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Investigation of leucine‐rich repeat kinase 2
Author(s) -
Anand Vasanti S.,
Reichling Laurie J.,
Lipinski Kerri,
Stochaj Wayne,
Duan Weili,
Kelleher Kerry,
Pungaliya Pooja,
Brown Eugene L.,
Reinhart Peter H.,
Somberg Richard,
Hirst Warren D.,
Riddle Steven M.,
Braithwaite Steven P.
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06789.x
Subject(s) - lrrk2 , autophosphorylation , kinase , map3k7 , leucine rich repeat , förster resonance energy transfer , cyclin dependent kinase 9 , mutant , mutation , map2k7 , protein kinase a , biology , cyclin dependent kinase 2 , biochemistry , chemistry , gene , fluorescence , physics , quantum mechanics
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 (LanthaScreen™) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR‐FRET assays 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 LRRKtide as the substrate. This mutation also affects binding of ATP to LRRK2, with wild‐type binding being tighter ( K m,app of 57 μ m ) than with the G2019S mutant ( K m,app of 134 μ m ). 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.