Protein phosphatase 2A holoenzymes regulate leucine-rich repeat kinase 2 phosphorylation and accumulation.

Abstract

LRRK2 is a highly phosphorylated multidomain protein and mutations in the gene encoding LRRK2 are a major genetic determinant of Parkinson's disease (PD). Dephosphorylation at LRRK2's S910/S935/S955/S973 phosphosite cluster is observed in several conditions including in sporadic PD brain, in several disease mutant forms of LRRK2 and after pharmacological LRRK2 kinase inhibition. However, the mechanism of LRRK2 dephosphorylation is poorly understood. We performed a phosphatome-wide reverse genetics screen to identify phosphatases involved in the dephosphorylation of the LRRK2 phosphosite S935. Candidate phosphatases selected from the primary screen were tested in mammalian cells, Xenopus oocytes and in vitro. Effects of PP2A on endogenous LRRK2 phosphorylation were examined via expression modulation with CRISPR/dCas9. Our screening revealed LRRK2 phosphorylation regulators linked to the PP1 and PP2A holoenzyme complexes as well as CDC25 phosphatases. We showed that dephosphorylation induced by different kinase inhibitor triggered relocalisation of phosphatases PP1 and PP2A in LRRK2 subcellular compartments in HEK-293 T cells. We also demonstrated that LRRK2 is an authentic substrate of PP2A both in vitro and in Xenopus oocytes. We singled out the PP2A holoenzyme PPP2CA:PPP2R2 as a powerful phosphoregulator of pS935-LRRK2. Furthermore, we demonstrated that this specific PP2A holoenzyme induces LRRK2 relocalization and triggers LRRK2 ubiquitination, suggesting its involvement in LRRK2 clearance. The identification of the PPP2CA:PPP2R2 complex regulating LRRK2 S910/S935/S955/S973 phosphorylation paves the way for studies refining PD therapeutic strategies that impact LRRK2 phosphorylation.

• Authors: Matthieu Drouyer, Marc F Bolliger, Evy Lobbestael, Chris Van den Haute, Marco Emanuele, Réginald Lefebvre, William Sibran, Tina De Wit, Coline Leghay, Eugénie Mutez, Nicolas Dzamko, Glenda M Halliday, Shigeo Murayama, Alain Martoriati, Katia Cailliau, Jean-François Bodart, Marie-Christine Chartier-Harlin, Veerle Baekelandt, R Jeremy Nichols, Jean-Marc Taymans
• Journal: Neurobiology of disease (Neurobiol Dis) Vol. 157 Pg. 105426 (09 2021) ISSN: 1095-953X [Electronic] United States
• PMID: 34144124 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2021. Published by Elsevier Inc.

Chemical References

• Holoenzymes
• Nerve Tissue Proteins
• PPP2R2A protein, human
• PPP2R2C protein, human
• PPP2R2D protein, human
• Protein Kinase Inhibitors
• Xenopus Proteins
• Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
• PPP2CA protein, human
• PPP2R2B protein, human
• Protein Phosphatase 1
• Protein Phosphatase 2
• ppp2r2a protein, Xenopus laevis

Topics

• Animals
• HEK293 Cells
• Holoenzymes (metabolism)
• Humans
• In Vitro Techniques
• Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 (genetics, metabolism)
• Nerve Tissue Proteins (metabolism)
• Oocytes (metabolism)
• Phosphorylation
• Protein Kinase Inhibitors (pharmacology)
• Protein Phosphatase 1 (metabolism)
• Protein Phosphatase 2 (metabolism)
• Protein Transport (drug effects)
• Xenopus Proteins (metabolism)
• Xenopus laevis