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/ http://www.ebi.ac.uk/ dani_ls-2011-3 Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. 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 predetermined predetermined participant predetermined Daniëls V., Vancraenenbroeck R., Law B.M., Greggio E., Lobbestael E., Gao F., De Maeyer M., Cookson M.R., Harvey K., Baekelandt V., Taymans J.M. Parkinsons - Interactions investigated in the context of Parkinsons disease Construct details when appearing to differ from those described in the publication (for FLAG-LRRK2 + mutants), were supplied by the authors. jean-marc.taymans@med.kuleuven.be. jean-marc.taymans@med.kuleuven.be J. Neurochem. (0022-3042) 2011 Only protein-protein interactions imex curation Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. An enzyme coupled assay measuring the depletion of NADH at 340nm, using purified proteins, as shown in the SDS-PAGE gel in figure 1. dani_ls-2011-2 Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. in vitro In vitro affinity chrom affinity chromatography technology Affinity purification Affinity purification affinity chrom western blot western blot Immuno blot Immuno blot Daniëls V., Vancraenenbroeck R., Law B.M., Greggio E., Lobbestael E., Gao F., De Maeyer M., Cookson M.R., Harvey K., Baekelandt V., Taymans J.M. Parkinsons - Interactions investigated in the context of Parkinsons disease Construct details when appearing to differ from those described in the publication (for FLAG-LRRK2 + mutants), were supplied by the authors. jean-marc.taymans@med.kuleuven.be. jean-marc.taymans@med.kuleuven.be J. Neurochem. (0022-3042) 2011 Only protein-protein interactions imex curation Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. An enzyme coupled assay measuring the depletion of NADH at 340nm, using purified proteins, as shown in the SDS-PAGE gel in figure 1. dani_ls-2011-5 Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Daniëls V., Vancraenenbroeck R., Law B.M., Greggio E., Lobbestael E., Gao F., De Maeyer M., Cookson M.R., Harvey K., Baekelandt V., Taymans J.M. Parkinsons - Interactions investigated in the context of Parkinsons disease Construct details when appearing to differ from those described in the publication (for FLAG-LRRK2 + mutants), were supplied by the authors. jean-marc.taymans@med.kuleuven.be. jean-marc.taymans@med.kuleuven.be J. Neurochem. (0022-3042) 2011 Only protein-protein interactions imex curation Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. An enzyme coupled assay measuring the depletion of NADH at 340nm, using purified proteins, as shown in the SDS-PAGE gel in figure 1. dani_ls-2011-1 Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. in vitro In vitro gtpase assay gtpase assay GTPase gtp hydrolisis gtp hydrolisis GTPase predetermined predetermined participant predetermined Daniëls V., Vancraenenbroeck R., Law B.M., Greggio E., Lobbestael E., Gao F., De Maeyer M., Cookson M.R., Harvey K., Baekelandt V., Taymans J.M. Parkinsons - Interactions investigated in the context of Parkinsons disease An enzyme coupled assay measuring the depletion of NADH at 340nm, using purified proteins, as shown in the SDS-PAGE gel in figure 1. Construct details when appearing to differ from those described in the publication (for FLAG-LRRK2 + mutants), were supplied by the authors. jean-marc.taymans@med.kuleuven.be. jean-marc.taymans@med.kuleuven.be J. Neurochem. (0022-3042) 2011 Only protein-protein interactions imex curation Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. dani_ls-2011-6 Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant. 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 tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Daniëls V., Vancraenenbroeck R., Law B.M., Greggio E., Lobbestael E., Gao F., De Maeyer M., Cookson M.R., Harvey K., Baekelandt V., Taymans J.M. Parkinsons - Interactions investigated in the context of Parkinsons disease Construct details when appearing to differ from those described in the publication (for FLAG-LRRK2 + mutants), were supplied by the authors. jean-marc.taymans@med.kuleuven.be. jean-marc.taymans@med.kuleuven.be J. Neurochem. (0022-3042) 2011 Only protein-protein interactions imex curation Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. An enzyme coupled assay measuring the depletion of NADH at 340nm, using purified proteins, as shown in the SDS-PAGE gel in figure 1. lrrk2_human Leucine-rich repeat serine/threonine-protein