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/ PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell. Sci. (0021-9533) 2005 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk Accepted 2007-APR-19 by DAVET. 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA J. Cell. Sci. (0021-9533) 2005 PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell. Sci. (0021-9533) 2005 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA canlf-mdck Canis familiaris (dog) kidney epithelial cells Canis lupus familiaris Dog Canis familiaris mdck Canine Cocker Spaniel kidney epithelial cell line anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk Accepted 2007-APR-19 by DAVET. 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA J. Cell. Sci. (0021-9533) 2005 PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell. Sci. (0021-9533) 2005 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk Accepted 2007-APR-19 by DAVET. 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA J. Cell. Sci. (0021-9533) 2005 PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell. Sci. (0021-9533) 2005 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA 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 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk Accepted 2007-APR-19 by DAVET. 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA J. Cell. Sci. (0021-9533) 2005 PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. PCTAIRE protein kinases interact directly with the COPII complex and modulate secretory cargo transport. J. Cell. Sci. (0021-9533) 2005 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA 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 Palmer KJ., Konkel JE., Stephens DJ. david.stephens@bristol.ac.uk Accepted 2007-APR-19 by DAVET. 10-APR-2007: Contacted by JYOTI. Adult human brain cDNA J. Cell. Sci. (0021-9533) 2005 cdk18_human Cyclin-dependent kinase 18 CDK18 PCTAIRE-motif protein kinase 3 PCTK3 Serine/threonine-protein kinase PCTAIRE-3 Cell division protein kinase 18 PCTAIRE3 protein protein human Homo sapiens Human MIMNKMKNFKRRFSLSVPRTETIEESLAEFTEQFNQLHNRRNENLQLGPLGRDPPQECSTFSPTDSGEEPGQLSPGVQFQRRQNQRRFSMEDVSKRLSLPMDIRLPQEFLQKLQMESPDLPKPLSRMSRRASLSDIGFGKLETYVKLDKLGEGTYATVFKGRSKLTENLVALKEIRLEHEEGAPCTAIREVSLLKNLKHANIVTLHDLIHTDRSLTLVFEYLDSDLKQYLDHCGNLMSMHNVKIFMFQLLRGLAYCHHRKILHRDLKPQNLLINERGELKLADFGLARAKSVPTKTYSNEVVTLWYRPPDVLLGSTEYSTPIDMWGVGCIHYEMATGRPLFPGSTVKEELHLIFRLLGTPTEETWPGVTAFSEFRTYSFPCYLPQPLINHAPRLDTDGIHLLSSLLLYESKSRMSAEAALSHSYFRSLGERVHQLEDTASIFSLKEIQLQKDPGYRGLAFQQPGRGKNRRQSIF 43C83A573A595476 f1pby2_canlf CDK18 protein protein canlf Canis familiaris (dog) Canis lupus familiaris Dog Canis familiaris LTMNKMKNFKRRLSLSVPRTETIEESLTEFTEQFNQLHNRRNEDLQLGPLSRDPQPETSTFSPTDSGEDPGQPSPGMQYRRQNQRRFSMEDISKRLSLPMDIRLPQEFLQKLQLESPDLPKPLSRMSRRASLSDIGFGKLETYVKLDKLGEGTYATVFKGRSKLTENLVALKEIRLEHEEGAPCTAIREVSLLKNLKHANIVTLHDLIHTERSLTLVFEYLDSDLKQYLDHCGNLMSMHNVKIFMFQLLRGLAYCHRRKILHRDLKPQNLLISERGELKLADFGLARAKSVPTKTYSNEVVTLWYRPPDVLLGSTEYSTPIDMWGVGCIHYEMATGRPLFPGTVKEELQLIILTLLDTDGLHLLSSLLLYESKSRMSAEAALSHPYFRSLGERVHQLEDTASIFSLKEIQLQKDPGYRGLAFQQPGRGKSRRQSIF 