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/ PINOID-Mediated Signaling Involves Calcium-Binding Proteins PINOID-Mediated Signaling Involves Calcium-Binding Proteins Plant Physiology 2003 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. in vitro In vitro in gel kinase assay in-gel kinase assay in gel kinase assay predetermined predetermined participant predetermined Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl Accepted 2008-FEB-9 by CDEROW. 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. Plant Physiology 2003 PINOID-Mediated Signaling Involves Calcium-Binding Proteins PINOID-Mediated Signaling Involves Calcium-Binding Proteins Plant Physiology 2003 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl Accepted 2008-FEB-9 by CDEROW. 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. Plant Physiology 2003 PINOID-Mediated Signaling Involves Calcium-Binding Proteins PINOID-Mediated Signaling Involves Calcium-Binding Proteins Plant Physiology 2003 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. 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 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl Accepted 2008-FEB-9 by CDEROW. 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. Plant Physiology 2003 PINOID-Mediated Signaling Involves Calcium-Binding Proteins PINOID-Mediated Signaling Involves Calcium-Binding Proteins Plant Physiology 2003 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. 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 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl Accepted 2008-FEB-9 by CDEROW. 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. Plant Physiology 2003 PINOID-Mediated Signaling Involves Calcium-Binding Proteins PINOID-Mediated Signaling Involves Calcium-Binding Proteins Plant Physiology 2003 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. 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 Rene Benjamins, Carlos S. Galva�n Ampudia, Paul J.J. Hooykaas, and Remko Offringa offringa@rulbim.leidenuniv.nl Accepted 2008-FEB-9 by CDEROW. 01-April-2008: Contacted by JYOTI. Arabidopsis cDNA library was constructed using mRNA isolated from green parts of 6-week-old flowering Arabidopsis plants. Plant Physiology 2003 pid_arath Protein kinase PINOID Protein kinase ABRUPTUS T31E10.1 PID ABR At2g34650 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MLRESDGEMSLGTTNSPISSGTESCSSFSRLSFDAPPSTIPEEESFLSLKPHRSSDFAYAEIRRRKKQGLTFRDFRLMRRIGAGDIGTVYLCRLAGDEEESRSSYFAMKVVDKEALALKKKMHRAEMEKTILKMLDHPFLPTLYAEFEASHFSCIVMEYCSGGDLHSLRHRQPHRRFSLSSARFYAAEVLVALEYLHMLGIIYRDLKPENILVRSDGHIMLSDFDLSLCSDSIAAVESSSSSPENQQLRSPRRFTRLARLFQRVLRSKKVQTLEPTRLFVAEPVTARSGSFVGTHEYVAPEVASGGSHGNAVDWWAFGVFLYEMIYGKTPFVAPTNDVILRNIVKRQLSFPTDSPATMFELHARNLISGLLNKDPTKRLGSRRGAAEVKVHPFFKGLNFALIRTLTPPEIPSSVVKKPMKSATFSGRSSNKPAAFDYF DIP protein O64682 original sequence version: 112 DIP protein O64682 original sequence version: 79 29A33E80FF96B032 mbp_bovin Myelin basic protein MBP Myelin A1 protein 20 kDa microtubule-stabilizing protein protein protein bovin Bos taurus (Bovine) Bovine AAQKRPSQRSKYLASASTMDHARHGFLPRHRDTGILDSLGRFFGSDRGAPKRGSGKDGHHAARTTHYGSLPQKAQGHRPQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGLKGHDAQGTLSKIFKLGGRDSRSGSPMARR DIP protein P02687 original sequence version: 75 8E1157B7A1978484 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 cml12_arath Calmodulin-like protein 12 At2g41100 CML12 CAL4 TCH3 Touch-induced calmodulin-related protein 3 T3K9.13 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MADKLTDDQITEYRESFRLFDKNGDGSITKKELGTMMRSIGEKPTKADLQDLMNEADLDGDGTIDFPEFLCVMAKNQGHDQAPRHTKKTMADKLTDDQITEYRESFRLFDKNGDGSITKKELRTVMFSLGKNRTKADLQDMMNEVDLDGDGTIDFPEFLYLMAKNQGHDQAPRHTKKTMVDYQLTDDQILEFREAFRVFDKNGDGYITVNELRTTMRSLGETQTKAELQDMINEADADGDGTISFSEFVCVMTGKMIDTQSKKETYRVVNQGQGQVQRHTRNDRAGGTNWERDIAVGVASNIIASPISDFMKDRFKDLFEALLS 94277BFBC2D2C5CE pid-pbp1 4 6 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association The second gene we identified, PINOID BINDING PROTEIN 1 (PBP1, At5g54490), encodes a previously uncharacterized protein of 127 amino acids with three possible EF-hand calcium-binding motifs. Table 1 pid-tch3 4 9 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait physical association physical association One of the CBP genes, TCH3 (At2g41100), was identified previously by Braam and Davis (1990) in a screen for genes that are up-regulated in response to mechanical stimuli such as wind and touch. Table 1 pid-pbp1-1 3 6 unspecified role unspecified role bait bait region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 8 unspecified role unspecified role prey prey physical association physical association PID interacts with PBP1 and TCH3 in yeast. Figure 2 pid-tch3-1 3 6 unspecified role unspecified role bait bait region required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey physical association physical association PID interacts with PBP1 and TCH3 in yeast. Figure 2 pid-tch3-2 2 9 unspecified role unspecified role prey prey c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecobb Escherichia coli (strain B / BL21) 6 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 ecobb Escherichia coli (strain B / BL21) physical association physical association Calcium dependency of the in vitro interaction between PBP1 or TCH3 and PID. Figure 3 and 4 The interaction was dependent on the presence of calcium in the binding buffer. pid-pbp1-2 2 6 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 ecobb Escherichia coli (strain B / BL21) 8 unspecified role unspecified role prey prey c-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range ecobb Escherichia coli (strain B / BL21) physical association physical association The interaction was enhanced in the presence of calcium in the binding buffer. Figure 3 and 4 Calcium dependency of the in vitro interaction between PBP1 or TCH3 and PID. pid-mbp 1 6 enzyme enzyme 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 7 enzyme target enzyme target substrate substrate neutral component neutral component phosphorylation phosphorylation reaction phosphorylation 6 GST:PID phosphorylates MBP, phosphorylation of myelin basic protein. pid-pid 1 6 enzyme target enzyme target substrate substrate unspecified role unspecified role 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 6 enzyme enzyme unspecified role unspecified role 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 phosphorylation phosphorylation reaction phosphorylation 6 PID protein is also active as a protein kinase (Fig. 6). Next, we determined whether PID phosphorylates TCH3 and PBP1 by coincubating GST:PID with the His-tagged versions of TCH3 or PBP1 in the presence of calcium. GST:PID did not phosphorylate TCH3 or PBP1, whereas autophosphorylation of GST:PID was still detectable. The authors comment that "Interestingly, autophosphorylation of GST:PID was reproducibly enhanced in the presence of PBP1." autophosphorylation of PID protein. pid-pbp1-3 5 6 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey physical association physical association Table 1 pid-tch3-3 5 6 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association Table 1