1. Biochemistry and Chemical Biology
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Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

  1. Martin Steger
  2. Federico Diez
  3. Herschel S Dhekne
  4. Pawel Lis
  5. Raja S Nirujogi
  6. Ozge Karayel
  7. Francesca Tonelli
  8. Terina N Martinez
  9. Esben Lorentzen
  10. Suzanne R Pfeffer
  11. Dario R Alessi  Is a corresponding author
  12. Matthias Mann  Is a corresponding author
  1. Max Planck Institute of Biochemistry, Germany
  2. University of Dundee, United Kingdom
  3. Stanford University School of Medicine, United States
  4. The Michael J. Fox Foundation for Parkinson's Research, United States
  5. Aarhus University, Denmark
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Cite this article as: eLife 2017;6:e31012 doi: 10.7554/eLife.31012

Abstract

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, Tonelli 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.

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. Federico Diez

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  3. Herschel S Dhekne

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  4. Pawel Lis

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  5. Raja S Nirujogi

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  6. Ozge Karayel

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

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
  8. Terina N Martinez

    The Michael J. Fox Foundation for Parkinson's Research, New York, United States
    Competing interests
    No competing interests declared.
  9. Esben Lorentzen

    Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6493-7220
  10. Suzanne R Pfeffer

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    Suzanne R Pfeffer, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X
  11. Dario R Alessi

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    For correspondence
    d.r.alessi@dundee.ac.uk
    Competing interests
    No competing interests declared.
  12. 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1292-4799

Funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Ivan Dikic, Goethe University Frankfurt, Germany

Publication history

  1. Received: August 4, 2017
  2. Accepted: November 9, 2017
  3. Accepted Manuscript published: November 10, 2017 (version 1)
  4. Version of Record published: November 20, 2017 (version 2)

Copyright

© 2017, 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

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Martin Steger et al.
    Research Article Updated

    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 threefold. 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.

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