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/ Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. J. Biol. Chem. (0021-9258) 2006 imex curation Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. bardwell@uci.edu 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. Additional information on participants taken from manufacturers webstes. bardwell@uci.edu Accepted 2006-DEC-11 by JYOTI. 21-Dec-2006: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. J. Biol. Chem. (0021-9258) 2006 imex curation Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. bardwell@uci.edu 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. bardwell@uci.edu Accepted 2006-DEC-11 by JYOTI. 21-Dec-2006: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. J. Biol. Chem. (0021-9258) 2006 imex curation Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. bardwell@uci.edu 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. Additional information on participants taken from manufacturers webstes. bardwell@uci.edu Accepted 2006-DEC-11 by JYOTI. 21-Dec-2006: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. Interacting JNK-docking sites in MKK7 promote binding and activation of JNK mitogen-activated protein kinases. J. Biol. Chem. (0021-9258) 2006 imex curation Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. bardwell@uci.edu 21-Dec-2006: Contacted by JYOTI. Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Ho DT., Bardwell AJ., Grewal S., Iverson C., Bardwell L. Apoptosis - Interactions involving proteins with a function related to apoptosis Binding reactions were analyzed by scintillation counting and SDS-PAGE. SDS-PAGE gels were first Coomassie stained and photographed, then treated with fluorography solution dried down, and autoradiographed. Binding constants were calculated based on the known input concentrations and a determination of the amount complex formed, assuming a simple bimolecular binding reaction. bardwell@uci.edu Accepted 2006-DEC-11 by JYOTI. 21-Dec-2006: Contacted by JYOTI. J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation o14733-2 Dual specificity mitogen-activated protein kinase kinase 7 B MAP2K7 JNKK2 MEK7 MKK7 PRKMK7 JNK-activating kinase 2 MAPK/ERK kinase 7 c-Jun N-terminal kinase kinase 2 Stress-activated protein kinase kinase 4 SKK4 protein protein human Homo sapiens Human MAASSLEQKLSRLEAKLKQENREARRRIDLNLDISPQRPRPTLQLPLANDGGSRSPSSESSPQHPTPPARPRHMLGLPSTLFTPRSMESIEIDQKLQEIMKQTGYLTIGGQVPPSLWRGEGGGPARLDPSWERQWGAGGGGRAPGTLQPSLSSQRYQAEINDLENLGEMGSGTCGQVWKMRFRKTGHVIAVKQMRRSGNKEENKRILMDLDVVLKSHDCPYIVQCFGTFITNTDVFIAMELMGTCAEKLKKRMQGPIPERILGKMTVAIVKALYYLKEKHGVIHRDVKPSNILLDERGQIKLCDFGISGRLVDSKAKTRSAGCAAYMAPERIDPPDPTKPDYDIRADVWSLGISLVELATGQFPYKNCKTDFEVLTKVLQEEPPLLPGHMGFSGDFQSFVKDCLTKDHRKRPKYNKLLEHSFIKRYETLEVDVASWFKDVMAKTESPRTSGVLSQPHLPFFR May be due to intron retention. DF3496066961B0C7 atf2_human Cyclic AMP-dependent transcription factor ATF-2 ATF2 CREB2 CREBP1 Activating transcription factor 2 cAMP response element-binding protein CRE-BP1 HB16 Cyclic AMP-responsive element-binding protein 2 protein protein human Homo sapiens Human MKFKLHVNSARQYKDLWNMSDDKPFLCTAPGCGQRFTNEDHLAVHKHKHEMTLKFGPARNDSVIVADQTPTPTRFLKNCEEVGLFNELASPFENEFKKASEDDIKKMPLDLSPLATPIIRSKIEEPSVVETTHQDSPLPHPESTTSDEKEVPLAQTAQPTSAIVRPASLQVPNVLLTSSDSSVIIQQAVPSPTSSTVITQAPSSNRPIVPVPGPFPLLLHLPNGQTMPVAIPASITSSNVHVPAAVPLVRPVTMVPSVPGIPGPSSPQPVQSEAKMRLKAALTQQHPPVTNGDTVKGHGSGLVRTQSEESRPQSLQQPATSTTETPASPAHTTPQTQSTSGRRRRAANEDPDEKRRKFLERNRAAASRCRQKRKVWVQSLEKKAEDLSSLNGQLQSEVTLLRNEVAQLKQLLLAHKDCPVTAMQKKSGYHTADKDDSSEDISVPSSPHTEAIQHSSVSTSNGVSSTSKAEAVATSVLTQMADQSTEPALSQIVMAPSSQSQPSGS 