kinase 2 LRRK2 PARK8 Dardarin protein protein human Homo sapiens Human MASGSCQGCEEDEETLKKLIVRLNNVQEGKQIETLVQILEDLLVFTYSERASKLFQGKNIHVPLLIVLDSYMRVASVQQVGWSLLCKLIEVCPGTMQSLMGPQDVGNDWEVLGVHQLILKMLTVHNASVNLSVIGLKTLDLLLTSGKITLLILDEESDIFMLIFDAMHSFPANDEVQKLGCKALHVLFERVSEEQLTEFVENKDYMILLSALTNFKDEEEIVLHVLHCLHSLAIPCNNVEVLMSGNVRCYNIVVEAMKAFPMSERIQEVSCCLLHRLTLGNFFNILVLNEVHEFVVKAVQQYPENAALQISALSCLALLTETIFLNQDLEEKNENQENDDEGEEDKLFWLEACYKALTWHRKNKHVQEAACWALNNLLMYQNSLHEKIGDEDGHFPAHREVMLSMLMHSSSKEVFQASANALSTLLEQNVNFRKILLSKGIHLNVLELMQKHIHSPEVAESGCKMLNHLFEGSNTSLDIMAAVVPKILTVMKRHETSLPVQLEALRAILHFIVPGMPEESREDTEFHHKLNMVKKQCFKNDIHKLVLAALNRFIGNPGIQKCGLKVISSIVHFPDALEMLSLEGAMDSVLHTLQMYPDDQEIQCLGLSLIGYLITKKNVFIGTGHLLAKILVSSLYRFKDVAEIQTKGFQTILAILKLSASFSKLLVHHSFDLVIFHQMSSNIMEQKDQQFLNLCCKCFAKVAMDDYLKNVMLERACDQNNSIMVECLLLLGADANQAKEGSSLICQVCEKESSPKLVELLLNSGSREQDVRKALTISIGKGDSQIISLLLRRLALDVANNSICLGGFCIGKVEPSWLGPLFPDKTSNLRKQTNIASTLARMVIRYQMKSAVEEGTASGSDGNFSEDVLSKFDEWTFIPDSSMDSVFAQSDDLDSEGSEGSFLVKKKSNSISVGEFYRDAVLQRCSPNLQRHSNSLGPIFDHEDLLKRKRKILSSDDSLRSSKLQSHMRHSDSISSLASEREYITSLDLSANELRDIDALSQKCCISVHLEHLEKLELHQNALTSFPQQLCETLKSLTHLDLHSNKFTSFPSYLLKMSCIANLDVSRNDIGPSVVLDPTVKCPTLKQFNLSYNQLSFVPENLTDVVEKLEQLILEGNKISGICSPLRLKELKILNLSKNHISSLSENFLEACPKVESFSARMNFLAAMPFLPPSMTILKLSQNKFSCIPEAILNLPHLRSLDMSSNDIQYLPGPAHWKSLNLRELLFSHNQISILDLSEKAYLWSRVEKLHLSHNKLKEIPPEIGCLENLTSLDVSYNLELRSFPNEMGKLSKIWDLPLDELHLNFDFKHIGCKAKDIIRFLQQRLKKAVPYNRMKLMIVGNTGSGKTTLLQQLMKTKKSDLGMQSATVGIDVKDWPIQIRDKRKRDLVLNVWDFAGREEFYSTHPHFMTQRALYLAVYDLSKGQAEVDAMKPWLFNIKARASSSPVILVGTHLDVSDEKQRKACMSKITKELLNKRGFPAIRDYHFVNATEESDALAKLRKTIINESLNFKIRDQLVVGQLIPDCYVELEKIILSERKNVPIEFPVIDRKRLLQLVRENQLQLDENELPHAVHFLNESGVLLHFQDPALQLSDLYFVEPKWLCKIMAQILTVKVEGCPKHPKGIISRRDVEKFLSKKRKFPKNYMSQYFKLLEKFQIALPIGEEYLLVPSSLSDHRPVIELPHCENSEIIIRLYEMPYFPMGFWSRLINRLLEISPYMLSGRERALRPNRMYWRQGIYLNWSPEAYCLVGSEVLDNHPESFLKITVPSCRKGCILLGQVVDHIDSLMEEWFPGLLEIDICGEGETLLKKWALYSFNDGEEHQKILLDDLMKKAEEGDLLVNPDQPRLTIPISQIAPDLILADLPRNIMLNNDELEFEQAPEFLLGDGSFGSVYRAAYEGEEVAVKIFNKHTSLRLLRQELVVLCHLHHPSLISLLAAGIRPRMLVMELASKGSLDRLLQQDKASLTRTLQHRIALHVADGLRYLHSAMIIYRDLKPHNVLLFTLYPNAAIIAKIADYGIAQYCCRMGIKTSEGTPGFRAPEVARGNVIYNQQADVYSFGLLLYDILTTGGRIVEGLKFPNEFDELEIQGKLPDPVKEYGCAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRNASIWLGCGHTDRGQLSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINTEDGKKRHTLEKMTDSVTCLYCNSFSKQSKQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIGNVSTPLMCLSESTNSTERNVMWGGCGTKIFSFSNDFTIQKLIETRTSQLFSYAAFSDSNIITVVVDTALYIAKQNSPVVEVWDKKTEKLCGLIDCVHFLREVMVKENKESKHKMSYSGRVKTLCLQKNTALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQKEIQSCLTVWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE gdp guanosine 5-diphosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) 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 lrrk2-lrrk2-66 1363676 n/a 1363681 unspecified role unspecified role bait bait roc-cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role prey prey purified purified Q5S007:p.Arg1441Gly mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Tyr1699Cys mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Lys1347Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Arg1441His mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Arg1441Cys mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false roc-cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Thr1348Asn mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false 2 cor_roc-1 1363676 n/a 1363681 unspecified role unspecified role prey prey purified purified roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role bait bait cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false S2-1 The COR domain does not dimerize with the RocCOR domain - for WT or mutants. lrrk2-lrrk2-54 1363676 n/a 1363681 unspecified role unspecified role prey prey purified purified roc-cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Arg1441Gly mutation decreasing mutation decreasing interaction hotspot hotspot mutation decreasing certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role bait bait roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false S2-2 lrrk2-lrrk2-56 1363676 n/a 1363681 unspecified role unspecified role bait bait purified purified roc-cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Lys1347Ala mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Thr1348Asn mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role prey prey roc-cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false S2-3 lrrk2-lrrk2-62 1363676 n/a 1363681 