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/ The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2. The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2. Biochem. Biophys. Res. Commun. (0006-291X) 2005 imex curation Horn J., Lopez I., Miller MW., Gomez-Cambronero J. Cancer - Interactions investigated in the context of cancer COS-7 cells stably transfected with and over-expressing human myc-PLD2 were used. The PTP1B is stated to be from Calbiochem. The Calbiochem site describes the recombinant PTP1B as human (Cat. No. 539735) and expressed in E. coli. julian.cambronero@wright.edu 29-OCT-2007: Contacted by JYOTI. Only protein-protein interactions chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Horn J., Lopez I., Miller MW., Gomez-Cambronero J. Cancer - Interactions investigated in the context of cancer COS-7 cells stably transfected with and over-expressing human myc-PLD2 were used. julian.cambronero@wright.edu Accepted 2007-JUL-20 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2005 Only protein-protein interactions imex curation The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2. The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2. Biochem. Biophys. Res. Commun. (0006-291X) 2005 imex curation Horn J., Lopez I., Miller MW., Gomez-Cambronero J. Cancer - Interactions investigated in the context of cancer COS-7 cells stably transfected with and over-expressing human myc-PLD2 were used. The PTP1B is stated to be from Calbiochem. The Calbiochem site describes the recombinant PTP1B as human (Cat. No. 539735) and expressed in E. coli. julian.cambronero@wright.edu 29-OCT-2007: Contacted by JYOTI. Only protein-protein interactions in vitro In vitro phosphatase assay phosphatase assay predetermined predetermined participant predetermined Horn J., Lopez I., Miller MW., Gomez-Cambronero J. Cancer - Interactions investigated in the context of cancer COS-7 cells stably transfected with and over-expressing human myc-PLD2 were used. The PTP1B is stated to be from Calbiochem. The Calbiochem site describes the recombinant PTP1B as human (Cat. No. 539735) and expressed in E. coli. julian.cambronero@wright.edu Accepted 2007-JUL-20 by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2005 Only protein-protein interactions imex curation The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2. The uncovering of a novel regulatory mechanism for PLD2: formation of a ternary complex with protein tyrosine phosphatase PTP1B and growth factor receptor-bound protein GRB2. Biochem. Biophys. Res. Commun. (0006-291X) 2005 imex curation Horn J., Lopez I., Miller MW., Gomez-Cambronero J. Cancer - Interactions investigated in the context of cancer COS-7 cells stably transfected with and over-expressing human myc-PLD2 were used. The PTP1B is stated to be from Calbiochem. The Calbiochem site describes the recombinant PTP1B as human (Cat. No. 539735) and expressed in E. coli. julian.cambronero@wright.edu 29-OCT-2007: Contacted by JYOTI. Only protein-protein interactions chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Horn J., Lopez I., Miller MW., Gomez-Cambronero J. Cancer - Interactions investigated in the context of cancer COS-7 cells stably transfected with and over-expressing human myc-PLD2 were used. julian.cambronero@wright.edu Accepted 2007-JUL-20 by JYOTI. 29-OCT-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2005 Only protein-protein interactions imex curation pld2_human Phospholipase D2 Choline phosphatase 2 Phosphatidylcholine-hydrolyzing phospholipase D2 PLD1C PLD2 protein protein human Homo sapiens Human MTATPESLFPTGDELDSSQLQMESDEVDTLKEGEDPADRMHPFLAIYELQSLKVHPLVFAPGVPVTAQVVGTERYTSGSKVGTCTLYSVRLTHGDFSWTTKKKYRHFQELHRDLLRHKVLMSLLPLARFAVAYSPARDAGNREMPSLPRAGPEGSTRHAASKQKYLENYLNRLLTMSFYRNYHAMTEFLEVSQLSFIPDLGRKGLEGMIRKRSGGHRVPGLTCCGRDQVCYRWSKRWLVVKDSFLLYMCLETGAISFVQLFDPGFEVQVGKRSTEARHGVRIDTSHRSLILKCSSYRQARWWAQEITELAQGPGRDFLQLHRHDSYAPPRPGTLARWFVNGAGYFAAVADAILRAQEEIFITDWWLSPEVYLKRPAHSDDWRLDIMLKRKAEEGVRVSILLFKEVELALGINSGYSKRALMLLHPNIKVMRHPDQVTLWAHHEKLLVVDQVVAFLGGLDLAYGRWDDLHYRLTDLGDSSESAASQPPTPRPDSPATPDLSHNQFFWLGKDYSNLITKDWVQLDRPFEDFIDRETTPRMPWRDVGVVVHGLPARDLARHFIQRWNFTKTTKAKYKTPTYPYLLPKSTSTANQLPFTLPGGQCTTVQVLRSVDRWSAGTLENSILNAYLHTIRESQHFLYIENQFFISCSDGRTVLNKVGDEIVDRILKAHKQGWCYRVYVLLPLLPGFEGDISTGGGNSIQAILHFTYRTLCRGEYSILHRLKAAMGTAWRDYISICGLRTHGELGGHPVSELIYIHSKVLIADDRTVIIGSANINDRSLLGKRDSELAVLIEDTETEPSLMNGAEYQAGRFALSLRKHCFGVILGANTRPDLDLRDPICDDFFQLWQDMAESNANIYEQIFRCLPSNATRSLRTLREYVAVEPLATVSPPLARSELTQVQGHLVHFPLKFLEDESLLPPLGSKEGMIPLEVWT