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/ KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell (1040-4651) 2004 imex curation Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu 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 Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Accepted 2009-AUG-23 by ORCHARD. Arabidopsis cDNA library Plant Cell (1040-4651) 2004 orchard Only protein-protein interactions imex curation KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell (1040-4651) 2004 imex curation Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Arabidopsis cDNA library 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 by comassie molecular weight estimation by coomasie staining weight by comassie Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Accepted 2009-AUG-23 by ORCHARD. Arabidopsis cDNA library Plant Cell (1040-4651) 2004 orchard Only protein-protein interactions imex curation KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell (1040-4651) 2004 imex curation Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Arabidopsis cDNA library orchard Only protein-protein interactions in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Accepted 2009-AUG-23 by ORCHARD. Arabidopsis cDNA library Plant Cell (1040-4651) 2004 orchard Only protein-protein interactions imex curation KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell (1040-4651) 2004 imex curation Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu 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 Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Accepted 2009-AUG-23 by ORCHARD. Arabidopsis cDNA library Plant Cell (1040-4651) 2004 orchard Only protein-protein interactions imex curation KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell (1040-4651) 2004 imex curation Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Arabidopsis cDNA library orchard Only protein-protein interactions in vitro In vitro filter binding filter binding Filter overlay assay Filter overlay assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Accepted 2009-AUG-23 by ORCHARD. Arabidopsis cDNA library Plant Cell (1040-4651) 2004 orchard Only protein-protein interactions imex curation KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis. Plant Cell (1040-4651) 2004 imex curation Reddy VS., Day IS., Thomas T., Reddy AS. reddy@colostate.edu Arabidopsis cDNA library orchard Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Reddy VS., Day IS., Thomas T., Reddy AS. Crude protein isolated from Arabidopsis flower, pollen, and young seedlings was passed through a CaM Sepharose column to enrich the KCBP fraction in the extract prior to assay. reddy@colostate.edu Accepted 2009-AUG-23 by ORCHARD. Arabidopsis cDNA library Plant Cell (1040-4651) 2004 orchard Only protein-protein interactions imex curation calcium(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) pbp1_arath Calcium-binding protein PBP1 PINOID-binding protein 1 F24B18.11 KRP2 KIC-related protein 2 PBP1 At5g54490 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MASPKSSTRPNQENQEPQFQDFFPTMAGKLGGEGLIEEICKGFELLMDKDKGVITFESLRRNASTVLGLGDLTDDDVRYMINEGDFDRDGALNQMEFCVLMFRLSPELMEASRCVVTEVIEEEFYRH 21ABDD4955EB21A5 carp1_arath Calcium-binding protein KRP1 KRP1 KIC-related protein 1 M4I22.90 At4g27280 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MASPKSPTRPTQQNPQPNFHDFLPTMAGNLGGEGLIGELCNGFELLMDREKGVITFESLRRNAAAVLGLGDLTDEDVRCMIKEGDFDCDGALNQMEFCVLMFRLSPDLMEASRCLVTEVIEEEFGFTRRH 543443C26D0657DE calm2_arath Calmodulin-2/3/5 CAM2 CAL1 At2g41110 T3K9.12 CAM3 At3g56800 T8M16.130 CAM5 TCH1 At2g27030 T20P8.8 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MADQLTDDQISEFKEAFSLFDKDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIKEADVDGDGQINYEEFVKVMMAK 722B188A90AD423A kic_arath Calcium-binding protein KIC KIC At2g46600 KCBP-interacting calcium-binding protein F13A10.13 