1. Cell Biology

Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase

  1. Herschel S Dhekne
  2. Francesca Tonelli
  3. Wondwossen M Yeshaw
  4. Claire Y Chiang
  5. Charles Limouse
  6. Ebsy Jaimon
  7. Elena Purlyte
  8. Dario R Alessi
  9. Suzanne R Pfeffer  Is a corresponding author
  1. Stanford University, United States
  2. University of Dundee, United Kingdom
https://doi.org/10.7554/eLife.87098
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Cite this article
  1. Herschel S Dhekne
  2. Francesca Tonelli
  3. Wondwossen M Yeshaw
  4. Claire Y Chiang
  5. Charles Limouse
  6. Ebsy Jaimon
  7. Elena Purlyte
  8. Dario R Alessi
  9. Suzanne R Pfeffer
(2023)
Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase
eLife 12:e87098.
https://doi.org/10.7554/eLife.87098
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Abstract

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. Alphafold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

Data availability

All primary data associated with each figure has been deposited in a repository and can be found at 10.5281/zenodo.8020979, https://zenodo.org/record/8035448, and https://zenodo.org/record/7659210.

The following data sets were generated

Article and author information

Author details

  1. Herschel S Dhekne

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2240-1230

  2. Francesca Tonelli

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4600-6630

  3. Wondwossen M Yeshaw

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3134-3458

  4. Claire Y Chiang

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Charles Limouse

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ebsy Jaimon

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Elena Purlyte

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  8. Dario R Alessi

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2140-9185

  9. Suzanne R Pfeffer

    Department of Biochemistry, Stanford University, Stanford, United States
    For correspondence
    pfeffer@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

Funding

Aligning Science Across Parkinson's Disease (000463)

  • Dario R Alessi
  • Suzanne R Pfeffer

Michael J. Fox Foundation for Parkinson's Research (009258)

  • Dario R Alessi
  • Suzanne R Pfeffer

Michael J. Fox Foundation for Parkinson's Research (021132)

  • Suzanne R Pfeffer

National Institutes of Health (5T32 GM007276)

  • Claire Y Chiang

Medical Research Council (MC_UU_00018/1)

  • Dario R Alessi

Boehringer Ingelheim

  • Dario R Alessi

GlaxoSmithKline

  • Dario R Alessi

Merck KGaA

  • Dario R Alessi

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

Ethics

Animal experimentation: All animal studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 and regulations set by the University of Dundee and the U.K. Home Office.

Reviewing Editor

  1. J. Wade Harper, Harvard Medical School, United States

Version history

  1. Preprint posted: February 18, 2023 (view preprint)
  2. Received: February 23, 2023
  3. Accepted: June 22, 2023
  4. Accepted Manuscript published: October 24, 2023 (version 1)

Copyright

© 2023, Dhekne 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. Herschel S Dhekne
  2. Francesca Tonelli
  3. Wondwossen M Yeshaw
  4. Claire Y Chiang
  5. Charles Limouse
  6. Ebsy Jaimon
  7. Elena Purlyte
  8. Dario R Alessi
  9. Suzanne R Pfeffer
(2023)
Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase
eLife 12:e87098.
https://doi.org/10.7554/eLife.87098

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