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/ Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell (0092-8674) 2009 imex curation He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xiaodong.wang@utsouthwestern.edu 01-Sep-2009: Contacted by JYOTI. davet Only protein-protein interactions 5 human-ht_29 Homo sapiens HT-29 caucasian women with colon adenocarcinoma ht_29 HT-29 confirmed as human with IEF of AST, LDH established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma in 1964; described to be heterotransplantable forming well-differentiated grade I tumors anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xiaodong.wang@utsouthwestern.edu Accepted 2009-AUG-24 by DAVET. 01-Sep-2009: Contacted by JYOTI. Cell (0092-8674) 2009 davet Only protein-protein interactions 5 imex curation Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell (0092-8674) 2009 imex curation He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xiaodong.wang@utsouthwestern.edu 01-Sep-2009: Contacted by JYOTI. davet Only protein-protein interactions 5 human-ht_29 Homo sapiens HT-29 caucasian women with colon adenocarcinoma ht_29 HT-29 confirmed as human with IEF of AST, LDH established from the primary tumor of a 44-year-old Caucasian woman with colon adenocarcinoma in 1964; described to be heterotransplantable forming well-differentiated grade I tumors anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xiaodong.wang@utsouthwestern.edu Accepted 2009-AUG-24 by DAVET. 01-Sep-2009: Contacted by JYOTI. Cell (0092-8674) 2009 davet Only protein-protein interactions 5 imex curation Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell (0092-8674) 2009 imex curation He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xiaodong.wang@utsouthwestern.edu 01-Sep-2009: Contacted by JYOTI. davet Only protein-protein interactions 5 human-t98g Human caucasian glioblastoma line T98G t98g Human Caucasian glioblastoma T98G anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot He S., Wang L., Miao L., Wang T., Du F., Zhao L., Wang X. Apoptosis - Interactions involving proteins with a function related to apoptosis xiaodong.wang@utsouthwestern.edu Accepted 2009-AUG-24 by DAVET. 01-Sep-2009: Contacted by JYOTI. Cell (0092-8674) 2009 davet Only protein-protein interactions 5 imex curation casp8_human Caspase-8 CASP8 MCH5 ICE-like apoptotic protease 5 MORT1-associated ced-3 homolog FADD-homologous ICE/ced-3-like protease FADD-like ICE Apoptotic cysteine protease Apoptotic protease Mch-5 CAP4 protein protein human Homo sapiens Human MDFSRNLYDIGEQLDSEDLASLKFLSLDYIPQRKQEPIKDALMLFQRLQEKRMLEESNLSFLKELLFRINRLDLLITYLNTRKEEMERELQTPGRAQISAYRVMLYQISEEVSRSELRSFKFLLQEEISKCKLDDDMNLLDIFIEMEKRVILGEGKLDILKRVCAQINKSLLKIINDYEEFSKERSSSLEGSPDEFSNGEELCGVMTISDSPREQDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD DIP protein Q14790 original sequence version: 184 DIP protein Q14790 original sequence version: 135 DIP protein Q14790 original sequence version: 136 DIP protein Q14790 original sequence version: 147 DIP protein Q14790 original sequence version: 146 DIP protein Q14790 original sequence version: 170 DIP protein Q14790 original sequence version: 198 DIP protein Q14790-1 original sequence version: 197 DIP protein Q14790 original sequence version: 158 7A5FEAA6B39B582F fadd_human FAS-associated death domain protein FAS-associating death domain-containing protein Mediator of receptor induced toxicity FADD MORT1 GIG3 Growth-inhibiting gene 3 protein protein protein human Homo sapiens Human MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHTELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQLKVSDTKIDSIEDRYPRNLTERVRESLRIWKNTEKENATVAHLVGALRSCQMNLVADLVQEVQQARDLQNRSGAMSPMSWNSDASTSEAS DIP protein Q13158 original sequence version: 155 DIP protein Q13158 original sequence version: 130 DIP protein Q13158 original sequence version: 110 DIP protein Q13158 original sequence version: 118 DIP protein Q13158 original sequence version: 141 DIP protein Q13158 original sequence version: 166 0E65E2F852E83507 ripk3_human Receptor-interacting serine/threonine-protein kinase 3 RIPK3 RIP3 RIP-like protein kinase 3 Receptor-interacting protein 3 protein protein human Homo sapiens Human MSCVKLWPSGAPAPLVSIEELENQELVGKGGFGTVFRAQHRKWGYDVAVKIVNSKAISREVKAMASLDNEFVLRLEGVIEKVNWDQDPKPALVTKFMENGSLSGLLQSQCPRPWPLLCRLLKEVVLGMFYLHDQNPVLLHRDLKPSNVLLDPELHVKLADFGLSTFQGGSQSGTGSGEPGGTLGYLAPELFVNVNRKASTASDVYSFGILMWAVLAGREVELPTEPSLVYEAVCNRQNRPSLAELPQAGPETPGLEGLKELMQLCWSSEPKDRPSFQECLPKTDEVFQMVENNMNAAVSTVKDFLSQLRSSNRRFSIPESGQGGTEMDGFRRTIENQHSRNDVMVSEWLNKLNLEEPPSSVPKKCPSLTKRSRAQEEQVPQAWTAGTSSDSMAQPPQTPETSTFRNQMPSPTSTGTPSPGPRGNQGAERQGMNWSCRTPEPNPVTGRPLVNIYNCSGVQVGDNNYLTMQQTTALPTWGLAPSGKGRGLQHPPPVGSQEGPKDPEAWSRPQGWYNHSGK DIP protein Q9Y572 original sequence version: 134 BF755F8A0B1810A1 