIP protein P04637 original sequence version: 218 DIP protein P04637-1 original sequence version: 218 AD5C149FD8106131 uba1_human Ubiquitin-like modifier-activating enzyme 1 Ubiquitin-activating enzyme E1 Protein A1S9 UBA1 A1S9T UBE1 protein protein human Homo sapiens Human MSSSPLSKKRRVSGPDPKPGSNCSPAQSVLSEVPSVPTNGMAKNGSEADIDEGLYSRQLYVLGHEAMKRLQTSSVLVSGLRGLGVEIAKNIILGGVKAVTLHDQGTAQWADLSSQFYLREEDIGKNRAEVSQPRLAELNSYVPVTAYTGPLVEDFLSGFQVVVLTNTPLEDQLRVGEFCHNRGIKLVVADTRGLFGQLFCDFGEEMILTDSNGEQPLSAMVSMVTKDNPGVVTCLDEARHGFESGDFVSFSEVQGMVELNGNQPMEIKVLGPYTFSICDTSNFSDYIRGGIVSQVKVPKKISFKSLVASLAEPDFVVTDFAKFSRPAQLHIGFQALHQFCAQHGRPPRPRNEEDAAELVALAQAVNARALPAVQQNNLDEDLIRKLAYVAAGDLAPINAFIGGLAAQEVMKACSGKFMPIMQWLYFDALECLPEDKEVLTEDKCLQRQNRYDGQVAVFGSDLQEKLGKQKYFLVGAGAIGCELLKNFAMIGLGCGEGGEIIVTDMDTIEKSNLNRQFLFRPWDVTKLKSDTAAAAVRQMNPHIRVTSHQNRVGPDTERIYDDDFFQNLDGVANALDNVDARMYMDRRCVYYRKPLLESGTLGTKGNVQVVIPFLTESYSSSQDPPEKSIPICTLKNFPNAIEHTLQWARDEFEGLFKQPAENVNQYLTDPKFVERTLRLAGTQPLEVLEAVQRSLVLQRPQTWADCVTWACHHWHTQYSNNIRQLLHNFPPDQLTSSGAPFWSGPKRCPHPLTFDVNNPLHLDYVMAAANLFAQTYGLTGSQDRAAVATFLQSVQVPEFTPKSGVKIHVSDQELQSANASVDDSRLEELKATLPSPDKLPGFKMYPIDFEKDDDSNFHMDFIVAASNLRAENYDIPSADRHKSKLIAGKIIPAIATTTAAVVGLVCLELYKVVQGHRQLDSYKNGFLNLALPFFGFSEPLAAPRHQYYNQEWTLWDRFEVQGLQPNGEEMTLKQFLDYFKTEHKLEITMLSQGVSMLYSFFMPAAKLKERLDQPMTEIVSRVSKRKLGRHVRALVLELCCNDESGEDVEVPYVRYTIR DIP protein P22314 original sequence version: 157 DIP protein P22314 original sequence version: 152 4B413AAA75FAA562 ubiq_bovin Ubiquitin RPS27A UBA80 UBCEP1 UBA52 UBCEP2 UBB UBC protein protein bovin Bos taurus (Bovine) Bovine MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG C42A35397FFD9B52 ub2d2_human Ubiquitin-conjugating enzyme E2 D2 UBC5B UBCH4 UBE2D2 UBC4 UBCH5B Ubiquitin-protein ligase D2 Ubiquitin carrier protein D2 Ubiquitin-conjugating enzyme E2-17 kDa 2 Ubiquitin-conjugating enzyme E2(17)KB 2 PUBC1 p53-regulated ubiquitin-conjugating enzyme 1 (E3-independent) E2 ubiquitin-conjugating enzyme D2 E2 ubiquitin-conjugating enzyme D2 protein protein human Homo sapiens Human MALKRIHKELNDLARDPPAQCSAGPVGDDMFHWQATIMGPNDSPYQGGVFFLTIHFPTDYPFKPPKVAFTTRIYHPNINSNGSICLDILRSQWSPALTISKVLLSICSLLCDPNPDDPLVPEIARIYKTDREKYNRIAREWTQKYAM DIP protein P62837 original sequence version: 81 DIP protein P62837-1 original sequence version: 134 DIP protein P62837 original sequence version: 95 DIP protein P62837 original sequence version: 139 DIP protein P62837 original sequence version: 37 DIP protein P62837 original sequence version: 115 DIP protein P62837 original sequence version: 58 DIP protein P62837 original sequence version: 74 DIP protein P62837 original sequence version: 103 DIP protein P62837 original sequence version: 101 C942BE7853CBC355 mdm2_human E3 ubiquitin-protein ligase Mdm2 MDM2 p53-binding protein Mdm2 Oncoprotein Mdm2 Double minute 2 protein RING-type E3 ubiquitin transferase Mdm2 protein protein human Homo sapiens Human MCNTNMSVPTDGAVTTSQIPASEQETLVRPKPLLLKLLKSVGAQKDTYTMKEVLFYLGQYIMTKRLYDEKQQHIVYCSNDLLGDLFGVPSFSVKEHRKIYTMIYRNLVVVNQQESSDSGTSVSENRCHLEGGSDQKDLVQELQEEKPSSSHLVSRPSTSSRRRAISETEENSDELSGERQRKRHKSDSISLSFDESLALCVIREICCERSSSSESTGTPSNPDLDAGVSEHSGDWLDQDSVSDQFSVEFEVESLDSEDYSLSEEGQELSDEDDEVYQVTVYQAGESDTDSFEEDPEISLADYWKCTSCNEMNPPLPSHCNRCWALRENWLPEDKGKDKGEISEKAKLENSTQAEEGFDVPDCKKTIVNDSRESCVEENDDKITQASQSQESEDYSQPSTSSSIIYSSQEDVKEFEREETQDKEESVESSLPLNAIEPCVICQGRPKNGCIVHGKTGHLMACFTCAKKLKKRNKPCPVCRQPIQMIVLTYFP DIP protein Q00987 original sequence version: 93 DIP protein Q00987-1 original sequence version: 192 DIP protein Q00987 original sequence version: 88 DIP protein Q00987 original sequence version: 180 DIP protein Q00987 original sequence version: 188 DIP protein Q00987 original sequence version: 189 DIP protein Q00987 original sequence version: 115 DIP protein Q00987-1 original sequence version: null DIP protein Q00987 original sequence version: 149 DIP protein Q00987 original sequence version: 146 DIP protein Q00987 original sequence version: 140 DIP protein Q00987 original sequence version: 183 DIP protein Q00987 original sequence version: 145 DIP protein Q00987 original sequence version: 182 DIP protein Q00987 original sequence version: 151 DIP protein Q00987 original sequence version: 156 DIP protein Q00987-1 original sequence version: 160 DIP protein Q00987 original