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/ Interaction of elongation factor 1-alpha with leucine-rich repeat kinase 2 impairs kinase activity and microtubule bundling in vitro. Interaction of elongation factor 1-alpha with leucine-rich repeat kinase 2 impairs kinase activity and microtubule bundling in vitro. Neuroscience (0306-4522) 2009 imex curation Gillardon F. Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Parkinsons - Interactions investigated in the context of Parkinsons disease Fig 2 antibody = mouse monoclonal anti-EF1A antibody (clone CBP-KK1, Millipore). It looks to me if it most likely reacts with both EEF1A1 AND EEF1A2 - the immunogen is "Crude calmodulin-binding proteins from Trypanosoma brucei isolated using calmodulin-affinity chromatography. " - so not much help... Not added the co-loc. in fig S3 due to specificity problems with antibodies, and no such reactions demonstrated elsewhere in the paper. Figure 2/S3 results have not been included as the specificity of the anti-EF1A antibody used is unclear; similalry for figure S3 for Beta-tubulin. frank.gillardon@bc.boehringer-ingelheim.com 01-March-2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gillardon F. Parkinsons - Interactions investigated in the context of Parkinsons disease Fig 2 antibody = mouse monoclonal anti-EF1A antibody (clone CBP-KK1, Millipore). It looks to me if it most likely reacts with both EEF1A1 AND EEF1A2 - the immunogen is "Crude calmodulin-binding proteins from Trypanosoma brucei isolated using calmodulin-affinity chromatography. " - so not much help... Not added the co-loc. in fig S3 due to specificity problems with antibodies, and no such reactions demonstrated elsewhere in the paper. TLC based, using radiolabeled a-33P-GTP. Figure 2/S3 results have not been included as the specificity of the anti-EF1A antibody used is unclear; similalry for figure S3 for Beta-tubulin. frank.gillardon@bc.boehringer-ingelheim.com Accepted 2013-FEB-14 by ORCHARD 01-March-2013: Contacted by IntAct-Help. Neuroscience (0306-4522) 2009 Only protein-protein interactions imex curation Interaction of elongation factor 1-alpha with leucine-rich repeat kinase 2 impairs kinase activity and microtubule bundling in vitro. Interaction of elongation factor 1-alpha with leucine-rich repeat kinase 2 impairs kinase activity and microtubule bundling in vitro. Neuroscience (0306-4522) 2009 imex curation Gillardon F. Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Parkinsons - Interactions investigated in the context of Parkinsons disease Fig 2 antibody = mouse monoclonal anti-EF1A antibody (clone CBP-KK1, Millipore). It looks to me if it most likely reacts with both EEF1A1 AND EEF1A2 - the immunogen is "Crude calmodulin-binding proteins from Trypanosoma brucei isolated using calmodulin-affinity chromatography. " - so not much help... Not added the co-loc. in fig S3 due to specificity problems with antibodies, and no such reactions demonstrated elsewhere in the paper. Figure 2/S3 results have not been included as the specificity of the anti-EF1A antibody used is unclear; similalry for figure S3 for Beta-tubulin. frank.gillardon@bc.boehringer-ingelheim.com 01-March-2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Gillardon F. Parkinsons - Interactions investigated in the context of Parkinsons disease Fig 2 antibody = mouse monoclonal anti-EF1A antibody (clone CBP-KK1, Millipore). It looks to me if it most likely reacts with both EEF1A1 AND EEF1A2 - the immunogen is "Crude calmodulin-binding proteins from Trypanosoma brucei isolated using calmodulin-affinity chromatography. " - so not much help... Not added the co-loc. in fig S3 due to specificity problems with antibodies, and no such reactions demonstrated