Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites.
Animals
Biomarkers
/ analysis
Fibroblasts
/ metabolism
Humans
Immunoprecipitation
/ methods
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
/ antagonists & inhibitors
Limit of Detection
Mice, Mutant Strains
Mutation
Parkinson Disease
/ genetics
Phosphorylation
Serine
/ metabolism
Tandem Mass Spectrometry
/ methods
Vesicular Transport Proteins
/ genetics
rab GTP-Binding Proteins
/ metabolism
Rab GTPases
leucine rich repeat kinase
mass spectrometry
Journal
The Biochemical journal
ISSN: 1470-8728
Titre abrégé: Biochem J
Pays: England
ID NLM: 2984726R
Informations de publication
Date de publication:
29 01 2021
29 01 2021
Historique:
received:
26
11
2020
revised:
23
12
2020
accepted:
23
12
2020
pubmed:
29
12
2020
medline:
30
4
2021
entrez:
28
12
2020
Statut:
ppublish
Résumé
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, multiplexed 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 LRRK2-phosphorylated at the 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, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and 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 endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.
Identifiants
pubmed: 33367571
pii: 227421
doi: 10.1042/BCJ20200930
pmc: PMC7833208
doi:
Substances chimiques
Biomarkers
0
Vesicular Transport Proteins
0
Vps35 protein, mouse
0
Serine
452VLY9402
LRRK2 protein, human
EC 2.7.11.1
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
EC 2.7.11.1
Lrrk2 protein, mouse
EC 2.7.11.1
rab GTP-Binding Proteins
EC 3.6.5.2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
299-326Subventions
Organisme : Medical Research Council
ID : G0700656
Pays : United Kingdom
Organisme : Chief Scientist Office
ID : SCAF/18/01
Pays : United Kingdom
Organisme : Medical Research Council
ID : MR/P00704X/1
Pays : United Kingdom
Organisme : Parkinson's UK
ID : H-1701
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_12016/2
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UU_00018/1
Pays : United Kingdom
Informations de copyright
© 2021 The Author(s).
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