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/ E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. (0008-5472) 2005 imex curation Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. (0008-5472) 2005 imex curation Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-prostate_carcinoma Homo sapiens prostate carcinoma prostate_carcinoma Prostate carcinoma (cancer) confocal microscopy confocal microscopy immunostaining immunostaining Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. (0008-5472) 2005 imex curation Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma confocal microscopy confocal microscopy immunostaining immunostaining Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu Accepted 2007-NOV-8 by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. (0008-5472) 2005 imex curation Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. (0008-5472) 2005 imex curation Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu 13-Nov-2007: Contacted by JYOTI. Only protein-protein interactions human-c4_2 Homo sapiens c4-2 androgen-independent prostate cancer cells c4_2 Human androgen-independent prostate cancer cells C4-2 This cell line is also known as C4-2B. confocal microscopy confocal microscopy immunostaining immunostaining Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer. Cancer Res. (0008-5472) 2005 imex curation Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu 13-Nov-2007: 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 Jaggi M., Rao PS., Smith DJ., Wheelock MJ., Johnson KR., Hemstreet GP., Balaji KC. Cancer - Interactions investigated in the context of cancer PKD1 and E-cadherin were isolated from cell extracts. Phosphorylation was carried out in the presence of [32P]ATP. [32P]-labeled proteins were identified by autoradiography. A search on the internet revealed the details regarding the synthetic peptide Syntide-2. kcbalaji@unmc.edu Accepted 2007-NOV-8 by JYOTI. 13-Nov-2007: Contacted by JYOTI. Cancer Res. (0008-5472) 2005 Only protein-protein interactions imex curation cadh1_human Cadherin-1 Epithelial cadherin Uvomorulin CAM 120/80 CDH1 CDHE UVO protein protein human Homo sapiens Human MGPWSRSLSALLLLLQVSSWLCQEPEPCHPGFDAESYTFTVPRRHLERGRVLGRVNFEDCTGRQRTAYFSLDTRFKVGTDGVITVKRPLRFHNPQIHFLVYAWDSTYRKFSTKVTLNTVGHHHRPPPHQASVSGIQAELLTFPNSSPGLRRQKRDWVIPPISCPENEKGPFPKNLVQIKSNKDKEGKVFYSITGQGADTPPVGVFIIERETGWLKVTEPLDRERIATYTLFSHAVSSNGNAVEDPMEILITVTDQNDNKPEFTQEVFKGSVMEGALPGTSVMEVTATDADDDVNTYNAAIAYTILSQDPELPDKNMFTINRNTGVISVVTTGLDRESFPTYTLVVQAADLQGEGLSTTATAVITVTDTNDNPPIFNPTTYKGQVPENEANVVITTLKVTDADAPNTPAWEAVYTILNDDGGQFVVTTNPVNNDGILKTAKGLDFEAKQQYILHVAVTNVVPFEVSLTTSTATVTVDVLDVNEAPIFVPPEKRVEVSEDFGVGQEITSYTAQEPDTFMEQKITYRIWRDTANWLEINPDTGAISTRAELDREDFEHVKNSTYTALIIATDNGSPVATGTGTLLLILSDVNDNAPIPEPRTIFFCERNPKPQVINIIDADLPPNTSPFTAELTHGASANWTIQYNDPTQESIILKPKMALEVGDYKINLKLMDNQNKDQVTTLEVSVCDCEGAAGVCRKAQPVEAGLQIPAILGILGGILALLILILLLLLFLRRRAVVKEPLLPPEDDTRDNVYYYDEEGGGEEDQDFDLSQLHRGLDARPEVTRNDVAPTLMSVPRYLPRPANPDEIGNFIDENLKAADTDPTAPPYDSLLVFDYEGSGSEAASLSSLNSSESDKDQDYDYLNEWGNRFKKLADMYGGGEDD DIP protein P12830 original sequence version: 191 DIP protein P12830 original sequence version: 185 DIP protein P12830 original sequence version: 108 DIP protein P12830 original sequence version: null DIP protein P12830 original sequence version: 134 DIP protein P12830 original sequence version: 147 DIP protein P12830 original sequence version: 174 DIP protein P12830 original sequence version: 189 DIP protein P12830 original sequence version: 156 DIP protein P12830 original sequence version: 201 DIP protein P12830 original sequence version: 117 E427118043A13C67 syntide_2_peptide Syntide-2 synthetic peptide peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) PLARTLSVAGLPGKK An amino acid sequence which is not represented in UniProt The native peptide PLARTLSVAGLPGKK is a selective Substrate for CaM kinase II and PKC. it is also a poor substrate for phosphorylase kinase and is not phosphorylated by myosin light chain kinase. The sequence of PLARTLSVAGLPGKK is homologous to phosphorylation site 2 in glycogen synthase. CBB20424AD7344D2 kpcd1_human Serine/threonine-protein kinase D1 nPKC-D1 Protein kinase D Protein kinase C mu type nPKC-mu PRKD1 PKD PKD1 PRKCM protein protein human Homo sapiens Human