0190EEFAEC8891A7 jip1_mouse C-Jun-amino-terminal kinase-interacting protein 1 Mapk8ip1 Ib1 Jip1 Mapk8ip Prkm8ip Islet-brain-1 Mitogen-activated protein kinase 8-interacting protein 1 JNK MAP kinase scaffold protein 1 protein protein mouse Mus musculus Mouse MAERESGLGGGAASPPAASPFLGLHIASPPNFRLTHDISLEEFEDEDLSEITDECGISLQCKDTLSLRPPRAGLLSAGSSGSAGSRLQAEMLQMDLIDAAGDTPGAEDDEEEEDDELAAQRPGVGPPKAESNQDPAPRSQGQGPGTGSGDTYRPKRPTTLNLFPQVPRSQDTLNNNSLGKKHSWQDRVSRSSSPLKTGEQTPPHEHICLSDELPPQGSPVPTQDRGTSTDSPCRRSAATQMAPPSGPPATAPGGRGHSHRDRIHYQADVRLEATEEIYLTPVQRPPDPAEPTSTFMPPTESRMSVSSDPDPAAYSVTAGRPHPSISEEDEGFDCLSSPERAEPPGGGWRGSLGEPPPPPRASLSSDTSALSYDSVKYTLVVDEHAQLELVSLRPCFGDYSDESDSATVYDNCASASSPYESAIGEEYEEAPQPRPPTCLSEDSTPDEPDVHFSKKFLNVFMSGRSRSSSAESFGLFSCVINGEEHEQTHRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIEVTKEPEHMAALAKNSDWIDQFRVKFLGSVQVPYHKGNDVLCAAMQKIATTRRLTVHFNPPSSCVLEISVRGVKIGVKADDALEAKGNKCSHFFQLKNISFCGYHPKNNKYFGFITKHPADHRFACHVFVSEDSTKALAESVGRAFQQFYKQFVEYTCPTEDIYLE 274013B12D91049D p45984-1 Mitogen-activated protein kinase 9 MAPK9 JNK2 PRKM9 JNK-55 Stress-activated protein kinase JNK2 c-Jun N-terminal kinase 2 Stress-activated protein kinase 1a SAPK1A protein protein human Homo sapiens Human MSDSKCDSQFYSVQVADSTFTVLKRYQQLKPIGSGAQGIVCAAFDTVLGINVAVKKLSRPFQNQTHAKRAYRELVLLKCVNHKNIISLLNVFTPQKTLEEFQDVYLVMELMDANLCQVIHMELDHERMSYLLYQMLCGIKHLHSAGIIHRDLKPSNIVVKSDCTLKILDFGLARTACTNFMMTPYVVTRYYRAPEVILGMGYKENVDIWSVGCIMGELVKGCVIFQGTDHIDQWNKVIEQLGTPSAEFMKKLQPTVRNYVENRPKYPGIKFEELFPDWIFPSESERDKIKTSQARDLLSKMLVIDPDKRISVDEALRHPYITVWYDPAEAEAPPPQIYDAQLEEREHAIEEWKELIYKEVMDWEERSKNGVVKDQPSDAAVSSNATPSQSSSINDISSMSTEQTLASDTDSSLDASTGPLEGCR 9C15DA79981290AF jun_human Transcription factor AP-1 JUN Activator protein 1 Proto-oncogene c-Jun V-jun avian sarcoma virus 17 oncogene homolog p39 protein protein human Homo sapiens Human MTAKMETTFYDDALNASFLPSESGPYGYSNPKILKQSMTLNLADPVGSLKPHLRAKNSDLLTSPDVGLLKLASPELERLIIQSSNGHITTTPTPTQFLCPKNVTDEQEGFAEGFVRALAELHSQNTLPSVTSAAQPVNGAGMVAPAVASVAGGSGSGGFSASLHSEPPVYANLSNFNPGALSSGGGAPSYGAAGLAFPAQPQQQQQPPHHLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNHVNSGCQLMLTQQLQTF DIP protein P05412 original sequence version: 119 DIP protein P05412 original sequence version: 114 DIP protein P05412 original sequence version: 185 DIP protein P05412 original sequence version: 145 DIP protein P05412 original sequence version: 202 DIP protein P05412 original sequence version: 206 DIP protein P05412 original sequence version: 163 DIP protein P05412 original sequence version: 175 DIP protein P05412 original sequence version: 189 DIP protein P05412 original sequence version: 169 0695E23AC4D33561 mk01_mouse Mitogen-activated protein kinase 1 Mapk1 Erk2 Mapk Prkm1 Extracellular signal-regulated kinase 2 Mitogen-activated protein kinase 2 MAP kinase isoform p42 ERT1 protein protein mouse Mus musculus Mouse MAAAAAAGPEMVRGQVFDVGPRYTNLSYIGEGAYGMVCSAYDNLNKVRVAIKKISPFEHQTYCQRTLREIKILLRFRHENIIGINDIIRAPTIEQMKDVYIVQDLMETDLYKLLKTQHLSNDHICYFLYQILRGLKYIHSANVLHRDLKPSNLLLNTTCDLKICDFGLARVADPDHDHTGFLTEYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGKHYLDQLNHILGILGSPSQEDLNCIINLKARNYLLSLPHKNKVPWNRLFPNADSKALDLLDKMLTFNPHKRIEVEQALAHPYLEQYYDPSDEPIAEAPFKFDMELDDLPKEKLKELIFEETARFQPGYRS DIP protein P63085 original sequence version: 120 DIP protein P63085 original sequence version: 94 DIP protein P63085 original sequence version: 88 DIP protein P63085 original sequence version: 103 DIP protein P63085 original