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/ LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status. LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status. Front Mol Neurosci 2014 imex curation Reynolds A., Doggett E.A., Riddle S.M., Lebakken C.S., Nichols R.J. Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Parkinsons - Interactions investigated in the context of Parkinsons disease Results have not been annotated when LRRK2 has only been purified by IP alone. The authors confirm that " all of the LRRK2 constructs, as used in Figure 1, are FULL length, FLAG tagged, and Human in origin. The variants used in figure 1 were supplied in a collaboration (with LifeTech), and appear to be no longer available. jnichols@parkinsonsinstitute.org 20-Oct-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay antibody detection identification by antibody antibody detection Reynolds A., Doggett E.A., Riddle S.M., Lebakken C.S., Nichols R.J. Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. For Figure 1(A) it is stated that:- "We next employed full length recombinant LRRK2 [wild-type, Gly2019Ser, Arg1441Cys, and Asp1994Ala] (LifeTechnologies)". However Life Technologies do not sell full length R1441C nor D1994A mutants, so all these mutants have been entered as having FLAG tags, as these tags are stated to be used for the detection of reaction products - see Figures 1 D-G and Methods section. The authors have not replied to a clarification request. Results have not been annotated when LRRK2 has only been purified by IP alone. Not sure what a "Dot-blot Immunoblot" might be, but I would not think it would qualify as "Western Blotting"... jnichols@parkinsonsinstitute.org Accepted 2014-OCT-07 by ORCHARD Front Mol Neurosci 2014 Only protein-protein interactions imex curation LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status. LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status. Front Mol Neurosci 2014 imex curation Reynolds A., Doggett E.A., Riddle S.M., Lebakken C.S., Nichols R.J. Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Parkinsons - Interactions investigated in the context of Parkinsons disease Results have not been annotated when LRRK2 has only been purified by IP alone. The authors confirm that " all of the LRRK2 constructs, as used in Figure 1, are FULL length, FLAG tagged, and Human in origin. The variants used in figure 1 were supplied in a collaboration (with LifeTech), and appear to be no longer available. jnichols@parkinsonsinstitute.org 20-Oct-2014: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay tag visualisation tag visualisation Reynolds A., Doggett E.A., Riddle S.M., Lebakken C.S., Nichols R.J. Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. For Figure 1(A) it is stated that:- "We next employed full length recombinant LRRK2 [wild-type, Gly2019Ser, Arg1441Cys, and Asp1994Ala] (LifeTechnologies)". However Life Technologies do not sell full length R1441C nor D1994A mutants, so all these mutants have been entered as having FLAG tags, as these tags are stated to be used for the detection of reaction products - see Figures 1 D-G and Methods section. The authors have not replied to a clarification request. Results have not been annotated when LRRK2 has only been purified by IP alone. jnichols@parkinsonsinstitute.org Accepted 2014-OCT-07 by ORCHARD Front Mol Neurosci 2014 Only protein-protein interactions imex curation 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 moes_human Moesin Membrane-organizing extension spike protein MSN protein protein human Homo sapiens Human MPKTISVRVTTMDAELEFAIQPNTTGKQLFDQVVKTIGLREVWFFGLQYQDTKGFSTWLKLNKKVTAQDVRKESPLLFKFRAKFYPEDVSEELIQDITQRLFFLQVKEGILNDDIYCPPETAVLLASYAVQSKYGDFNKEVHKSGYLAGDKLLPQRVLEQHKLNKDQWEERIQVWHEEHRGMLREDAVLEYLKIAQDLEMYGVNYFSIKNKKGSELWLGVDALGLNIYEQNDRLTPKIGFPWSEIRNISFNDKKFVIKPIDKKAPDFVFYAPRLRINKRILALCMGNHELYMRRRKPDTIEVQQMKAQAREEKHQKQMERAMLENEKKKREMAEKEKEKIEREKEELMERLKQIEEQTKKAQQELEEQTRRALELEQERKRAQSEAEKLAKERQEAEEAKEALLQASRDQKKTQEQLALEMAELTARISQLEMARQKKESEAVEWQQKAQMVQEDLEKTRAELKTAMSTPHVAEPAENEQDEQDENGAEASADLRADAMAKDRSEEERTTEAEKNERVQKHLKALTSELANARDESKKTANDMIHAENMRLGRDKYKTLRQIRQGNTKQRIDEFESM DIP protein P26038 original sequence version: 168 865A6C5CB14AE586 lrrk2-msn-14 2 4 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 flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined phosphorylation phosphorylation reaction phosphorylation 1C P26038 opser Specific Activity of WT LRRK2 =52 nmol/mg/min, R1441C mutant - 64 nmol/mg/min, G2019S mutant = 128 nmol/mg/min, (Kinase Dead mutant = not detected). lrrk2-msn-15 2 3 enzyme enzyme neutral component neutral component region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q5S007:p.Gly2019Ser mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain G S Wed May 02 15:36:11 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly2019ser' Tue Jun 30 19:57:49 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly2019Ser' Q5S007:p.Arg1441Cys mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain R C Wed May 02 20:08:19 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg1441cys' Tue Jun 30 19:44:15 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg1441Cys' Q5S007:p.Asp1994Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain D A Wed May 02 14:33:11 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1994ala' Tue Jun 30 17:51:11 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1994Ala' 4 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 1Ci P26038 opser Specific Activity of WT LRRK2 =52 nmol/mg/min, R1441C mutant - 64 nmol/mg/min, G2019S mutant = 128 nmol/mg/min, (Kinase Dead mutant = not detected). lrrk2-3 1 3 self self self self Q5S007:p.Arg1441Cys mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain R C Wed May 02 16:05:49 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'arg1441cys' Tue Jun 30 17:18:25 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Arg1441Cys' Q5S007:p.Gly2019Ser mutation increasing rate mutation increasing interaction rate mutation increasing rate certain certain sequence position certain certain certain sequence position certain G S Wed May 02 16:08:54 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly2019ser' Tue Jun 30 15:38:50 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly2019Ser' Q5S007:p.Asp1994Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain D A Wed May 02 20:30:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'asp1994ala' Tue Jun 30 19:15:49 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Asp1994Ala' region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined phosphorylation phosphorylation reaction phosphorylation true 1D-G Q5S007 ser-1292 O-phospho-L-serine Q5S007 thr-1503 O-phospho-L-serine Q5S007 thr-1491 O-phospho-L-serine Q5S007 thr-2483 O-phospho-L-serine For the Gly2019Ser and Arg1441Cys LRRK2 mutants, Ser1292 is already phosphorylated at the beginning of the reaction. A twofold increase in autophosphorylation can be seen using the Gly2019Ser mutant; the autophosphorylation activity of the Arg1441Cys mutant is increased relative to wild-type. lrrk2-3 1 3 self self self self region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined phosphorylation phosphorylation reaction phosphorylation true 1D-G Q5S007 ser-1292 O-phospho-L-serine Q5S007 thr-1503 O-phospho-L-serine Q5S007 thr-1491 O-phospho-L-serine Q5S007 thr-2483 O-phospho-L-serine Q5S007 increases activity of Q5S007