Development of a multiplex targeted mass spectrometry assay to quantify levels of LRRK2 phosphorylated Rab substrates and Ser910 and Ser935 biomarker sites

ABSTRACT: Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplex targeted mass spectrometry assay to quantify endogenous levels of LRRK2 phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and phosphorylation of the LRRK2 Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts & human neutrophils) and mouse tissues (brain, lung, kidney and spleen). We define how each of the different pRab proteins are impacted by Parkinson’s pathogenic LRRK2[R1441C] and VPS35[D620N] mutations as well as LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that LRRK2 can phosphorylate Rab1 physiologically. We argue that this targeted mass spectrometry assay can replace immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Spleen, Brain, Lung, Cell Culture, Neutrophil, Fibroblast, Kidney

DISEASE(S): Parkinson's Disease

SUBMITTER: Raja Sekhar Nirujogi  

LAB HEAD: Dario R. Alessi

PROVIDER: PXD022662 | Pride | 2021-03-17

REPOSITORIES: Pride