DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis. Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2006 imex curation Zegzouti H., Anthony RG., Jahchan N., Bogre L., Christensen SK. The source of the MBP protein used in this study is not given and therefore not entered here. sioux@ucla.edu 22-Jan-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Zegzouti H., Anthony RG., Jahchan N., Bogre L., Christensen SK. The source of the MBP protein used in this study is not given and therefore not entered here. sioux@ucla.edu Accepted 2007-DEC-14 by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2006 IMEx Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis. Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2006 imex curation Zegzouti H., Anthony RG., Jahchan N., Bogre L., Christensen SK. The source of the MBP protein used in this study is not given and therefore not entered here. sioux@ucla.edu 22-Jan-2008: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Zegzouti H., Anthony RG., Jahchan N., Bogre L., Christensen SK. The source of the MBP protein used in this study is not given and therefore not entered here. sioux@ucla.edu Accepted 2007-DEC-14 by JYOTI. 22-Jan-2008: Contacted by JYOTI. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2006 IMEx 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 pdpk1_arath 3-phosphoinositide-dependent protein kinase 1 PDPK1 PDK1 T32M21.110 At5g04510 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MLAMEKEFDSKLVLQGNSSNGANVSRSKSFSFKAPQENFTSHDFEFGKIYGVGSYSKVVRAKKKETGTVYALKIMDKKFITKENKTAYVKLERIVLDQLEHPGIIKLYFTFQDTSSLYMALESCEGGELFDQITRKGRLSEDEARFYTAEVVDALEYIHSMGLIHRDIKPENLLLTSDGHIKIADFGSVKPMQDSQITVLPNAASDDKACTFVGTAAYVPPEVLNSSPATFGNDLWALGCTLYQMLSGTSPFKDASEWLIFQRIIARDIKFPNHFSEAARDLIDRLLDTEPSRRPGAGSEGYVALKRHPFFNGVDWKNLRSQTPPKLAPDPASQTASPERDDTHGSPWNLTHIGDSLATQNEGHSAPPTSSESSGSITRLASIDSFDSRWQQFLEPGESVLMISAVKKLQKITSKKVQLILTNKPKLIYVDPSKLVVKGNIIWSDNSNDLNVVVTSPSHFKICTPKKVLSFEDAKQRASVWKKAIETLQNR 2319EE69CDB5CB38 pid-3 1 3 putative self putative self putative self putative self O64682:p.[Ser288Glu;Ser290Glu] mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain S E certain certain sequence position certain certain certain sequence position certain S E Wed May 02 15:41:07 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser288glu,ser290glu' Tue Jun 30 16:55:15 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser288Glu;Ser290Glu]' region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined O64682:p.Asp205Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain D A Wed May 02 19:01:13 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp205ala' Tue Jun 30 19:39:05 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp205Ala' ecobb Escherichia coli (strain B / BL21) phosphorylation phosphorylation reaction phosphorylation true 1C 1D 3C These data provide evidence that intramolecular autophosphorylation positively regulates the ability of PID to transphosphorylate substrates in vitro. autophosphorylation of PID protein. Increasing levels of Magnesium ions stimulated autophosphorylation of PID Calcium ions The authors concluded that "These data suggest that Ca ions may inhibit PID activity by competitive binding and displacement of the Mg2 ions". pdk1-pid-3 2 4 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) 3 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 3A Western blots of bound proteins probed with anti-His antibody show that both PID and MPID bind PDK1 pdk1-pid-1 1 3 enzyme target enzyme target substrate substrate 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 O64682:p.[Ser288Glu;Ser290Glu] mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain S E certain certain sequence position certain certain certain sequence position certain S E Wed May 02 16:23:50 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser288glu,ser290glu' Tue Jun 30 20:16:07 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser288Glu;Ser290Glu]' O64682:p.[Phe435Val;Phe438Val] mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain F V certain certain sequence position certain certain certain sequence position certain F V Wed May 02 19:25:26 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'phe435val,phe438val' Tue Jun 30 18:06:21 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Phe435Val;Phe438Val]' ecobb Escherichia coli (strain B / BL21) 4 enzyme enzyme neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecobb Escherichia coli (strain B / BL21) phosphorylation phosphorylation reaction phosphorylation 3B, 3C, 4B, Supplementary information Figure 8 The interaction between PID and PDK1 results in a dramatic increase in PID phosphorylation (Fig. 3B). PID showed a 6.5-fold increase in measured phosphate incorporation as compared with MPID. This difference must represent increased PID autocatalysis in response to activation by PDK1, suggesting that transphosphorylation by PDK1 activates PID through up-regulation of autophosphorylation activity The authors concluded that "transphosphorylation by PDK1 activates PID through up-regulation of autophosphorylation activity". In addition that "PDK1 activates PID by phosphorylating one or more of the serines within the PID activation loop". phosphorylation of PID Time course experiments show that the increase in PDK1-dependent PID autophosphorylation is rapid, with peak levels of activation occurring after 30 min of coincubation pdk1-pid-2 1 3 enzyme target enzyme target substrate substrate 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 arath-protoplast Arabidopsis thaliana protoplast Protoplast defined as a membrane-bound cell from which the outer wall has been partially or completely removed. 4 enzyme enzyme neutral component neutral component region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath-protoplast Arabidopsis thaliana protoplast Protoplast defined as a membrane-bound cell from which the outer wall has been partially or completely removed. phosphorylation phosphorylation reaction phosphorylation 5 These results demonstrate that At- PDK1 is a potent upstream activator of PID in vivo. phosphorylation of PID