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEPTEKSMLLETTSTTKMETKYEDMLPVMAEKMDVEEFVSELCKGFSLLADPERHLITAESLRRNSGILGIEGMSKEDAQGMVREGDLDGDGALNQTEFCVLMVRLSPEMMEDAETWLEKALTQELCNHNLSSMP DIP protein Q9ZPX9 original sequence version: 96 2D7A9E3971F3697B kn14e_arath Kinesin-like protein KIN-14E KIN14E KCBP Kinesin-like calmodulin-binding protein Protein ZWICHEL ZWI K14B20.10 At5g65930 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEGQRGSNSSLSSGNGTEVATDVSSCFYVPNPSGTDFDAESSSLPPLSPAPQVALSIPAELAAAIPLIDRFQVEAFLRLMQKQIQSAGKRGFFYSKKSSGSNVRERFTFEDMLCFQKDPIPTSLLKINSDLVSRATKLFHLILKYMGVDSSDRSTPPSLDERIDLVGKLFKKTLKRVELRDELFAQISKQTRHNPDRQYLIKAWELMYLCASSMPPSKDIGGYLSEYIHNVAHDATIEPDAQVLAVNTLKALKRSIKAGPRHTTPGREEIEALLTGRKLTTIVFFLDETFEEISYDMATTVSDAVEELAGTIKLSAFSSFSLFECRKVVSSSKSSDPGNEEYIGLDDNKYIGDLLAEFKAIKDRNKGEILHCKLVFKKKLFRESDEAVTDLMFVQLSYVQLQHDYLLGNYPVGRDDAAQLCALQILVGIGFVNSPESCIDWTSLLERFLPRQIAITRAKREWELDILARYRSMENVTKDDARQQFLRILKALPYGNSVFFSVRKIDDPIGLLPGRIILGINKRGVHFFRPVPKEYLHSAELRDIMQFGSSNTAVFFKMRVAGVLHIFQFETKQGEEICVALQTHINDVMLRRYSKARSAANSLVNGDISCSSKPQNFEVYEKRLQDLSKAYEESQKKIEKLMDEQQEKNQQEVTLREELEAIHNGLELERRKLLEVTLDRDKLRSLCDEKGTTIQSLMSELRGMEARLAKSGNTKSSKETKSELAEMNNQILYKIQKELEVRNKELHVAVDNSKRLLSENKILEQNLNIEKKKKEEVEIHQKRYEQEKKVLKLRVSELENKLEVLAQDLDSAESTIESKNSDMLLLQNNLKELEELREMKEDIDRKNEQTAAILKMQGAQLAELEILYKEEQVLRKRYYNTIEDMKGKIRVYCRIRPLNEKESSEREKQMLTTVDEFTVEHPWKDDKRKQHIYDRVFDMRASQDDIFEDTKYLVQSAVDGYNVCIFAYGQTGSGKTFTIYGHESNPGLTPRATKELFNILKRDSKRFSFSLKAYMVELYQDTLVDLLLPKSARRLKLEIKKDSKGMVFVENVTTIPISTLEELRMILERGSERRHVSGTNMNEESSRSHLILSVVIESIDLQTQSAARGKLSFVDLAGSERVKKSGSAGCQLKEAQSINKSLSALGDVIGALSSGNQHIPYRNHKLTMLMSDSLGGNAKTLMFVNVSPAESNLDETYNSLLYASRVRTIVNDPSKHISSKEMVRLKKLVAYWKEQAGKKGEEEDLVDIEEDRTRKDEADS EE6B562CAA6201EF cml19_arath Calcium-binding protein CML19 CML19 Calmodulin-like protein 19 CEN2 Centrin 2 AP22.11 C7A10.350 At4g37010 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MSEAAQLRRGLKPKGKTYGLTNQKRREIREIFDLFDIDGSGSIDASELNVAMRSLGFEMNNQQINELMAEVDKNQSGAIDFDEFVHMMTTKFGERDSIDELSKAFKIIDHDNNGKISPRDIKMIAKELGENFTDNDIEEMIEEADRDKDGEVNLEEFMKMMKRTSYG 8BF97BB1AD7B9E6C kcbp-kic 1 11 unspecified role unspecified role prey prey 12 unspecified role unspecified role bait bait 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 the authors describe the bait construct as containing the C-terminal region (860-1261 amino acids) of Q9FHN8, however the UniProt record for this protein shows the protein is 1260 amino acids in length. The UniProt record is used in this entry. Because KIC was isolated using the C-terminal region of KCBP, additional two-hybrid assays were performed to determine the specificity of the KIC-KCBP interaction. kcbp-kic-1 4 12 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 11 unspecified role unspecified role prey prey physical association physical association the authors describe the bait construct as containing the C-terminal region (860-1261 amino acids) of Q9FHN8, however the UniProt record for this protein shows the protein is 1260 amino acids in length. The UniProt record is used in this entry. Yeast transformed with both KIC and full-length KCBP were grown on His− medium and showed β-galactosidase activity, whereas yeast transformed with both KIC and the N-terminal KCBP did not grow on His− medium and showed no β-galactosidase activity on yeast complete medium plates Details of the bait construct (N-terminal version) were obtained from references provided in the text. Figure 1 kic-calcium 5 11 unspecified role unspecified role prey prey region S-tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 7 unspecified role unspecified role bait bait direct interaction direct interaction Calcium bound only to the bands containing KIC or CaM (used as a positive control) and did not bind any other proteins from the crude fraction or BSA Figure 4A The calcium was isotope 45 labelled. cam-calcium-1 5 7 unspecified role unspecified role bait bait 10 unspecified role unspecified role prey prey region S protein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Calcium bound only to the