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1.
J Pharm Biomed Anal ; 76: 49-58, 2013 Mar 25.
Article in English | MEDLINE | ID: mdl-23313773

ABSTRACT

After the discovery of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson's disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme's activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds. In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC). To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Protein Serine-Threonine Kinases/metabolism , Animals , Blotting, Western , HEK293 Cells , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Leukocytes, Mononuclear/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Rats , Rats, Long-Evans , Sensitivity and Specificity
2.
Neuron ; 75(6): 1008-21, 2012 Sep 20.
Article in English | MEDLINE | ID: mdl-22998870

ABSTRACT

LRRK2 is a kinase mutated in Parkinson's disease, but how the protein affects synaptic function remains enigmatic. We identified LRRK2 as a critical regulator of EndophilinA. Using genetic and biochemical studies involving Lrrk loss-of-function mutants and Parkinson-related LRRK2(G2019S) gain-of-kinase function, we show that LRRK2 affects synaptic endocytosis by phosphorylating EndoA at S75, a residue in the BAR domain. We show that LRRK2-mediated EndoA phosphorylation has profound effects on EndoA-dependent membrane tubulation and membrane association in vitro and in vivo and on synaptic vesicle endocytosis at Drosophila neuromuscular junctions in vivo. Our work uncovers a regulatory mechanism that indicates that reduced LRRK2 kinase activity facilitates EndoA membrane association, while increased kinase activity inhibits membrane association. Consequently, both too much and too little LRRK2-dependent EndoA phosphorylation impedes synaptic endocytosis, and we propose a model in which LRRK2 kinase activity is part of an EndoA phosphorylation cycle that facilitates efficient vesicle formation at synapses.


Subject(s)
Acyltransferases/metabolism , Drosophila Proteins/metabolism , Endocytosis/physiology , Neuromuscular Junction/physiology , Protein Serine-Threonine Kinases/metabolism , Acyltransferases/genetics , Animals , Animals, Genetically Modified , Brain/cytology , Brain/metabolism , CHO Cells , Calcium/metabolism , Clathrin/metabolism , Cricetinae , Drosophila , Drosophila Proteins/genetics , Endocytosis/genetics , Gene Expression Regulation/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Mass Spectrometry , Mice , Microscopy, Electron, Transmission , Models, Molecular , Mutation/genetics , Neuromuscular Junction/drug effects , Neuromuscular Junction/ultrastructure , Phosphorylation/genetics , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Sequence Alignment , Serine/genetics , Serine/metabolism , Synaptic Potentials/drug effects , Synaptic Potentials/genetics , Synaptic Vesicles/drug effects , Synaptic Vesicles/physiology , Transfection
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