Substrate specificity and inhibitors of LRRK2, a protein kinase mutated in Parkinson's disease
Résumé
The Leucine Rich Repeat Protein Kinase-2 (LRRK2) is mutated in a significant number of Parkinson's disease patients, but little is known about its regulation and function. A common mutation changing Gly2019 to Ser enhances catalytic activity, suggesting small molecule inhibitors might have utility in treating Parkinson's Disease. We utilised various approaches to explore the substrate specificity requirements of LRRK2 and elaborated a peptide substrate termed Nictide, that had 20-fold lower Km and nearly 2-fold higher Vmax than the widely deployed LRRKtide substrate. We demonstrate that LRRK2 has marked preference for phosphorylating Thr over Ser. We also observed that several Rho kinase (ROCK) 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, Y-27632 as well as sunitinib, a structurally unrelated multikinase inhibitor that in contrast to 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 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. Our findings reported in this study will aid with the investigation of the LRRK2 kinase.
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