bands containing KIC or CaM (used as a positive control) and did not bind any other proteins from the crude fraction or BSA Figure 4A The calcium was isotope 45. kic-kcbp 6 12 unspecified role unspecified role prey prey arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress 11 unspecified role unspecified role bait bait region S protein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association Full-length KCBP (and some degraded protein) was pulled down only in the presence of KIC and Ca2+ Figure 5B Calcium 2+ ions kic-kcbp-1 3 12 unspecified role unspecified role bait bait region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role prey prey region S protein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association As shown in Figure 5A, KIC coprecipitated with KCBP in the presence of Ca2+ but not in the absence of Ca2+ Figure 5A Calcium 2+ ions kic-kcbp-2 2 11 unspecified role unspecified role prey prey region S protein tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Purified KIC was incubated with the synthetic peptide in 4 M urea in ratios of 1:0.5, 1:1, and 1:2 (KIC:peptide) in the presence or absence (plus EGTA) of Ca2+. CaM, which is known to bind the peptide, was used as a control. In the presence of Ca2+, there was a shift upward for both KIC and CaM (Figure 6A). In the absence of Ca2+ and the presence of EGTA, there was no shift Figure 6 Calcium 2+ ions cam-kcbp-3 2 10 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) direct interaction direct interaction Purified KIC was incubated with the synthetic peptide in 4 M urea in ratios of 1:0.5, 1:1, and 1:2 (KIC:peptide) in the presence or absence (plus EGTA) of Ca2+. CaM, which is known to bind the peptide, was used as a control. In the presence of Ca2+, there was a shift upward for both KIC and CaM (Figure 6A). In the absence of Ca2+ and the presence of EGTA, there was no shift Figure 6 Calcium 2+ ions kic-kcbp-cam 2 12 unspecified role unspecified role bait bait c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 10 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 12 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association Because both CaM and KIC bind the peptide, a competition assay was performed. Equal concentrations of KIC and CaM were incubated with the peptide. Both proteins again showed a shift; however, the amount of protein shifted at each ratio was decreased for both proteins Figure 6 Calcium 2+ ions krp1-calcium-2 5 7 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Therefore, we tested the microtubule binding and microtubule-stimulated ATPase activities of KCBP by the KRPs (At4g27280 and At5g54490) and a centrin (At4g37010). The three proteins were cloned into pET28 as T7-tag fusions, induced, and purified on a Phenyl Sepharose column (Figure 9A). All three proteins were shown to bind Ca2+ using a 45Ca2+-overlay assay Figure 9A The calcium was isotope 45. krp2-calcium-3 5 7 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Therefore, we tested the microtubule binding and microtubule-stimulated ATPase activities of KCBP by the KRPs (At4g27280 and At5g54490) and a centrin (At4g37010). The three proteins were cloned into pET28 as T7-tag fusions, induced, and purified on a Phenyl Sepharose column (Figure 9A). All three proteins were shown to bind Ca2+ using a 45Ca2+-overlay assay Figure 9A The calcium was isotope 45. cml19-calcium-4 5 7 unspecified role unspecified role bait bait 13 unspecified role unspecified role prey prey region t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction Therefore, we tested the microtubule binding and microtubule-stimulated ATPase activities of KCBP by the KRPs (At4g27280 and At5g54490) and a centrin (At4g37010). The three proteins were cloned into pET28 as T7-tag fusions, induced, and purified on a Phenyl Sepharose column (Figure 9A). All three proteins were shown to bind Ca2+ using a 45Ca2+-overlay assay Figure 9A The calcium was isotope 45.