'Up with the LRRK': a phosphorylated Rab10 assay for evaluation of LRRK2 activity and inhibitor engagement.

Patrick A Eyers

doi:10.1042/bcj20160671c

Abstract

Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both in vitro and in vivo. A new study published in the Biochemical Journal by Ito et al. establishes that a ‘Phos-tag’™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory.

Highlights

Protein phosphorylation is a reversible covalent modification that has the useful consequence of changing both the acidity and the chemical nature of modified amino acid(s) [1]
The kinetics of leucine-rich repeat kinase 2 (LRRK2) dephosphorylation were markedly slower using this assay. These data, which were itself internally validated with a ‘drug-resistant’ G2019S LRRK2 mutant and the compound GSK2578215A [31], might be useful for evaluating the appropriate ‘window’ of target engagement for other LRRK2 inhibitors in vivo and might have a profound effect on the choice of selection of Phos-tag Ras-related protein in brain 10 (Rab10) assays over LRRK2 phospho-specific antibodies, especially under conditions where Rab10 phospho-specific antibodies are ineffective or limiting
Cell signalling researchers are working in a golden age of chemical biology

Summary

Introduction

Protein phosphorylation is a reversible covalent modification that has the useful consequence of changing both the acidity (through introduction of negative charge) and the chemical nature of modified amino acid(s) [1]. These physiochemical changes can be exploited to measure the context, extent and lability of phosphorylation in vast number of ways, most classically through polyacrylamide gel-based quantification (typically western blotting and phospho-specific antibody procedures) or by using mass spectrometry (MS)-based approaches.

Results

Conclusion