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/ A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1997 imex curation Williams RW., Wilson JM., Meyerowitz EM. meyerow@cco.caltech.edu 14-Nov-2008: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro phosphatase assay phosphatase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Williams RW., Wilson JM., Meyerowitz EM. meyerow@cco.caltech.edu meyerow@cco.caltech.edu. Accepted 2008-DEC-5 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1997 jyoti Only protein-protein interactions imex curation A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1997 imex curation Williams RW., Wilson JM., Meyerowitz EM. meyerow@cco.caltech.edu 14-Nov-2008: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Williams RW., Wilson JM., Meyerowitz EM. The GST-KAPP protein was phosphorylated with heart muscle kinase prior to filter binding assay. meyerow@cco.caltech.edu meyerow@cco.caltech.edu. Accepted 2008-DEC-5 by JYOTI. 14-Nov-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1997 jyoti Only protein-protein interactions imex curation A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. A possible role for kinase-associated protein phosphatase in the Arabidopsis CLAVATA1 signaling pathway. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1997 imex curation Williams RW., Wilson JM., Meyerowitz EM. meyerow@cco.caltech.edu 14-Nov-2008: Contacted by JYOTI. jyoti Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Williams RW., Wilson JM., Meyerowitz EM. meyerow@cco.caltech.edu meyerow@cco.caltech.edu. Accepted 2008-DEC-5 by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1997 jyoti Only protein-protein interactions imex curation clv1_arath Receptor protein kinase CLAVATA1 CLV1 T4O12.5 At1g75820 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAMRLLKTHLLFLHLYLFFSPCFAYTDMEVLLNLKSSMIGPKGHGLHDWIHSSSPDAHCSFSGVSCDDDARVISLNVSFTPLFGTISPEIGMLTHLVNLTLAANNFTGELPLEMKSLTSLKVLNISNNGNLTGTFPGEILKAMVDLEVLDTYNNNFNGKLPPEMSELKKLKYLSFGGNFFSGEIPESYGDIQSLEYLGLNGAGLSGKSPAFLSRLKNLREMYIGYYNSYTGGVPPEFGGLTKLEILDMASCTLTGEIPTSLSNLKHLHTLFLHINNLTGHIPPELSGLVSLKSLDLSINQLTGEIPQSFINLGNITLINLFRNNLYGQIPEAIGELPKLEVFEVWENNFTLQLPANLGRNGNLIKLDVSDNHLTGLIPKDLCRGEKLEMLILSNNFFFGPIPEELGKCKSLTKIRIVKNLLNGTVPAGLFNLPLVTIIELTDNFFSGELPVTMSGDVLDQIYLSNNWFSGEIPPAIGNFPNLQTLFLDRNRFRGNIPREIFELKHLSRINTSANNITGGIPDSISRCSTLISVDLSRNRINGEIPKGINNVKNLGTLNISGNQLTGSIPTGIGNMTSLTTLDLSFNDLSGRVPLGGQFLVFNETSFAGNTYLCLPHRVSCPTRPGQTSDHNHTALFSPSRIVITVIAAITGLILISVAIRQMNKKKNQKSLAWKLTAFQKLDFKSEDVLECLKEENIIGKGGAGIVYRGSMPNNVDVAIKRLVGRGTGRSDHGFTAEIQTLGRIRHRHIVRLLGYVANKDTNLLLYEYMPNGSLGELLHGSKGGHLQWETRHRVAVEAAKGLCYLHHDCSPLILHRDVKSNNILLDSDFEAHVADFGLAKFLVDGAASECMSSIAGSYGYIAPEYAYTLKVDEKSDVYSFGVVLLELIAGKKPVGEFGEGVDIVRWVRNTEEEITQPSDAAIVVAIVDPRLTGYPLTSVIHVFKIAMMCVEEEAAARPTMREVVHMLTNPPKSVANLIAF 2B2DC41AFF733B08 p2c70_arath Protein phosphatase 2C 70 KAPP Kinase-associated protein phosphatase Protein ROOT ATTENUATED GROWTH 1 RAG1 F7K24.30 T24G5.3 At5g19280 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAMIGMNIIGLFMVLMLLLISLIILFACKPWRYFSRFRSSSRFSSTFKVGDLQRPLISDDGNLIQGQTSEVTREYDLEGACYQNDGLLHSSLTEGRFYKQRLPSSSPHFSQGESFVLEVISEPSDNALVGQTLKLPAEKGSLAEVQTYDWQNNRNENLQYNLEKDRLINLSPRLVEDQRSWLFLEVIAGPAIGLQHAVNSTSSSKLPVKLGRVSPSDLALKDSEVSGKHAQITWNSTKFKWELVDMGSLNGTLVNSHSISHPDLGSRKWGNPVELASDDIITLGTTTKVYVRISSQNEFQIPFKIGVASDPMAMRRGGRKLPMEDVCHYKWPLPGANKFGLFCVCDGHGGSGAAQSAIKIIPEVLANILSDSLRKEKVLSKRDASDVLRDMFAKTEARLEEHQYEGCTATVLLVWKDNEENFFAQCANLGDSACVIHLGGRYIQMTEDHRVVSLSERKRFQEAGLALRDGETRLFGINLARMLGDKFPKQQDSRFSAEPYISEPLRIDQSSKDVFAVLASDGLWDVVSPKKAVQLVLQMRDKERGRESSAEKIANGLLNEARAMRTKDNTSIIYLDFDTSL EE0C2A41E55E100D clv1-3 3 4 putative self putative self putative self putative self 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 Q9SYQ8:p.Lys720Glu mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K E Wed May 02 19:30:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys720glu' Tue Jun 30 19:10:23 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys720Glu' region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q9SYQ8:p.Gly856Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain G D Wed May 02 14:38:04 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly856asp' Tue Jun 30 18:20:32 