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

Lis, P., Burel, S., Steger, M., Mann, M., Brown, F., Diez, F., et al. (2018). Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase. Biochemical Journal, 475, 1-22. doi:10.1042/BCJ20170802.

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© 2018 The Author(s) This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).
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Lis, Pawel1, Author
Burel, Sophie1, Author
Steger, Martin2, Author              
Mann, Matthias2, Author              
Brown, Fiona1, Author
Diez, Federico1, Author
Tonelli, Francesca1, Author
Holton, Janice L.1, Author
Ho, Philip Winglok1, Author
Ho, Shu-Leong1, Author
Chou, Meng-Yun1, Author
Polinski, Nicole K.1, Author
Martinez, Terina N.1, Author
Davies, Paul1, Author
Alessi, Dario R.1, Author
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1external, ou_persistent22              
2Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565159              

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Free keywords: CYTOPLASMIC LOCALIZATION; MONOCLONAL-ANTIBODIES; 14-3-3 BINDING; PHOSPHORYLATION; MUTATIONS; INHIBITORS; MLI-2; MICEBiochemistry & Molecular Biology;
 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-controlled 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.

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Language(s): eng - English
 Dates: 2018-012018
 Publication Status: Published in print
 Pages: 22
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: ISI: 000428084900001
DOI: 10.1042/BCJ20170802
 Degree: -

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Title: Biochemical Journal
Source Genre: Journal
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Publ. Info: London : Published by Portland Press on behalf of the Biochemical Society.
Pages: - Volume / Issue: 475 Sequence Number: - Start / End Page: 1 - 22 Identifier: ISSN: 0264-6021
CoNE: https://pure.mpg.de/cone/journals/resource/110992357308158