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 Ser935 and Ser1292 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 Ser910 and Ser935 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.
Highlights
Our knowledge of the origins of Parkinson’s disease has been transformed by the identification of genes whose mutation in humans leads to Mendelian inherited disease [1,2]
We first explored the effect of phosphorylation of recombinant bacterial expressed Rab10 with leucine-rich repeat kinase 2 (LRRK2)[G2019S] on the electrophoretic mobility of Rab10 on Phos-tag-containing polyacrylamide gels
Immunoblot analysis with a phospho-specific antibody confirmed that the slower migrating Rab10 species that appears following LRRK2 phosphorylation is Rab10 phosphorylated at Thr73 (Figure 1A)
Summary
Our knowledge of the origins of Parkinson’s disease has been transformed by the identification of genes whose mutation in humans leads to Mendelian inherited disease [1,2] One of these genes encodes the leucine-rich repeat kinase 2 (LRRK2) protein kinase where autosomal dominant mutations account for ∼1 % of sporadic Parkinson’s disease [3,4]. The most common LRRK2 mutation converts Gly2019 into a serine within the kinase domain magnesium ion-binding motif [5] This mutation enhances in vitro protein kinase activity ∼3-fold [6,7], indicating that abnormal increase in the kinase activity of LRRK2 is involved in the pathogenesis of Parkinson’s disease, suggesting that LRRK2 kinase inhibitors have therapeutic benefit for the treatment of Parkinson’s disease. Unlike the G2019S mutation, the R1441G/H/C and Y1699C mutations do not directly enhance LRRK2 in vitro kinase activity [12]
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