unspecified role unspecified role prey prey purified purified roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role bait bait cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Tyr1699Cys mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false 4C lrrk2-lrrk2-67 1363676 n/a 1363681 unspecified role unspecified role bait bait cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role prey prey purified purified roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false 3D ROC dimerisation was almost undetectable. gtp-lrrk2-10 1363677 n/a 1363682 competitor competitor competitor unspecified role unspecified role chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1363681 unspecified role unspecified role prey prey purified purified roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false Q5S007:p.Thr1348Asn mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false 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. n/a 1363683 unspecified role unspecified role bait bait chemical synthesis Chemical synthesis (Chemical synthesis) physical association physical association false false false S1D, 5. gtp-lrrk2-13 1363677 n/a 1363683 unspecified role unspecified role bait bait chemical synthesis Chemical synthesis (Chemical synthesis) n/a 1363681 unspecified role unspecified role prey prey purified purified Q5S007:p.Thr1348Asn mutation disrupting mutation disrupting interaction mutation disrupting certain certain sequence position certain certain certain sequence position certain false n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false false false S1D, 5 -2 lrrk2-lrrk2-57 1363678 n/a 1363681 unspecified role unspecified role prey prey n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false kinase_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Tyr1699Cys mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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. n/a 1363681 unspecified role unspecified role bait bait roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false ecoli Escherichia coli (strain K12) physical association physical association false false false 3A/B, 4A/B, 6A/B The interaction between ROC and COR tended to be strengthened by inclusion of the kinase domain. GDP, GTPγS lrrk2-lrrk2-60 1363678 n/a 1363681 competitor competitor competitor unspecified role unspecified role cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false 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. n/a 1363681 unspecified role unspecified role bait bait roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false ecoli Escherichia coli (strain K12) n/a 1363681 unspecified role unspecified role prey prey n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false 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 false false false 3C, S3 The ROC:COR interaction could not be outcompeted by addition of ROC. gtp-lrrk2-6 1363679 n/a 1363681 enzyme enzyme neutral component neutral component purified purified Q5S007:p.Thr1348Asn mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false Q5S007:p.Tyr1699Cys mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false Q5S007:p.Arg1441Cys mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false 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. n/a 1363683 enzyme target enzyme target substrate substrate neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) gtpase reaction gtpase reaction false false false Fig 1 and S1 Protein fragments (ROC, ROC-COR-kinase-WD40, ROC-COR-kinase and ROC-COR domains, both WT and with T1348N mutation, showed GTPase activity at only background levels. lrrk2-lrrk2-64 1363680 n/a 1363681 competitor competitor competitor prey prey Q5S007:p.Tyr1699Cys mutation increasing mutation increasing interaction mutation increasing certain certain sequence position certain certain certain sequence position certain false n_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged n-term range n-terminal range n-term range n-term range n-terminal range n-term range false cor_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false n/a 1363681 unspecified role unspecified role bait bait n_terminus myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range false roc_domain sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false physical association physical association false false false S4 The stabilizing effect of the Y1699C mutation was preserved when the COR domain was expanded with the kinase domain.