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/ Expression of the SNT-1/FRS2 phosphotyrosine binding domain inhibits activation of MAP kinase and PI3-kinase pathways and antiestrogen resistant growth induced by FGF-1 in human breast carcinoma cells. Expression of the SNT-1/FRS2 phosphotyrosine binding domain inhibits activation of MAP kinase and PI3-kinase pathways and antiestrogen resistant growth induced by FGF-1 in human breast carcinoma cells. Oncogene (0950-9232) 2006 imex curation Manuvakhova M., Thottassery JV., Hays S., Qu Z., Rentz SS., Westbrook L., Kern FG. Cancer - Interactions investigated in the context of cancer Could not find the species of the Raf-1 RBD used so did not curate Fig. 3. went to the Upstate Biotech website for the p13Suc1 species. p13Suc1 agarose was used to precipitate endogenous SNT-1 from cellular lysates. The p13Suc1 agarose captured fractions from total cell lysates were analysed by electrophoresis on polyacrylamide gel followed by immunoblotting with anti-phosphotyrosine, anti-FRS2, Gab1 and Sos1 antibodies.the cell line used contains a Dox inducible myc tagged SNT-1 PTB fusion protein. fkern@lexgen.com 01-DEC-2006: Contacted by JYOTI. Only protein-protein interactions human-c9 Homo sapiens C9 cell line c9 Human C9 cell line C9 cells are are a stable ML20 line transfected with CMV-driven rtTA2S-M2. The breast cancer cell line ML20 is a clonally derived subline of MCF-7 that is stably transfected with a LacZ expression vector. pull down pull down western blot western blot Immuno blot Immuno blot Manuvakhova M., Thottassery JV., Hays S., Qu Z., Rentz SS., Westbrook L., Kern FG. Cancer - Interactions investigated in the context of cancer Could not find the species of the Raf-1 RBD used so did not curate Fig. 3. went to the Upstate Biotech website for the p13Suc1 species. p13Suc1 agarose was used to precipitate endogenous SNT-1 from cellular lysates. The p13Suc1 agarose captured fractions from total cell lysates were analysed by electrophoresis on polyacrylamide gel followed by immunoblotting with anti-phosphotyrosine, anti-FRS2, Gab1 and Sos1 antibodies.the cell line used contains a Dox inducible myc tagged SNT-1 PTB fusion protein. fkern@lexgen.com Accepted 2006-NOV-20 by DAVET. 01-DEC-2006: Contacted by JYOTI. Oncogene (0950-9232) 2006 Only protein-protein interactions imex curation sos1_human Son of sevenless homolog 1 SOS1 protein protein human Homo sapiens Human