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/ Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Plant Cell (1040-4651) 2002 imex curation Cherel I., Michard E., Platet N., Mouline K., Alcon C., Sentenac H., Thibaud JB. cherel@ensam.inra.fr Arabidopsis cDNA orchard Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Cherel I., Michard E., Platet N., Mouline K., Alcon C., Sentenac H., Thibaud JB. cherel@ensam.inra.fr Accepted 2009-SEP-27 by ORCHARD. Arabidopsis cDNA Plant Cell (1040-4651) 2002 orchard Only protein-protein interactions imex curation Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Plant Cell (1040-4651) 2002 imex curation Cherel I., Michard E., Platet N., Mouline K., Alcon C., Sentenac H., Thibaud JB. cherel@ensam.inra.fr Arabidopsis cDNA 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 nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Cherel I., Michard E., Platet N., Mouline K., Alcon C., Sentenac H., Thibaud JB. cherel@ensam.inra.fr Accepted 2009-SEP-27 by ORCHARD. Arabidopsis cDNA Plant Cell (1040-4651) 2002 orchard Only protein-protein interactions imex curation Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA. Plant Cell (1040-4651) 2002 imex curation Cherel I., Michard E., Platet N., Mouline K., Alcon C., Sentenac H., Thibaud JB. cherel@ensam.inra.fr Arabidopsis cDNA 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 Cherel I., Michard E., Platet N., Mouline K., Alcon C., Sentenac H., Thibaud JB. cherel@ensam.inra.fr Accepted 2009-SEP-27 by ORCHARD. Arabidopsis cDNA Plant Cell (1040-4651) 2002 orchard Only protein-protein interactions imex curation akt2_arath Potassium channel AKT2/3 AKT2 AKT3 T10I14.30 At4g22200 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDLKYSASHCNLSSDMKLRRFHQHRGKGREEEYDASSLSLNNLSKLILPPLGVASYNQNHIRSSGWIISPMDSRYRCWEFYMVLLVAYSAWVYPFEVAFLNSSPKRNLCIADNIVDLFFAVDIVLTFFVAYIDERTQLLVREPKQIAVRYLSTWFLMDVASTIPFDAIGYLITGTSTLNITCNLLGLLRFWRLRRVKHLFTRLEKDIRYSYFWIRCFRLLSVTLFLVHCAGCSYYLIADRYPHQGKTWTDAIPNFTETSLSIRYIAAIYWSITTMTTVGYGDLHASNTIEMVFITVYMLFNLGLTAYLIGNMTNLVVEGTRRTMEFRNSIEAASNFVNRNRLPPRLKDQILAYMCLRFKAESLNQQHLIDQLPKSIYKSICQHLFLPSVEKVYLFKGVSREILLLLVSKMKAEYIPPREDVIMQNEAPDDVYIIVSGEVEIIDSEMERESVLGTLRCGDIFGEVGALCCRPQSYTFQTKSLSQLLRLKTSFLIETMQIKQQDNATMLKNFLQHHKKLSNLDIGDLKAQQNGENTDVVPPNIASNLIAVVTTGNAALLDELLKAKLSPDITDSKGKTPLHVAASRGYEDCVLVLLKHGCNIHIRDVNGNSALWEAIISKHYEIFRILYHFAAISDPHIAGDLLCEAAKQNNVEVMKALLKQGLNVDTEDHHGVTALQVAMAEDQMDMVNLLATNGADVVCVNTHNEFTPLEKLRVVEEEEEEERGRVSIYRGHPLERRERSCNEAGKLILLPPSLDDLKKIAGEKFGFDGSETMVTNEDGAEIDSIEVIRDNDKLYFVVNKII 6F138F9149ED3CD7 p2c37_arath Protein phosphatase 2C 37 F24K9.8 At3g11410 PP2CA AHG3 Protein ABA-HYPERSENSITIVE GERMINATION 3 Protein phosphatase 2C A protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAGICCGVVGETEPAAPVDSTSRASLRRRLDLLPSIKIVADSAVAPPLENCRKRQKRETVVLSTLPGNLDLDSNVRSENKKARSAVTNSNSVTEAESFFSDVPKIGTTSVCGRRRDMEDAVSIHPSFLQRNSENHHFYGVFDGHGCSHVAEKCRERLHDIVKKEVEVMASDEWTETMVKSFQKMDKEVSQRECNLVVNGATRSMKNSCRCELQSPQCDAVGSTAVVSVVTPEKIIVSNCGDSRAVLCRNGVAIPLSVDHKPDRPDELIRIQQAGGRVIYWDGARVLGVLAMSRAIGDNYLKPYVIPDPEVTVTDRTDEDECLILASDGLWDVVPNETACGVARMCLRGAGAGDDSDAAHNACSDAALLLTKLALARQSSDNVSVVVVDLRKRRNNQASS DIP protein P49598 original sequence version: 79 DIP protein P49598 original sequence version: 80 DIP protein P49598 original sequence version: 95 DIP protein P49598 original sequence version: 130 83B82E32FEC71D4D kat1_arath Potassium channel KAT1 KAT1 MPL12.2 At5g46240 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSISWTRNFFERFCVEEYNIDTIKQSSFLSADLLPSLGARINQSTKLRKHIISPFNPRYRAWEMWLVLLVIYSAWICPFQFAFITYKKDAIFIIDNIVNGFFAIDIILTFFVAYLDSHSYLLVDSPKKIAIRYLSTWFAFDVCSTAPFQPLSLLFNYNGSELGFRILSMLRLWRLRRVSSLFARLEKDIRFNYFWIRCTKLISVTLFAIHCAGCFNYLIADRYPNPRKTWIGAVYPNFKEASLWNRYVTALYWSITTLTTTGYGDFHAENPREMLFDIFFMMFNLGLTAYLIGNMTNLVVHWTSRTRTFRDSVRAASEFASRNQLPHDIQDQMLSHICLKFKTEGLKQQETLNNLPKAIRSSIANYLFFPIVHNIYLFQGVSRNFLFQLVSDIDAEYFPPKEDIILQNEAPTDLYILVSGAVDFTVYVDGHDQFQGKAVIGETFGEVGVLYYRPQPFTVRTTELSQILRISRTSLMSAMHAHADDGRVIMNNLFMKLRGQQSIAIDDSNTSGHENRDFKSMGWEEWRDSRKDGYGLDVTNPTSDTALMDAIHKEDTEMVKKILKEQKIERAKVERSSSETAGRSYANDSSKKDPYCSSSNQIIKPCKREEKRVTIHMMSESKNGKLILLPSSIEELLRLASEKFGGCNFTKITNADNAEIDDLDVIWDGDHLYFSSN 7F9C8285ED702338 kt2-kat1 2 6 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association kt2-pp2c 2 5 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 4 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association kt2-pp2c-1 3 5 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 4 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 18 20 physical association physical association Figure 1B We further checked the specificity of the AKT2-AtPP2CA interaction by testing the C-terminal domains of five different Arabidopsis Shaker channels (including AKT2) with the AtPP2CA construct. Hybrid constructs encoding the C-terminal region (the whole cytoplasmic domain downstream ofthe S6 segment) of AKT2 (from Met-324; see Methods), AKT1 (from His 294; Daram et al., 1997), KAT1 (from Thr- 303; Urbach et al., 2000), KAT2 (from Arg-316; Pilot et al., 2001), or AtKC1 (from His-330) were used. With the AtPP2CA catalytic domain fused to the Gal4 activator domain, none of the DNA binding domain constructs except the one corresponding to AKT2 gave a positive signal kt2-pp2c-2 1 5 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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) 4 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 23 26 physical association physical association Figure 2B AKT2-CT Binding on Nickel-Affinity Agarose Beads Loaded with His-Tagged AtPP2CA Fusion Polypeptide.