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly856Asp' ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation true Figure 3A Figure 3B Figure 3D Autophosphorylation of the CLV1 kinase domain CLV1 was autophosphorylated. The authors noted that "GST-CKD autophosphorylation is reduced when incubated with increasing amounts of CKDK720E". They concluded "this finding indicates that CLV1 can interact intermolecularly and undergo transphosphorylation". clv1-2 3 4 enzyme target enzyme target substrate substrate neutral component neutral component Q9SYQ8:p.Lys720Glu mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K E Wed May 02 18:12:44 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys720glu' Tue Jun 30 19:31:35 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys720Glu' ecoli Escherichia coli (strain K12) 4 enzyme enzyme unspecified role unspecified role region 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) phosphorylation phosphorylation reaction phosphorylation Figure 3C We therefore tested the ability of CLV1 to transphosphorylate. The inactive CKDK720E protein, when removed from the GST-moiety by thrombin cleavage, could be phosphorylated in the presence of the active GST-CKD protein The mutant form of CLV1 (K720E) was phosphorylated. kapp-clv1 2 5 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 4 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 Q9SYQ8:p.Lys720Glu mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K E Wed May 02 16:59:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys720glu' Tue Jun 30 18:47:04 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys720Glu' Q9SYQ8:p.Gly856Asp mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain G D Wed May 02 19:48:49 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly856asp' Tue Jun 30 16:08:17 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly856Asp' region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 4A The authors noted that "GST-KI had a reduced interaction with the dephosphorylated GST-CKD as compared with phosphorylated GST-CKD". The resulting GST-KI protein interacted with GST-CKD (Fig. 4A) in a filter binding assay. GST-KI did not bind to either GST-CKD1-1 or GST-CKDK720E at detectable levels (Fig. 4A), suggesting that the introduced mutations alter residues or structures that are required for the interaction, or that the CLV1/KAPP interaction is dependent on phosphorylation of the CLV1 kinase domain. clv1-kapp-2 3 4 enzyme enzyme neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q9SYQ8:p.Lys720Glu mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K E Wed May 02 14:45:19 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys720glu' Tue Jun 30 17:06:48 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys720Glu' Q9SYQ8:p.Gly856Asp mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain G D Wed May 02 15:28:03 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly856asp' Tue Jun 30 16:48:27 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly856Asp' 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) 5 enzyme target enzyme target substrate substrate 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 ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation Figure 4B The ability of the CLV1 kinase domain to phosphorylate KAPP was also tested. The GST-CKD protein was able to phosphorylate the full-length KAPP protein fused to GST (Fig. 4B). Consistent with their abilities to autophosphorylate, GST-CKD1-1 could phosphorylate GST-KAPP at reduced levels and the GST-CKDK720E protein could not phosphorylate GST-KAPP at any detectable level ( KAPP was phosphorylated. clv1-kapp-3 1 5 enzyme enzyme 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 ecoli Escherichia coli (strain K12) 4 enzyme target enzyme target substrate substrate neutral component neutral component region 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) dephosphorylation dephosphorylation reaction dephosphorylation Figure 4C The CKD protein is dephosphorylated by KAPP, and like other type 2C protein phosphatases, KAPP activity is dependent on Mg2+ and/or Mn2+ CVL1 was dephosphorylated. The authors noted "KAPP activity is dependent on Mg2+ and/or Mn2+".