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/ The Parkinson's disease-linked LRRK2 mutation I2020T stabilizes an active state conformation leading to increased kinase activity. The Parkinson's disease-linked LRRK2 mutation I2020T stabilizes an active state conformation leading to increased kinase activity. J. Biol. Chem. (0021-9258) 2014 imex curation Ray S., Bender S., Kang S., Lin R., Glicksman M.A., Liu M. Parkinsons - Interactions investigated in the context of Parkinsons disease Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Invitrogen supplied LRRK2 (amino acids 970–2527) and LRRK2 mutation I2020T (also amino acids 970–2527) expressed in a baculovirus system. Both proteins are GST tagged, the mutant with an N-terminal tag, but no tag location is given for the WT protein. 21-Jul-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay tag visualisation tag visualisation Ray S., Bender S., Kang S., Lin R., Glicksman M.A., Liu M. Parkinsons - Interactions investigated in the context of Parkinsons disease I have not entered anything under "Parameters" apart from IC 50s- as I am not sure what "alpha" is -(?? the ratio of the two Kms) - and the Segal reference (very long) is not searchable... Invitrogen supplied LRRK2 (amino acids 970–2527) and LRRK2 mutation I2020T (also amino acids 970–2527) expressed in a baculovirus system. Both proteins are GST tagged, the mutant with an N-terminal tag, but no tag location is given for the WT protein. Accepted 2014-JUN-06 by ORCHARD J. Biol. Chem. (0021-9258) 2014 imex curation gsk3-xiii GSK3-XIII small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) 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 imatinib Imatinib small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) Protein kinase inhibitor Protein kinase inhibitor moes_human Moesin Membrane-organizing extension spike protein MSN protein protein human Homo sapiens Human MPKTISVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWFFGLQYQDTKGFSTWLKLNKKVTAQDVRKESPLLFKFRAKFYPEDVSEELIQDITQRLFFLQVKEGILNDDIYCPPETAVLLASYAVQSKYGDFNKEVHKSGYLAGDKLLPQRVLEQHKLNKDQWEERIQVWHEEHRGMLREDAVLEYLKIAQDLEMYGVNYFSIKNKKGSELWLGVDALGLNIYEQNDRLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILALCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQMERAMLENEKKKREMAEKEKEKIEREKEELMERLKQIEEQTKKAQQELEEQTRRALELEQERKRAQSEAEKLAKERQEAEEAKEALLQASRDQKKTQEQLALEMAELTARISQLEMARQKKESEAVEWQQKAQMVQEDLEKTRAELKTAMSTPHVAEPAENEQDEQDENGAEASADLRADAMAKDRSEEERTTEAEKNERVQKHLKALTSELANARDESKKTANDMIHAENMRLGRDKYKTLRQIRQGNTKQRIDEFESM DIP protein P26038 original sequence version: 168 865A6C5CB14AE586 ponatinib Ponatinib small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) lrrk2-msn-6 1 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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) 3 enzyme enzyme neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 phosphorylation phosphorylation reaction phosphorylation Figure 1,2,3 Table 1. kcat = 8.1/min Ka (ATP) = 69uM Kb = 87uM alpha (Km)= 1.8 The t-I2020T mutant has lower kinase activity than t-WT but ATP binds to the mutant much more tightly than t-WT, with a K_ATP value 40-fold lower than that of t-WT. Incubation at room temperature for 150 min. Initial velocities were measured as a function of [ATP] at a saturating concentration of LRRKtide. I have assumed that Ka is substrate dissociation constant for ATP - as this value is about X40 for mutant as compared to WT. lrrk2-msn-7 1 3 enzyme enzyme neutral component neutral component Q5S007:p.Ile2020Thr mutation mutation certain certain sequence position certain certain certain sequence position certain I T Wed May 02 14:27:52 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile2020thr' Tue Jun 30 19:45:39 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile2020Thr' 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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 Figure 1,2,3 Table 1.-2 kcat = 4.4/min Ka (ATP) = 1.6uM Kb = 110uM alpha (Km)= 7.3 The t-I2020T mutant has lower kinase activity than t-WT but ATP binds to the mutant much more tightly than t-WT, with a K_ATP value 40-fold lower than that of t-WT. Incubation at room temperature for 150 min. Initial velocities were measured as a function of [ATP] at a saturating concentration of LRRKtide. I have assumed that Ka is the substrate dissociation constant for ATP - as this value is about X40 for mutant as compared to WT. lrrk2-msn-8 1 3 enzyme enzyme neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 5 enzyme target enzyme target substrate substrate neutral component neutral component P26038:p.Thr558Ser mutation mutation certain certain sequence position certain certain certain sequence position certain T S Wed May 02 19:40:27 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr558ser' Tue Jun 30 17:40:05 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr558Ser' region_lrrktide 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 Figure 1, Table 1.-3 kcat = 0.31/min Ka (ATP) = 77uM Kb = 554uM alpha (Km)= 2.6 Unlike LRRKtide, when LRRKtideS is the phosphoryl acceptor, the t-I2020T mutant is more active than t-WT. Incubation at room temperature for 150 min. Initial velocities were measured as a function of [ATP] at a saturating concentration of LRRKtideS. I have assumed that Ka is substrate dissociation constant for ATP - as this value is about X40 for mutant as compared to WT. lrrk2-msn-9 1 3 enzyme enzyme neutral component neutral component 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q5S007:p.Ile2020Thr mutation mutation certain certain sequence position certain certain certain sequence position certain I T Wed May 02 18:46:16 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile2020thr' Tue Jun 30 17:40:15 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile2020Thr' 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain P26038:p.Thr558Ser mutation mutation certain certain sequence position certain certain certain sequence position certain T S Wed May 02 15:10:09 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'thr558ser' Tue Jun 30 16:19:32 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Thr558Ser' chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Figure 1,2,3 Table 1.