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/ Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay predetermined predetermined participant predetermined Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis When possible to add x-refs to GO add x-ref to GO term for autophosphorylation to both interactions. scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard Only protein-protein interactions human-b-cell Homo sapiens B cell b-cell B-cell [B lymphocyte; B-lymphocyte; B cell; B-cells] anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis A web search revealed that DeFew cells are a human B lymphoma cell line. PMID 7787756 states that MC3 cells are a chronic myeloid leukemia cell line. This experiment covers the data regarding MC3 and DeFew cells in Figure 3 E. This data has been merged as both cell lines are B cell lines. scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard Only protein-protein interactions human-b-cell Homo sapiens B cell b-cell B-cell [B lymphocyte; B-lymphocyte; B cell; B-cells] confocal microscopy confocal microscopy immunostaining immunostaining Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard 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 Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard Only protein-protein interactions 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. anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein. Nat. Immunol. (1529-2908) 2001 imex curation Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis Cancer - Interactions investigated in the context of cancer scala@dbbm.unina.it 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. orchard Only protein-protein interactions human-b-cell Homo sapiens B cell b-cell B-cell [B lymphocyte; B-lymphocyte; B cell; B-cells] pull down pull down western blot western blot Immuno blot Immuno blot Liu W., Quinto I., Chen X., Palmieri C., Rabin RL., Schwartz OM., Nelson DL., Scala G. Apoptosis - Interactions involving proteins with a function related to apoptosis scala@dbbm.unina.it Accepted 2008-MAY-11 by ORCHARD. 14-May-2008: Contacted by JYOTI. A human B cell cDNA library was used. Nat. Immunol. (1529-2908) 2001 orchard Only protein-protein interactions imex curation ibtk_human Inhibitor of Bruton tyrosine kinase IBTK BTKI KIAA1417 protein protein human Homo sapiens Human MSSPMPDCTSKCRSLKHALDVLSVVTKGSENQIKAFLSSHCYNAATIKDVFGRNALHLVSSCGKKGVLDWLIQKGVDLLVKDKESGWTALHRSIFYGHIDCVWSLLKHGVSLYIQDKEGLSALDLVMKDRPTHVVFKNTDPTDVYTWGDNTNFTLGHGSQNSKHHPELVDLFSRSGIYIKQVVLCKFHSVFLSQKGQVYTCGHGPGGRLGHGDEQTCLVPRLVEGLNGHNCSQVAAAKDHTVVLTEDGCVYTFGLNIFHQLGIIPPPSSCNVPRQIQAKYLKGRTIIGVAAGRFHTVLWTREAVYTMGLNGGQLGCLLDPNGEKCVTAPRQVSALHHKDIALSLVAASDGATVCVTTRGDIYLLADYQCKKMASKQLNLKKVLVSGGHMEYKVDPEHLKENGGQKICILAMDGAGRVFCWRSVNSSLKQCRWAYPRQVFISDIALNRNEILFVTQDGEGFRGRWFEEKRKSSEKKEILSNLHNSSSDVSYVSDINSVYERIRLEKLTFAHRAVSVSTDPSGCNFAILQSDPKTSLYEIPAVSSSSFFEEFGKLLREADEMDSIHDVTFQVGNRLFPAHKYILAVHSDFFQKLFLSDGNTSEFTDIYQKDEDSAGCHLFVVEKVHPDMFEYLLQFIYTDTCDFLTHGFKPRIHLNKNPEEYQGTLNSHLNKVNFHEDDNQKSAFEVYKSNQAQTVSERQKSKPKSCKKGKNIREDDPVRMLQTVAKKFDFSNLSSRLDGVRFENEKINVIAKNTGNKLKLSQKKCSFLCDVTMKSVDGKEFPCHKCVLCARLEYFHSMLSSSWIEASSCAALEMPIHSDILKVILDYLYTDEAVVIKESQNVDFICSVLVVADQLLITRLKEICEVALTEKLTLKNAAMLLEFAAMYSAKQLKLSCLQFIGLNMAALLEARSLDVLSDGVLKDLSEFYRKMIPAMDRRVITPYQDGPDISYLEVEDGDIFLKEEINMEQNHSETMFKKAKTKAKKKPRKRSDSSGGYNLSDIIQSPSSTGLLKSGKTNSVESLPELLTSDSEGSYAGVGSPRDLQSPDFTTGFHSDKIEAKVKPYVNGTSPVYSREDLKPWEKSPILKISAPQPIPSNRIDTTSSASWVAGSFSPVSPPVVDLRTIMEIEESRQKCGATPKSHLGKTVSHGVKLSQKQRKMIALTTKENNSGMNSMETVLFTPSKAPKPVNAWASSLHSVSSKSFRDFLLEEKKSVTSHSSGDHVKKVSFKGIENSQAPKIVRCSTHGTPGPEGNHISDLPLLDSPNPWLSSSVTAPSMVAPVTFASIVEEELQQEAALIRSREKPLALIQIEEHAIQDLLVFYEAFGNPEEFVIVERTPQGPLAVPMWNKHGC 67B2D11E276A6AF1 ebi-1521221 Inhibitor of Bruton tyrosine kinase protein protein human Homo sapiens Human MLIDIISSKMIALTTKENNSGMNSMETVLFTPSKAPKPVNAWASSLHSVSSKSFRDFLLEEKKSVTSHSSGDHVKKVSFKGIENSQAPKIVRCSTHGTPGPEGNHISDLPLLDSPNPWLSSSVTAPSMVAPVTFASIVEEELQQEAALIRSREKPLALIQIEEHAIQDLLVFYEAFGNPEEFVIVERTPQGPLAVPMWNKHGC The sequence has been withdrawn from uniprot. sequence is significantly different from parent. 3CA64BAB21CAD20B btk_human Tyrosine-protein kinase BTK Bruton tyrosine kinase B-cell progenitor kinase BPK BTK AGMX1 ATK Agammaglobulinemia tyrosine kinase protein protein human Homo sapiens Human MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES DIP protein Q06187 original sequence version: 118 DIP protein Q06187 original sequence version: 143 DF06B5D1FEC257CC btk-ibtk 5 9 unspecified role unspecified role prey prey 10 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind range range range range physical association physical association The coordinates given for the BTK construct used, do not match with the length of the protein nor the coordinates of the specified domains as given by the UniProtKB entry. The range given is taken from the ranges of these domains in the UniProtKB entry. 