Investigation of leucine-rich repeat kinase 2 : enzymological properties and novel assays.
Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity,may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used,for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2.Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (Lantha-ScreenTM) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRETassays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation,G2019S, results in a robust enhancement of kinase activity with LRRKtideas the substrate. This mutation also affects binding of ATP to LRRK2,with wild-type binding being tighter (Km,app of 57 lm) than with theG2019S mutant (Km,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.
Anand
Vasanti S
VS
Wyeth Research, Discovery Neuroscience, Princeton, NJ 08543, USA.
Reichling
Laurie J
LJ
Lipinski
Kerri
K
Stochaj
Wayne
W
Duan
Weili
W
Kelleher
Kerry
K
Pungaliya
Pooja
P
Brown
Eugene L
EL
Reinhart
Peter H
PH
Somberg
Richard
R
Hirst
Warren D
WD
Riddle
Steven M
SM
Braithwaite
Steven P
SP
D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation; D015870:Gene Expression; D020128:Inhibitory Concentration 50; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D047428:Protein Kinase Inhibitors; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases