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/ Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Cell. Signal. (0898-6568) 2007 imex curation Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte 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 Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu Accepted 2007-JUL-13 by JYOTI. Human peripheral blood leukocyte Cell. Signal. (0898-6568) 2007 Only protein-protein interactions imex curation Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Cell. Signal. (0898-6568) 2007 imex curation Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu Accepted 2007-JUL-13 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Cell. Signal. (0898-6568) 2007 Only protein-protein interactions imex curation Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Cell. Signal. (0898-6568) 2007 imex curation Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Only protein-protein interactions crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. confocal microscopy confocal microscopy predetermined predetermined participant predetermined Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu Accepted 2007-JUL-13 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Time after treatment with 1uM Phorbol 12-myristate 13-acetate (mins) Human peripheral blood leukocyte Cell. Signal. (0898-6568) 2007 Only protein-protein interactions imex curation Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Cell. Signal. (0898-6568) 2007 imex curation Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Only protein-protein interactions crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu Accepted 2007-JUL-13 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Cell. Signal. (0898-6568) 2007 Only protein-protein interactions imex curation Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Cell. Signal. (0898-6568) 2007 imex curation Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Only protein-protein interactions rat-l6 Rattus norvegicus l6 Rat L6 myotube cell line anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu Accepted 2007-JUL-13 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Cell. Signal. (0898-6568) 2007 Only protein-protein interactions imex curation Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2. Cell. Signal. (0898-6568) 2007 imex curation Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Lu G., Chen J., Espinoza LA., Garfield S., Toshiyuki S., Akiko H., Huppler A., Wang QJ. Synapse - Interactions of proteins with an established role in the presynapse. qjw1@pitt.edu Accepted 2007-JUL-13 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Human peripheral blood leukocyte Cell. Signal. (0898-6568) 2007 Only protein-protein interactions imex curation vamp2_rat Vesicle-associated membrane protein 2 Vamp2 Syb2 Synaptobrevin-2 protein protein rat Rattus norvegicus (Rat) Rat MSATAATVPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST DIP protein P63045 original sequence version: 95 DIP protein P63045 original sequence version: 90 DIP protein P63045 original sequence version: 64 DIP protein P63045 original sequence version: 62 DIP protein P63045 original sequence version: 85 DIP protein P63045 original sequence version: 104 DIP protein P63045 original sequence version: 77 DIP protein P63045 original sequence version: 75 DIP protein P63045 original sequence version: 106 4A0D0D56B5409D0A d4a229_rat Serine/threonine-protein kinase Prkd3 protein protein rat Rattus norvegicus (Rat) Rat