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/ The MAP kinase substrate MKS1 is a regulator of plant defense responses. The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. (0261-4189) 2005 Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library EMBO J. (0261-4189) 2005 orchard The MAP kinase substrate MKS1 is a regulator of plant defense responses. The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. (0261-4189) 2005 Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. Transgenic arabidopsis plants expressing epitope tagged MAP4 were used in this study. mundy@biobase.dk Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library EMBO J. (0261-4189) 2005 orchard The MAP kinase substrate MKS1 is a regulator of plant defense responses. The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. (0261-4189) 2005 Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library EMBO J. (0261-4189) 2005 orchard The MAP kinase substrate MKS1 is a regulator of plant defense responses. The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. (0261-4189) 2005 Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk Accepted 2007-DEC-12 by ORCHARD. Arabidopsis cDNA library EMBO J. (0261-4189) 2005 orchard The MAP kinase substrate MKS1 is a regulator of plant defense responses. The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J. (0261-4189) 2005 Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Andreasson E., Jenkins T., Brodersen P., Thorgrimsen S., Petersen NH., Zhu S., Qiu JL., Micheelsen P., Rocher A., Petersen M., Newman MA., Bjorn Nielsen H., Hirt H., Somssich I., Mattsson O., Mundy J. mundy@biobase.dk Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library EMBO J. (0261-4189) 2005 orchard wrk33_arath Probable WRKY transcription factor 33 WRKY33 At2g38470 T19C21.4 WRKY DNA-binding protein 33 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAASFLTMDNSRTRQNMNGSANWSQQSGRTSTSSLEDLEIPKFRSFAPSSISISPSLVSPSTCFSPSLFLDSPAFVSSSANVLASPTTGALITNVTNQKGINEGDKSNNNNFNLFDFSFHTQSSGVSAPTTTTTTTTTTTTTNSSIFQSQEQQKKNQSEQWSQTETRPNNQAVSYNGREQRKGEDGYNWRKYGQKQVKGSENPRSYYKCTFPNCPTKKKVERSLEGQITEIVYKGSHNHPKPQSTRRSSSSSSTFHSAVYNASLDHNRQASSDQPNSNNSFHQSDSFGMQQEDNTTSDSVGDDEFEQGSSIVSRDEEDCGSEPEAKRWKGDNETNGGNGGGSKTVREPRIVVQTTSDIDILDDGYRWRKYGQKVVKGNPNPRSYYKCTTIGCPVRKHVERASHDMRAVITTYEGKHNHDVPAARGSGYATNRAPQDSSSVPIRPAAIAGHSNYTTSSQAPYTLQMLHNNNTNTGPFGYAMNNNNNNSNLQTQQNFVGGGFSRAKEEPNEETSFFDSFMP A90A6A8A2DF56DB1 mpk6_arath Mitogen-activated protein kinase 6 MPK6 At2g43790 F18O19.10 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDGGSGQPAADTEMTEAPGGFPAAAPSPQMPGIENIPATLSHGGRFIQYNIFGNIFEVTAKYKPPIMPIGKGAYGIVCSAMNSETNESVAIKKIANAFDNKIDAKRTLREIKLLRHMDHENIVAIRDIIPPPLRNAFNDVYIAYELMDTDLHQIIRSNQALSEEHCQYFLYQILRGLKYIHSANVLHRDLKPSNLLLNANCDLKICDFGLARVTSESDFMTEYVVTRWYRAPELLLNSSDYTAAIDVWSVGCIFMELMDRKPLFPGRDHVHQLRLLMELIGTPSEEELEFLNENAKRYIRQLPPYPRQSITDKFPTVHPLAIDLIEKMLTFDPRRRITVLDALAHPYLNSLHDISDEPECTIPFNFDFENHALSEEQMKELIYREALAFNPEYQQ 296D2BD753C6DDA4 wrk25_arath Probable WRKY transcription factor 25 WRKY25 At2g30250 T9D9.6 WRKY DNA-binding protein 25 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSSTSFTDLLGSSGVDCYEDDEDLRVSGSSFGGYYPERTGSGLPKFKTAQPPPLPISQSSHNFTFSDYLDSPLLLSSSHSLISPTTGTFPLQGFNGTTNNHSDFPWQLQSQPSNASSALQETYGVQDHEKKQEMIPNEIATQNNNQSFGTERQIKIPAYMVSRNSNDGYGWRKYGQKQVKKSENPRSYFKCTYPDCVSKKIVETASDGQITEIIYKGGHNHPKPEFTKRPSQSSLPSSVNGRRLFNPASVVSEPHDQSENSSISFDYSDLEQKSFKSEYGEIDEEEEQPEMKRMKREGEDEGMSIEVSKGVKEPRVVVQTISDIDVLIDGFRWRKYGQKVVKGNTNPRSYYKCTFQGCGVKKQVERSAADERAVLTTYEGRHNHDIPTALRRS 9057D65B69E12A0C mks1_arath Protein MKS1 MKS1 Protein MAP kinase 4 substrate 1 VQ motif-containing protein 21 VQ21 MVE11.5 At3g18690 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDPSEYFAGGNPSDQQNQKRQLQICGPRPSPLSVHKDSHKIKKPPKHPAPPPNRDQPPPYIPREPVVIYAVSPKVVHATASEFMNVVQRLTGISSGVFLESGGGGDVSPAARLASTENASPRGGKEPAARDETVEINTAMEEAAEFGGYAPGILSPSPALLPTASTGIFSPMYHQGGMFSPAIPLGLFSPAGFMSPFRSPGFTSLVASPTFADFFSHIWDQD 54CB8449CD5AC2B5 mpk4_arath Mitogen-activated protein kinase 4 MPK4 At4g01370 F2N1.1 F2N1_2-t protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSAESCFGSSGDQSSSKGVATHGGSYVQYNVYGNLFEVSRKYVPPLRPIGRGAYGIVCAATNSETGEEVAIKKIGNAFDNIIDAKRTLREIKLLKHMDHENVIAVKDIIKPPQRENFNDVYIVYELMDTDLHQIIRSNQPLTDDHCRFFLYQLLRGLKYVHSANVLHRDLKPSNLLLNANCDLKLGDFGLARTKSETDFMTEYVVTRWYRAPELLLNCSEYTAAIDIWSVGCILGETMTREPLFPGKDYVHQLRLITELIGSPDDSSLGFLRSDNARRYVRQLPQYPRQNFAARFPNMSAGAVDLLEKMLVFDPSRRITVDEALCHPYLAPLHDINEEPVCVRPFNFDFEQPTLTEENIKELIYRETVKFNPQDSV 448F65C25C95AAEB mpk4-msk1 4 10 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association