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/ http://www.ebi.ac.uk/ fuglsang-2007-4 Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein. in vitro In vitro protein kinase assay protein kinase assay autoradiography autoradiography Fuglsang AT., Guo Y., Cuin TA., Qiu Q., Song C., Kristiansen KA., Bych K., Schulz A., Shabala S., Schumaker KS., Palmgren MG., Zhu JK. palmgren@life.ku.dk Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation fuglsang-2007-1 Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein. 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 Fuglsang AT., Guo Y., Cuin TA., Qiu Q., Song C., Kristiansen KA., Bych K., Schulz A., Shabala S., Schumaker KS., Palmgren MG., Zhu JK. palmgren@life.ku.dk Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation fuglsang-2007-2 Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein. arath-mesophyll Arabidopsis thaliana mesophyll mesophyll Mesophyll of plant leaf The photosynthetic parenchyma between the epidermal layers of a foliage leaf. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Fuglsang AT., Guo Y., Cuin TA., Qiu Q., Song C., Kristiansen KA., Bych K., Schulz A., Shabala S., Schumaker KS., Palmgren MG., Zhu JK. Mesophyll protoplasts were prepared and transfected using Arabidopsis leaves as described in Sheen, 2001. palmgren@life.ku.dk Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation fuglsang-2007-3 Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein. in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay autoradiography autoradiography Fuglsang AT., Guo Y., Cuin TA., Qiu Q., Song C., Kristiansen KA., Bych K., Schulz A., Shabala S., Schumaker KS., Palmgren MG., Zhu JK. palmgren@life.ku.dk Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation fuglsang-2007-5 Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein. in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting western blot western blot Immuno blot Immuno blot Fuglsang AT., Guo Y., Cuin TA., Qiu Q., Song C., Kristiansen KA., Bych K., Schulz A., Shabala S., Schumaker KS., Palmgren MG., Zhu JK. palmgren@life.ku.dk Plant Cell (1040-4651) 2007 Only protein-protein interactions imex curation cipkb_arath CBL-interacting serine/threonine-protein kinase 11 SOS2-like protein kinase PKS5 SNF1-related kinase 3.22 SOS3-interacting protein 4 CIPK11 PKS5 SIP4 SnRK3.22 At2g30360 T9D9.17 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MPEIEIAAGSGDNNDALFGKYELGKLLGCGAFAKVFHARDRRTGQSVAVKILNKKKLLTNPALANNIKREISIMRRLSHPNIVKLHEVMATKSKIFFAMEFVKGGELFNKISKHGRLSEDLSRRYFQQLISAVGYCHARGVYHRDLKPENLLIDENGNLKVSDFGLSALTDQIRPDGLLHTLCGTPAYVAPEILSKKGYEGAKVDVWSCGIVLFVLVAGYLPFNDPNVMNMYKKIYKGEYRFPRWMSPDLKRFVSRLLDINPETRITIDEILKDPWFVRGGFKQIKFHDDEIEDQKVESSLEAVKSLNAFDLISYSSGLDLSGLFAGCSNSSGESERFLSEKSPEMLAEEVEGFAREENLRMKKKKEEEYGFEMEGQNGKFGIGICISRLNDLLVVVEARRRGGDGDCYKEMWNGKLRVQLIRVCDQTSSTNAAI pma2_arath ATPase 2, plasma membrane-type AHA2 Proton pump 2 F9N11.40 At4g30190 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSSLEDIKNETVDLEKIPIEEVFQQLKCSREGLTTQEGEDRIQIFGPNKLEEKKESKLLKFLGFMWNPLSWVMEMAAIMAIALANGDGRPPDWQDFVGIICLLVINSTISFIEENNAGNAAAALMAGLAPKTKVLRDGKWSEQEAAILVPGDIVSIKLGDIIPADARLLEGDPLKVDQSALTGESLPVTKHPGQEVFSGSTCKQGEIEAVVIATGVHTFFGKAAHLVDSTNQVGHFQKVLTAIGNFCICSIAIGMVIEIIVMYPIQRRKYRDGIDNLLVLLIGGIPIAMPTVLSVTMAIGSHRLSQQGAITKRMTAIEEMAGMDVLCSDKTGTLTLNKLSVDKNLVEVFCKGVEKDQVLLFAAMASRVENQDAIDAAMVGMLADPKEARAGIREVHFLPFNPVDKRTALTYIDGSGNWHRVSKGAPEQILELAKASNDLSKKVLSIIDKYAERGLRSLAVARQVVPEKTKESPGAPWEFVGLLPLFDPPRHDSAETIRRALNLGVNVKMITGDQLAIGKETGRRLGMGTNMYPSSALLGTHKDANLASIPVEELIEKADGFAGVFPEHKYEIVKKLQERKHIVGMTGDGVNDAPALKKADIGIAVADATDAARGASDIVLTEPGLSVIISAVLTSRAIFQRMKNYTIYAVSITIRIVFGFMLIALIWEFDFSAFMVLIIAILNDGTIMTISKDRVKPSPTPDSWKLKEIFATGVVLGGYQAIMTVIFFWAAHKTDFFSDTFGVRSIRDNNHELMGAVYLQVSIISQALIFVTRSRSWSFVERPGALLMIAFLIAQLIATLIAVYANWEFAKIRGIGWGWAGVIWLYSIVTYFPLDVFKFAIRYILSGKAWLNLFENKTAFTMKKDYGKEEREAQWALAQRTLHGLQPKEAVNIFPEKGSYRELSEIAEQAKRRAEIARLRELHTLKGHVESVVKLKGLDIETPSHYTV cnbl2_arath Calcineurin