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/ Arabidopsis Mitogen-Activated Protein Kinase MPK18 Mediates Cortical Microtubule Functions in Plant Cells. Arabidopsis Mitogen-Activated Protein Kinase MPK18 Mediates Cortical Microtubule Functions in Plant Cells. Plant J. (0960-7412) 2009 imex curation Walia A., Lee JS., Wasteneys G., Ellis B. bee@interchange.ubc.ca 21-Dec-2010: Contacted by JYOTI. orchard Only protein-protein interactions nicbe Nicotiana benthamiana bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Walia A., Lee JS., Wasteneys G., Ellis B. bee@interchange.ubc.ca Accepted 2009-JUL-8 by ORCHARD. 21-Dec-2010: Contacted by JYOTI. Plant J. (0960-7412) 2009 orchard Only protein-protein interactions imex curation Arabidopsis Mitogen-Activated Protein Kinase MPK18 Mediates Cortical Microtubule Functions in Plant Cells. Arabidopsis Mitogen-Activated Protein Kinase MPK18 Mediates Cortical Microtubule Functions in Plant Cells. Plant J. (0960-7412) 2009 imex curation Walia A., Lee JS., Wasteneys G., Ellis B. bee@interchange.ubc.ca 21-Dec-2010: Contacted by JYOTI. orchard Only protein-protein interactions 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 Walia A., Lee JS., Wasteneys G., Ellis B. bee@interchange.ubc.ca Accepted 2009-JUL-8 by ORCHARD. Plant J. (0960-7412) 2009 orchard Only protein-protein interactions imex curation Arabidopsis Mitogen-Activated Protein Kinase MPK18 Mediates Cortical Microtubule Functions in Plant Cells. Arabidopsis Mitogen-Activated Protein Kinase MPK18 Mediates Cortical Microtubule Functions in Plant Cells. Plant J. (0960-7412) 2009 imex curation Walia A., Lee JS., Wasteneys G., Ellis B. bee@interchange.ubc.ca 21-Dec-2010: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro phosphatase assay phosphatase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Walia A., Lee JS., Wasteneys G., Ellis B. Note the myelin basic protein mentioned in Fig 1d is not described in enough detail to allow it to be curated. bee@interchange.ubc.ca Accepted 2009-JUL-8 by ORCHARD. 21-Dec-2010: Contacted by JYOTI. Plant J. (0960-7412) 2009 orchard Only protein-protein interactions imex curation mpk18_arath Mitogen-activated protein kinase 18 MPK18 At1g53510 F22G10.12 T3F20.17 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MQQNQVKKGTKEMEFFTEYGDANRYRILEVIGKGSYGVVCAAIDTHTGEKVAIKKINDVFEHISDALRILREVKLLRLLRHPDIVEIKSIMLPPSKREFKDIYVVFELMESDLHQVIKANDDLTREHHQFFLYQMLRALKFMHTANVYHRDLKPKNILANANCKLKVCDFGLARVAFNDTPTTVFWTDYVATRWYRAPELCGSFFSKYTPAIDVWSIGCIFAEVLTGKPLFPGKSVVHQLELITDLLGTPKSETISGVRNDKARKYLTEMRKKNPVTFSQKFSKADPLALRLLQRLLAFDPKDRPTPAEALADPYFKGLSKIEREPSSQQISKMEFEFERRRLTKDDIRELIYREILEYHPQLLKDYMSGSEGSNFVYPSAIGHLRQQFTYLEENSSRNGPVIPLERKHASLPRSTVHSTVVHSTSQPNLGATDSRRVSFEPSKNGASSAGHPSTSAYPTKSIGPPPRVPPSGRPGRVVESSVSYENGRNLKEAYFRSAVSSPHCYFRPNTMTNPENRNIEASSFPPKPQNPVHQFSPTEPPAATTNQADVETMNHPNPYFQPQLPKTDQLNNNTHMAIDAKLLQAQSQFGPAGAAAVAVAAHRNIGTISYSAAS 1439ED0B3C82210E mpk12_arath Mitogen-activated protein kinase 12 MPK12 T3F17.28 At2g46070 