1 btk-ibtk-1 3 10 unspecified role unspecified role bait bait region PHBM sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range range range region SH2 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 9 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined in vitro In vitro direct interaction direct interaction The coordinates given for the BTK construct used, do not match with the coordinates of the specified domains as given by the UniProtKB entry. The ranges given are taken from the ranges of these domains in the UniProtKB entry. 3 A IBtk was efficiently bound by GST-Btk (aa 1-659) and GSTPHBM (aa 25-173; this region includes the PH domain and the unique Btk motif (BM) from aa 139 to 173, which is common to the Btk family of tyrosine kinases22) (Fig. 3a, lanes 1, 4). IBtk was bound weakly by GST-SH2 (aa 281-372, Fig. 3a, lane 2) and not at all by GST-SH3 (aa 219-268) or by GST ibtk-btk 7 10 unspecified role unspecified role prey prey human Homo sapiens Human 9 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 physical association physical association 3 C We next tested GST-IBtk fusion proteins for binding to endogenous Btk using a pull-down assay. Btk proteins were specifically recovered from MC3 lysates after binding to GST-IBtk ibtk-btk-1 6 10 unspecified role unspecified role prey prey human Homo sapiens Human 9 unspecified role unspecified role bait bait physical association physical association 3 D We next tested GST-IBtk fusion proteins for binding to endogenous Btk using a pull-down assay. Btk proteins were specifically recovered from MC3 lysates after binding to GST-IBtk Q9P2D0-4; Q06187 transfection IBTK peptide - An IBTK peptide displaced the immunoprecipitation activity of the anti-IBTK serum. ibtk-btk-2 2 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait physical association physical association 3 E we subjected cell lysates of DeFew and MC3 B cells to immunoprecipitation with IBtk antiserum to peptide 1 followed by immunoblotting with anti-Btk. Btk was detected in the immunoprecipitated proteins, indicating that IBtk associated with Btk in B cells under physiologic conditions btk-ibtk-2 4 8 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component colocalization colocalization 3 F, G In unstimulated cells, confocal analysis revealed a diffuse localization of IBtk and Btk at discrete cytoplasmic regions (Fig. 3f, A,B); an overlay of the two images together with the differential interference contrast (DIC) image showed some colocalization of the two proteins (Fig. 3f, C). Upon BCR cross-linking, the cells showed a predominant submembrane localization of both Btk and IBtk In unstimulated cells, colocalization of IBtk and Btk took place in the cytoplasm. Upon BCR cross-linking, colocalization took place in the sub-membrane region. btk-btk_substrat 1 10 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) 10 enzyme enzyme unspecified role unspecified role 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) phosphorylation phosphorylation reaction phosphorylation 4 A we assayed the enzyme activity of Btk on a peptide, KKVVALYDYMPMN, that corresponds to aa 217-229 of human Btk and includes the principal tyrosine autophosphorylation site28. This peptide functions as a good substrate of Btk kinase activity29. First, we tested the activity of GST-IBtk on the kinase activity of purified GST-Btk (Fig. 4a,b); in these experiments, IBtk inhibited Btk tyrosine activity in a dose-dependent manner IBTK - GST-IBTK inhibition of BTK kinase activity increases with increasing concentration of GST-IBTK. The BTK substrate peptide (BTK 217-229) is autophosphorylated on Tyr-223. btk-btk_substrat-1 1 10 enzyme enzyme unspecified role unspecified role human-b-cell Homo sapiens B cell b-cell B-cell [B lymphocyte; B-lymphocyte; B cell; B-cells] 10 enzyme target enzyme target substrate substrate unspecified role unspecified role region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation 4 C, E, F IBTK - GST-IBTK inhibition of BTK kinase activity increases with increasing concentration of GST-IBTK. Deletion analysis revealed that the C-terminal construct of IBTK retained inhibitory activty but the N-terminal construct did not. The BTK substrate peptide (BTK 217-229) is autophosphorylated on Tyr-223. we recovered endogenous Btk from MC3 B cells by immunoprecipitation and tested it for tyrosine kinase activity in the presence of either full-length or truncated IBtk. IBtk efficiently downregulated Btk tyrosine kinase activity IBTK causes nearly complete inhibition of BTK from unstimulated cells and only a substantial decrease in BCR-stimulated cells.