9E4653A445CDF3D0 cdk16_human Cyclin-dependent kinase 16 PCTAIRE-motif protein kinase 1 CDK16 PCTK1 Serine/threonine-protein kinase PCTAIRE-1 Cell division protein kinase 16 PCTAIRE1 protein protein human Homo sapiens Human MDRMKKIKRQLSMTLRGGRGIDKTNGAPEQIGLDESGGGGGSDPGEAPTRAAPGELRSARGPLSSAPEIVHEDLKMGSDGESDQASATSSDEVQSPVRVRMRNHPPRKISTEDINKRLSLPADIRLPEGYLEKLTLNSPIFDKPLSRRLRRVSLSEIGFGKLETYIKLDKLGEGTYATVYKGKSKLTDNLVALKEIRLEHEEGAPCTAIREVSLLKDLKHANIVTLHDIIHTEKSLTLVFEYLDKDLKQYLDDCGNIINMHNVKLFLFQLLRGLAYCHRQKVLHRDLKPQNLLINERGELKLADFGLARAKSIPTKTYSNEVVTLWYRPPDILLGSTDYSTQIDMWGVGCIFYEMATGRPLFPGSTVEEQLHFIFRILGTPTEETWPGILSNEEFKTYNYPKYRAEALLSHAPRLDSDGADLLTKLLQFEGRNRISAEDAMKHPFFLSLGERIHKLPDTTSIFALKEIQLQKEASLRSSSMPDSGRPAFRVVDTEF 9A5DDD34A5E5CBBB sc23a_human Protein transport protein Sec23A SEC23A SEC23-related protein A protein protein human Homo sapiens Human MTTYLEFIQQNEERDGVRFSWNVWPSSRLEATRMVVPVAALFTPLKERPDLPPIQYEPVLCSRTTCRAVLNPLCQVDYRAKLWACNFCYQRNQFPPSYAGISELNQPAELLPQFSSIEYVVLRGPQMPLIFLYVVDTCMEDEDLQALKESMQMSLSLLPPTALVGLITFGRMVQVHELGCEGISKSYVFRGTKDLSAKQLQEMLGLSKVPLTQATRGPQVQQPPPSNRFLQPVQKIDMNLTDLLGELQRDPWPVPQGKRPLRSSGVALSIAVGLLECTFPNTGARIMMFIGGPATQGPGMVVGDELKTPIRSWHDIDKDNAKYVKKGTKHFEALANRAATTGHVIDIYACALDQTGLLEMKCCPNLTGGYMVMGDSFNTSLFKQTFQRVFTKDMHGQFKMGFGGTLEIKTSREIKISGAIGPCVSLNSKGPCVSENEIGTGGTCQWKICGLSPTTTLAIYFEVVNQHNAPIPQGGRGAIQFVTQYQHSSGQRRIRVTTIARNWADAQTQIQNIAASFDQEAAAILMARLAIYRAETEEGPDVLRWLDRQLIRLCQKFGEYHKDDPSSFRFSETFSLYPQFMFHLRRSSFLQVFNNSPDESSYYRHHFMRQDLTQSLIMIQPILYAYSFSGPPEPVLLDSSSILADRILLMDTFFQILIYHGETIAQWRKSGYQDMPEYENFRHLLQAPVDDAQEILHSRFPMPRYIDTEHGGSQARFLLSKVNPSQTHNNMYAWGQESGAPILTDDVSLQVFMDHLKKLAVSSAA 128DF9964B253313 sc24d_human Protein transport protein Sec24D KIAA0755 SEC24D SEC24-related protein D protein protein human Homo sapiens Human MSQQGYVATPPYSQPQPGIGLSPPHYGHYGDPSHTASPTGMMKPAGPLGATATRGMLPPGPPPPGPHQFGQNGAHATGHPPQRFPGPPPVNNVASSHAPYQPSAQSSYPGPISTSSVTQLGSQLSAMQINSYGSGMAPPSQGPPGPLSATSLQTPPRPPQPSILQPGSQVLPPPPTTLNGPGASPLPLPMYRPDGLSGPPPPNAQYQPPPLPGQTLGAGYPPQQANSGPQMAGAQLSYPGGFPGGPAQMAGPPQPQKKLDPDSIPSPIQVIENDRASRGGQVYATNTRGQIPPLVTTDCMIQDQGNASPRFIRCTTYCFPCTSDMAKQAQIPLAAVIKPFATIPSNESPLYLVNHGESGPVRCNRCKAYMCPFMQFIEGGRRYQCGFCNCVNDVPPFYFQHLDHIGRRLDHYEKPELSLGSYEYVATLDYCRKSKPPNPPAFIFMIDVSYSNIKNGLVKLICEELKTMLEKIPKEEQEETSAIRVGFITYNKVLHFFNVKSNLAQPQMMVVTDVGEVFVPLLDGFLVNYQESQSVIHNLLDQIPDMFADSNENETVFAPVIQAGMEALKAADCPGKLFIFHSSLPTAEAPGKLKNRDDKKLVNTDKEKILFQPQTNVYDSLAKDCVAHGCSVTLFLFPSQYVDVASLGLVPQLTGGTLYKYNNFQMHLDRQQFLNDLRNDIEKKIGFDAIMRVRTSTGFRATDFFGGILMNNTTDVEMAAIDCDKAVTVEFKHDDKLSEDSGALIQCAVLYTTISGQRRLRIHNLGLNCSSQLADLYKSCETDALINFFAKSAFKAVLHQPLKVIREILVNQTAHMLACYRKNCASPSAASQLILPDSMKVLPVYMNCLLKNCVLLSRPEISTDERAYQRQLVMTMGVADSQLFFYPQLLPIHTLDVKSTMLPAAVRCSESRLSEEGIFLLANGLHMFLWLGVSSPPELIQGIFNVPSFAHINTDMTLLPEVGNPYSQQLRMIMGIIQQKRPYSMKLTIVKQREQPEMVFRQFLVEDKGLYGGSSYVDFLCCVHKEICQLLN B46E566F096F37F0 sec23a-pctk3-1 4 9 unspecified role unspecified role bait bait 6 unspecified role unspecified role prey prey region sufficient to bind sufficient binding region sufficient to bind full