Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Genta Ito (Lead / Corresponding author), Kristina Katsemonova, Francesca Tonelli, Pawel Lis, Marco Baptista, Natalia Shpiro, Graham Duddy, Steve Wilson, Philip Wing-Lok Ho, Shu-Leong Ho, Alastair D. Reith, Dario Alessi (Lead / Corresponding author)

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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 Thr/Ser 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] knock-in 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) knock-in 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] knock-in 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 aide with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.
Original languageEnglish
Pages (from-to)2671-2685
Number of pages15
JournalBiochemical Journal
Volume473
Issue number17
Early online date29 Jul 2016
DOIs
Publication statusPublished - 30 Aug 2016

Keywords

  • Protein kinases
  • Signal transduction
  • Rab GTPase
  • Parkinson's disease

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