sequence version: 121 DIP protein P63085 original sequence version: 101 3BBCF22471EDBA0B p45983-1 Mitogen-activated protein kinase 8 JNK1-alpha-2 MAPK8 JNK1 PRKM8 SAPK1 JNK-46 Stress-activated protein kinase JNK1 c-Jun N-terminal kinase 1 Stress-activated protein kinase 1c SAPK1C protein protein human Homo sapiens Human MSRSKRDNNFYSVEIGDSTFTVLKRYQNLKPIGSGAQGIVCAAYDAILERNVAIKKLSRPFQNQTHAKRAYRELVLMKCVNHKNIIGLLNVFTPQKSLEEFQDVYIVMELMDANLCQVIQMELDHERMSYLLYQMLCGIKHLHSAGIIHRDLKPSNIVVKSDCTLKILDFGLARTAGTSFMMTPYVVTRYYRAPEVILGMGYKENVDLWSVGCIMGEMVCHKILFPGRDYIDQWNKVIEQLGTPCPEFMKKLQPTVRTYVENRPKYAGYSFEKLFPDVLFPADSEHNKLKASQARDLLSKMLVIDASKRISVDEALQHPYINVWYDPSEAEAPPPKIPDKQLDEREHTIEEWKELIYKEVMDLEERTKNGVIRGQPSPLGAAVINGSQHPSSSSSVNDVSSMSTDPTLASDTDSSLEAAAGPLGCCR 94FB6BE0358B9B60 mp2k4_human Dual specificity mitogen-activated protein kinase kinase 4 JNK-activating kinase 1 c-Jun N-terminal kinase kinase 1 SAPK/ERK kinase 1 MAP2K4 JNKK1 MKK4 PRKMK4 SERK1 MEK4 MAPK/ERK kinase 4 SKK1 SEK1 Stress-activated protein kinase kinase 1 protein protein human Homo sapiens Human MAAPSPSGGGGSGGGSGSGTPGPVGSPAPGHPAVSSMQGKRKALKLNFANPPFKSTARFTLNPNPTGVQNPHIERLRTHSIESSGKLKISPEQHWDFTAEDLKDLGEIGRGAYGSVNKMVHKPSGQIMAVKRIRSTVDEKEQKQLLMDLDVVMRSSDCPYIVQFYGALFREGDCWICMELMSTSFDKFYKYVYSVLDDVIPEEILGKITLATVKALNHLKENLKIIHRDIKPSNILLDRSGNIKLCDFGISGQLVDSIAKTRDAGCRPYMAPERIDPSASRQGYDVRSDVWSLGITLYELATGRFPYPKWNSVFDQLTQVVKGDPPQLSNSEEREFSPSFINFVNLCLTKDESKRPKYKELLKHPFILMYEERAVEVACYVCKILDQMPATPSSPMYVD A472537F2F26770B elk1_human ETS domain-containing protein Elk-1 ELK1 protein protein human Homo sapiens Human MDPSVTLWQFLLQLLREQGNGHIISWTSRDGGEFKLVDAEEVARLWGLRKNKTNMNYDKLSRALRYYYDKNIIRKVSGQKFVYKFVSYPEVAGCSTEDCPPQPEVSVTSTMPNVAPAAIHAAPGDTVSGKPGTPKGAGMAGPGGLARSSRNEYMRSGLYSTFTIQSLQPQPPPHPRPAVVLPSAAPAGAAAPPSGSRSTSPSPLEACLEAEEAGLPLQVILTPPEAPNLKSEELNVEPGLGRALPPEVKVEGPKEELEVAGERGFVPETTKAEPEVPPQEGVPARLPAVVMDTAGQAGGHAASSPEISQPQKGRKPRDLELPLSPSLLGGPGPERTPGSGSGSGLQAPGPALTPSLLPTHTLTPVLLTPSSLPPSIHFWSTLSPIAPRSPAKLSFQFPSSGSAQVHIPSISVDGLSTPVVLSPGPQKP 68F71F8ADB9D38CA mk08_human Mitogen-activated protein kinase 8 MAPK8 JNK1 PRKM8 Stress-activated protein kinase JNK1 c-Jun N-terminal kinase 1 JNK-46 SAPK1 Stress-activated protein kinase 1c SAPK1C protein protein human Homo sapiens Human MSRSKRDNNFYSVEIGDSTFTVLKRYQNLKPIGSGAQGIVCAAYDAILERNVAIKKLSRPFQNQTHAKRAYRELVLMKCVNHKNIIGLLNVFTPQKSLEEFQDVYIVMELMDANLCQVIQMELDHERMSYLLYQMLCGIKHLHSAGIIHRDLKPSNIVVKSDCTLKILDFGLARTAGTSFMMTPYVVTRYYRAPEVILGMGYKENVDLWSVGCIMGEMVCHKILFPGRDYIDQWNKVIEQLGTPCPEFMKKLQPTVRTYVENRPKYAGYSFEKLFPDVLFPADSEHNKLKASQARDLLSKMLVIDASKRISVDEALQHPYINVWYDPSEAEAPPPKIPDKQLDEREHTIEEWKELIYKEVMDLEERTKNGVIRGQPSPLGAAVINGSQHPSSSSSVNDVSSMSTDPTLASDTDSSLEAAAGPLGCCR DIP protein P45983 original sequence version: 96 DIP protein P45983 original sequence version: 185 DIP protein P45983 original sequence version: 124 DIP protein P45983 original sequence version: 103 94FB6BE0358B9B60 map2k7-mapk8-1 2 5 unspecified role unspecified role bait bait n-terminus 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 n-terminal region 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) 14 unspecified role unspecified role prey prey 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 direct interaction direct interaction To confirm that the MKK7beta N terminus was sufficient to bind JNK, and to evaluate whether systematic removal of the putative docking sites would compromise binding, fusions of GST to portions of the N terminus of MKK7 were tested for their ability to bind in vitro to 35S-labeled JNK1 (Fig. 2A). 