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.

Reviewing Editor

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

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.

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)
  5. Version of Record published: December 8, 2023 (version 2)

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

Share this article

https://doi.org/10.7554/eLife.87098

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Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation

    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
    2. Cell Biology

    Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    Martin Steger, Francesca Tonelli ... Matthias Mann
    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.

    1. Cell Biology

    Rab12 is a regulator of LRRK2 and its activation by damaged lysosomes

    Xiang Wang, Vitaliy V Bondar ... Anastasia G Henry
    Short Report Updated

    Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson’s disease (PD) and Crohn’s disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.