MSANNSPPSAQKSVFPATVSAVLPTPSPCSSPKTGLSARLSNGSFSAASLTNSRGSVHTVSFLLQIGLTRESVTIEAQELSLSAVKDLVCSIVYQKFPECGFFGMYDKILLFRHNMNSENILQLITSADEIHEGDLVEVVLSALATVEDFQIRPHALYVHSYKAPTFCDYCGEMLWGLVRQGLKCEGCGLNYHKRCAFKIPNNCSGVRKRRLSNVSLPGPGLSVPRPLQPECVPLLSEESHTHQEPSKRIPSWSGRPIWMEKMVMCRVKVPHTFAVHSYGRPTICQYCKRLLKGLFRQGLQCKDCKFNCHKRCASKVPRDCLGEVTFNGEPSSVGTDTDMPMDIDSNDVNSDGSRGLDDSEEPSPPEDKMFFLDAADLDVERDEEPVKTISPSTSNNIPLMRVVQSIKHTKRKSSTVVKEGWMVHYTSRDNLRKRHYWRLDSKCLTLFQNESGSKYYKEIPLSEILRISSPQDFTSISQGSNPHCFEIITDTVVYFVGENNGNSTHNPVLAATGVGLDVAQSWEKAIRQALMPVTPQASVCTSPGQGKDHKDLATSISVSNCQIQENVDISSVYQIFADEVLGSGQFGIVYGGKHRKTGRDVAIKVIDKMRFPTKQESQLRNEVAILQNLHHPGIVNLECMFETPERVFVVMEKLHGDMLEMILSSEKSRLPERITKFMVTQILVALRNLHFKNIVHCDLKPENVLLASAEPFPQVKLCDFGFARIIGEKSFRRSVVGTPAYLAPEVLRSKGYNRSLDMWSVGVIVYVSLSGTFPFNEDEDINDQIQNAAFMYPPNPWREISSEAIDLINNLLQVKMRKRYSVDKSLSHPWLQDYQTWLDLREFETRIGERYITHESDDARWEIHAYTHNLEYPKHFIMAPNPDDMEEGP EB8BA70DF389659A g3hx91_crigr Serine/threonine-protein kinase I79_015610 protein protein crigr Cricetulus griseus (Chinese hamster) Cricetulus barabensis griseus Chinese hamster MSANNSPPSAQKSVLPATVSAVLPTPSPCSSPKTGLSARLSNGSCSAPSLTNSRGSVHTVSFLLQIGLTRESVTIEAQELSLSAVKDLVCSIVYQKFPECGFFGMYDKILLFRHDMNSENILQLITSADDIHEGDLVEVVLSALATVEDFQIRPHTLYVHSYKAPTFCDYCGEMLWGLVRQGLKCEGCGLNYHKRCAFKIPNNCSGVRKRRLSNVSLPGPGLSVPRPLQPECVPLLSEESHTHQEPSKRIPSWSGRPIWMEKMVMCRVKVPHTFAVHSYGRPTICQYCKRLLKGLFRQGMQCKDCKFNCHKRCASKVPRDCLGEVTFNGEPSSLGTDTDIPMDIDSNDMNSDGSRGLDDSEEPSPPEDKMFFLDPTDLDVERDEETVKTISPSTSNNIPLMRVVQSIKHTKRRSSTMVKEGWMVHYTSRDNLRKRHYWRLDSKCLTLFQNESGSKYYKEIPLSEILRVSSPQDLSSISQGSNPHCFEIITDTVVYFVGENNGSSSHNPVLAATGVGLDVAQSWEKAIRQALMPVTPQASICTSPGQGKDHKDLATSISVSNSQVQENVDISSVYQIFADEVLGSGQFGIVYGGKHRKTGRDVAIKVIDKMRFPTKQESQLRNEVAILQNLHHPGIVNLECMFETPERVFVVMEKLHGDMLEMILSSEKSRLPERITKFMVTQILVALRNLHFKNIVHCDLKPENVLLASAEPFPQVKLCDFGFARIIGEKSFRRSVVGTPAYLAPEVLRSKGYNRSLDMWSVGVIVYVSLSGTFPFNEDEDINDQIQNAAFMYPPNPWREISSEAIDLINNLLQVKMRKRFSVDKSLSHPWLQDYQTWLDLREFETRIGERYITHESDDARWEIHAYTHNLEYPKHFIMAPNPDDMEEDP BE53781193040070 vamp2_human Vesicle-associated membrane protein 2 Synaptobrevin-2 VAMP2 SYB2 protein protein human Homo sapiens Human MSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWWKNLKMMIILGVICAIILIIIIVYFST DIP protein P63027 original sequence version: 108 DIP protein P63027 original sequence version: 60 DIP protein P63027 original sequence version: 74 9CD679C4F6F1B5A8 kpcd3_human Serine/threonine-protein kinase D3 PRKD3 EPK2 PRKCN Protein kinase C nu type nPKC-nu Protein kinase EPK2 protein protein human Homo sapiens Human MSANNSPPSAQKSVLPTAIPAVLPAASPCSSPKTGLSARLSNGSFSAPSLTNSRGSVHTVSFLLQIGLTRESVTIEAQELSLSAVKDLVCSIVYQKFPECGFFGMYDKILLFRHDMNSENILQLITSADEIHEGDLVEVVLSALATVEDFQIRPHTLYVHSYKAPTFCDYCGEMLWGLVRQGLKCEGCGLNYHKRCAFKIPNNCSGVRKRRLSNVSLPGPGLSVPRPLQPEYVALPSEESHVHQEPSKRIPSWSGRPIWMEKMVMCRVKVPHTFAVHSYTRPTICQYCKRLLKGLFRQGMQCKDCKFNCHKRCASKVPRDCLGEVTFNGEPSSLGTDTDIPMDIDNNDINSDSSRGLDDTEEPSPPEDKMFFLDPSDLDVERDEEAVKTISPSTSNNIPLMRVVQSIKHTKRKSSTMVKEGWMVHYTSRDNLRKRHYWRLDSKCLTLFQNESGSKYYKEIPLSEILRISSPRDFTNISQGSNPHCFEIITDTMVYFVGENNGDSSHNPVLAATGVGLDVAQSWEKAIRQALMPVTPQASVCTSPGQGKDHKDLSTSISVSNCQIQENVDISTVYQIFADEVLGSGQFGIVYGGKHRKTGRDVAIKVIDKMRFPTKQESQLRNEVAILQNLHHPGIVNLECMFETPERVFVVMEKLHGDMLEMILSSEKSRLPERITKFMVTQILVALRNLHFKNIVHCDLKPENVLLASAEPFPQVKLCDFGFARIIGEKSFRRSVVGTPAYLAPEVLRSKGYNRSLDMWSVGVIIYVSLSGTFPFNEDEDINDQIQNAAFMYPPNPWREISGEAIDLINNLLQVKMRKRYSVDKSLSHPWLQDYQTWLDLREFETRIGERYITHESDDARWEIHAYTHNLVYPKHFIMAPNPDDMEEDP 66D5E7E7235064F5 prkd3-vamp2-1 1 10 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait physical association physical association VAMP2, the vesicle-localized v-SNARE, was later identified as a novel binding partner of PKD3 through yeast two-hybrid screening. prkd3-vamp2-2 6 11 unspecified role unspecified role prey prey 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 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range