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/ Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1998 imex curation Clark KL., Larsen PB., Wang X., Chang C. CC203@umail.umd.edu 17-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 Clark KL., Larsen PB., Wang X., Chang C. CC203@umail.umd.edu CC203@umail.umd.ed Accepted 2008-MAR-18 by CDEROW. 17-April-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1998 Only protein-protein interactions imex curation Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1998 imex curation Clark KL., Larsen PB., Wang X., Chang C. CC203@umail.umd.edu 17-April-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Clark KL., Larsen PB., Wang X., Chang C. CC203@umail.umd.edu CC203@umail.umd.ed Accepted 2008-MAR-18 by CDEROW. 17-April-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1998 Only protein-protein interactions imex curation Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1998 imex curation Clark KL., Larsen PB., Wang X., Chang C. CC203@umail.umd.edu 17-April-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Clark KL., Larsen PB., Wang X., Chang C. CC203@umail.umd.edu CC203@umail.umd.ed Accepted 2008-MAR-18 by CDEROW. 17-April-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 1998 Only protein-protein interactions imex curation ctr1_arath Serine/threonine-protein kinase CTR1 Protein CONSTITUTIVE TRIPLE RESPONSE1 F17C15_150 CTR1 At5g03730 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEMPGRRSNYTLLSQFSDDQVSVSVTGAPPPHYDSLSSENRSNHNSGNTGKAKAERGGFDWDPSGGGGGDHRLNNQPNRVGNNMYASSLGLQRQSSGSSFGESSLSGDYYMPTLSAAANEIESVGFPQDDGFRLGFGGGGGDLRIQMAADSAGGSSSGKSWAQQTEESYQLQLALALRLSSEATCADDPNFLDPVPDESALRTSPSSAETVSHRFWVNGCLSYYDKVPDGFYMMNGLDPYIWTLCIDLHESGRIPSIESLRAVDSGVDSSLEAIIVDRRSDPAFKELHNRVHDISCSCITTKEVVDQLAKLICNRMGGPVIMGEDELVPMWKECIDGLKEIFKVVVPIGSLSVGLCRHRALLFKVLADIIDLPCRIAKGCKYCNRDDAASCLVRFGLDREYLVDLVGKPGHLWEPDSLLNGPSSISISSPLRFPRPKPVEPAVDFRLLAKQYFSDSQSLNLVFDPASDDMGFSMFHRQYDNPGGENDALAENGGGSLPPSANMPPQNMMRASNQIEAAPMNAPPISQPVPNRANRELGLDGDDMDIPWCDLNIKEKIGAGSFGTVHRAEWHGSDVAVKILMEQDFHAERVNEFLREVAIMKRLRHPNIVLFMGAVTQPPNLSIVTEYLSRGSLYRLLHKSGAREQLDERRRLSMAYDVAKGMNYLHNRNPPIVHRDLKSPNLLVDKKYTVKVCDFGLSRLKASTFLSSKSAAGTPEWMAPEVLRDEPSNEKSDVYSFGVILWELATLQQPWGNLNPAQVVAAVGFKCKRLEIPRNLNPQVAAIIEGCWTNEPWKRPSFATIMDLLRPLIKSAVPPPNRSDL 2922D3DCD0CC15BC cki1_arath Histidine kinase CKI1 CKI1 At2g47430 Protein CYTOKININ-INDEPENDENT 1 T30B22.27 