Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease

R. Jeremy Nichols, Nicolas Dzamko, Jessica E. Hutti, Lewis C. Cantley, Maria Deak, Jennifer Moran, Paul Bamborough, Alastair D. Reith, Dario R. Alessi

    Research output: Contribution to journalArticlepeer-review

    172 Citations (Scopus)

    Abstract

    The LRRK2 (leucine-rich repeat protein kinase-2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly(2019) to serine enhances catalytic activity, suggesting that small-molecule inhibitors might have utility in treating Parkinson's disease. We employed various approaches to explore the substrate-specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower K-m and nearly 2-fold higher V-max than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating threonine over serine. We also observed that several ROCK (Rho kinase) inhibitors such as Y-27632 and H-1152, suppressed LRRK2 with similar potency to which they inhibited ROCK2. In contrast, GSK429286A, a selective ROCK inhibitor, did not significantly inhibit LRRK2. We also identified a mutant LRRK2[A2016T] that was normally active, but resistant to H-1152 and Y-27632, as well as sunitinib, a structurally unrelated multikinase inhibitor that, in contrast with other compounds, suppresses LRRK2, but not ROCK. We have also developed the first sensitive antibody that enables measurement of endogenous LRRK2 protein levels and kinase activity as well as shRNA (short hairpin RNA) methods to reduce LRRK2 expression. Finally, we describe a pharmacological approach to validate whether substrates are phosphorylated by LRRK2 and use this to provide evidence that LRRK2 may not be rate-limiting for the phosphorylation of the proposed substrate moesin. The findings of the present study will aid with the investigation of LRRK2.

    Original languageEnglish
    Pages (from-to)47-60
    Number of pages14
    JournalBiochemical Journal
    Volume424
    Early online date9 Sept 2009
    DOIs
    Publication statusPublished - 15 Nov 2009

    Keywords

    • Leucine-rich repeat protein kinase-2 (LRRK2)
    • Moesin
    • Parkinson's disease
    • Phosphorylation
    • Rho kinase (ROCK)

    Fingerprint

    Dive into the research topics of 'Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease'. Together they form a unique fingerprint.

    Cite this