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Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
Published on: September 18, 2013
Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors
Genta Ito1, Kristina Katsemonova2, Francesca Tonelli2
1MRC 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.
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.
Insights
A new Phos-tag assay effectively measures Rab10 phosphorylation by leucine-rich repeat kinase 2 (LRRK2), crucial for Parkinson's disease research. This method aids in evaluating LRRK2 inhibitors and mutations impacting the LRRK2 signaling pathway.
Area of Science:
- Neuroscience
- Molecular Biology
- Biochemistry
Background:
- Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are a primary genetic cause of inherited Parkinson's disease.
- LRRK2 kinase activity leads to the phosphorylation of Rab GTPase proteins, including Rab10, at specific residues.
- Understanding LRRK2 signaling is critical for developing targeted Parkinson's disease therapies.
Purpose of the Study:
- To develop a robust method for assessing endogenous Rab10 phosphorylation.
- To validate the role of LRRK2 as the primary kinase for Rab10.
- To evaluate the impact of LRRK2 mutations and inhibitors on Rab10 phosphorylation.
Main Methods:
- Development of a Phos-tag based assay to detect electrophoretic mobility shifts of phosphorylated Rab10.
- Application of the assay in mouse embryonic fibroblasts (MEFs) and B-cells.
- Utilized kinase-inactive LRRK2 and LRRK2 inhibitor treatments to confirm LRRK2's role and assess inhibitor efficacy.
Main Results:
- The Phos-tag assay successfully detected LRRK2-mediated Rab10 phosphorylation in various cell types and tissues.
- Rab10 phosphorylation was abolished in kinase-inactive LRRK2 models, confirming LRRK2 as the major Rab10 kinase.
- Pathogenic LRRK2 mutations (G2019S, R1441G) increased Rab10 phosphorylation, while inhibitors rapidly decreased it (1-2 min).
- Phosphorylation of Rab10 was suppressed in LRRK2[S910A+S935A] MEFs, suggesting a role for Ser910/935 phosphorylation and 14-3-3 binding.
Conclusions:
- The Rab Phos-tag assay provides a sensitive and reliable tool for studying LRRK2 kinase activity.
- This assay can effectively monitor the effects of LRRK2 inhibitors and pathogenic mutations on Rab10 phosphorylation.
- Findings advance the understanding of LRRK2 signaling pathways relevant to Parkinson's disease pathogenesis and drug development.
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