dna sequence full identification by DNA sequencing full dna sequence certain certain sequence position certain certain certain sequence position certain physical association physical association We screened a human brain cDNA library using the two-hybrid method to identify proteins that interact with Sec23Ap sec23a-pctk3-2 5 6 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 9 unspecified role unspecified role prey prey physical association physical association Thus, the central regions of both PCTAIRE-3a and -3b (Herskovits and Davies, 2004Go) interact with Sec23Ap The central region of PCTK3 shown to interact with SEC23A is common to all isoforms of PCTK3 known at time of publication. The interaction was not observed with full length PTCK3. Fig 1A, B, C sec24d-pctk3-1 5 10 unspecified role unspecified role prey prey 6 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association A weaker interaction was also detectable with Sec24Dp The interaction was observed to weaker than that between PCTK3 and SEC24A. The interaction was not observed with full length PTCK3. Fig 1A, B, C sec23a-pctk1-1 5 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey physical association physical association A specific interaction was seen between Sec23Ap and the region of PCTAIRE-1 corresponding to that of PCTAIRE-3 identified in the initial screen (exons 4-8). The interacting region of PCTK1 has been inferred by alignment to PCTK3. The interaction was not observed with full length PTCK3. Fig 1C sec24d-pctk1-1 5 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association Again, an interaction with Sec24Dp was also detectable (Fig. 1C). The interacting region of PCTK1 has been inferred by alignment to PCTK3. The interaction was not observed with full length PTCK1. Fig 1C sec23a-pctk1-2 1 9 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 spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection 8 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 Fig. 2 shows that hSec23Ap can be specifically captured by immobilized GST-PCTAIRE (lane 2). Fig 2 sec23a-pctk1-3 3 8 unspecified role unspecified role bait bait ecobb Escherichia coli (strain B / BL21) 9 unspecified role unspecified role prey prey spofr-sf_9 Spodoptera frugiperda insect cells Fall armyworm sf_9 insect cells Derived from pupal ovarian tissue of the fall armyworm Cloned from the parent cell line, IPLB-SF-21-AE (SF-21) derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae cells are susceptible to Baculovirus infection physical association physical association We were able to co-immunoprecipitate hSec23Ap with PCTAIRE-3 from MDCK cell lysates (Fig. 3B, lanes 1-3), as well as with PCTAIRE-1 from HeLa cell lysates (Fig. 3B, lanes 4-6). Fig 3B sec23a-pctk3-3 2 7 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey physical association physical association We were able to co-immunoprecipitate hSec23Ap with PCTAIRE-3 from MDCK cell lysates (Fig. 3B, lanes 1-3), as well as with PCTAIRE-1 from HeLa cell lysates (Fig. 3B, lanes 4-6). Fig 3C