MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. Molecular oncology 2013 imex curation Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se 27-Mar-2018: Contacted by IntAct-Help. Only protein-protein interactions imex curation chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se Accepted 2018-MAR-05 AT 16:35 GMT AT 16:35 GMT by LUANA Molecular oncology 2013 Only protein-protein interactions imex curation imex curation FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. Molecular oncology 2013 imex curation Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se 27-Mar-2018: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se Accepted 2018-MAR-05 AT 16:35 GMT AT 16:35 GMT by LUANA Molecular oncology 2013 Only protein-protein interactions imex curation imex curation FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. Molecular oncology 2013 imex curation Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se 27-Mar-2018: Contacted by IntAct-Help. Only protein-protein interactions imex curation human-oci_aml5 human-oci_aml5 oci_aml5 oci_aml5 anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se Accepted 2018-MAR-05 AT 16:35 GMT AT 16:35 GMT by LUANA Molecular oncology 2013 Only protein-protein interactions imex curation imex curation FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. FLT3 signals via the adapter protein Grb10 and overexpression of Grb10 leads to aberrant cell proliferation in acute myeloid leukemia. Molecular oncology 2013 imex curation Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se 27-Mar-2018: Contacted by IntAct-Help. Only protein-protein interactions imex curation chlae-cos_1 Chlorocebus aethiops COS-1 cells Grivet cos_1 CV-1 Simian cells (cercopithecus aethiops) transformed by SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Kazi JU., Rönnstrand L. Cancer - Interactions investigated in the context of cancer Lars.Ronnstrand@med.lu.se Accepted 2018-MAR-05 AT 16:35 GMT AT 16:35 GMT by LUANA Molecular oncology 2013 Only protein-protein interactions imex curation imex curation flt3_human Receptor-type tyrosine-protein kinase FLT3 FLK2 FL cytokine receptor Fetal liver kinase-2 FLT3 STK1 Fms-like tyrosine kinase 3 Stem cell tyrosine kinase 1 CD135 protein protein human Homo sapiens Human MPALARDGGQLPLLVVFSAMIFGTITNQDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVEVDVSASITLQVLVDAPGNISCLWVFKHSSLNCQPHFDLQNRGVVSMVILKMTETQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQGESCKEESPAVVKKEEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNISFYATIGVCLLFIVVLTLLICHKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKEKADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLHGNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDSNYVVRGNARLPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDGRVSECPHTYQNRRPFSREMDLGLLSPQAQVEDS DIP protein P36888 original sequence version: 118 grb10_human Growth factor receptor-bound protein 10 GRB10 GRBIR KIAA0207 GRB10 adapter protein Insulin receptor-binding protein Grb-IR protein protein human Homo sapiens Human MALAGCPDSFLHHPYYQDKVEQTPRSQQDPAGPGLPAQSDRLANHQEDDVDLEALVNDMNASLESLYSACSMQSDTVPLLQNGQHARSQPRASGPPRSIQPQVSPRQRVQRSQPVHILAVRRLQEEDQQFRTSSLPAIPNPFPELCGPGSPPVLTPGSLPPSQAAAKQDVKVFSEDGTSKVVEILADMTARDLCQLLVYKSHCVDDNSWTLVEHHPHLGLERCLEDHELVVQVESTMASESKFLFRKNYAKYEFFKNPMNFFPEQMVTWCQQSNGSQTQLLQNFLNSSSCPEIQGFLHVKELGKKSWKKLYVCLRRSGLYCSTKGTSKEPRHLQLLADLEDSNIFSLIAGRKQYNAPTDHGLCIKPNKVRNETKELRLLCAEDEQTRTCWMTAFRLLKYGMLLYQNYRIPQQRKALLSPFSTPVRSVSENSLVAMDFSGQTGRVIENPAEAQSAALEEGHAWRKRSTRMNILGSQSPLHPSTLSTVIHRTQHWFHGRISREESHRIIKQQGLVDGLFLLRDSQSNPKAFVLTLCHHQKIKNFQILPCEDDGQTFFSLDDGNTKFSDLIQLVDFYQLNKGVLPCKLKHHCIRVAL DIP protein Q13322 original sequence version: 114 53A5F885E17C6C6B p85a_human Phosphatidylinositol 3-kinase regulatory subunit alpha PIK3R1 GRB1 Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha protein protein human Homo sapiens Human MSAEGYQYRALYDYKKEREEDIDLHLGDILTVNKGSLVALGFSDGQEARPEEIGWLNGYNETTGERGDFPGTYVEYIGRKKISPPTPKPRPPRPLPVAPGSSKTEADVEQQALTLPDLAEQFAPPDIAPPLLIKLVEAIEKKGLECSTLYRTQSSSNLAELRQLLDCDTPSVDLEMIDVHVLADAFKRYLLDLPNPVIPAAVYSEMISLAPEVQSSEEYIQLLKKLIRSPSIPHQYWLTLQYLLKHFFKLSQTSSKNLLNARVLSEIFSPMLFRFSAASSDNTENLIKVIEILISTEWNERQPAPALPPKPPKPTTVANNGMNNNMSLQDAEWYWGDISREEVNEKLRDTADGTFLVRDASTKMHGDYTLTLRKGGNNKLIKIFHRDGKYGFSDPLTFSSVVELINHYRNESLAQYNPKLDVKLLYPVSKYQQDQVVKEDNIEAVGKKLHEYNTQFQEKSREYDRLYEEYTRTSQEIQMKRTAIEAFNETIKIFEEQCQTQERYSKEYIEKFKREGNEKEIQRIMHNYDKLKSRISEIIDSRRRLEEDLKKQAAEYREIDKRMNSIKPDLIQLRKTRDQYLMWLTQKGVRQKKLNEWLGNENTEDQYSLVEDDEDLPHHDEKTWNVGSSNRNKAENLLRGKRDGTFLVRESSKQGCYACSVVVDGEVKHCVINKTATGYGFAEPYNLYSSLKELVLHYQHTSLVQHNDSLNVTLAYPVYAQQRR DIP protein P27986 original sequence version: 181 DIP protein P27986 original sequence version: 184 DIP protein P27986-1 original sequence version: 135 DIP protein P27986 original sequence version: 135 DIP protein P27986-1 original sequence version: 145 DIP protein P27986-1 original sequence version: 147 DIP protein P27986 original sequence version: 191 B9DAD8416C33140F flt3-grb10-1 4 5 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell P36888:p.Lys644Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K A Wed May 02 19:25:28 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys644ala' Tue Jun 30 17:57:16 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys644Ala' 6 