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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., Ho, Philip Winglok, Ho, Shu-Leong, Chou, Meng-Yun, Polinski, Nicole K., Martinez, Terina N., Davies, Paul, Alessi, Dario R.·2018·Biochemical Journal
DOI 10.1042/bcj20170802PMC PMC5748839
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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-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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  • Rab10 was identified as phosphorylated by LRRK2 combining two independent genetic and chemical phosphoproteomic screens and has been validated in subsequent studies.

    “Rab10 specifically was identified as phosphorylated by LRRK2 combining two independent genetic and chemical phosphoproteomic screens and has been validated in subsequent studies [19, 28, 32, 34, 35, 41, 79].”

    From Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain (2023) · cites this paper as [41]

    The Introduction identifies Rab10 as an LRRK2 substrate, and the study's antibody experiments provide further evidence of LRRK2-dependent Rab10 phosphorylation. Our copy does not say Rab10 was identified by combining two independent genetic and chemical phosphoproteomic screens. The indexed passages have section labels but no page numbers. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • The G2019S-LRRK2 mutation increases LRRK2 kinase activity.

    “The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates.”

    From Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain (2023) · cites this paper as [41]

  • Multiple studies show that a subset of Rab GTPases are LRRK2 kinase substrates, and LRRK2 can phosphorylate these Rab GTPases in vivo and in vitro.

    “Multiple studies show that a subset of Rab GTPases are LRRK2 kinase substrates, and LRRK2 can phosphorylate these Rab GTPases in vivo and in vitro [41, 79].”

    From Cellular and subcellular localization of Rab10 and phospho-T73 Rab10 in the mouse and human brain (2023) · cites this paper as [41]

  • RAB10 serves as a prominent LRRK2 kinase substrate in all cell lines and tissues analyzed.

    “RAB10 serves as a prominent LRRK2 kinase substrate in all cell lines and tissues analyzed [12,18,32,33,34,35].”

    From Distinct Roles for RAB10 and RAB29 in Pathogenic LRRK2-Mediated Endolysosomal Trafficking Alterations (2020) · cites this paper as [33]

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Citations

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Research works printed but not linked: 27

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  2. Paisán-Ruı´z, 2004, Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease, Neuron, 44, 595, 10.1016/j.neuron.2004.10.023 · DOI
  3. Simón-Sánchez, 2009, Genome-wide association study reveals genetic risk underlying Parkinson's disease, Nat. Genet., 41, 1308, 10.1038/ng.487 · DOI · Paper page
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  9. West, 2017, Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease, Exp. Neurol., 298, 236, 10.1016/j.expneurol.2017.07.019 · DOI
  10. Steger, 2016, Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases, eLife, 5, e12813, 10.7554/eLife.12813 · DOI · Paper page
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  12. Steger, 2017, Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis, eLife, 6, e31012, 10.7554/eLife.31012 · DOI · Paper page
  13. Thirstrup, 2017, Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells, Sci. Rep., 7, 10300, 10.1038/s41598-017-10501-z · DOI
  14. Levin, 2016, Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen, Proc. Natl Acad. Sci. U.S.A., 113, E4776, 10.1073/pnas.1608355113 · DOI
  15. Shinde, 2016, PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7, Nat. Commun., 7, 10689, 10.1038/ncomms10689 · DOI
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  17. Scott, 2017, Discovery of a 3-(4-pyrimidinyl) indazole (MLi-2), an orally available and selective leucine-rich repeat kinase 2 (LRRK2) inhibitor that reduces brain kinase activity, J. Med. Chem., 60, 2983, 10.1021/acs.jmedchem.7b00045 · DOI
  18. Harlow, 1988, Antibodies A Laboratory Manual
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  21. Fan, Y., Howden, A.J.M., Sarhan, A.R., Lis, P., Martinez, T.N., Brockmann, K.et al. (2017) Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils. Biochem. J.475, 1–2210.1042/BCJ20170803 · DOI · Paper page
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Metadata

Authors
Lis, P., Burel, S., Steger, M., Mann, M., Brown, F., Diez, F., Tonelli, F., Holton, J. L., Ho, P. W., Ho, S.-L., Chou, M.-Y., Polinski, N. K., Martinez, T. N., Davies, P., & Alessi, D. R.
Venue
Biochemical Journal
Publisher
Portland Press Ltd.
Published
2018-01-02
Volume
475
Issue
1
Pages
1-22
ISSN
  • 0264-6021
  • 1470-8728
DOI
10.1042/bcj20170802
PMC
PMC5748839
Cited by
151
References
3
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor, 569 d)

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