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

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.

Reviewing Editor

  1. Ivan Dikic, Goethe University Medical School, Germany

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.

Version 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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  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
(2016)
Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases
eLife 5:e12813.
https://doi.org/10.7554/eLife.12813

Share this article

https://doi.org/10.7554/eLife.12813

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Edmundo G Vides, Ayan Adhikari ... Suzanne R Pfeffer
    Research Advance Updated

    Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease, and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here, we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360–450 and show that this domain, termed ‘site #1,’ can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher-affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed ‘site #2’ that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

    1. Biochemistry and Chemical Biology
    Martin Steger, Federico Diez ... Matthias Mann
    Research Advance Updated

    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.