DIP protein O14939 original sequence version: 128 F25F6B73B45F57ED ptn1_human Tyrosine-protein phosphatase non-receptor type 1 PTPN1 PTP1B Protein-tyrosine phosphatase 1B protein protein human Homo sapiens Human MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEKGSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDEDHALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT DIP protein P18031 original sequence version: 141 DIP protein P18031 original sequence version: 191 802377DCD33F41FD 4-nitrophenyl dihydrogen phosphate 4-nitrophenyl dihydrogen phosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) ptpn1_cerae_protein Cercopithecus aethiops PTP1B protein protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey An amino acid sequence which is not represented in UniProt Similar to P18031 grb2_cerae_protein Cercopithecus aethiops Grb2 protein protein protein chlae Chlorocebus aethiops Cercopithecus aethiops Grivet Green monkey An amino acid sequence which is not represented in UniProt Similar to P62993 ptpn1-c6h6no6p 2 6 enzyme target enzyme target substrate substrate unspecified role unspecified role 5 enzyme enzyme unspecified role unspecified role ecoli Escherichia coli (strain K12) dephosphorylation dephosphorylation reaction dephosphorylation 4 we assayed PTP1B activity using PNPP as a substrate to determine if phosphatase activity was lost or altered under the experimental conditions. The results presented in Fig. 4A indicate that PTP1B was active in a concentration-dependent manner in the buffers used above. Moreover, under these conditions, PTP1B exhibited its normal range of regulation: stimulation by millimolar concentrations of DTT and EDTA, and inhibition by micromolar concentrations of ZnCl2 DTT - the presence of DTT (2.0 E-4 M or 5.0 E-4 M) stimulates the phosphatase activity of PTP1B. EDTA - the presence of EDTA (1.0 E-3 M or 2.0 E-3 M) stimulates the phosphatase activity of PTP1B. ZnCl2 - the presence of ZnCl2 (5.0 E-2 M or 1.0 E-1 M) inhibits the phosphatase activity of PTP1B. grb2-pld2 3 4 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role bait bait physical association physical association 8 A Fig. 8A shows that PLD2 was immunoprecipitated with Grb2-specific antibodies; an interaction that was stimulated by addition of recombinant Grb2 to the lysates prior to immunoprecipitation. O14939 transfection O14939 over-expressed Grb2 - addition of recombinant Grb2 to the lysates prior to immunoprecipitation stimulated the interaction. grb2-ptpn1 3 7 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association 9 A PTP1B - addition of recombinant PTP1B to the lysates prior to immunoprecipitation stimulated the interaction. Similarly, PTP1B and Grb2 were reciprocally pulled down with Grb2 and PTP1B-specific antibodies, respectively (Fig. 9A). ptpn1-pld2 3 4 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role bait bait physical association physical association 9 B PTP1B - addition of recombinant PTP1B to the lysates prior to immunoprecipitation stimulated the interaction. Fig. 9B shows that PTP1B and PLD2 were reciprocally co-immunoprecipitable. O14939 transfection O14939 over-expressed pld2-grb2 1 8 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 8 B O14939 transfection O14939 over-expressed Grb2 - addition of recombinant Grb2 to the lysates prior to immunoprecipitation stimulated the interaction. Grb2 was pulled down with myc-specific antibodies pld2-ptp1b 1 4 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey physical association physical association 9 B PTP1B - addition of recombinant PTP1B to the lysates prior to immunoprecipitation stimulated the interaction. Fig. 9B shows that PTP1B and PLD2 were reciprocally co-immunoprecipitable. O14939 transfection O14939 over-expressed