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/ CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis. CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2004 imex curation Daniel X., Sugano S., Tobin EM. etobin@ucla.edu 16-April-2008: Contacted by JYOTI. 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 Daniel X., Sugano S., Tobin EM. etobin@ucla.edu etobin@ucla.edu. Accepted 2008-APR-18 by JYOTI. 16-April-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2004 Only protein-protein interactions imex curation CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis. CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2004 imex curation Daniel X., Sugano S., Tobin EM. etobin@ucla.edu 16-April-2008: Contacted by JYOTI. Only protein-protein interactions 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. Accepted 2008-APR-18 by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2004 Only protein-protein interactions imex curation 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 35F553B9CDA53D33 csk2d_arath Casein kinase II subunit beta-3 CKB3 F27H5_40 At3g60250 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MYKERSGGGGGGSSRSEILGGAIDRKRINDALNKKLEKSSTSTTTSRVFSSKDKDPFSFTSTKTQLPDVESETDSEGSDVSGSEGDDTSWISWFCNLRGNDFFCEVDEDYIQDDFNLCGLSGQVPYYDYALDLILDVDASNSEMFTDEQHEMVESAAEMLYGLIHVRYILTTKGMAAMTEKYKNCDFGRCPRVFCCGQSCLPVGQSDIPRSSTVKIYCPKCEDISYPRSKFQGNIDGAYFGTTFPHLFLMTYGNLKPQKPTQSYVPKIFGFKVHKP 611FFC1222B2CC55 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 083C963EE6DD53A6 cca1-cka1-3 2 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 n-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P92973:p.Ser5Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 16:57:24 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser5ala' Tue Jun 30 17:21:34 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser5Ala' P92973:p.Ser6Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 17:17:54 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser6ala' Tue Jun 30 16:19:00 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser6Ala' P92973:p.Ser431Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 14:45:52 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser431ala' Tue Jun 30 17:29:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser431Ala' P92973:p.Ser432Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 15:43:32 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser432ala' Tue Jun 30 17:10:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser432Ala' P92973:p.Ser433Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 15:57:39 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser433ala' Tue Jun 30 16:59:21 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser433Ala' P92973:p.Ser484Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 15:35:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser484ala' Tue Jun 30 19:27:45 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser484Ala' ecoli Escherichia coli (strain K12) 3 enzyme enzyme neutral component neutral component ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation Figure 1 We identified the sites in CCA1 that are phosphorylated by CK2 in vitro. Phosphorylation of P92973 Details of production of recombinant proteins were obtained from references provided in text. 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". The two smallest c-termnal orientated region shown to be phosphorylated were 317-525 and 437-608 , therefore the the interacting region was taken to be 437-525. cca1-cbk3-4 1 4 unspecified role unspecified role bait bait 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 physical association physical association Figure 4A Fig. 4A shows that mCCA1 can interact physically with CKB3; the domain responsible for the interaction with CKB3 is in both CCA1 and mCCA1 comprised between amino acids 317 and 608. cca1-cka1-4 1 5 unspecified role unspecified role bait bait 5 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 P92973:p.Ser5Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 16:33:26 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser5ala' Tue Jun 30 20:24:58 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser5Ala' P92973:p.Ser6Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 18:38:43 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser6ala' Tue Jun 30 15:57:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser6Ala' P92973:p.Ser431Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 18:29:25 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser431ala' Tue Jun 30 16:44:17 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser431Ala' P92973:p.Ser432Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 17:49:16 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser432ala' Tue Jun 30 19:33:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser432Ala' P92973:p.Ser433Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 16:59:38 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser433ala' Tue Jun 30 20:13:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser433Ala' P92973:p.Ser484Ala mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain S A Wed May 02 15:01:18 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser484ala' Tue Jun 30 17:23:26 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser484Ala' physical association physical association Figure 4B To address the importance of a CCA1 CCA1 homodimer in the regulation of the circadian clock, we performed yeast two-hybrid assays between the full-length CCA1 protein, and full-length or deletions of CCA1 and mCCA1 proteins. Fig. 4B shows that CCA1 can interact physically with itself. A very strong -galactosidase activity was monitored with two copies of the full-length CCA1 protein. The domain responsible for this interaction is between amino acids 100 and 317. When these experiments were performed with mCCA1, the interaction strength was reduced dramatically; it was 7.7-fold weaker with full-length mCCA1 than with full-length CCA1.