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSGESSSGSTEHCIKVVPTHGGRYVQYNVYGQLFEVSRKYVPPIRPIGRGACGIVCAAVNSVTGEKVAIKKIGNAFDNIIDAKRTLREIKLLRHMDHENVITIKDIVRPPQRDIFNDVYIVYELMDTDLQRILRSNQTLTSDQCRFLVYQLLRGLKYVHSANILHRDLRPSNVLLNSKNELKIGDFGLARTTSDTDFMTEYVVTRWYRAPELLLNCSEYTAAIDIWSVGCILGEIMTGQPLFPGKDYVHQLRLITELVGSPDNSSLGFLRSDNARRYVRQLPRYPKQQFAARFPKMPTTAIDLLERMLVFDPNRRISVDEALGHAYLSPHHDVAKEPVCSTPFSFDFEHPSCTEEHIKELIYKESVKFNPDH 1A8FF099D1DC80B9 dphs1_arath Dual specificity protein phosphatase PHS1 PHS1 Protein PROPYZAMIDE-HYPERSENSITIVE 1 MQM1.1 At5g23720 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAEPEKKRDQPFSQEKDEEKDLYLVHDEHESPLPLTVTSRVLYMLGDIASGPAYRFTQWLDLVRKRSATYGSSGFPHRLHRIDDMVTSAGERNTDPKSPPSRQSSEISLWERLGKASTVDIDSSCFSWNMLSSLHHTEHSSSTDHSEEDQSKPLEVTVNSGGVVFFALFNSSSSEDASRKEEAAVIKFASSRMATQSERLGYEFSKWLGVQIPQARVIHSCNPEWTLIKEATEKAQAKATSEGDEVGEMTCSELLEALELSRCLLLMSYVHGCPMLESMSSFETEEKAERAAAALGRILVLDLVIRNEDRLPCRQLRWRGNPANLLLTDRIVSSAKHHECSFDEAFDSAIKRYHPKDYRSIQRERRASSVDSRSRLSISDQMLVSQASDFSDITESPRSYDTGLMSPMSDRSVAADFHLVAIDSGVPRRPPAGKRASDQEIYPRLVELLLNSSQYSSNLLHEITEGSLGYPQAEDGEETSNVRSVVTPVVREFRNGFRAGLRDLQEFHIFLVTLHQKLDVLLRAFFSMMDKTMCADFDREDFAVPESPSHTHGHEVNHYPSPSKDRVPSDNSSDHSESDMQKSVPRTPNSENKEDGSSPKSRESWHGRSGKGGESLSSQRLAAKLRDFHKFAKVDAESNKELDQWNETLRNEVMKLCQENGFNTGFFEGSDNNSCTDAYELKVRLEHILERISLISKAANTEKPSMIQENLFIGGGLAARSIYTLQHLGITHVLCLCANEIGQSDTQYPDLFEYQNFSITDDEDSNIESIFQEALDFIKHGEETGGKILVHCFEGRSRSATVVLAYLMLQKKLTLLEAWSKLRKVHRRAQPNDGFARILINLDKKCHGKVSMEWRQRKPTMKVCPVCGKNAGLSSSSLKLHLQKSHRKLSSGSVDSAMNMEIQKALEALKLSTGRGSSASSNSFQSHPG 5FE10C006DD39A07 phs1-mpk12 2 5 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait physical association physical association Figure 1a For these assays, PHS1 fused to the DNAbinding domain of GAL4 was used as the bait protein, and individual MPKs fused to the transcriptional activation domain of GAL4 were used as the prey proteins. Of the 20 MPKs tested, only MPK12 and MPK18 interacted strongly with PHS1 phs1-mpk18 2 6 unspecified role unspecified role bait bait 4 unspecified role unspecified role prey prey physical association physical association Figure 1a phs1-mpk18-1 1 4 unspecified role unspecified role neutral component neutral component region N-terminal region of YFP protein yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role neutral component neutral component region C-terminal region of YFP protein yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction Figure 1b To test whether PHS1 could associate with MPK12 and MPK18 in vivo, we performed bimolecular fluorescence complementation (BiFC) assays in Nicotiana leaf epidermal cells. In BiFC, the putative bait and prey partners are fused separately to the N- and C-terminal regions of the yellow fluorescent protein (YFP) (Walter et al., 2004). A physical association between bait and prey brings the N- and C-terminal fragments of the split YFP together, and allows functional reassembly of the fluorescent protein. When PHS1-YC and MPK18-YN were co expressed, a positive YFP signal was detected in the cytoplasm phs1-mpk18-2 3 6 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 residue phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 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 1c Treatment of 32P-autophosphorylated MPK18 with purified recombinant PHS1 results in removal of most of the incorporated label MPK18 was phosphorylated by autophosphorylation