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/ Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. Additional detail on Src-related kinases taken from manufacturers website ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Only protein-protein interactions human-jurkat Homo sapiens jurkat Human T cell leukemia anti bait coip anti bait coimmunoprecipitation anti bait coip anti tag western anti tag western blot anti tag western Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Only protein-protein interactions in vitro In vitro far western blotting far western blotting Affinity blotting Affinity blotting predetermined predetermined participant predetermined Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. Identity of Grb2 assumed to be the same as in related paper PMID: 15467741 ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. Additional details of Src-related kinases taken from manufacturers website ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Only protein-protein interactions mouse-cad Mouse neuronal cell line cad Mouse neuronal cell line anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation. Biochem. Biophys. Res. Commun. (0006-291X) 2006 imex curation Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA 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 Lee MF., Beauchamp RL., Beyer KS., Gusella JF., Ramesh V. ramesh@helix.mgh.harvard.edu Accepted 2007-APR-1 by JYOTI. 12-MAR-2007: Contacted by JYOTI. Human fetal frontal cortex cDNA Biochem. Biophys. Res. Commun. (0006-291X) 2006 Only protein-protein interactions imex curation fyn_human Tyrosine-protein kinase Fyn p59-Fyn Proto-oncogene c-Fyn Src-like kinase FYN Proto-oncogene Syn protein protein human Homo sapiens Human MGCVQCKDKEATKLTEERDGSLNQSSGYRYGTDPTPQHYPSFGVTSIPNYNNFHAAGGQGLTVFGGVNSSSHTGTLRTRGGTGVTLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYVAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIRESETTKGAYSLSIRDWDDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQHYSERAAGLCCRLVVPCHKGMPRLTDLSVKTKDVWEIPRESLQLIKRLGNGQFGEVWMGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEYMNKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANILVGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQSFLEDYFTATEPQYQPGENL DIP protein P06241 original sequence version: 195 DIP protein P06241 original sequence version: 147 DIP protein P06241 original sequence version: 164 4A1E443A4B5A0977 fyn_mouse Tyrosine-protein kinase Fyn p59-Fyn Fyn Proto-oncogene c-Fyn protein protein mouse Mus musculus Mouse MGCVQCKDKEAAKLTEERDGSLNQSSGYRYGTDPTPQHYPSFGVTSIPNYNNFHAAGGQGLTVFGGVNSSSHTGTLRTRGGTGVTLFVALYDYEARTEDDLSFHKGEKFQILNSSEGDWWEARSLTTGETGYIPSNYVAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTFLIRESETTKGAYSLSIRDWDDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQHYSERAAGLCCRLVVPCHKGMPRLTDLSVKTKDVWEIPRESLQLIKRLGNGQFGEVWMGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANILVGNGLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELVTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEYLQGFLEDYFTATEPQYQPGENL 99558702596DAEE0 grb2_human Growth factor receptor-bound protein 2 GRB2 ASH Adapter protein GRB2 SH2/SH3 adapter GRB2 Protein Ash protein protein human Homo sapiens Human MEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNV 83A4B0BA1B248DC4 med28_human Mediator of RNA polymerase II transcription subunit 28 Mediator complex subunit 28 Tumor angiogenesis marker EG-1 Endothelial-derived protein 1 Merlin and Grb2-interacting cytoskeletal protein MED28 EG1 FKSG20 protein protein human Homo sapiens Human MAAPLGGMFSGQPPGPPQAPPGLPGQASLLQAAPGAPRPSSSTLVDELESSFEACFASLVSQDYVNGTDQEEIRTGVDQCIQKFLDIARQTECFFLQKRLQLSVQKPEQVIKEDVSELRNELQRKDALVQKHLTKLRHWQQVLEDINVQHKKPADIPQGSLAYLEQASANIPAPLKPT 63DE023972908066 src_human Proto-oncogene tyrosine-protein kinase Src pp60c-src SRC SRC1 Proto-oncogene c-Src protein protein human Homo sapiens Human MGSNKSKPKDASQRRRSLEPAENVHGAGGGAFPASQTPSKPASADGHRGPSAAFAPAAAEPKLFGGFNSSDTVTSPQRAGPLAGGVTTFVALYDYESRTETDLSFKKGERLQIVNNTEGDWWLAHSLSTGQTGYIPSNYVAPSDSIQAEEWYFGKITRRESERLLLNAENPRGTFLVRESETTKGAYCLSVSDFDNAKGLNVKHYKIRKLDSGGFYITSRTQFNSLQQLVAYYSKHADGLCHRLTTVCPTSKPQTQGLAKDAWEIPRESLRLEVKLGQGCFGEVWMGTWNGTTRVAIKTLKPGTMSPEAFLQEAQVMKKLRHEKLVQLYAVVSEEPIYIVTEYMSKGSLLDFLKGETGKYLRLPQLVDMAAQIASGMAYVERMNYVHRDLRAANILVGENLVCKVADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTELTTKGRVPYPGMVNREVLDQVERGYRMPCPPECPESLHDLMCQCWRKEPEERPTFEYLQAFLEDYFTSTEPQYQPGENL DIP protein P12931 original sequence version: 188 DIP protein P12931 original sequence version: 189 DIP protein P12931 original sequence version: 190 DIP protein P12931 original sequence version: 191 DIP protein P12931 original sequence version: 115 DIP protein P12931 original sequence version: 116 DIP protein P12931 original sequence version: 164 DIP protein P12931 original sequence version: 142 DIP protein P12931 original sequence version: 143 DIP protein P12931 original sequence version: 150 DIP protein P12931 original sequence version: 160 DIP protein P12931 original sequence version: 184 DIP protein P12931 original sequence version: 155 DIP protein P12931-1 original sequence version: 155 DIP protein P12931 original sequence version: 202 C1908084683E5DE8 med28_mouse Mediator of RNA polymerase II transcription subunit 28 Med28 Fksg20 Mediator complex subunit 28 protein protein mouse Mus musculus Mouse MAASLGGMFTGQPPGPPPPPPGLPGQASLLQAAPGAPRPSNSTLVDELESSFEACFASLVSQDYVNGTDQEEIRTGVDQCIQKFLDIARQTECFFLQKRLQLSVQKPDQVIKEDVSELRSELQRKDALVQKHLTKLRHWQQVLEDINVQHKKPADMPQGSLAFLEQASANIPAPLKQT 