Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif

et al. (2010) Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif. PLoS ONE 5(10): e13672. doi:10.1371/journal.pone.0013672 Identification and Characterization of a Leucine-Rich Repeat Kinase 2 (LRRK2) Consensus Phosphorylation Motif Pooja P. Pungaliya 0 1 Yuchen Bai 0 1 Kerri Lipinski 0 1 Vasanti S. Anand 0 1 Saurabh Sen 0 1 Eugene L. 0 1 Brown 0 1 Brian Bates 0 1 Peter H. Reinhart 0 1 Andrew B. West 0 1 Warren D. Hirst 0 1 Steven P. 0 1 Braithwaite 0 1 Mel B. Feany, Brigham and Women's Hospital, Harvard Medical School, United States of America 0 a Current address: Center of Innovative Biology and Biotherapeutics, Pfizer Incorporated, Pearl River, New York, United States of America b Current address: Quicksilver Consultants, Newton Highlands, Massachusetts, United States of America c Current address: Proteostasis Therapeutics , Cambridge, Massachusetts , United States of America d Current address: Neuroscience Research Unit, Pfizer Incorporated, Groton, Connecticut, United States of America e Current address: Signum Biosciences , Monmouth Junction, New Jersey , United States of America 1 1 Global Biotherapeutic Technologies , Pfizer Incorporated, Cambridge, Massachusetts , United States of America, 2 Neuroscience Research Unit, Pfizer Incorporated, Princeton, New Jersey, United States of America, 3 Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham , Birmingham, Alabama , United States of America 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. - Funding: The authors are grateful for grant support from the Michael J. Fox Foundation. The Michael J. Fox Foundation had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was funded by Pfizer, Inc. The company had a role in this study due to employment of one or more authors of this study. Competing Interests: One or more of the authors are employees of Pfizer, Inc. The authors confirm that this does not alter their adherence to all the PloS ONE policies on sharing data and material. Parkinsons disease (PD) is a neurodegenerative disorder that is associated with progressive loss of dopaminergic neurons in the substantia nigra. Demise of dopaminergic neurons leads to the clinical features of PD which include bradykinesia, rigidity, and resting tremor [1]. Though most PD cases are sporadic, there are several genetic determinants such as mutations in SNCA, Parkin, PINK1, DJ-1 and LRRK2 (reviewed in [2]). Linkage analysis studies have shown that missense mutations in leucine-rich repeat kinase 2 (LRRK2) lead to an autosomal late-onset form of PD [3,4]. LRRK2 is a 286 kDa cytoplasmic protein with GTPase and kinase domains as well as multiple protein-protein interaction domains. Mutations have been identified throughout the protein with the most common mutation, G2019S, residing in its kinase domain [5,6]. In vitro data has suggested that the G2019S substitution augments kinase activity by at least 2-fold [79], therefore making the study of LRRK2s kinase activity important for understanding disease aetiology and for development of therapeutics. PD is characterized pathologically by presence of Lewy bodies composed of a-synuclein and degeneration primarily of dopaminergic neurons. Several cellular mechanisms underlie these pathological features including mitochondrial dysfunction, protein aggregation, oxidative stress, and synaptic dysregulation [1014]. LRRK2 has been implicated in many of these pathophysiological functions. Patients carrying LRRK2 mutations develop pathological inclusions composed of a-synuclein and ubiquitin aggregates within dopaminergic neurons of the substantia nigra [4], and LRRK2 can potentiate a-synuclein pathology in transgenic mouse models [15]. Drosophila expressing mutant forms of LRRK2 have higher levels of hydroxyl-free radicals than dLRRK2 null and heterozygote flies suggesting a function for LRRK2 in oxidative stress [16]. A fraction of LRRK2 co-localizes in synaptic vesicles and overexpression of LRRK2 mutants impairs synaptic vesicle endocytosis [17]. These studies suggest a role for LRRK2 in the pathogenic pathways of PD, but the exact pathways linking the kinase to these functions remain unknown. Fundamental to addressing these questions is the identification of LRRK2s physiological substrate(s). In this study, two oriented degenerate peptide library scans were employed to identify a LRRK2 G2019S phosphorylation motif as F/Y-x-T-x-R/K. This motif resides within a number of proteins including LRRK2 itself at Thr1410. This Thr residue is found in the ROC domain of LRRK2 and was demonstrated to be a site of autophosphorylation by mass spectrometry. Potential functional relevance of this phosphorylation was evidenced by the finding that a T1410A mutation reduced GTPase activity but not GTP binding, autophosphorylation, or kinase activity. The identification of this motif and potential substrates suggests mechanisms by which LRRK2 can function in PD relevant pathways. Materials Biotinylated peptide substrates were purchased from Anaspec, Inc. Positional scanning peptide library was designed and prepared as described by Turk et al. [18]. Peptide concentration was calculated based on the molar absorptivity of the peptides at 280 nm. Recombinant GST-tagged LRRK2 protein 970 (...truncated)