Krumova, Petranka, Reyniers, Lauran, Meyer, Marc, Stauffer, Daniela, Gerrits, Bertran, Hoving, Sjouke, Kaupmann, Klemens, Voshol, Johannes, Fabbrio, Dario, Bauer, Andreas, Rovelli, Giorgio, Taymans, Jean-Marc, Bouwmeester, Antonius and Baekelandt, Veerle (2015) Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate. FASEB Journal, 29 (7). pp. 2980-2992. ISSN 1530-6860

Abstract

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the most common cause of autosomal dominant forms of Parkinson’s disease. LRRK2 is a modular, multi-domain protein containing two enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling, however knowledge of LRRK2’s upstream regulators and downstream effectors is incomplete. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. To this end, we first generated a catalytically active LRRK2 protein capable of employing bulky bio-orthogonal ATPγS analogues (M1947A/G2019S). This engineered, bioactive and functional LRRK2 kinase was used to thiophosphorylate putative substrates from brain extracts of LRRK2 KO mice. Isolation of thiophosphorylated peptides led to the identification of putative LRRK2 substrates. Several of these are involved in the regulation of microtubule (MT) dynamics including MAP/Microtubule Affinity-Regulating Kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate microtubule-binding proteins such as Tau, MAP2 and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic labelling experiments in living cells confirmed MARK1 as a LRRK2 substrate. In addition, we show that LRRK2 and MARK1 are interacting in eukaryotic cells. Taken together, we used a chemical genetic approach to identify LRRK2 substrates in the context of a complex cellular environment and have identified and validated MARK1 as a substrate. Our findings contribute to the identification of physiological LRRK2 substrates, and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morphology

Item Type:	Article
Date Deposited:	26 Apr 2016 23:45
Last Modified:	26 Apr 2016 23:45
URI:	https://oak.novartis.com/id/eprint/22544