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MMVKVTKLVASRPIVVFCVLAFLVVVFECIWISNWRTTTENLVKEVASFTEDLRTSLVSEIENIGKFTYAKTNLSTIGLARVIDSYITNNDTGFTEIQTQIAPLLFVAYSTILQVSQVSYISRDGLMFSYIAESNTSVAVFANSSSNSSRGDYTWYTQTVDQLTGRLNGNSTKSQSLDVTHTDWFQAAQSNNYTTAFVGTSLGGEDNETLIQSVVSLYSKKGLVSLGFPVKTLTEVLNSLNLHGEELYMWTKDGTVLVREGSLNDSFFISNGSICFGRESNSLWSQCIPENCSSSGYEVEIKRLRYQAFCSVIEVSGVPLRYTLMFPNKGGATRIKHQAEKAKYQLIVVMIFLGFGWPVWFVWFMMQATRREMHMRATLINQMEATQQAERKSMNKSQAFANASHDIRGALAGMKGLIDICRDGVKPGSDVDTTLNQVNVCAKDLVALLNSVLDMSKIESGKMQLVEEDFNLSKLLEDVIDFYHPVAMKKGVDVVLDPHDGSVFKFSNVRGDSGRLKQILNNLVSNAVKFTVDGHIAVRAWAQRPGSNSSVVLASYPKGVSKFVKSMFCKNKEESSTYETEISNSIRNNANTMEFVFEVDDTGKGIPMEMRKSVFENYVQVRETAQGHQGTGLGLGIVQSLVRLMGGEIRITDKAMGEKGTCFQFNVLLTTLESPPVSDMKVRQEIEAGGDYVSTPNLGLTINTSLGGSMNIRNLSPRFNNCLSSSPKQEGSRVVLLLKNEERRRVTEKYIKNLGIKVTVVEKWEHLSYALERLFGFSPQSSMGRAECSLSCPSSRELPFIGMDGIDSRSQLPKRRSISFSAVVLLVIDAKTGPFFELCDIVKQFRRGLPHGISCKVVWLNESSTRVSERGDISCSRPLHGSRLMEVLKMLPEFGGTVLKEPPTELQRESLLRHSFVAERSPKHKVQEEGPSSMFNKKLGKRIMASTDSESETRVKSVRTGRKPIGNPEDEQETSKPSDDEFLRGKRVLVVDDNFISRKVATGKLKKMGVSEVEQCDSGKEALRLVTEGLTQREEQGSVDKLPFDYIFMDCQMPEMDGYEATREIRKVEKSYGVRTPIIAVSGHDPGSEEARETIQAGMDAFLDKSLNQLANVIREIESKRH C6833F731DA84781 ers1_arath Ethylene response sensor 1 ERS1 ERS At2g40940 Protein ERS1 T20B5.14 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MESCDCFETHVNQDDLLVKYQYISDALIALAYFSIPLELIYFVQKSAFFPYKWVLMQFGAFIILCGATHFINLWMFFMHSKAVAIVMTIAKVSCAVVSCATALMLVHIIPDLLSVKNRELFLKKKADELDREMGLILTQEETGRHVRMLTHGIRRTLDRHTILRTTLVELGKTLCLEECALWMPSQSGLYLQLSHTLSHKIQVGSSVPINLPIINELFNSAQAMHIPHSCPLAKIGPPVGRYSPPEVVSVRVPLLHLSNFQGSDWSDLSGKGYAIMVLILPTDGARKWRDHELELVENVADQVAVALSHAAILEESMHARDQLMEQNFALDKARQEAEMAVHARNDFLAVMNHEMRTPMHAIISLSSLLLETELSPEQRVMIETILKSSNLVATLISDVLDLSRLEDGSLLLENEPFSLQAIFEEVISLIKPIASVKKLSTNLILSADLPTYAIGDEKRLMQTILNIMGNAVKFTKEGYISIIASIMKPESLQELPSPEFFPVLSDSHFYLCVQVKDTGCGIHTQDIPLLFTKFVQPRTGTQRNHSGGGLGLALCKRFVGLMGGYMWIESEGLEKGCTASFIIRLGICNGPSSSSGSMALHLAAKSQTRPWNW E4F5DB384E6727B2 etr1_arath Ethylene receptor 1 ETR1 T27F4.9 At1g66340 Protein ETHYLENE RESPONSE 1 Protein ETR1 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MEVCNCIEPQWPADELLMKYQYISDFFIAIAYFSIPLELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFTTHSRTVALVMTTAKVLTAVVSCATALMLVHIIPDLLSVKTRELFLKNKAAELDREMGLIRTQEETGRHVRMLTHEIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLRHQHPVEYTVPIQLPVINQVFGTSRAVKISPNSPVARLRPVSGKYMLGEVVAVRVPLLHLSNFQINDWPELSTKRYALMVLMLPSDSARQWHVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNVALDLARREAETAIRARNDFLAVMNHEMRTPMHAIIALSSLLQETELTPEQRLMVETILKSSNLLATLMNDVLDLSRLEDGSLQLELGTFNLHTLFREVLNLIKPIAVVKKLPITLNLAPDLPEFVVGDEKRLMQIILNIVGNAVKFSKQGSISVTALVTKSDTRAADFFVVPTGSHFYLRVKVKDSGAGINPQDIPKIFTKFAQTQSLATRSSGGSGLGLAISKRFVNLMEGNIWIESDGLGKGCTAIFDVKLGISERSNESKQSGIPKVPAIPRHSNFTGLKVLVMDENGVSRMVTKGLLVHLGCEVTTVSSNEECLRVVSHEHKVVFMDVCMPGVENYQIALRIHEKFTKQRHQRPLLVALSGNTDKSTKEKCMSFGLDGVLLKPVSLDNIRDVLSDLLEPRVLYEGM D6ED3C4BBA87E96E ers-ctr1-2 1 6 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 4 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 physical association physical association Figure 1B and 2A We tested for a two-hybrid interaction between AD�CTR153�568 and a fusion of DB to the ERS histidine kinase domain (DB�ERS261�613) as described for ETR1. Again, the