B-like protein 2 CBL2 SCABP1 At5g55990 MDA7.3 SOS3-like calcium-binding protein 1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSQCVDGIKHLCTSVLGCFDLDLYKQSGGLGDPELLARDTVFSVSEIEALYELFKKISSAVIDDGLINKEEFQLALFKTNKKESLFADRVFDLFDTKHNGILGFEEFARALSVFHPNAPIDDKIHFSFQLYDLKQQGFIERQEVKQMVVATLAESGMNLKDTVIEDIIDKTFEEADTKHDGKIDKEEWRSLVLRHPSLLKNMTLQYLKDITTTFPSFVFHSQVEDT calcium(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) 14332_arath 14-3-3-like protein GF14 omega GRF2 GF14 At1g78300 F3F9.16 General regulatory factor 2 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MASGREEFVYMAKLAEQAERYEEMVEFMEKVSAAVDGDELTVEERNLLSVAYKNVIGARRASWRIISSIEQKEESRGNDDHVTAIREYRSKIETELSGICDGILKLLDSRLIPAAASGDSKVFYLKMKGDYHRYLAEFKTGQERKDAAEHTLAAYKSAQDIANAELAPTHPIRLGLALNFSVFYYEILNSPDRACNLAKQAFDEAIAELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQDDAADEIKEAAAPKPTEEQQ pks5-aha2 828278 n/a 828283 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 false ecoli Escherichia coli (strain K12) n/a 828284 enzyme target enzyme target substrate substrate neutral component neutral component arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress phosphorylation phosphorylation reaction phosphorylation false false false Figure 9A Phosphorylation of P19456 pks5-aha2-1 828278 n/a 828284 enzyme target enzyme target substrate substrate neutral component neutral component c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast n/a 828283 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 false ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false false false Figure 9B Phosphorylation of P19456 It is not clear from the paper how the AHA2 protein was purified from Yeast , that is if a Histidine tag was attached (and subsequently removed) from the protein. The authors concluded that the "results strongly indicate that the target site for PKS5 phosphorylation is located within the C-terminal 73 residues of AHA2". pks5-aha2-2 828278 n/a 828284 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 false P19456:p.Ser931Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) n/a 828283 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 false ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false false false Figure 9C Figure 9D Phosphorylation of P19456 The authors concluded that the phosphorylation site was " located within the last 30 amino acids of the regulatory C-terminal region of the H+-ATPase". pks5 828278 n/a 828283 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 false ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false true false Figure 9A autophosphorylation cbl2-pks5 828279 n/a 828283 unspecified role unspecified role bait bait n/a 828285 unspecified role unspecified role prey prey physical association physical association false false false Figure 7B cbl2-pks5-1 828280 n/a 828283 unspecified role unspecified role bait bait c-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false n/a 828285 unspecified role unspecified role prey prey c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false physical association physical association false false false Figure 7C cbl2-calcium 828281 n/a 828286 unspecified role unspecified role prey prey n/a 828285 unspecified role unspecified role bait bait c-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figure 7D The Ca45 isotope was used in this assay. 14_3_3-aha2-3 828282 n/a 828284 unspecified role unspecified role bait bait P19456:p.Ser931Ala mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false P19456:p.[Arg913Ala;Ser931Asp] mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P19456:p.Ser931Asp mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n/a 828287 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false Figure 10C Figure 10E The authors do not give details of how the proteins in the assay were produced or purified.. A peptide covering the e 24 C-terminal residues of AHA2 which was phosphorylated at residue Threonine 947 (Thr-947)