947FDA98E806EA13 lck_human Tyrosine-protein kinase Lck Protein YT16 Proto-oncogene Lck T cell-specific protein-tyrosine kinase LCK Lymphocyte cell-specific protein-tyrosine kinase p56-LCK Leukocyte C-terminal Src kinase protein protein human Homo sapiens Human MGCGCSSHPEDDWMENIDVCENCHYPIVPLDGKGTLLIRNGSEVRDPLVTYEGSNPPASPLQDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWKAQSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHELVRHYTNASDGLCTRLSRPCQTQKPQKPWWEDEWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPDAFLAEANLMKQLQHQRLVRLYAVVTQEPIYIITEYMENGSLVDFLKTPSGIKLTINKLLDMAAQIAEGMAFIEERNYIHRDLRAANILVSDTLSCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEAINYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEVIQNLERGYRMVRPDNCPEELYQLMRLCWKERPEDRPTFDYLRSVLEDFFTATEGQYQPQP DIP protein P06239 original sequence version: 164 DIP protein P06239-1 original sequence version: 193 44BFF0D43FFB420D med28-fyn-1 7 8 unspecified role unspecified role prey prey 11 unspecified role unspecified role bait bait c-terminus 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 Table 1 Identification of Fyn as a magicin-specific binding protein in the yeast two-hybrid assay med28-fyn-2 6 11 unspecified role unspecified role bait bait c-terminus sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag certain certain sequence position certain certain certain sequence position certain 9 unspecified role unspecified role prey prey mouse-cad Mouse neuronal cell line cad Mouse neuronal cell line physical association physical association fig. 2A Pull-down (Fig 2A) and coimmunoprecipitation (Fig. 2B) experiments in CAD cells confirmed the interaction of CAD and Fyn med28-fyn-3 5 13 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey mouse-cad Mouse neuronal cell line cad Mouse neuronal cell line physical association physical association fig. 2B Pull-down (Fig 2A) and coimmunoprecipitation (Fig. 2B) experiments in CAD cells confirmed the interaction of CAD and Fyn fyn-med28-1 1 11 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 8 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. phosphorylation phosphorylation reaction phosphorylation Magicin is phosphorylated by Fyn kinase in vitro. 3 Phosphorylation of MED28 fyn-med28-2 4 11 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 8 unspecified role unspecified role bait bait sh3 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range 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) physical association physical association Magicin interacts with the SH3 domains of Src family proteins. 4A lck-med28-1 4 11 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. 14 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 sh3 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range ecoli Escherichia coli (strain K12) physical association physical association Magicin interacts with the SH3 domains of Src family proteins. 4A src-med28-1 4 12 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 sh3 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined human-293t Homo sapiens 293 cells transformed with SV40 large T antigen 293t 293 cells expressing SV40 large T antigen. The original designation of this cell line was 293tsA1609neo. It is not in Cabri,ATCC or HyperCLDB. physical association physical association Magicin interacts with the SH3 domains of Src family proteins. 4A fyn-med28-3 2 8 unspecified role unspecified role bait bait 11 unspecified role unspecified role prey prey physical association physical association Coimmunoprecipitations performed with Fyn, Lck or anti-magicin (Tim3) antibodies revealed anin vivo association of magicin with both Fyn and Lck at the endogenous levels in Jurkat cells (Fig. 4B). 4B fyn-med28-4 2 11 unspecified role unspecified role bait bait 14 unspecified role unspecified role prey prey 8 unspecified role unspecified role prey prey association association Coimmunoprecipitations performed with Fyn, Lck or anti-magicin (Tim3) antibodies revealed anin vivo association of magicin with both Fyn and Lck at the endogenous levels in Jurkat cells (Fig. 4B). 4B lck-med28-3 1 14 enzyme enzyme unspecified role unspecified role n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range spofr-sf_21 Spodoptera frugiperda insect cells sf_21 Insect cells derived from immature ovaries of fall armyworm Spodoptera frugiperda pupae. full name: IPLB-SF-21-AE cells are susceptible to Baculovirus infection. 11 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) phosphorylation phosphorylation reaction phosphorylation We mutated these residues (Y64F or Y163F) in His-tagged magicin by site-directed mutagenesis and performed in vitro kinase assays in the presence of Lck. 3 Phosphorylation of MED28 - primarily on Tyr-64 (mutational analysis) med28-grb2-4 3 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 sh2 region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role prey prey region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region (required) 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 ecoli Escherichia coli (strain K12) 65 68 direct interaction direct interaction In order to test whether phospho-magicin and the SH2 domain of Grb2 directly interact, and to avoid phosphorylation-independent interaction with the SH3 domains of Grb2, we carried out blot overlay experiments employing phosphorylated magicin and GST-Grb2(SH2). 5D