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Updated: Nov 24, 2025
Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935
Raja S Nirujogi1, Francesca Tonelli1, Matthew Taylor1
1Medical Research Council (MRC) Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.
Abstract:
Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.
Insights
A new assay measures LRRK2-phosphorylated Rab proteins, crucial for Parkinson's disease research. This method reveals elevated Rab phosphorylation in patients with VPS35 mutations, offering insights into disease mechanisms.
Area of Science:
- Neuroscience
- Biochemistry
- Genetics
Background:
- Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are a common cause of familial Parkinson's disease.
- LRRK2 phosphorylates Rab GTPases, affecting their interactions and cellular functions.
- Understanding LRRK2's substrates and signaling pathways is critical for Parkinson's disease (PD) research.
Purpose of the Study:
- To develop and validate a novel multiplexed targeted mass spectrometry assay for quantifying LRRK2-phosphorylated Rab proteins.
- To measure endogenous levels of specific Rab substrates and LRRK2 phosphorylation sites.
- To investigate the impact of PD-associated mutations and LRRK2 inhibitors on Rab phosphorylation.
Main Methods:
- Development of a multiplexed targeted mass spectrometry assay.
- Quantification of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35, Rab43) and total Rabs.
- Analysis of LRRK2 phosphorylation at Ser910 and Ser935.
- Application of the assay in cell lines (MEFs, neutrophils) and mouse tissues (brain, kidney, lung, spleen).
- Assessment of LRRK2[R1441C] and VPS35[D620N] mutations and LRRK2 inhibitors.
Main Results:
- The assay successfully quantified endogenous pRab levels and LRRK2 phosphorylation sites.
- VPS35[D620N] mutation, but not LRRK2[R1441C], increased Rab1 phosphorylation, which was blocked by LRRK2 inhibitors.
- This study provides the first evidence of endogenous Rab1 as a physiological LRRK2 substrate.
- Elevated phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10, Rab43) was observed in Parkinson's patients with VPS35[D620N] mutations.
- The developed assay offers advantages over traditional immunoblotting methods for assessing LRRK2 Rab signaling.
Conclusions:
- A novel mass spectrometry assay enables precise quantification of LRRK2-mediated Rab phosphorylation.
- The VPS35[D620N] mutation dysregulates LRRK2 signaling, leading to increased Rab phosphorylation in Parkinson's disease patients.
- This assay is a valuable tool for studying LRRK2 in PD pathogenesis and for evaluating LRRK2 inhibitors.
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