2 map2k4-mapk8-1 2 14 unspecified role unspecified role prey prey 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 12 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined d region 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) direct interaction direct interaction In contrast, the isolated D-site from MKK4 (MKK4-(36�49)) bound reasonably well to JNK1 when tested in the same assay (Fig. 3, B and C). 3B, 3C map2k7-mapk8-2 2 14 unspecified role unspecified role prey prey 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 5 unspecified role unspecified role bait bait n-terminus 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 n-terminal region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain d1m region RRRIDLNL to EEEIDANA binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain d3m region RPRHMLGL to EPEHMAGL binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain d2m region RPRPTLQL to EPEPTAQA binding region binding-associated region binding region mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction To ascertain if any of the D-sites were necessary for high affinity JNK binding, substitution mutations were introduced into D1, D2, and D3, either alone or in combination, in the context of MKK7-(1�85). 4 and 6 Double mutants were more effective than single in inhibiting the interaction, particularly D1 + D2, and the triple mutant almost completely lost the ability to bind JNK. map2k7-mapk8-3 4 5 unspecified role unspecified role bait bait n-terminal region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain d1m region RRRIDLNL to EEEIDANA required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain d2m region RPRPTLQL to EPEPTAQA required to bind necessary binding region required to bind mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain c-terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecoli Escherichia coli (strain K12) 14 unspecified role unspecified role prey prey 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 physical association physical association 5B D1 + D2 introduced as a double mutant. The resulting mutant protein was expressed in human kidney epithelial (HEK293) cells and compared with wild-type, FLAG-tagged MKK7 for its ability to bind to co-overexpressed JNK1, as assessed by co-immunoprecipitation (Fig. 5A). mapk8-cjun-2 1 11 enzyme enzyme unspecified role unspecified role 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 spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. 9 enzyme target enzyme target substrate substrate unspecified role unspecified role 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 region 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) phosphorylation phosphorylation reaction phosphorylation Phosphorylation of c-jun MAP2K7. Phosphorylates and activates MAPK8. Stimulation inhibited by D1 and D2 mutants, or by adding kinase-dead MAP2K7. To assess the ability of the D1/D2 double mutant to phosphorylate and activate JNK1, wild-type or mutant MKK7 was expressed in HEK293 cells, immunoprecipitated, and mixed with purified JNK1 and c-Jun proteins in a coupled kinase assay (Fig. 5C). 5D map2k7-mapk8-4 2 12 inhibitor inhibitor prey prey d-region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 5 unspecified role unspecified role prey prey 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 11 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) association association A, D-site peptides (triangle) were used to inhibit the ability of MKK7 to bind to GST-JNK1. 