Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase

Pawel Lis, Sophie Burel, Martin Steger, Matthias Mann, Fiona Brown, Federico Diez, Francesca Tonelli, Janice L. Holton, Philip Winglok Ho, Shu-Leong Ho, Meng-Yun Chou, Nicole K. Polinski, Terina N. Martinez, Paul Davies, Dario R. Alessi (Lead / Corresponding author)

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Abstract

Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson’s disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-con-trolled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo. These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.

Original languageEnglish
Pages (from-to)1-22
Number of pages22
JournalBiochemical Journal
Volume475
Issue number1
Early online date10 Nov 2017
DOIs
Publication statusPublished - 2 Jan 2018

Keywords

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal/biosynthesis
  • Antibodies, Phospho-Specific/biosynthesis
  • Antibody Specificity
  • Gene Expression Regulation
  • Genetic Predisposition to Disease
  • Gyrus Cinguli/enzymology
  • Humans
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics
  • Mice
  • Multigene Family
  • Mutation
  • Parkinson Disease/enzymology
  • Phosphorylation
  • Protein Isoforms/chemistry
  • Rabbits
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • rab GTP-Binding Proteins/chemistry

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    Lis, P., Burel, S., Steger, M., Mann, M., Brown, F., Diez, F., Tonelli, F., Holton, J. L., Winglok Ho, P., Ho, S-L., Chou, M-Y., Polinski, N. K., Martinez, T. N., Davies, P., & Alessi, D. R. (2018). Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase. Biochemical Journal, 475(1), 1-22. https://doi.org/10.1042/BCJ20170802