certain certain sequence position certain ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. direct interaction direct interaction Only the full-length GST-PKD3 showed positive interaction with Flag-VAMP2, GST and GST-PKD3-C1 domains did not show any binding activities to Flag-VAMP2 Fig. 4A prkd3-vamp2-3 6 10 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 ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role prey prey 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 crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. direct interaction direct interaction Our results show hat GCT-VAMP2 was able to specifically pull down PKD3-GFP expressed in CHO cells (Fig. 4a). Fig. 4A prkd3-vamp2-4 4 10 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 9 unspecified role unspecified role prey prey direct interaction direct interaction the association of PKD3 and VAMP2 was confirmed in vivo in mammalian cells by Immunoprecipitation (IP). Fig. 4b prkd3-vamp2-5 4 11 unspecified role unspecified role bait bait 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 10 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction the association of PKD3 and VAMP2 was confirmed in vivo in mammalian cells by Immunoprecipitation (IP). Fig. 4b prkd3-vamp2-6 4 11 unspecified role unspecified role prey prey 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 10 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) direct interaction direct interaction the association of PKD3 and VAMP2 was confirmed in vivo in mammalian cells by Immunoprecipitation (IP). Fig. 4b prkd3-vamp2-7 5 8 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association As shown in Fig. 4b (lower right panels), PKD3 was detected in VAMP2 immunoprecipitates Fig. 4b prkd3-vamp2-8 5 8 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey physical association physical association In contrast, VAMP2 was only minimally detected in the PKD3 immunoprecipitates Fig. 4b Weak interaction - possibly due to limited VAMP2 expression prkd3-1 2 11 putative self putative self putative self putative self 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 O94806:p.[Ser731Ala;Ser735Glu] mutation mutation mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain S E certain certain sequence position certain certain certain sequence position certain S A Wed May 02 18:47:50 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser731ala,ser735glu' Tue Jun 30 16:46:01 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser731Ala;Ser735Glu]' phosphorylation phosphorylation reaction phosphorylation true ser731ala_ser754glu mutant is constitutively active As shown in Fig. 5a, wild-type, PMA-activated and constitutive active PKD3-GFP all possessed kinase activity as indicated by their significant levels of autophosphorylation Fig. 5a prkd3-vamp2-9 3 10 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 11 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization PKD3 partially colocalized with VAMP2, and translocated with RFP-PKD3 in response to 1uM PMA. Fig. 6 0 prkd3-vamp2-10 3 10 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 11 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization PKD3 partially colocalized with VAMP2, and translocated with RFP-PKD3 in response to 1uM PMA. Fig. 6 10 prkd3-2 2 11 putative self putative self putative self putative self phosphorylation phosphorylation reaction phosphorylation true As shown in Fig. 5a, wild-type, PMA-activated and constitutive active PKD3-GFP all possessed kinase activity as indicated by their significant levels of autophosphorylation Fig. 5a