sequence version: 160 DIP protein Q00987 original sequence version: 168 DIP protein Q00987 original sequence version: 186 DIP protein Q00987 original sequence version: 206 F37CE163876BC983 mdm4_human Protein Mdm4 MDM4 MDMX p53-binding protein Mdm4 Mdm2-like p53-binding protein Protein Mdmx Double minute 4 protein protein protein human Homo sapiens Human MTSFSTSAQCSTSDSACRISPGQINQVRPKLPLLKILHAAGAQGEMFTVKEVMHYLGQYIMVKQLYDQQEQHMVYCGGDLLGELLGRQSFSVKDPSPLYDMLRKNLVTLATATTDAAQTLALAQDHSMDIPSQDQLKQSAEESSTSRKRTTEDDIPTLPTSEHKCIHSREDEDLIENLAQDETSRLDLGFEEWDVAGLPWWFLGNLRSNYTPRSNGSTDLQTNQDVGTAIVSDTTDDLWFLNESVSEQLGVGIKVEAADTEQTSEEVGKVSDKKVIEVGKNDDLEDSKSLSDDTDVEVTSEDEWQCTECKKFNSPSKRYCFRCWALRKDWYSDCSKLTHSLSTSDITAIPEKENEGNDVPDCRRTISAPVVRPKDAYIKKENSKLFDPCNSVEFLDLAHSSESQETISSMGEQLDNLSEQRTDTENMEDCQNLLKPCSLCEKRPRDGNIIHGRTGHLVTCFHCARRLKKAGASCPICKKEIQLVIKVFIA DIP protein O15151 original sequence version: 141 DIP protein O15151 original sequence version: 109 DIP protein O15151 original sequence version: 129 DIP protein O15151 original sequence version: 147 415E6FA5A0C69857 mdm4-ube2d2 1 9 unspecified role unspecified role neutral component neutral component ring_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 O15151:p.Lys435Glu mutation mutation certain certain sequence position certain certain certain sequence position certain K E Wed May 02 14:40:42 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys435glu' Tue Jun 30 18:17:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys435Glu' ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role neutral component neutral component c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) direct interaction direct interaction S1 mdm2-ube2d2-1 1 7 unspecified role unspecified role neutral component neutral component c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ring_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 ecoli Escherichia coli (strain K12) direct interaction direct interaction S1 mdm4-tp53-4 2 9 enzyme enzyme neutral component neutral component O15151:p.Asn448Cys mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain N C Wed May 02 15:44:19 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asn448cys' Tue Jun 30 16:26:44 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asn448Cys' O15151:p.Arg453Lys mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain R K Wed May 02 15:59:48 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg453lys' Tue Jun 30 18:21:01 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg453Lys' O15151:p.Lys435Glu mutation mutation certain certain sequence position certain certain certain sequence position certain K E Wed May 02 18:46:12 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys435glu' Tue Jun 30 19:00:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys435Glu' 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 ring_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain O15151:p.Ile450Val mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain I V Wed May 02 17:07:59 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile450val' Tue Jun 30 17:30:32 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile450Val' ecoli Escherichia coli (strain K12) 5 unspecified role unspecified role ancillary ancillary 4 enzyme target enzyme target substrate substrate neutral component neutral component region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 7 unspecified role unspecified role ancillary ancillary c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role ancillary ancillary ubiquitination ubiquitination reaction ubiquitination 1B p53 ubiquitination Replacement of the asparagine residue at position 448 of MdmX by cysteine, the respective residue of Mdm2, results in an MdmX mutant that can efficiently ubiquitinate p53 in vitro. ubiq-tp53-1 3 4 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait n_terminus 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 physical association physical association Fig 3 p53 ubiquitination Mdm2 is required for p53 ubiquitylation and degradation within cells Mdm4. Wild-type Mdm4 is not, but a construct containing Asn448Cys, a region spanning amino acids 465–480 (numbering according to human Mdm4) is required for p53 ubiquitination.