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

NEUROBIOLOGY OF DISEASE

卷:157

Article

Drouyer, Matthieu^1,2,11  Bolliger, Marc F.^3,12  Lobbestael, Evy⁴  Van den Haute, Chris^4,5  Emanuele, Marco^1,2  Lefebvre, Reginald^1,2  Sibran, William^1,2  De Wit, Tina⁴  Leghay, Coline^1,2  Mutez, Eugenie^1,2  Dzamko, Nicolas^6,7  Halliday, Glenda M.^6,7  Murayama, Shigeo⁸  Martoriati, Alain⁹  Cailliau, Katia⁹  Bodart, Jean-Francois⁹  Chartier-Harlin, Marie-Christine^1,2  Baekelandt, Veerle⁴  Nichols, R. Jeremy^3,10  Taymans, Jean-Marc^1,2,4 

[1] Univ Lille, CHU Lille, INSERM, UMR S1172,LilNCog,Lille Neurosci & Cognit, F-59000 Lille, France

[2] INSERM, Team Brain Biol & Chem, UMR S 1172, F-59000 Lille, France

[3] Parkinsons Inst & Clin Ctr, Sunnyvale, CA 94085 USA

[4] Katholieke Univ Leuven, Dept Neurosci, Lab Neurobiol & Gene Therapy, B-3000 Leuven, Belgium

[5] Katholieke Univ Leuven, Leuven Viral Vector Core, Leuven, Belgium

[6] Univ Sydney, Cent Clin Sch, Camperdown, NSW 2050, Australia

[7] Univ NSW, Sch Med Sci, Kensington, NSW 2033, Australia

[8] Tokyo Metropolitan Geriatr Hosp, Dept Neuropathol, Brain Bank Aging Res, Tokyo 1730015, Japan

[9] Univ Lille, CNRS, UMR 8576 UGSF, Unite Glycobiol Struct & Fonct, F-59000 Lille, France

[10] Stanford Univ, Sch Med, Dept Pathol, Palo Alto, CA 94305 USA

[11] Childrens Med Res Inst, Westmead, NSW, Australia

[12] ESCAPE Bio, San Francisco, CA 94080 USA

关键词: LRRK2;    Phosphatases;    Phosphorylation;    Ubiquitination;    Parkinson's disease;    PP2A;    CRISPRi;   

DOI  :  10.1016/j.nbd.2021.105426

来源: Elsevier

【 摘 要 】

LRRK2 is a hig h l y phosphorylated multidomain protein and mutations in the gene encoding LRRK2 ar e a major genetic determinant of Parkinson's disease (PD). Dephosphorylation at LRRK2's S910/S935/S955/S973 phos-phosite 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 dephos-phorylation is poorly understood. We performed a phosphatome-wide reverse genetics screen to identi f y phosphatases involved in the dephosphorylation of the LRRK2 phosphosite S935. Candidate phosphatases selected from the prima r y screen were tested in mammalian cells, Xenopus oocytes and in vitro. Effects of PP2A on endogenous LRRK2 phos-phorylation were examined via expression modulation with CRISPR/dCas9. Our screening revealed LRRK2 phosphorylation regulators linked to the PP1 and PP2A holoenzyme complexes as we l l 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.