MPK4 interacts in yeast and in vitro with MKS1 msk1-mpk4 1 10 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association MPK4 interacts in yeast and in vitro with MKS1 mpk4-msk1-2 1 9 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait physical association physical association MPK4 interacts in yeast and in vitro with MKS1 msk1-mpk4-1 3 9 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) 10 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 The MKS1 and MPK4 interaction was confirmed by in vitro binding assays. This showed that recombinant, N-terminal 6xHis-tagged MKS1 from Escherichia coli could pull down labeled MPK4, but not a control human lamin 1 mpk4-msk1-1 5 10 enzyme enzyme neutral component neutral component region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 enzyme target enzyme target substrate substrate neutral component neutral component Q8LGD5:p.Ser30Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 16:28:23 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser30ala' Tue Jun 30 18:27:14 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser30Ala' 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 phosphorylation phosphorylation reaction phosphorylation HA-tagged MPK4 from plants phosphorylated recombinant His-tagged MKS1 1 The author's state that "This indicates that MKS1 is an in vitro substrate of MPK4, and that MKS1 Ser30 and/or Ser72, both contained in the C3 truncation, may be phosphorylation sites." phosphorylation of amino acid residue serine 30 msk1-mpk4-2 2 10 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role bait bait physical association physical association These results indicate that MKS1 and MPK4 interact in vivo, and that MPK4 is the major kinase activity that phosphorylates MKS1 under default conditions. 3 The authors state that "In addition, the levels of phosphorylated MKS1 detected with a phosphoserine/phosphothreonine-specific antibody were markedly higher in wild-type plants than in mpk4 mutants" msk1-wrky33 4 6 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association To further investigate MKS1 function, we conducted a second Y2H library screen to identify additional MKS1 interacting proteins. Of 6 107 clones screened, four encoding WRKY25 (W25) and 11 encoding WRKY33 (W33) were identified and confirmed by b-galactosidase assays. msk1-wrky25 4 9 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association To further investigate MKS1 function, we conducted a second Y2H library screen to identify additional MKS1 interacting proteins. Of 6 107 clones screened, four encoding WRKY25 (W25) and 11 encoding WRKY33 (W33) were identified and confirmed by b-galactosidase assays. msk1-wrky33-1 3 9 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) 6 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 The interactions of MKS1 with W25 and W33 were also confirmed in vitro using His-tagged MKS1 1 msk1-wrky25-1 3 8 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 9 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) physical association physical association The interactions of MKS1 with W25 and W33 were also confirmed in vitro using His-tagged MKS1 1 mpk4-wrky25 5 10 enzyme enzyme neutral component neutral component 8 enzyme target enzyme target substrate substrate neutral component neutral component phosphorylation phosphorylation reaction phosphorylation Kinase assays with MPK6 or MPK4 immunoprecipitated from plant cells showed that fulllength W25 was preferentially phosphorylated by MPK4 5 flagellin Phosphorylation of WRKY25 protein. mpk6-wrky25 5 8 enzyme target enzyme target substrate substrate neutral component neutral component 7 enzyme enzyme neutral component neutral component arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress phosphorylation phosphorylation reaction phosphorylation Kinase assays with MPK6 or MPK4 immunoprecipitated from plant cells showed that fulllength W25 was preferentially phosphorylated by MPK4 5 flagellin Phosphorylation of WRKY25 protein. msk1-wrky25-2 2 10 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role prey prey physical association physical association In addition, the anti-MPK4 antibody preferentially precipitated, from wild type versus mpk4, MPK4 that phosphorylated the N-terminal regions of W25 and W33 5D The authors state that "MPK4 that phosphorylated the N-terminal regions of W25 and W33". The assay used and coordinates of this region of the WRKY25 protein are not clearly set out in the text.