Identification and characterization of a leucine-rich repeat kinase 2 (LRRK2) consensus phosphorylation motif.
Published online
Journal Article
Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.
Full Text
Duke Authors
Cited Authors
- Pungaliya, PP; Bai, Y; Lipinski, K; Anand, VS; Sen, S; Brown, EL; Bates, B; Reinhart, PH; West, AB; Hirst, WD; Braithwaite, SP
Published Date
- October 27, 2010
Published In
Volume / Issue
- 5 / 10
Start / End Page
- e13672 -
PubMed ID
- 21060682
Pubmed Central ID
- 21060682
Electronic International Standard Serial Number (EISSN)
- 1932-6203
Digital Object Identifier (DOI)
- 10.1371/journal.pone.0013672
Language
- eng
Conference Location
- United States