MQAQQLPYEFFSEENAPKWRGLLVPALKKVQGQVHPTLESNDDALQYVEELILQLLNMLCQAQPRSASDVEERVQKSFPHPIDKWAIADAQSAIEKRKRRNPLSLPVEKIHPLLKEVLGYKIDHQVSVYIVAVLEYISADILKLVGNYVRNIRHYEITKQDIKVAMCADKVLMDMFHQDVEDINILSLTDEEPSTSGEQTYYDLVKAFMAEIRQYIRELNLIIKVFREPFVSNSKLFSANDVENIFSRIVDIHELSVKLLGHIEDTVEMTDEGSPHPLVGSCFEDLAEELAFDPYESYARDILRPGFHDRFLSQLSKPGAALYLQSIGEGFKEAVQYVLPRLLLAPVYHCLHYFELLKQLEEKSEDQEDKECLKQAITALLNVQSGMEKICSKSLAKRRLSESACRFYSQQMKGKQLAIKKMNEIQKNIDGWEGKDIGQCCNEFIMEGTLTRVGAKHERHIFLFDGLMICCKSNHGQPRLPGASNAEYRLKEKFFMRKVQINDKDDTNEYKHAFEIILKDENSVIFSAKSAEEKNNWMAALISLQYRSTLERMLDVTMLQEEKEEQMRLPSADVYRFAEPDSEENIIFEENMQPKAGIPIIKAGTVIKLIERLTYHMYADPNFVRTFLTTYRSFCKPQELLSLIIERFEIPEPEPTEADRIAIENGDQPLSAELKRFRKEYIQPVQLRVLNVCRHWVEHHFYDFERDAYLLQRMEEFIGTVRGKAMKKWVESITKIIQRKKIARDNGPGHNITFQSSPPTVEWHISRPGHIETFDLLTLHPIEIARQLTLLESDLYRAVQPSELVGSVWTKEDKEINSPNLLKMIRHTTNLTLWFEKCIVETENLEERVAVVSRIIEILQVFQELNNFNGVLEVVSAMNSSPVYRLDHTFEQIPSRQKKILEEAHELSEDHYKKYLAKLRSINPPCVPFFGIYLTNILKTEEGNPEVLKRHGKELINFSKRRKVAEITGEIQQYQNQPYCLRVESDIKRFFENLNPMGNSMEKEFTDYLFNKSLEIEPRNPKPLPRFPKKYSYPLKSPGVRPSNPRPGTMRHPTPLQQEPRKISYSRIPESETESTASAPNSPRTPLTPPPASGASSTTDVCSVFDSDHSSPFHSSNDTVFIQVTLPHGPRSASVSSISLTKGTDEVPVPPPVPPRRRPESAPAESSPSKIMSKHLDSPPAIPPRQPTSKAYSPRYSISDRTSISDPPESPPLLPPREPVRTPDVFSSSPLHLQPPPLGKKSDHGNAFFPNSPSPFTPPPPQTPSPHGTRRHLPSPPLTQEVDLHSIAGPPVPPRQSTSQHIPKLPPKTYKREHTHPSMHRDGPPLLENAHSS DIP protein Q07889 original sequence version: 117 DIP protein Q07889 original sequence version: 116 DIP protein Q07889 original sequence version: 124 DIP protein Q07889 original sequence version: 171 C6B99CCA11A8DE45 cks1_schpo Cyclin-dependent kinases regulatory subunit suc1 SPBC1734.14c P13 protein protein schpo Schizosaccharomyces pombe (strain ATCC 38366 / 972) Fission yeast MSKSGVPRLLTASERERLEPFIDQIHYSPRYADDEYEYRHVMLPKAMLKAIPTDYFNPETGTLRILQEEEWRGLGITQSLGWEMYEVHVPEPHILLFKREKDYQMKFSQQRGG 1CE50666AAD8264F frs2_human Fibroblast growth factor receptor substrate 2 FRS2 Suc1-associated neurotrophic factor target 1 FGFR-signaling adaptor SNT protein protein human Homo sapiens Human MGSCCSCPDKDTVPDNHRNKFKVINVDDDGNELGSGIMELTDTELILYTRKRDSVKWHYLCLRRYGYDSNLFSFESGRRCQTGQGIFAFKCARAEELFNMLQEIMQNNSINVVEEPVVERNNHQTELEVPRTPRTPTTPGFAAQNLPNGYPRYPSFGDASSHPSSRHPSVGSARLPSVGEESTHPLLVAEEQVHTYVNTTGVQEERKNRTSVHVPLEARVSNAESSTPKEEPSSIEDRDPQILLEPEGVKFVLGPTPVQKQLMEKEKLEQLGRDQVSGSGANNTEWDTGYDSDERRDAPSVNKLVYENINGLSIPSASGVRRGRLTSTSTSDTQNINNSAQRRTALLNYENLPSLPPVWEARKLSRDEDDNLGPKTPSLNGYHNNLDPMHNYVNTENVTVPASAHKIEYSRRRDCTPTVFNFDIRRPSLEHRQLNYIQVDLEGGSDSDNPQTPKTPTTPLPQTPTRRTELYAVIDIERTAAMSNLQKALPRDDGTSRKTRHNSTDLPM 833714EE12097C75 gab1_human GRB2-associated-binding protein 1 Growth factor receptor bound protein 2-associated protein 1 GRB2-associated binder 1 GAB1 protein protein human Homo sapiens Human