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/ Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. J. Biol. Chem. (0021-9258) 2002 imex curation Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA davet Only protein-protein interactions human-hs68 Homo sapiens Hs68 normal diploid foreskin fibroblasts hs68 Human Hs68 normal diploid foreskin fibroblasts confocal microscopy confocal microscopy immunostaining immunostaining Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr Accepted 2008-Not a month!-29 by DAVET. 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA J. Biol. Chem. (0021-9258) 2002 davet Only protein-protein interactions imex curation Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. J. Biol. Chem. (0021-9258) 2002 imex curation Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA davet Only protein-protein interactions in vitro In vitro elisa enzyme linked immunosorbent assay ELISA ELISA elisa antibody detection identification by antibody antibody detection Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr Accepted 2008-Not a month!-29 by DAVET. 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA J. Biol. Chem. (0021-9258) 2002 davet Only protein-protein interactions imex curation Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. J. Biol. Chem. (0021-9258) 2002 imex curation Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA davet Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr Accepted 2008-Not a month!-29 by DAVET. 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA J. Biol. Chem. (0021-9258) 2002 davet Only protein-protein interactions imex curation Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. J. Biol. Chem. (0021-9258) 2002 imex curation Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA davet 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 Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr Accepted 2008-Not a month!-29 by DAVET. 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA J. Biol. Chem. (0021-9258) 2002 davet Only protein-protein interactions imex curation Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. J. Biol. Chem. (0021-9258) 2002 imex curation Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA davet Only protein-protein interactions crigr-cho chinese hamster ovary cell line cho chinese hamster ovary cell line Also called LR73 cells. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Fournier HN., Dupe-Manet S., Bouvard D., Lacombe ML., Marie C., Block MR., Albiges-Rizo C. NDPK - Interactions involving proteins containing InterPro domain IPR001564, Nucleoside diphosphate kinase, core. block@ujf-grenoble.fr Accepted 2008-Not a month!-29 by DAVET. 09-April-2009: Contacted by JYOTI. Human placenta MATCHMAKER cDNA J. Biol. Chem. (0021-9258) 2002 davet Only protein-protein interactions imex curation ndkb_human Nucleoside diphosphate kinase B nm23-H2 C-myc purine-binding transcription factor PUF NME2 NM23B Histidine protein kinase NDKB protein protein human Homo sapiens Human MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE DIP protein P22392 original sequence version: 182 1A5C3F84D7AD272C itb1_human Integrin beta-1 Fibronectin receptor subunit beta VLA-4 subunit beta ITGB1 FNRB MDF2 MSK12 Glycoprotein IIa protein protein human Homo sapiens Human MNLQPIFWIGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPNCGWCTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGTAEKLKPEDITQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLENVKSLGTDLMNEMRRITSDFRIGFGSFVEKTVMPYISTTPAKLRNPCTSEQNCTSPFSYKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVLPNDGQCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEGVTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGFTEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVNSEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGLICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPKFQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQPVQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGIVLIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK DIP protein P05556 original sequence version: 139 DIP protein P05556-1 original sequence version: 143 DIP protein P05556 original sequence version: 154 DIP protein P05556 original sequence version: 167 DE35979C1625578C o14713-1 Integrin beta-1-binding protein 1 ITGB1BP1 ICAP1 Integrin cytoplasmic domain-associated protein 1 ICAP1-alpha protein protein human Homo sapiens Human MFRKGKKRHSSSSSQSSEISTKSKSVDSSLGGLSRSSTVASLDTDSTKSSGQSNNNSDTCAEFRIKYVGAIEKLKLSEGKGLEGPLDLINYIDVAQQDGKLPFVPPEEEFIMGVSKYGIKVSTSDQYDVLHRHALYLIIRMVCYDDGLGAGKSLLALKTTDASNEEYSLWVYQCNSLEQAQAICKVLSTAFDSVLTSEKP May be produced at very low levels due to a premature stop codon in the mRNA, leading to nonsense-mediated mRNA decay. 0F041238E68FBE23 itgb1bp1-nme2-1 4 6 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association To search for potential ICAP-1alpha -binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1alpha . itgb1bp1-itgb1-1 4 8 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey physical association physical association Forty-two positive clones were obtained and sequenced. BLAST searches in cDNA data bases revealed that four inserts overlap with the cytoplasmic domain of beta 1 integrin, confirming the reported ICAP-1alpha /beta 1 integrin interaction already described itgb1bp1-nme2-2 2 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role bait bait 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction To confirm the direct interaction of nm23-H2 with ICAP-1alpha , we carried out an ELISA-based solid phase binding assay Fig. 2A itgb1bp1-nme2-3 2 8 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 ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction To confirm the direct interaction of nm23-H2 with ICAP-1alpha , we carried out an ELISA-based solid phase binding assay Fig. 2A itgb1bp1-nme2-4 3 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 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 ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction In an independent approach to corroborate these results, we incubated recombinant His-tagged ICAP-1alpha bound to a cobalt chelating resin with a solution of purified recombinant nm23-H2 protein or with HeLa cell lysates containing endogenous nm23-H2 (Fig. 2B). Fig. 2B Both monomeric and dimeric ITGB1BP1 bound to NME2 itgb1bp1-nme2-5 3 6 unspecified role unspecified role prey prey human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 8 unspecified role unspecified role bait bait 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 sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association In an independent approach to corroborate these results, we incubated recombinant His-tagged ICAP-1alpha bound to a cobalt chelating resin with a solution of purified recombinant nm23-H2 protein or with HeLa cell lysates containing endogenous nm23-H2 (Fig. 2B). Fig. 2B Only the dimeric form of NME2 was bound to ITGB1BP1 itgb1bp1-nme2-6 5 8 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association nly in ICAP-1alpha -transfected cells, immunoprecipitation of ICAP-1alpha using the anti-ICAP-1alpha 9B10 monoclonal antibody resulted in a co-immunoprecipitation of endogenous nm23-H2 as detected by Western blot analysis using an affinity-purified polyclonal antibody (Fig. 2D). Fig. 2D O14713-1 transfection itgb1bp1-nme2-7 1 6 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component ecoli Escherichia coli (strain K12) colocalization colocalization ICAP-1alpha co-localizes with nm23-H2 in peripheral ruffles in spreading Hs68 cells. Fig. 3