title: Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.
abstract: 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 1-2 min, markedly more rapidly than the Ser(935) and Ser(1292) biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser(910) and Ser(935) and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo 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.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K. genta-ito@umin.ac.jp d.r.alessi@dundee.ac.uk.
authors.forename: Genta
authors.initials: G
authors.lastname: Ito
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
authors.forename: Kristina
authors.initials: K
authors.lastname: Katsemonova
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
authors.forename: Francesca
authors.initials: F
authors.lastname: Tonelli
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
authors.forename: Pawel
authors.initials: P
authors.lastname: Lis
authors.affiliation: The Michael J. Fox Foundation for Parkinson's Research, Grand Central Station, P.O. Box 4777, New York, NY 10163, U.S.A.
authors.forename: Marco A S
authors.initials: MA
authors.lastname: Baptista
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
authors.forename: Natalia
authors.initials: N
authors.lastname: Shpiro
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals R&D, New Frontiers Science Park, Harlow, Essex CM19 5AD, U.K.
authors.forename: Graham
authors.initials: G
authors.lastname: Duddy
authors.affiliation: RD Platform Technology & Science, GlaxoSmithKline, U.K.
authors.forename: Steve
authors.initials: S
authors.lastname: Wilson
authors.affiliation: Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong.
authors.forename: Philip Wing-Lok
authors.initials: PW
authors.lastname: Ho
authors.affiliation: Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong.
authors.forename: Shu-Leong
authors.initials: SL
authors.lastname: Ho
authors.affiliation: Neurodegeneration Discovery Performance Unit, RD Neurosciences, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, U.K.
authors.forename: Alastair D
authors.initials: AD
authors.lastname: Reith
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K. genta-ito@umin.ac.jp d.r.alessi@dundee.ac.uk.
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D000818:Animals; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; C520736:Rab10 protein, mouse; D020691:rab GTP-Binding Proteins
keywords: Parkinson's disease; Rab GTPase; protein kinases; signal transduction