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

  1. Edmundo G Vides
  2. Ayan Adhikari
  3. Claire Y Chiang
  4. Pawel Lis
  5. Elena Purlyte
  6. Charles Limouse
  7. Justin L Shumate
  8. Elena Spinola-Lasso
  9. Herschel S Dhekne
  10. Dario R Alessi
  11. Suzanne R Pfeffer  Is a corresponding author
  1. Stanford University, United States
  2. University of Dundee, United Kingdom
  3. Universidad de Las Palmas de Gran Canaria, Spain

Abstract

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.

Data availability

All primary data associated with each figure has been deposited in a repository and can be found at https://doi.org/10.5061/dryad.3tx95x6j7; quantitation data of the blots in Figure 3--Fig. Supp. 4 (for the bar graphs in Figures 3C and 3D) can be found at doi (10.5281/zenodo.7057419).

The following data sets were generated

Article and author information

Author details

  1. Edmundo G Vides

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  2. Ayan Adhikari

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  3. Claire Y Chiang

    Department of Biochemistry, Stanford University, 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4978-7671
  5. Elena Purlyte

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

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  7. Justin L Shumate

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
  8. Elena Spinola-Lasso

    Departamento de Bioquímica y Biología Molecular, Universidad de Las Palmas de Gran Canaria, Gran Canaria, Spain
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2207-5786
  9. Herschel S Dhekne

    Department of Biochemistry, Stanford University, Stanford, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2240-1230
  10. Dario R Alessi

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2140-9185
  11. Suzanne R Pfeffer

    Department of Biochemistry, Stanford University, Stanford, United States
    For correspondence
    pfeffer@stanford.edu
    Competing interests
    Suzanne R Pfeffer, Senior editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

Funding

Aligning Science Across Parkinson's Disease (ASAP-000463)

  • Dario R Alessi
  • Suzanne R Pfeffer

Michael J Fox Foundation (17298)

  • Dario R Alessi
  • Suzanne R Pfeffer

Michael J Fox Foundation (6986)

  • Dario R Alessi
  • Suzanne R Pfeffer

Medical Research Council (MC_UU_00018/1)

  • 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. Wade Harper, Harvard Medical School, United States

Version history

  1. Preprint posted: April 25, 2022 (view preprint)
  2. Received: April 26, 2022
  3. Accepted: September 22, 2022
  4. Accepted Manuscript published: September 23, 2022 (version 1)
  5. Version of Record published: October 17, 2022 (version 2)

Copyright

© 2022, Vides 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.

Metrics

  • 2,340
    Page views
  • 634
    Downloads
  • 8
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Edmundo G Vides
  2. Ayan Adhikari
  3. Claire Y Chiang
  4. Pawel Lis
  5. Elena Purlyte
  6. Charles Limouse
  7. Justin L Shumate
  8. Elena Spinola-Lasso
  9. Herschel S Dhekne
  10. Dario R Alessi
  11. Suzanne R Pfeffer
(2022)
A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation
eLife 11:e79771.
https://doi.org/10.7554/eLife.79771

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    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. Biochemistry and Chemical Biology
    2. Epidemiology and Global Health
    Takashi Sasaki, Yoshinori Nishimoto ... Yasumichi Arai
    Research Article

    Background: High levels of circulating adiponectin are associated with increased insulin sensitivity, low prevalence of diabetes, and low body mass index (BMI); however, high levels of circulating adiponectin are also associated with increased mortality in the 60-70 age group. In this study, we aimed to clarify factors associated with circulating high-molecular-weight (cHMW) adiponectin levels and their association with mortality in the very old (85-89 years old) and centenarians.

    Methods: The study included 812 (women: 84.4%) for centenarians and 1,498 (women: 51.7%) for the very old. The genomic DNA sequence data were obtained by whole genome sequencing or DNA microarray-imputation methods. LASSO and multivariate regression analyses were used to evaluate cHMW adiponectin characteristics and associated factors. All-cause mortality was analyzed in three quantile groups of cHMW adiponectin levels using Cox regression.

    Results: The cHMW adiponectin levels were increased significantly beyond 100 years of age, were negatively associated with diabetes prevalence, and were associated with SNVs in CDH13 (p = 2.21 × 10-22) and ADIPOQ (p = 5.72 × 10-7). Multivariate regression analysis revealed that genetic variants, BMI, and high-density lipoprotein cholesterol (HDLC) were the main factors associated with cHMW adiponectin levels in the very old, whereas the BMI showed no association in centenarians. The hazard ratios for all-cause mortality in the intermediate and high cHMW adiponectin groups in very old men were significantly higher rather than those for all-cause mortality in the low level cHMW adiponectin group, even after adjustment with BMI. In contrast, the hazard ratios for all-cause mortality were significantly higher for high cHMW adiponectin groups in very old women, but were not significant after adjustment with BMI.

    Conclusions: cHMW adiponectin levels increased with age until centenarians, and the contribution of known major factors associated with cHMW adiponectin levels, including BMI and HDLC, varies with age, suggesting that its physiological significance also varies with age in the oldest old.

    Funding: This study was supported by grants from the Ministry of Health, Welfare, and Labour for the Scientific Research Projects for Longevity; a Grant-in-Aid for Scientific Research (No 21590775, 24590898, 15KT0009, 18H03055, 20K20409, 20K07792, 23H03337) from the Japan Society for the Promotion of Science; Keio University Global Research Institute (KGRI), Kanagawa Institute of Industrial Science and Technology (KISTEC), Japan Science and Technology Agency (JST) Research Complex Program 'Tonomachi Research Complex' Wellbeing Research Campus: Creating new values through technological and social innovation (JP15667051), the Program for an Integrated Database of Clinical and Genomic Information from the Japan Agency for Medical Research and Development (No. 16kk0205009h001, 17jm0210051h0001, 19dk0207045h0001); the medical-welfare-food-agriculture collaborative consortium project from the Japan Ministry of Agriculture, Forestry, and Fisheries; and the Biobank Japan Program from the Ministry of Education, Culture, Sports, and Technology.