{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T23:09:47Z","timestamp":1792710587552,"version":"4.5.0"},"reference-count":43,"publisher":"Portland Press Ltd.","issue":"17","license":[{"start":{"date-parts":[[2019,7,25]],"date-time":"2019-07-25T00:00:00Z","timestamp":1564012800000},"content-version":"vor","delay-in-days":1059,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2016,9,1]]},"abstract":"<jats:p>Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine\/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells\/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1\u20132\u00a0min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40\u201380\u00a0min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in\u00a0vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.<\/jats:p>","DOI":"10.1042\/bcj20160557","type":"journal-article","created":{"date-parts":[[2016,7,30]],"date-time":"2016-07-30T01:19:39Z","timestamp":1469841579000},"page":"2671-2685","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":182,"title":["Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors"],"prefix":"10.1042","volume":"473","author":[{"given":"Genta","family":"Ito","sequence":"first","affiliation":[{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristina","family":"Katsemonova","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Tonelli","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pawel","family":"Lis","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco A.S.","family":"Baptista","sequence":"additional","affiliation":[{"name":"The Michael J. Fox Foundation for Parkinson's Research, Grand Central Station, P.O. Box 4777, New York, NY 10163, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalia","family":"Shpiro","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Graham","family":"Duddy","sequence":"additional","affiliation":[{"name":"Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals R&D, New Frontiers Science Park, Harlow, Essex CM19 5AD, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steve","family":"Wilson","sequence":"additional","affiliation":[{"name":"RD Platform Technology & Science, GlaxoSmithKline, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip\u00a0Wing-Lok","family":"Ho","sequence":"additional","affiliation":[{"name":"Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shu-Leong","family":"Ho","sequence":"additional","affiliation":[{"name":"Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alastair\u00a0D.","family":"Reith","sequence":"additional","affiliation":[{"name":"Neurodegeneration Discovery Performance Unit, RD Neurosciences, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dario\u00a0R.","family":"Alessi","sequence":"additional","affiliation":[{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2016,8,30]]},"reference":[{"key":"2021112216314380100_B1","doi-asserted-by":"publisher","first-page":"570","DOI":"10.1016\/j.cell.2015.01.019","article-title":"SnapShot: genetics of Parkinson's disease","volume":"160","author":"Bras","year":"2015","journal-title":"Cell"},{"key":"2021112216314380100_B2","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1002\/mds.25249","article-title":"The genetics of Parkinson's disease: progress and therapeutic implications","volume":"28","author":"Singleton","year":"2013","journal-title":"Mov. 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