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/ daniel-2004-2 CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis. 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 Daniel X., Sugano S., Tobin EM. etobin@ucla.edu etobin@ucla.edu. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2004 Only protein-protein interactions imex curation daniel-2004-1 CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis. in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Daniel X., Sugano S., Tobin EM. etobin@ucla.edu etobin@ucla.edu. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2004 Only protein-protein interactions imex curation csk2d_arath Casein kinase II subunit beta-3 CKB3 F27H5_40 At3g60250 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MYKERSGGGGGGSSRSEILGGAIDRKRINDALNKKLEKSSTSTTTSRVFSSKDKDPFSFTSTKTQLPDVESETDSEGSDVSGSEGDDTSWISWFCNLRGNDFFCEVDEDYIQDDFNLCGLSGQVPYYDYALDLILDVDASNSEMFTDEQHEMVESAAEMLYGLIHVRYILTTKGMAAMTEKYKNCDFGRCPRVFCCGQSCLPVGQSDIPRSSTVKIYCPKCEDISYPRSKFQGNIDGAYFGTTFPHLFLMTYGNLKPQKPTQSYVPKIFGFKVHKP cca1_arath Protein CCA1 CCA1 At2g46830 Protein CIRCADIAN CLOCK ASSOCIATED 1 MYB-related transcription factor CCA1 F19D11 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress METNSSGEDLVIKTRKPYTITKQRERWTEEEHNRFIEALRLYGRAWQKIEEHVATKTAVQIRSHAQKFFSKVEKEAEAKGVAMGQALDIAIPPPRPKRKPNNPYPRKTGSGTILMSKTGVNDGKESLGSEKVSHPEMANEDRQQSKPEEKTLQEDNCSDCFTHQYLSAASSMNKSCIETSNASTFREFLPSREEGSQNNRVRKESNSDLNAKSLENGNEQGPQTYPMHIPVLVPLGSSITSSLSHPPSEPDSHPHTVAGDYQSFPNHIMSTLLQTPALYTAATFASSFWPPDSSGGSPVPGNSPPNLAAMAAATVAAASAWWAANGLLPLCAPLSSGGFTSHPPSTFGPSCDVEYTKASTLQHGSVQSREQEHSEASKARSSLDSEDVENKSKPVCHEQPSATPESDAKGSDGAGDRKQVDRSSCGSNTPSSSDDVEADASERQEDGTNGEVKETNEDTNKPQTSESNARRSRISSNITDPWKSVSDEGRIAFQALFSREVLPQSFTYREEHREEEQQQQEQRYPMALDLNFTAQLTPVDDQEEKRNTGFLGIGLDASKLMSRGRTGFKPYKRCSMEAKESRILNNNPIIHVEQKDPKRMRLETQAST csk21_arath Casein kinase II subunit alpha-1 CKA1 Casein kinase alpha 1 K8K14.10 At5g67380 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MIDTLFFLFFLFFDSPLRRLLLLCAVLALRAPTAHSPILRSSIVTPTARAVSEVSGCTTIDPDFLVEISDSNQTRAMSKARVYTEVNVIRPKDYWDYESLIVQWGEQDDYEVVRKVGRGKYSEVFEGINVNSKEKCIIKILKPVKKKKIRREIKILQNLCGGPNIVKLLDVVRDQHSKTPSLIFEYVNSTDFKVLYPTLTDYDIRYYIYELLKALDFCHSQGIMHRDVKPHNVMIDHELRKLRLIDWGLAEFYHPGKEYNVRVASRYFKGPELLVDLQDYDYSLDMWSLGCMFAGMIFRKEPFFYGHDNQDQLVKIAKVLGTDELNAYLNKYQLELDPQLEALVGRHSRKPWSKFINADNQHLVSPEAIDFLDKLLRYDHQDRLTAKEAMAHAYFAQVRAAETSRMRSQ cca1-cbk3-4 Interaction between CCA1 and CBK3 728757 N/A 728759 unspecified role unspecified role bait bait N/A 728760 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 false physical association physical association false false false Figure 4A cca1-cka1-4 CCA1 homodimer 728757 N/A 728760 unspecified role unspecified role bait bait N/A 728760 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser5Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser6Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser431Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser432Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser433Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser484Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false Figure 4B cca1-cka1-3 Phosphorylation of CCA1 by CKA1 728758 N/A 728760 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser5Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser6Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser431Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser432Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser433Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false P92973:p.Ser484Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) N/A 728761 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation false false false Figure 1 Phosphorylation of P92973 The authors note that "Using mass spectroscopy, we confirmed Ser-5 and -6, as well as one or more of four additional serine residues (Ser-431, -432, -433, and -484) as being phosphorylated by CK2 in vitro (data not shown). We substituted nucleotides in CCA1 so that these six serine residues were changed into alanine residues to make a new protein, mCCA1. We performed in vitro CK2 phosphorylation assays with increasing amounts of GST-CCA1 or GST-mCCA1 proteins as substrates. The results shown in Fig. 1C demonstrate that these substitutions virtually eliminated mCCA1 phosphorylation by CK2 in vitro".