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Updated: Sep 22, 2025
Assaying the Kinase Activity of LRRK2 in vitro
Published on: January 18, 2012
Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity
Belén Fernández1, Vinita G Chittoor-Vinod2, Jillian H Kluss3
1Institute of Parasitology and Biomedicine López-Neyra (IPBLN), Consejo Superior de Investigaciones Científicas (CSIC), Granada, Spain.
Background:
Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.
Objective:
To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.
Methods:
Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers.
Results:
Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated.
Conclusion:
Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.