-4 kcat = 0.75min Ka (ATP) = 7.6uM Kb = 263uM alpha (Km)= 1.7 Unlike LRRKtide, when LRRKtideS is the phosphoryl acceptor, the t-I2020T mutant is more active than t-WT. Incubation at room temperature for 150 min. Initial velocities were measured as a function of [ATP] at a saturating concentration of LRRKtideS. I have assumed that Ka is the substrate dissociation constant for ATP - as this value is about X40 for mutant as compared to WT. imatinib-lrrk2 1 4 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) 3 enzyme enzyme neutral component neutral component 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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 Figure 4 Table 3. Ponatinib and Imatinib show ATP non-competitive inhibition toward t-WT, but inhibit the t-I2020T mutant via an ATP competitive mechanism. GSK3-XIII shows competitive inhibition against both t-WT and the t-I2020T mutant. Initial velocities of LRRK2-catalyzed LRRKtide phosphorylation were measured as a function of inhibitor concentration at varied ATP concentrations and at a fixed LRRKtide concentration. lrrk2-msn-11 1 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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) 3 enzyme enzyme neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q5S007:p.Ile2020Thr mutation mutation certain certain sequence position certain certain certain sequence position certain I T Wed May 02 20:30:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile2020thr' Tue Jun 30 20:06:18 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile2020Thr' 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 6 inhibitor inhibitor neutral component neutral component phosphorylation phosphorylation reaction phosphorylation Figure 4 Table 3 Ponatinib and Imatinib show ATP non-competitive inhibition toward t-WT, but inhibit the t-I2020T mutant via an ATP competitive mechanism. GSK3-XIII shows competitive inhibition against both t-WT and the t-I2020T mutant. Initial velocities of LRRK2-catalyzed LRRKtide phosphorylation were measured as a function of inhibitor concentration at varied ATP concentrations and at a fixed LRRKtide concentration. imatinib-lrrk2-1 1 3 enzyme enzyme neutral component neutral component region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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) 2 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Figure 4 Table 3. Ponatinib and Imatinib show ATP non-competitive inhibition toward t-WT, but inhibit the t-I2020T mutant via an ATP competitive mechanism. GSK3-XIII shows competitive inhibition against both t-WT and the t-I2020T mutant. Initial velocities of LRRK2-catalyzed LRRKtide phosphorylation were measured as a function of inhibitor concentration at varied ATP concentrations and at a fixed LRRKtide concentration. lrrk2-msn-12 1 3 enzyme enzyme neutral component neutral component 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain Q5S007:p.Ile2020Thr mutation mutation certain certain sequence position certain certain certain sequence position certain I T Wed May 02 19:52:22 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile2020thr' Tue Jun 30 17:28:23 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile2020Thr' 4 inhibitor inhibitor neutral component neutral component 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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 Figure 4 Table 3 Ponatinib and Imatinib show ATP non-competitive inhibition toward t-WT, but inhibit the t-I2020T mutant via an ATP competitive mechanism. GSK3-XIII shows competitive inhibition against both t-WT and the t-I2020T mutant. Initial velocities of LRRK2-catalyzed LRRKtide phosphorylation were measured as a function of inhibitor concentration at varied ATP concentrations and at a fixed LRRKtide concentration. imatinib-lrrk2-2 1 3 enzyme enzyme neutral component neutral component 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.Ile2020Thr mutation mutation certain certain sequence position certain certain certain sequence position certain I T Wed May 02 16:29:34 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ile2020thr' Tue Jun 30 17:01:31 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ile2020Thr' region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 2 inhibitor inhibitor neutral component neutral component 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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 Figure 4 Table 3 Ponatinib and Imatinib show ATP non-competitive inhibition toward t-WT, but inhibit the t-I2020T mutant via an ATP competitive mechanism. GSK3-XIII shows competitive inhibition against both t-WT and the t-I2020T mutant. Initial velocities of LRRK2-catalyzed LRRKtide phosphorylation were measured as a function of inhibitor concentration at varied ATP concentrations and at a fixed LRRKtide concentration. imatinib-lrrk2-3 1 5 enzyme target enzyme target substrate substrate neutral component neutral component region_lrrktide 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) 3 enzyme enzyme neutral component neutral component 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 region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 6 inhibitor inhibitor neutral component neutral component chemical synthesis Chemical synthesis (Chemical synthesis) phosphorylation phosphorylation reaction phosphorylation Figure 4 Table 3. Ponatinib and Imatinib show ATP non-competitive inhibition toward t-WT, but inhibit the t-I2020T mutant via an ATP competitive mechanism. GSK3-XIII shows competitive inhibition against both t-WT and the t-I2020T mutant. Initial velocities of LRRK2-catalyzed LRRKtide phosphorylation were measured as a function of inhibitor concentration at varied ATP concentrations and at a fixed LRRKtide concentration.