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)

Research output: Contribution to journalArticle

20 Citations (Scopus)
205 Downloads (Pure)

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

Fingerprint

Protein Kinases
Parkinson Disease
Phosphotransferases
Antibodies
Proteins
Protein Kinase Inhibitors
Phospho-Specific Antibodies
Monoclonal Antibodies
Phosphorylation
rab3A GTP-Binding Protein
leucine-rich repeat proteins
Rabbits
Mutation
Gyrus Cinguli
Brain
Clinical Trials
Cells
Switches
Cell Line
Tissue

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

Cite this

Lis, Pawel ; Burel, Sophie ; Steger, Martin ; Mann, Matthias ; Brown, Fiona ; Diez, Federico ; Tonelli, Francesca ; Holton, Janice L. ; Winglok Ho, Philip ; Ho, Shu-Leong ; Chou, Meng-Yun ; Polinski, Nicole K. ; Martinez, Terina N. ; Davies, Paul ; Alessi, Dario R. / Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase. In: Biochemical Journal. 2018 ; Vol. 475, No. 1. pp. 1-22.
@article{3f0c373739c44624ac40e5157c428299,
title = "Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase",
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.",
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",
author = "Pawel Lis and Sophie Burel and Martin Steger and Matthias Mann and Fiona Brown and Federico Diez and Francesca Tonelli and Holton, {Janice L.} and {Winglok Ho}, Philip and Shu-Leong Ho and Meng-Yun Chou and Polinski, {Nicole K.} and Martinez, {Terina N.} and Paul Davies and Alessi, {Dario R.}",
note = "This work was supported by the Michael J. Fox Foundation for Parkinson's research [grant number 12938 (to M.M. and D.R.A.)]; the Medical Research Council [grant numbers MC_UU_12016/2 (to D.R.A.) and MR/P00704X/1 COEN award]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer-to D.R.A.) and the Henry G Leong Endowed Professorship in Neurology (to PWH & SLH).",
year = "2018",
month = "1",
day = "2",
doi = "10.1042/BCJ20170802",
language = "English",
volume = "475",
pages = "1--22",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press",
number = "1",

}

Lis, P, Burel, S, Steger, M, Mann, M, Brown, F, Diez, F, Tonelli, F, Holton, JL, Winglok Ho, P, Ho, S-L, Chou, M-Y, Polinski, NK, Martinez, TN, Davies, P & Alessi, DR 2018, 'Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase', Biochemical Journal, vol. 475, no. 1, pp. 1-22. https://doi.org/10.1042/BCJ20170802

Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase. / Lis, Pawel; Burel, Sophie; Steger, Martin; Mann, Matthias; Brown, Fiona ; Diez, Federico; Tonelli, Francesca; Holton, Janice L.; Winglok Ho, Philip; Ho, Shu-Leong; Chou, Meng-Yun ; Polinski, Nicole K.; Martinez, Terina N.; Davies, Paul; Alessi, Dario R. (Lead / Corresponding author).

In: Biochemical Journal, Vol. 475, No. 1, 02.01.2018, p. 1-22.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson’s disease kinase

AU - Lis, Pawel

AU - Burel, Sophie

AU - Steger, Martin

AU - Mann, Matthias

AU - Brown, Fiona

AU - Diez, Federico

AU - Tonelli, Francesca

AU - Holton, Janice L.

AU - Winglok Ho, Philip

AU - Ho, Shu-Leong

AU - Chou, Meng-Yun

AU - Polinski, Nicole K.

AU - Martinez, Terina N.

AU - Davies, Paul

AU - Alessi, Dario R.

N1 - This work was supported by the Michael J. Fox Foundation for Parkinson's research [grant number 12938 (to M.M. and D.R.A.)]; the Medical Research Council [grant numbers MC_UU_12016/2 (to D.R.A.) and MR/P00704X/1 COEN award]; the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer-to D.R.A.) and the Henry G Leong Endowed Professorship in Neurology (to PWH & SLH).

PY - 2018/1/2

Y1 - 2018/1/2

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Animals

KW - Antibodies, Monoclonal/biosynthesis

KW - Antibodies, Phospho-Specific/biosynthesis

KW - Antibody Specificity

KW - Gene Expression Regulation

KW - Genetic Predisposition to Disease

KW - Gyrus Cinguli/enzymology

KW - Humans

KW - Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics

KW - Mice

KW - Multigene Family

KW - Mutation

KW - Parkinson Disease/enzymology

KW - Phosphorylation

KW - Protein Isoforms/chemistry

KW - Rabbits

KW - Sequence Alignment

KW - Sequence Homology, Amino Acid

KW - Signal Transduction

KW - rab GTP-Binding Proteins/chemistry

UR - http://www.scopus.com/inward/record.url?scp=85036568749&partnerID=8YFLogxK

U2 - 10.1042/BCJ20170802

DO - 10.1042/BCJ20170802

M3 - Article

C2 - 29127256

VL - 475

SP - 1

EP - 22

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 1

ER -