ripk1_human Receptor-interacting serine/threonine-protein kinase 1 RIPK1 RIP Cell death protein RIP RIP1 Receptor-interacting protein 1 protein protein human Homo sapiens Human MQPDMSLNVIKMKSSDFLESAELDSGGFGKVSLCFHRTQGLMIMKTVYKGPNCIEHNEALLEEAKMMNRLRHSRVVKLLGVIIEEGKYSLVMEYMEKGNLMHVLKAEMSTPLSVKGRIILEIIEGMCYLHGKGVIHKDLKPENILVDNDFHIKIADLGLASFKMWSKLNNEEHNELREVDGTAKKNGGTLYYMAPEHLNDVNAKPTEKSDVYSFAVVLWAIFANKEPYENAICEQQLIMCIKSGNRPDVDDITEYCPREIISLMKLCWEANPEARPTFPGIEEKFRPFYLSQLEESVEEDVKSLKKEYSNENAVVKRMQSLQLDCVAVPSSRSNSATEQPGSLHSSQGLGMGPVEESWFAPSLEHPQEENEPSLQSKLQDEANYHLYGSRMDRQTKQQPRQNVAYNREEERRRRVSHDPFAQQRPYENFQNTEGKGTAYSSAASHGNAVHQPSGLTSQPQVLYQNNGLYSSHGFGTRPLDPGTAGPRVWYRPIPSHMPSLHNIPVPETNYLGNTPTMPFSSLPPTDESIKYTIYNSTGIQIGAYNYMEIGGTSSSLLDSTNTNFKEEPAAKYQAIFDNTTSLTDKHLDPIRENLGKHWKNCARKLGFTQSQIDEIDHDYERDGLKEKVYQMLQKWVMREGIKGATVGKLAQALHQCSRIDLLSSLIYVSQN DIP protein Q13546 original sequence version: 158 DIP protein Q13546-1 original sequence version: 140 DIP protein Q13546 original sequence version: 146 DIP protein Q13546-1 original sequence version: 146 DIP protein Q13546 original sequence version: 134 DIP protein Q13546 original sequence version: 109 976E2428D525A9B2 ripk1-ripk3-1 1 7 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait Q9Y572:p.Lys50Ala Catalytically inactive mutation mutation certain certain sequence position certain certain certain sequence position certain K A Wed May 02 16:33:29 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys50ala' Tue Jun 30 15:36:20 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys50Ala' 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 ser-199 opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain physical association physical association TNF-alpha plus Smac mimetic plus z-VAD - necrosis inducing agents. Interaction increases 4-8 hours following treatment with these agents. However, 4 hr after treatment of all three agents together, RIPK1 was found in the RIP3 K50A precipitate and the amount increased at 6 and 8 hr of treatment. Fig. 5E, 6B Necrostatin-1 ripk1-ripk3-2 2 7 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey Q9Y572:p.Lys50Ala Catalytically inactive mutation mutation certain certain sequence position certain certain certain sequence position certain K A Wed May 02 14:54:56 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys50ala' Tue Jun 30 16:32:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys50Ala' 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 physical association physical association TNF-alpha plus Smac mimetic plus z-VAD - necrosis inducing agents. Interaction increases 4-8 hours following treatment with these agents. The interaction between RIPK1 and RIP3 mutant was verified with reciprocal pull down with an anti-RIPK1 antibody (Figure 5F). Fig. 5F ripk1-ripk3-3 2 6 unspecified role unspecified role prey prey ser-199 (potential) opser O-phospho-L-serine (2S)-2-amino-3-(phosphonooxy)propanoic acid 2-amino-3-hydroxypropanoic acid 3-phosphate 2-azanyl-3-(phosphonooxy)propanoic acid ACT_SITE Phosphoserine intermediate MOD_RES Phosphoserine O-phospho-L-serine O-phosphonoserine O-phosphorylated L-serine O3-phosphoserine serine phosphate ester OPSer Phospho Phospho Seryl Phosphorylation phosphorylations certain certain sequence position certain certain certain sequence position certain 7 unspecified role unspecified role bait bait physical association physical association To test whether endogenous RIP3 indeed interacts with RIPK1 upon necrosis induction, we treated HT-29 cells with TNF-α, Smac mimetic and z-VAD, followed by immunoprecipitation using an anti-RIPK1 antibody at different time points. Fig. 6A, Supp. 5 The interaction was accompanied by a an increase in mol. wt. of RIPK3, apparently due to autophosphorylation of Ser-199. ripk1-ripk3-4 2 5 unspecified role unspecified role prey prey 7 unspecified role unspecified role prey prey 6 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 4 unspecified role unspecified role bait bait physical association physical association TNF-alpha plus Smac mimetic plus z-VAD - necrosis inducing agents. Recruitment of RIPK3 to the complex increases 4-6 hours following treatment with these agents. Fig. 6E As shown in Figure 6E, RIP3 was found to associate with the RIPK1/FADD/caspase-8 complex after 4 hr of treatment. Necrotatin-1, necrosis inhibitor. ripk1-ripk3-5 3 6 unspecified role unspecified role bait bait rhim region required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain 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 7 unspecified role unspecified role prey prey physical association physical association TNF-alpha plus Smac mimetic plus z-VAD - necrosis inducing agents. RIP3 RHIM mutant is unable to interact with RIPK1 upon necrotic stimuli. Supp. Fig. 6