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag 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 physical association physical association f1a f1b f1d To determine whether Grb10 can also interact with other PDGF receptor tyrosine kinases, we transiently expressed FLT3 and FLAG-tagged Grb10 in COS-1 cells. We were able to immunoprecipitate FLT3 from the cell lysates using an anti-FLAG antibody A constitutive Grb10eFLT3 association was observed in COS-1 cells overexpressing Grb10 and FLT3-ITD flt3-grb10-2 1 5 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell P36888:p.Lys644Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain K A Wed May 02 17:21:13 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys644ala' Tue Jun 30 17:10:52 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys644Ala' 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell flag tag flag tag 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 physical association physical association f1a f1b f1d To determine whether Grb10 can also interact with other PDGF receptor tyrosine kinases, we transiently expressed FLT3 and FLAG-tagged Grb10 in COS-1 cells. Furthermore Grb10 was also immunoprecipitated using an anti-FLT3 antibody (Figure 1A, lower panels) A constitutive Grb10eFLT3 association was observed in COS-1 cells overexpressing Grb10 and FLT3-ITD (Figure 1B). flt3-grb10-3 3 6 unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level 5 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f1c In addition we could show Grb10eFLT3 association in OCI-AML-5 cells using an anti- FLT3 antibody (Figure 1C). The OCI-AML-5 cell line that established from the peripheral blood of a 77-year-old man expresses both FLT3-WT and Grb10 endogenously and responds to G-CSF, GM-CSF, IL-3, and FL flt3-grb10-4 2 5 peptide synthesis peptide synthesis western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait purified purified phoTyr572 phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain region binding region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate physical association physical association f3a We screened for Grb10 binding sites in FLT3 by using synthetic peptides corresponding to the FLT3 phospho-tyrosine residues. We observed that phosphotyrosine peptides corresponding to FLT3 tyrosine residues 572 and 793 can pull-down Grb10 protein from cell lysates and that pY793 residue has higher affinity to the Grb10 protein flt3-grb10-5 4 5 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell P36888:p.Tyr572Phe mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain Y F Wed May 02 14:59:50 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'tyr572phe' Tue Jun 30 18:33:13 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr572Phe' P36888:p.Tyr793Phe mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain Y F Wed May 02 14:33:16 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'tyr793phe' Tue Jun 30 20:20:19 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Tyr793Phe' 6 unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell flag tag flag tag 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 physical association physical association f3b Then we generated FLT3-Y572F/Y793F mutant to further explore the binding specificity. Co-transfection of FLT3 mutant with Grb10 shows a decrease of binding in the cells expressing FLT3-Y572F/Y793F mutant grb10-pik3r1-1 1 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell 7 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level P85 is physiologically expressed in COS-1 cells, but its uniprotID needs to be update. Since the antibody, used to detect it, has been raised against the human isoform we have used Human P85 accession number. physical association physical association f7c We immunoprecipitated p85 with an anti-p85 antibody from FL stimulated and unstimulated cell lysates and observed that p85 is associated with Grb10 in response to the FLstimulation grb10-pik3r1-2 3 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell 7 unspecified role unspecified role bait bait living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f7d We immunoprecipitated p85 with an anti-p85 antibody from FL stimulated and unstimulated cell lysates and observed that p85 is associated with Grb10 in response to the FLstimulation flt3-grb10-6 2 5 peptide synthesis peptide synthesis western blot western blot Immuno blot Immuno blot unspecified role unspecified role bait bait purified purified phoTyr793 phosres phosphorylated residue PhosRes Phospho Phosphorylation Phosphorylation (O of Serine, Threonine, Tyrosine and Aspartate, N epsilon of Lysine) certain certain sequence position certain certain certain sequence position certain region binding region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 6 unspecified role unspecified role prey prey over-expressed over expressed level over-expressed cell lysate cell lysate physical association physical association f3a We screened for Grb10 binding sites in FLT3 by using synthetic peptides corresponding to the FLT3 phospho-tyrosine residues. We observed that phosphotyrosine peptides corresponding to FLT3 tyrosine residues 572 and 793 can pull-down Grb10 protein from cell lysates and that pY793 residue has higher affinity to the Grb10 protein