1. Biochemistry and Chemical Biology
  2. Cell Biology
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Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

  1. Martin Steger
  2. Francesca Tonelli
  3. Genta Ito
  4. Paul Davies
  5. Matthias Trost
  6. Melanie Vetter
  7. Stefanie Wachter
  8. Esben Lorentzen
  9. Graham Duddy
  10. Stephen Wilson
  11. Marco AS Baptista
  12. Brian K Fiske
  13. Matthew J Fell
  14. John A Morrow
  15. Alastair D Reith
  16. Dario R Alessi
  17. Matthias Mann  Is a corresponding author
  1. Max Planck Institute of Biochemistry, Germany
  2. University of Dundee, United Kingdom
  3. The Wellcome Trust Sanger Institute, United Kingdom
  4. GlaxoSmithKline Pharmaceuticals R&D, United Kingdom
  5. The Michael J. Fox Foundation for Parkinson's Research, United States
  6. Merck Research Laboratories, United States
Research Article
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Cite this article as: eLife 2016;5:e12813 doi: 10.7554/eLife.12813

Abstract

Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two to three-fold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD.

Article and author information

Author details

  1. Martin Steger

    Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  2. Francesca Tonelli

    Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  3. Genta Ito

    Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  4. Paul Davies

    Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  5. Matthias Trost

    Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  6. Melanie Vetter

    Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  7. Stefanie Wachter

    Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  8. Esben Lorentzen

    Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
    Competing interests
    No competing interests declared.
  9. Graham Duddy

    The Wellcome Trust Sanger Institute, Hinxton, United Kingdom
    Competing interests
    No competing interests declared.
  10. Stephen Wilson

    RD Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, United Kingdom
    Competing interests
    Stephen Wilson, Employees of GlaxoSmithKline, a global healthcare company that may conceivably benefit financially through this publication.
  11. Marco AS Baptista

    The Michael J. Fox Foundation for Parkinson's Research, New York, United States
    Competing interests
    No competing interests declared.
  12. Brian K Fiske

    The Michael J. Fox Foundation for Parkinson's Research, New York, United States
    Competing interests
    No competing interests declared.
  13. Matthew J Fell

    Early Discovery Neuroscience, Merck Research Laboratories, Boston, United States
    Competing interests
    Matthew J Fell, Employee of Merck, a global healthcare company that may conceivably benefit financially through this publication..
  14. John A Morrow

    Neuroscience, Merck Research Laboratories, Westpoint, United States
    Competing interests
    John A Morrow, employees of Merck Research Laboratories.
  15. Alastair D Reith

    Neurodegeneration Discovery Performance Unit, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, United Kingdom
    Competing interests
    Alastair D Reith, Employee of GlaxoSmithKline, a global healthcare company that may conceivably benefit financially through this publication.
  16. Dario R Alessi

    Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  17. Matthias Mann

    Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany
    For correspondence
    mmann@biochem.mpg.de
    Competing interests
    No competing interests declared.

Ethics

Animal experimentation: All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986, the GSK Policy on the Care, Welfare and Treatment of Animals, regulations set by the University of Dundee and the U.K. Home Office. Animal studies and breeding were approved by the University of Dundee ethical committee and performed under a U.K. Home Office project license.

Reviewing Editor

  1. Ivan Dikic, Goethe University Medical School, Germany

Publication history

  1. Received: November 3, 2015
  2. Accepted: January 21, 2016
  3. Accepted Manuscript published: January 29, 2016 (version 1)
  4. Version of Record published: February 16, 2016 (version 2)

Copyright

© 2016, Steger 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.

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Further reading

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    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.

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