6 mapk8-atf2-1 3 6 enzyme target enzyme target substrate substrate unspecified role unspecified role 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 region 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) 11 enzyme enzyme unspecified role unspecified role 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 spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. phosphorylation phosphorylation reaction phosphorylation Phosphorylation of ATF2 D-site Peptides from MKK7 Inhibit JNK1/2 Phosphorylation of c-Jun and ATF2 7 MAP2K4 - residues 37-52 MAP2K7 - residues 37-51 > 22-37 and 67-83 mapk9-cjun-1 3 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis 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) 8 enzyme enzyme unspecified role unspecified role 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 spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. phosphorylation phosphorylation reaction phosphorylation Phosphorylation of c-jun Compared with their potency in inhibiting JNK1, the ability of the MKK7-derived D-site peptides to inhibit JNK2 phosphorylation of c-Jun or ATF2 was about 5-fold lower (Fig. 7, F, 7 MAP2K4 - residues 37-52. Inhibition 5-fold less than that observed for MAPK08 (JNK1). MAP2K7 - residues 37-51 > 22-37 and 67-83. Inhibition 5-fold less than that observed for MAPK08 (JNK1). mapk9-atf2-1 3 6 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis 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) 8 enzyme enzyme unspecified role unspecified role 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 spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. phosphorylation phosphorylation reaction phosphorylation Phosphorylation of ATF2 D-site Peptides from MKK7 Inhibit JNK1/2 Phosphorylation of c-Jun and ATF2 7 MAP2K4 - residues 37-52, Inhibition 5-fold less than that observed for MAPK08 (JNK1). MAP2K7 - residues 37-51 > 22-37 and 67-83. Inhibition 5-fold less than that observed for MAPK08 (JNK1). mapk8-cjun-3 3 9 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis 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) 11 enzyme enzyme unspecified role unspecified role 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 spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. phosphorylation phosphorylation reaction phosphorylation Phosphorylation of c-jun Inhibition of JNK phosphorylation of transcription factors c-Jun and ATF2 with D-site peptides from MKK7. 7 MAP2K4 - residues 37-52 MAP2K7 - residues 37-51 > 22-37 and 67-83 mapk1-elk1-2 3 13 enzyme target enzyme target substrate substrate unspecified role unspecified role 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 region 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) 10 enzyme enzyme unspecified role unspecified role spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection phosphorylation phosphorylation reaction phosphorylation 8 Also, as shown in Fig. 8F, the MEK2-derived D-site peptide was a more effective ERK2 inhibitor than any of the three MKK7-derived peptides. MAP2K2 - residues 1-20 map2k7-mapk8-5 2 11 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 7 competitor competitor competitor prey prey d-region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain in vitro In vitro 5 unspecified role unspecified role prey prey 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 association association A, D-site peptides (triangle) were used to inhibit the ability of MKK7 to bind to GST-JNK1. 6