elsewhere in the paper. Figure 2/S3 results have not been included as the specificity of the anti-EF1A antibody used is unclear; similalry for figure S3 for Beta-tubulin. frank.gillardon@bc.boehringer-ingelheim.com Accepted 2013-FEB-14 by ORCHARD 01-March-2013: Contacted by IntAct-Help. Neuroscience (0306-4522) 2009 Only protein-protein interactions imex curation gtp guanosine triphosphate - GTP 5'-GTP GTP Guanosine 5'-triphosphate guanosine 5'-triphosphoric acid guanosine triphosphate GUANOSINE-5'-TRIPHOSPHATE H4gtp guanosine 5'-(tetrahydrogen triphosphate) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 InChI=1/C10H16N5O14P3/c11-10-13-7-4(8(18)14-10)12-2-15(7)9-6(17)5(16)3(27-9)1-26-31(22,23)29-32(24,25)28-30(19,20)21/h2-3,5-6,9,16-17H,1H2,(H,22,23)(H,24,25)(H2,19,20,21)(H3,11,13,14,18)/t3-,5-,6-,9-/m1/s1/f/h14,19-20,22,24H,11H2 ef1a2_human Elongation factor 1-alpha 2 EEF1A2 EEF1AL STN Eukaryotic elongation factor 1 A-2 Statin-S1 protein protein human Homo sapiens Human MGKEKTHINIVVIGHVDSGKSTTTGHLIYKCGGIDKRTIEKFEKEAAEMGKGSFKYAWVLDKLKAERERGITIDISLWKFETTKYYITIIDAPGHRDFIKNMITGTSQADCAVLIVAAGVGEFEAGISKNGQTREHALLAYTLGVKQLIVGVNKMDSTEPAYSEKRYDEIVKEVSAYIKKIGYNPATVPFVPISGWHGDNMLEPSPNMPWFKGWKVERKEGNASGVSLLEALDTILPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGILRPGMVVTFAPVNITTEVKSVEMHHEALSEALPGDNVGFNVKNVSVKDIRRGNVCGDSKSDPPQEAAQFTSQVIILNHPGQISAGYSPVIDCHTAHIACKFAELKEKIDRRSGKKLEDNPKSLKSGDAAIVEMVPGKPMCVESFSQYPPLGRFAVRDMRQTVAVGVIKNVEKKSGGAGKVTKSAQKAQKAGK DIP protein Q05639 original sequence version: 159 31E4E341CEE797EC lrrk2_human Leucine-rich repeat serine/threonine-protein kinase 2 LRRK2 PARK8 Dardarin protein protein human Homo sapiens Human MASGSCQGCEEDEETLKKLIVRLNNVQEGKQIETLVQILEDLLVFTYSERASKLFQGKNIHVPLLIVLDSYMRVASVQQVGWSLLCKLIEVCPGTMQSLMGPQDVGNDWEVLGVHQLILKMLTVHNASVNLSVIGLKTLDLLLTSGKITLLILDEESDIFMLIFDAMHSFPANDEVQKLGCKALHVLFERVSEEQLTEFVENKDYMILLSALTNFKDEEEIVLHVLHCLHSLAIPCNNVEVLMSGNVRCYNIVVEAMKAFPMSERIQEVSCCLLHRLTLGNFFNILVLNEVHEFVVKAVQQYPENAALQISALSCLALLTETIFLNQDLEEKNENQENDDEGEEDKLFWLEACYKALTWHRKNKHVQEAACWALNNLLMYQNSLHEKIGDEDGHFPAHREVMLSMLMHSSSKEVFQASANALSTLLEQNVNFRKILLSKGIHLNVLELMQKHIHSPEVAESGCKMLNHLFEGSNTSLDIMAAVVPKILTVMKRHETSLPVQLEALRAILHFIVPGMPEESREDTEFHHKLNMVKKQCFKNDIHKLVLAALNRFIGNPGIQKCGLKVISSIVHFPDALEMLSLEGAMDSVLHTLQMYPDDQEIQCLGLSLIGYLITKKNVFIGTGHLLAKILVSSLYRFKDVAEIQTKGFQTILAILKLSASFSKLLVHHSFDLVIFHQMSSNIMEQKDQQFLNLCCKCFAKVAMDDYLKNVMLERACDQNNSIMVECLLLLGADANQAKEGSSLICQVCEKESSPKLVELLLNSGSREQDVRKALTISIGKGDSQIISLLLRRLALDVANNSICLGGFCIGKVEPSWLGPLFPDKTSNLRKQTNIASTLARMVIRYQMKSAVEEGTASGSDGNFSEDVLSKFDEWTFIPDSSMDSVFAQSDDLDSEGSEGSFLVKKKSNSISVGEFYRDAVLQRCSPNLQRHSNSLGPIFDHEDLLKRKRKILSSDDSLRSSKLQSHMRHSDSISSLASEREYITSLDLSANELRDIDALSQKCCISVHLEHLEKLELHQNALTSFPQQLCETLKSLTHLDLHSNKFTSFPSYLLKMSCIANLDVSRNDIGPSVVLDPTVKCPTLKQFNLSYNQLSFVPENLTDVVEKLEQLILEGNKISGICSPLRLKELKILNLSKNHISSLSENFLEACPKVESFSARMNFLAAMPFLPPSMTILKLSQNKFSCIPEAILNLPHLRSLDMSSNDIQYLPGPAHWKSLNLRELLFSHNQISILDLSEKAYLWSRVEKLHLSHNKLKEIPPEIGCLENLTSLDVSYNLELRSFPNEMGKLSKIWDLPLDELHLNFDFKHIGCKAKDIIRFLQQRLKKAVPYNRMKLMIVGNTGSGKTTLLQQLMKTKKSDLGMQSATVGIDVKDWPIQIRDKRKRDLVLNVWDFAGREEFYSTHPHFMTQRALYLAVYDLSKGQAEVDAMKPWLFNIKARASSSPVILVGTHLDVSDEKQRKACMSKITKELLNKRGFPAIRDYHFVNATEESDALAKLRKTIINESLNFKIRDQLVVGQLIPDCYVELEKIILSERKNVPIEFPVIDRKRLLQLVRENQLQLDENELPHAVHFLNESGVLLHFQDPALQLSDLYFVEPKWLCKIMAQILTVKVEGCPKHPKGIISRRDVEKFLSKKRKFPKNYMSQYFKLLEKFQIALPIGEEYLLVPSSLSDHRPVIELPHCENSEIIIRLYEMPYFPMGFWSRLINRLLEISPYMLSGRERALRPNRMYWRQGIYLNWSPEAYCLVGSEVLDNHPESFLKITVPSCRKGCILLGQVVDHIDSLMEEWFPGLLEIDICGEGETLLKKWALYSFNDGEEHQKILLDDLMKKAEEGDLLVNPDQPRLTIPISQIAPDLILADLPRNIMLNNDELEFEQAPEFLLGDGSFGSVYRAAYEGEEVAVKIFNKHTSLRLLRQELVVLCHLHHPSLISLLAAGIRPRMLVMELASKGSLDRLLQQDKASLTRTLQHRIALHVADGLRYLHSAMIIYRDLKPHNVLLFTLYPNAAIIAKIADYGIAQYCCRMGIKTSEGTPGFRAPEVARGNVIYNQQADVYSFGLLLYDILTTGGRIVEGLKFPNEFDELEIQGKLPDPVKEYGCAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRNASIWLGCGHTDRGQLSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINTEDGKKRHTLEKMTDSVTCLYCNSFSKQSKQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIGNVSTPLMCLSESTNSTERNVMWGGCGTKIFSFSNDFTIQKLIETRTSQLFSYAAFSDSNIITVVVDTALYIAKQNSPVVEVWDKKTEKLCGLIDCVHFLREVMVKENKESKHKMSYSGRVKTLCLQKNTALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQKEIQSCLTVWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE DIP protein Q5S007 original sequence version: 36 DIP protein Q5S007 original sequence version: 68 DIP protein Q5S007 original sequence version: 64 26142A0CECBBC3F4 eef1a2-lrrk2-1 2 4 inhibitor inhibitor neutral component neutral component n_terminus t7 tag t7 tag MASMTGGQQMG epitope tag MASMTGGQQMG epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 5 putative self putative self putative self putative self region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n_terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range Q5S007:p.Gly2019Ser mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain G S Wed May 02 14:32:17 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly2019ser' Tue Jun 30 15:45:53 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly2019Ser' Q5S007:p.Lys1906Met mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain K M Wed May 02 17:47:37 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys1906met' Tue Jun 30 16:08:16 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys1906Met' physical association physical association 3, S1,.S4. EF1A significantly inhibited LRRK2 autophosphorylation when present in approximately fivefold molar excess, but at equimolar concentrations, however, EF1A only weakly (non-significantly) blocked kinase activity of LRRK2. lrrk2-gtp-1 1 5 enzyme enzyme neutral component neutral component c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range Q5S007:p.Gly2019Ser mutation mutation certain certain sequence position certain certain certain sequence position certain G S Wed May 02 15:06:05 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly2019ser' Tue Jun 30 17:19:41 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly2019Ser' c_terminus strep ii tag strep ii tag Strep (II) Strep II Strep (II) Strep II c-term range C-terminal range c-term range c-term range C-terminal range c-term range 3 enzyme target enzyme target substrate substrate neutral component neutral component region 33p radiolabel 33p radiolabel P33 33P undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chemical synthesis Chemical synthesis (Chemical synthesis) gtpase reaction gtpase reaction 4 Co-incubation with EF1A did not affect [α-33P]GTP hydrolysis by LRRK2. LRRK2 was expressed in Insect cells. lrrk2-2 2 5 putative self putative self putative self putative self region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q5S007:p.Lys1906Met mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain K M Wed May 02 15:17:33 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'lys1906met' Tue Jun 30 16:11:29 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Lys1906Met' n_terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range Q5S007:p.Gly2019Ser mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain G S Wed May 02 17:29:41 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly2019ser' Tue Jun 30 20:24:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly2019Ser' phosphorylation phosphorylation reaction phosphorylation true 3, S1, S4-2 EF1A significantly inhibited LRRK2 autophosphorylation when present in approximately fivefold molar excess, but at equimolar concentrations, however, EF1A only weakly (non-significantly) blocked kinase activity of LRRK2.