MSAPPVLRPPSPLLPVAAAAAAAAAALVPGSGPGPAPFLAPVAAPVGGISFHLQIGLSREPVLLLQDSSGDYSLAHVREMACSIVDQKFPECGFYGMYDKILLFRHDPTSENILQLVKAASDIQEGDLIEVVLSASATFEDFQIRPHALFVHSYRAPAFCDHCGEMLWGLVRQGLKCEGCGLNYHKRCAFKIPNNCSGVRRRRLSNVSLTGVSTIRTSSAELSTSAPDEPLLQKSPSESFIGREKRSNSQSYIGRPIHLDKILMSKVKVPHTFVIHSYTRPTVCQYCKKLLKGLFRQGLQCKDCRFNCHKRCAPKVPNNCLGEVTINGDLLSPGAESDVVMEEGSDDNDSERNSGLMDDMEEAMVQDAEMAMAECQNDSGEMQDPDPDHEDANRTISPSTSNNIPLMRVVQSVKHTKRKSSTVMKEGWMVHYTSKDTLRKRHYWRLDSKCITLFQNDTGSRYYKEIPLSEILSLEPVKTSALIPNGANPHCFEITTANVVYYVGENVVNPSSPSPNNSVLTSGVGADVARMWEIAIQHALMPVIPKGSSVGTGTNLHRDISVSISVSNCQIQENVDISTVYQIFPDEVLGSGQFGIVYGGKHRKTGRDVAIKIIDKLRFPTKQESQLRNEVAILQNLHHPGVVNLECMFETPERVFVVMEKLHGDMLEMILSSEKGRLPEHITKFLITQILVALRHLHFKNIVHCDLKPENVLLASADPFPQVKLCDFGFARIIGEKSFRRSVVGTPAYLAPEVLRNKGYNRSLDMWSVGVIIYVSLSGTFPFNEDEDIHDQIQNAAFMYPPNPWKEISHEAIDLINNLLQVKMRKRYSVDKTLSHPWLQDYQTWLDLRELECKIGERYITHESDDLRWEKYAGEQGLQYPTHLINPSASHSDTPETEETEMKALGERVSIL DIP protein Q15139 original sequence version: 79 DIP protein Q15139 original sequence version: 105 0BC9414C335D2DBB cdh1-prkd1 3 7 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component colocalization colocalization 1 Serial Z-sections of the LNCaP cells were taken at every micrometer using the LSM version 399 software and a gallery of serial Z-sections was constructed confirming the junctional localization and colocalization of PKD1 with E-cadherin in LNCaP prostate cancer cells. This colocalization was seen at cell borders. prkd1-cdh1 4 7 unspecified role unspecified role prey prey 9 unspecified role unspecified role bait bait physical association physical association 2 A Immunoblot analysis using E-cadherin-specific antibody showed the coimmunoprecipitation of E-cadherin with PKD1 in LNCaP cells cdh1-prkd1-1 4 9 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association 2 A we also did reciprocal immunoprecipitation assays with E-cadherin antibody and immunoblotted for PKD1. PKD1 was also immunoprecipitated with the E-cadherin antibody prkd1-cdh1-1 1 7 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role bait bait region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 2 A Furthermore, we carried out the immunoprecipitation experiments with [35S]-labeled methionine and cysteine LNCaP cell protein lysate, followed by analysis of all the immunoprecipitated proteins on SDS-PAGE and autoradiography. Under these conditions, relatively few proteins coimmunoprecipitated [(Fig. 2A(3) lane 1)] which provided additional evidence for specificity of the PKD1/E-cadherin interaction. prkd1-cdh1-2 6 9 enzyme enzyme unspecified role unspecified role human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma 7 enzyme target enzyme target substrate substrate unspecified role unspecified role human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma phosphorylation phosphorylation reaction phosphorylation 2 B, 3 B Analysis of phosphorylation by autoradiography (Fig. 2B) showed strong incorporation of radioactivity in the E-cadherin immunoprecipitate at 120 kDa, demonstrating phosphorylation of E-cadherin by PKD1 Go6976 - Phosphorylation of E-Cadherin by PKD1 is inhibited by Go6976. E-cadherin was phosphorylated. prkd1-syntide_2_pept 6 9 enzyme enzyme unspecified role unspecified role human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma 8 enzyme target enzyme target substrate substrate unspecified role unspecified role chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation 2 B a strong band in lane 3 below 6 kDa [Fig. 2B(1) lane 3)] indicates Syntide-2 phosphorylation by PKD1. cdh1-prkd1-2 5 7 unspecified role unspecified role neutral component neutral component 9 unspecified role unspecified role neutral component neutral component region gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization 5 A Figure 5 shows the merging of images taken in green (1, PKD1-GFP), red (2, E-cadherin), and DIC (3, DIC) channels indicating that PKD1 colocalizes with E-cadherin at cell junctions Colocalization takes place at cell junctions. cdh1-prkd1-3 2 9 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component colocalization colocalization 5 B Colocalization takes place at cell junctions. Merging of images 1 and 2 show that PKD1 and E-cadherin colocalized at the cell junctions of prostatic glandular epithelial cells