MSGGEVVCSGWLRKSPPEKKLKRYAWKRRWFVLRSGRLTGDPDVLEYYKNDHAKKPIRIIDLNLCQQVDAGLTFNKKEFENSYIFDINTIDRIFYLVADSEEEMNKWVRCICDICGFNPTEEDPVKPPGSSLQAPADLPLAINTAPPSTQADSSSATLPPPYQLINVPPHLETLGIQEDPQDYLLLINCQSKKPEPTRTHADSAKSTSSETDCNDNVPSHKNPASSQSKHGMNGFFQQQMIYDSPPSRAPSASVDSSLYNLPRSYSHDVLPKVSPSSTEADGELYVFNTPSGTSSVETQMRHVSISYDIPPTPGNTYQIPRTFPEGTLGQTSKLDTIPDIPPPRPPKPHPAHDRSPVETCSIPRTASDTDSSYCIPTAGMSPSRSNTISTVDLNKLRKDASSQDCYDIPRAFPSDRSSSLEGFHNHFKVKNVLTVGSVSSEELDENYVPMNPNSPPRQHSSSFTEPIQEANYVPMTPGTFDFSSFGMQVPPPAHMGFRSSPKTPPRRPVPVADCEPPPVDRNLKPDRKVKPAPLEIKPLPEWEELQAPVRSPITRSFARDSSRFPMSPRPDSVHSTTSSSDSHDSEENYVPMNPNLSSEDPNLFGSNSLDGGSSPMIKPKGDKQVEYLDLDLDSGKSTPPRKQKSSGSGSSVADERVDYVVVDQQKTLALKSTREAWTDGRQSTESETPAKSVK E0FAC78772B6677B suc1-gab1 1 4 unspecified role unspecified role prey prey region optyr O4'-phospho-L-tyrosine (2S)-2-amino-3-(4-phosphonooxyphenyl)propanoic acid 2-azanyl-3-(4-phosphonooxyphenyl)propanoic acid MOD_RES Phosphotyrosine O4-phosphotyrosine tyrosine phosphate 2-amino-3-(4-hydroxyphenyl)propanoic acid 4'-phosphate O4'-phospho-L-tyrosine O4'-phosphorylated L-tyrosine OPTyr Phospho Phospho Tyrosinyl Phosphorylation phosphorylationy undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 5 unspecified role unspecified role prey prey 2 unspecified role unspecified role prey prey 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 association association 2 A Fibroblast growth factor-1 supplementation of the media caused tyrosine phosphorylation of endogenous SNT-1 in control cells transfected with vector pTRE-Ins alone as evidenced by the increased intensity of two bands following Western blotting with antiphosphotyrosine antibody. Two bands were observed with this antibody because SNT-1/FRS2 undergoes an electrophoretic mobility shift that is caused by its phosphorylation on serine/threonine residues in response to FGF stimulation (Kouhara et al., 1997; Lax et al., 2002). Induction of SNT-1 PTB with Dox blocked the FGF-1-induced tyrosine phosphorylation of endogenous SNT-1 in Clone 29 cells upon Dox induction of SNT-1 PTB, the involvement of Gab1 and Sos1 in the multiprotein complex with SNT-1 in FGF-dependent conditions was significantly decreased in Clone 29 cells but not in control cells. Thus, abrogation of the recruitment of multiple proteins to the C-terminus of wild-type SNT-1 confirmed the dominant negative action of the SNT-1 PTB domain in MCF-7 cells. FGF-1 stimulates tyrosine phosphorylation of endogenous SNT-1. FGF-1 stimulates association of Sos1 and Gab1 with SNT-1. The induction of SNT-1 PTB with Dox inhibits the association of Sos1 and Gab1 with SNT-1. The induction of SNT-1 PTB with Dox inhibits the FGF-1-induced phosphorylation of endogenous SNT-1. Two bands were seen with the antiphosphotyrosine antibody as SNT-1 undergoes an electrophoretic mobility shift due to phosphorylation on serine or threonine residues in response to FGF stimulation.