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/ Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library 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 Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library Biochem. Biophys. Res. Commun. (0006-291X) 2006 orchard Only protein-protein interactions imex curation Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library Biochem. Biophys. Res. Commun. (0006-291X) 2006 orchard Only protein-protein interactions imex curation Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library 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 Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn Accepted 2007-DEC-12 by ORCHARD. Arabidopsis cDNA library Biochem. Biophys. Res. Commun. (0006-291X) 2006 orchard Only protein-protein interactions imex curation Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library Biochem. Biophys. Res. Commun. (0006-291X) 2006 orchard Only protein-protein interactions imex curation Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Li RJ., Hua W., Lu YT. yingtlu@whu.edu.cn 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library orchard Only protein-protein interactions arath-cell-culture Arabidopsis thaliana cell culture plant_cell_culture plant cellular culture The culture conditions should always be cited in the experimental modifications annotation Either whole plant or undifferentiated plant cells maintained in culture. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Li RJ., Hua W., Lu YT. Epitope tagged proteins were expressed in transformed arabidopsis cell culture cells in this study. yingtlu@whu.edu.cn Accepted 2007-DEC-12 by ORCHARD. 22-Jan-2008: Contacted by JYOTI. Arabidopsis cDNA library Biochem. Biophys. Res. Commun. (0006-291X) 2006 orchard Only protein-protein interactions imex curation camk3_arath CDPK-related kinase 3 CK CaMK4 Calcium/calmodulin-dependent protein kinase 4 T3A4.8 At2g46700 CRK3 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MGQCYGKVNQSKQNGEEEANTTTYVVSGDGNQIQPLTPVNYGRAKNTPARSSNPSPWPSPFPHGSASPLPSGVSPSPARTSTPRRFFRRPFPPPSPAKHIKASLIKRLGVKPKEGPIPEERGTEPEQSLDKSFGYGKNFGAKYELGKEVGRGHFGHTCSGRGKKGDIKDHPIAVKIISKAKMTTAIAIEDVRREVKLLKSLSGHKYLIKYYDACEDANNVYIVMELCDGGELLDRILARGGKYPEDDAKAIVVQILTVVSFCHLQGVVHRDLKPENFLFTSSREDSDLKLIDFGLSDFIRPDERLNDIVGSAYYVAPEVLHRSYSLEADIWSIGVITYILLCGSRPFWARTESGIFRTVLRTEPNYDDVPWPSCSSEGKDFVKRLLNKDYRKRMSAVQALTHPWLRDDSRVIPLDILIYKLVKAYLHATPLRRAALKALAKALTENELVYLRAQFMLLGPNKDGSVSLENFKTALMQNATDAMRESRVPEILHTMESLAYRKMYFEEFCAAAISIHQLEAVDAWEEIATAGFQHFETEGNRVITIEELARELNVGASAYGHLRDWVRSSDGKLSYLGFTKFLHGVTLRAAHARPR C6D9C158BA41A39C gln11_arath Glutamine synthetase cytosolic isozyme 1-1 GLN1-1 K12B20.50 At5g37600 Glutamate--ammonia ligase GLN1;1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSLVSDLINLNLSDSTDKIIAEYIWVGGSGMDMRSKARTLPGPVTDPSQLPKWNYDGSSTGQAPGEDSEVILYPQAIFKDPFRRGNNILVMCDAYTPAGEPIPTNKRHAAAKVFSNPDVAAEVPWYGIEQEYTLLQKDVKWPVGWPIGGYPGPQGPYYCGIGADKSFGRDVVDSHYKACLYAGINISGINGEVMPGQWEFQVGPAVGISAADEIWVARYILERITEIAGVVVSFDPKPIPGDWNGAGAHCNYSTKSMREEGGYEIIKKAIDKLGLRHKEHIAAYGEGNERRLTGHHETADINTFLWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYIVTSMIAETTILWNP AE54FB88287CFE9D crk3-gln1 3 7 unspecified role unspecified role prey prey region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain physical association physical association suggesting AtCRKP1 could interact with almost full length AtGLN1;1 protein in yeast. crk3-gln1-1 1 7 unspecified role unspecified role prey prey region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 15 17 physical association physical association Both full length and partial form (AtCRK3P1) of AtCRK3 could interact with AtGLN1;1 (full length) and AtGLNP2 (catalytic domain of AtGLN1;1) in yeast, but not with AtGLNP1 (the N-terminal beta-grasp domain of AtGLN1;1). 1 gln1-crk3 2 7 unspecified role unspecified role bait bait region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain spofr Spodoptera frugiperda (Fall armyworm) Fall armyworm 21 25 physical association physical association In vitro binding assays for AtGLN1;1 and AtCRK3. 2 10 milli molar MgCL and 100 micro molar ATP crk3-gln1-2 5 7 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association AtCRK3 was specifically co-immunoprecipitated with AtGLN1;1 and vice versa. These results suggest that AtCRK3 and AtGLN1;1 associate in Arabidopsis cells. 3 gln1-crk3-1 5 6 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association AtCRK3 was specifically co-immunoprecipitated with AtGLN1;1 and vice versa. These results suggest that AtCRK3 and AtGLN1;1 associate in Arabidopsis cells. 3 crk3-gln1-3 4 7 enzyme target enzyme target substrate substrate neutral component neutral component region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 6 enzyme enzyme neutral component neutral component n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 40 38 phosphorylation phosphorylation reaction phosphorylation Phosphorylation assays indicated that both AtCRK3 and AtCPK3P1 proteins were able to phosphorylate full length AtGLN1;1 protein and the truncated form AtGLNP2 (Fig. 4, lanes 1�4), which is consistent with the protein interaction results. 4 Km values were 20.4 +/- 1.1 micro molar. crk3-gln1-4 2 7 unspecified role unspecified role prey prey region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role bait bait n-terminal binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain n-terminal myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range spofr Spodoptera frugiperda (Fall armyworm) Fall armyworm 46 43 physical association physical association In vitro binding assays for AtGLN1;1 and AtCRK3. 2 10 milli molar MgCL and 100 micro molar ATP