PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins
- University of Dundee, United Kingdom
- Stanford University School of Medicine, United States
Abstract
Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A 'substrate-trapping' PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.
Data availability
We have deposited the mass spectrometry raw data and MaxQuant search output tables to ProteomeXchange, PRIDE database https://www.ebi.ac.uk/pride/archive/ unique identifier, PXD014794.We include the primary data for each of the 3 siRNA screens (Fig 2 Figure Supplement 1, 2 and 3), in addition to the quantitation of the pRab10/Total Rab10 ratios in Supplementary Excel File 1. The file also contains all RNA sequences of siRNA libraryAll Plasmids, antibodies and proteins (including datasheets and sequence information) that we have generated for this study can be requested and information downloaded from MRC PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk/).
Article and author information
Author details
Wondwossen M Yeshaw
Funding
Michael J. Fox Foundation for Parkinson's Research (17298)
- Suzanne R Pfeffer
- Dario R Alessi
Michael J. Fox Foundation for Parkinson's Research (6986)
- Suzanne R Pfeffer
- Dario R Alessi
Michael J. Fox Foundation for Parkinson's Research (Langston Award)
- Dario R Alessi
Medical Research Council (MC_UU_12016/2)
- Dario R Alessi
National Institutes of Health (DK37332)
- Suzanne R Pfeffer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Wade Harper, Harvard Medical School, United States
Publication history
- Received: July 22, 2019
- Accepted: October 30, 2019
- Accepted Manuscript published: October 30, 2019 (version 1)
- Version of Record published: November 12, 2019 (version 2)
Copyright
© 2019, Berndsen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 7,184
- Page views
-
- 994
- Downloads
-
- 55
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins
eLife 8:e50416.
https://doi.org/10.7554/eLife.50416
Further reading
-
- Biochemistry and Chemical Biology
We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.
-
- Cell Biology
Parkinson’s disease-associated LRRK2 kinase phosphorylates multiple Rab GTPases, including Rab8A and Rab10. We show here that LRRK2 kinase interferes with primary cilia formation in cultured cells, human LRRK2 G2019S iPS cells and in the cortex of LRRK2 R1441C mice. Rab10 phosphorylation strengthens its intrinsic ability to block ciliogenesis by enhancing binding to RILPL1. Importantly, the ability of LRRK2 to interfere with ciliogenesis requires both Rab10 and RILPL1 proteins. Pathogenic LRRK2 influences the ability of cells to respond to cilia-dependent, Hedgehog signaling as monitored by Gli1 transcriptional activation. Moreover, cholinergic neurons in the striatum of LRRK2 R1441C mice show decreased ciliation, which will decrease their ability to sense Sonic hedgehog in a neuro-protective circuit that supports dopaminergic neurons. These data reveal a molecular pathway for regulating cilia function that likely contributes to Parkinson’s disease-specific pathology.
Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
