Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity.
LRRK2
RAB protein
kinase
phosphorylation
proximity ligation assay
Journal
Journal of Parkinson's disease
ISSN: 1877-718X
Titre abrégé: J Parkinsons Dis
Pays: Netherlands
ID NLM: 101567362
Informations de publication
Date de publication:
2022
2022
Historique:
pubmed:
24
5
2022
medline:
14
7
2022
entrez:
23
5
2022
Statut:
ppublish
Résumé
Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.
Sections du résumé
BACKGROUND
Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.
OBJECTIVE
To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.
METHODS
Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers.
RESULTS
Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated.
CONCLUSION
Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.
Identifiants
pubmed: 35599495
pii: JPD213128
doi: 10.3233/JPD-213128
pmc: PMC9398093
doi:
Substances chimiques
LRRK2 protein, human
EC 2.7.11.1
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
EC 2.7.11.1
rab GTP-Binding Proteins
EC 3.6.5.2
Leucine
GMW67QNF9C
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1423-1447Subventions
Organisme : NINDS NIH HHS
ID : P50 NS108675
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS064934
Pays : United States
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