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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.
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•Our study provides the first broad survey of phosphatases in their ability to regulate LRRK2 phosphorylation in cells, revealing several PP2A subunits and confirming PP1 as regulators of LRRK2 phosphorylation.•Our validation experiments show that the PP2A phosphatase holoenzyme complex composed of the catalytic subunit PP2CA/B and the regulatory subunit PPP2R2A/B/C/D is a direct upstream regulator of LRRK2 phosphorylation.•The PP2A catalytic subunit PPP2CA/B alone is a poor dephosphorylator of LRRK2, and must be in complex with the regulatory subunit PPP2R2A/B/C/D to affect LRRK2 phosphorylation, both for WT LRRK2 and clinical mutant forms of LRRK2.•Induction of LRRK2 dephosphorylation with LRRK2 kinase inhibitors is associated to the recruitment of PP2A catalytic and regulatory subunits to the LRRK2 compartment.•PP2A holoenzyme mediated dephosphorylation of LRRK2 induces LRRK2 ubiquitination and affects LRRK2 expression levels.