A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation
- Stanford University, United States
- University of Dundee, United Kingdom
- 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).
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Data from: A feed-forward pathway drives LRRK2 kinase membrane recruitment and activationDryad Digital Repository, doi:10.5061/dryad.3tx95x6j7.
Article and author information
Author details
Elena Spinola-Lasso
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
- Wade Harper, Harvard Medical School, United States
Publication history
- Preprint posted: April 25, 2022 (view preprint)
- Received: April 26, 2022
- Accepted: September 22, 2022
- Accepted Manuscript published: September 23, 2022 (version 1)
- 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.
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A feed-forward pathway drives LRRK2 kinase membrane recruitment and activation
eLife 11:e79771.
https://doi.org/10.7554/eLife.79771
Further reading
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- Biochemistry and Chemical Biology
- Cell Biology
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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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
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