yeast transformants were His1 and blue (Fig. 1B). etr1-ctr1-3 1 7 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 4 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 physical association physical association Figure 1A, Figure 2A We found that yeast transformants containing either of the DB�ETR1 constructs together with the AD�CTR153�568 construct were His1 (histidine prototrophs) and produced blue color in the presence of the b-galactosidase substrate X-Gal, indicating that the histidine kinase region of ETR1 can interact with the amino-terminal domain of CTR1 etr1-ctr1-4 2 7 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 4 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 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 4 Additionally, we confirmed that both MBP�ETR1293�610 and MBP�ETR1293�729 could associate with one of the smaller AD�CTR1 fusions (CTR1 residues 171�521) (Fig. 4). ctr1-etr1 2 7 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 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 4 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 region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) physical association physical association Figure 3 We found that the purified MBP�CTR153�568 fusion could associate with DB�ETR1293�610, ctr1-ers1 2 4 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) 6 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 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 3 In vitro association of purified MBP�CTR153�568 with yeast DB�ETR1 and DB�ERS fusions in yeast cell extracts. etr1-ctr1-5 3 4 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 7 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 5A radiolabeled CTR1 amino-terminal domain (residues 53�568) could associate with MBP�ETR1293�610 and MBP� ETR1604�738 and only weakly with MBP�CKI1981�1122 and MBP alone cki1-ctr1-6 3 4 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 region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro 5 unspecified role unspecified role bait bait region mbp tag maltose binding protein tag maltose binding protein tag maltose binding protein tag mbp 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 ecobb Escherichia coli (strain B / BL21) direct interaction direct interaction Figure 5A radiolabeled CTR1 amino-terminal domain (residues 53�568) could associate with MBP�ETR1293�610 and MBP� ETR1604�738 and only weakly with MBP�CKI1981�1122 and MBP alone The authors state "Of note, when the amount of added radiolabeled CTR1 protein was increased 5-fold, the interaction with MBP-ETR increased whereas the apparent weak association with MBP alone was not enhanced significantly. The absence of association with the CKI1 receiver is indicative of the specificity of the CTR1 association with the ETR1 receiver".