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/ HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain. HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain. EMBO J. (0261-4189) 2000 imex curation Hardtke CS., Gohda K., Osterlund MT., Oyama T., Okada K., Deng XW. xingwang.deng@yale.edu 10-Jul-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Hardtke CS., Gohda K., Osterlund MT., Oyama T., Okada K., Deng XW. xingwang.deng@yale.edu Accepted 2009-JUN-11 by ORCHARD. EMBO J. (0261-4189) 2000 orchard Only protein-protein interactions imex curation HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain. HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain. EMBO J. (0261-4189) 2000 imex curation Hardtke CS., Gohda K., Osterlund MT., Oyama T., Okada K., Deng XW. xingwang.deng@yale.edu 10-Jul-2009: Contacted by JYOTI. orchard Only protein-protein interactions in vitro In vitro emsa electrophoretic mobility shift assay Gel retardation assay band shift band shift Gel retardation assay emsa weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Hardtke CS., Gohda K., Osterlund MT., Oyama T., Okada K., Deng XW. It was not possible to determine the species of origin of the RBCS1a promoter , therefore interactions involving this promoter were not curated. details of the CHS1 promoter were taken from a reference provided in the text. xingwang.deng@yale.edu Accepted 2009-JUN-11 by ORCHARD. 10-Jul-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2000 orchard Only protein-protein interactions imex curation HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain. HY5 stability and activity in arabidopsis is regulated by phosphorylation in its COP1 binding domain. EMBO J. (0261-4189) 2000 imex curation Hardtke CS., Gohda K., Osterlund MT., Oyama T., Okada K., Deng XW. xingwang.deng@yale.edu 10-Jul-2009: 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 Hardtke CS., Gohda K., Osterlund MT., Oyama T., Okada K., Deng XW. xingwang.deng@yale.edu Accepted 2009-JUN-11 by ORCHARD. 10-Jul-2009: Contacted by JYOTI. EMBO J. (0261-4189) 2000 orchard Only protein-protein interactions imex curation hy5_arath Transcription factor HY5 HY5 BZIP56 At5g11260 F2I11_150 Protein LONG HYPOCOTYL 5 bZIP transcription factor 56 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MQEQATSSLAASSLPSSSERSSSSAPHLEIKEGIESDEEIRRVPEFGGEAVGKETSGRESGSATGQERTQATVGESQRKRGRTPAEKENKRLKRLLRNRVSAQQARERKKAYLSELENRVKDLENKNSELEERLSTLQNENQMLRHILKNTTGNKRGGGGGSNADASL 1E3C4672B192D8A9 cop1_arath E3 ubiquitin-protein ligase COP1 COP1 Constitutive photomorphogenesis protein 1 RING-type E3 ubiquitin transferase COP1 T21L14.11 At2g32950 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEEISTDPVVPAVKPDPRTSSVGEGANRHENDDGGSGGSEIGAPDLDKDLLCPICMQIIKDAFLTACGHSFCYMCIITHLRNKSDCPCCSQHLTNNQLYPNFLLDKLLKKTSARHVSKTASPLDQFREALQRGCDVSIKEVDNLLTLLAERKRKMEQEEAERNMQILLDFLHCLRKQKVDELNEVQTDLQYIKEDINAVERHRIDLYRARDRYSVKLRMLGDDPSTRNAWPHEKNQIGFNSNSLSIRGGNFVGNYQNKKVEGKAQGSSHGLPKKDALSGSDSQSLNQSTVSMARKKRIHAQFNDLQECYLQKRRQLADQPNSKQENDKSVVRREGYSNGLADFQSVLTTFTRYSRLRVIAEIRHGDIFHSANIVSSIEFDRDDELFATAGVSRCIKVFDFSSVVNEPADMQCPIVEMSTRSKLSCLSWNKHEKNHIASSDYEGIVTVWDVTTRQSLMEYEEHEKRAWSVDFSRTEPSMLVSGSDDCKVKVWCTRQEASVINIDMKANICCVKYNPGSSNYIAVGSADHHIHYYDLRNISQPLHVFSGHKKAVSYVKFLSNNELASASTDSTLRLWDVKDNLPVRTFRGHTNEKNFVGLTVNSEYLACGSETNEVYVYHKEITRPVTSHRFGSPDMDDAEEEAGSYFISAVCWKSDSPTMLTANSQGTIKVLVLAA DIP protein P43254 original sequence version: 138 DIP protein P43254 original sequence version: 113 DIP protein P43254 original sequence version: 114 E6D9A1B702EAE8F0 chs-sinal-dna Chalcone synthase promoter Sinapis alba ds dna double stranded deoxyribonucleic acid ds DNA ds dna sinal Sinapis alba (White mustard) Brassica hirta White mustard The authors in the relevant paper (see PMID:9659918) describe this as "180 bp fragment of the CHS promoter that includes the Unit 1 region: 2247 to 268". They reference the promoter as the CHS promoter region from white mustard (Sinapis alba) and reference this with PMID 7599309. hy5-cop1-6 1 5 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region HMK (heart muscle kinase) tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region 32p radiolabel 32p radiolabel P32 32P 32P P32 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role bait bait 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) direct interaction direct interaction Figure 3C To test interactions, the capacity of the phosphorylated GSTHY5 (generated by kinase treatment) and dephosphorylated GSTHY5 (generated by a successive lambda protein phosphatase treatment) to pull down bacterially expressed and radioactively labeled FLAG-tagged COP1 in an in vitro protein interaction assay (Ang et al., 1998) was compared. Interestingly, the de-phosphorylated GSTHY5 interacted about four times stronger with COP1 than the phosphorylated protein, as determined by total radioactivity associated with the COP1 band The authors noted that " the de-phosphorylated GSTHY5 interacted about four times stronger with COP1 than the phosphorylated protein, as determined by total radioactivity associated with the COP1 band". However they added the caution that " As the in vitro phosphorylation and dephosphorylation of GSTHY5 may not be 100% efficient, the observed difference in COP1 binding capacity between the phosphorylated and dephosphorylated forms of GSTHY5 is very likely to be an underestimation". hy5-cop1-7 3 4 unspecified role unspecified role prey prey region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role bait bait physical association physical association Figure 4A Together, our results suggest that the CKII site is within the COP1-interactive domain of HY5. hy5-dna-1 2 4 unspecified role unspecified role prey prey 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) 6 unspecified role unspecified role bait bait direct interaction direct interaction Figure 4B We observed that for both the CHS1 and the RBCS1a minimal light inducible promoter fragments, dephosphorylation enhanced binding of HY5 to its DNA target in vitro ∼3- and 5-fold (as determined from scintillation counting of the retarded bands), respectively (Figure 4B and C). This result indicates that unphosphorylated HY5 is more effective in binding to these promoters. The authors concluded " unphosphorylated HY5 is more effective in binding to these promoters".