Investigation of leucine-rich repeat kinase Enzymological properties and novel assays Vasanti S Anand1, Laurie J Reichling2, Kerri Lipinski1, Wayne Stochaj3, Weili Duan3, Kerry Kelleher3, Pooja Pungaliya4, Eugene L Brown4, Peter H Reinhart1, Richard Somberg2, Warren D Hirst1, Steven M Riddle2 and Steven P Braithwaite1 Wyeth Research, Discovery Neuroscience, Princeton, NJ, USA Invitrogen Corporation, Discovery Sciences, Madison, WI, USA Wyeth Research, Chemical and Screening Sciences, Cambridge, MA, USA Wyeth Research, Biological Technologies, Cambridge, MA, USA Keywords LanthaScreenTM; LRRK2; LRRKtide; moesin; Parkinson’s disease Correspondence S P Braithwaite, Wyeth Research, Discovery Neuroscience, Princeton, CN8000, NJ 08543, USA Fax: +1 732 274 4020 Tel: +1 732 274 4556 E-mail: braiths@wyeth.com (Received 18 May 2008, revised 11 November 2008, accepted 12 November 2008) doi:10.1111/j.1742-4658.2008.06789.x Mutations in leucine-rich repeat kinase (LRRK2) comprise the leading cause of autosomal dominant Parkinson’s disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity, may lead to novel therapeutic strategies for treating Parkinson’s disease In this study, highly purified, baculovirus-expressed proteins have been used, for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2 Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (LanthaScreenTM) to characterize LRRK2 and test the effects of nonselective kinase inhibitors Finally, we have used both radiometric and TR-FRET assays to assess the role of clinical mutations affecting LRRK2’s kinase activity Our results suggest that only the most prevalent clinical mutation, G2019S, results in a robust enhancement of kinase activity with LRRKtide as the substrate This mutation also affects binding of ATP to LRRK2, with wild-type binding being tighter (Km,app of 57 lm) than with the G2019S mutant (Km,app of 134 lm) Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson’s disease may be identified Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder in humans, and has a relatively poorly understood etiology Linkage analysis studies in families with PD identified several mutations in the leucine-rich repeat kinase gene (LRRK2) [1,2] Moreover, epidemiological studies have shown that these mutations are the most prevalent cause of the autosomal form of the disorder, with high penetrance of certain mutations [3] The similarity in age of onset and clinical symptoms between familial and idiopathic forms may also provide insights into the pathways involved in sporadic cases of PD LRRK2 is a large, 286 kDa, multidomain protein [4] consisting of a number of putative protein–protein Abbreviations COR, C-terminus of Roc; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; LRRK2, leucine-rich repeat kinase 2; LRRK2-FL, full-length leucine-rich repeat kinase 2; PD, Parkinson’s disease; Roc, Ras of complex; TR-FRET, time-resolved fluorescence resonance energy transfer; 4E-BP, eukaryotic initiation factor 4E-binding protein 466 FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Anand et al interaction domains, including N-terminal ankyrin repeats, a leucine-rich repeat region, and a C-terminal WD40 domain It also contains a GTPase domain composed of Ras of complex (Roc) and C-terminus of Roc (COR) regions and a kinase domain Mutations linked to PD are found throughout the protein, including the kinase domain (G2019S and I2020T), the Roc– COR domain (R1441C and Y1699C), the leucine-rich repeats (I1122V), and the WD40 domain (R2385G) [4] The most prevalent of these mutations, G2019S [3,5– 7], within the Mg2+-binding region, has been shown to increase the kinase activity of LRRK2 [8], leading to neurodegeneration [9,10] and deficits in neurite outgrowth [11,12] The functional consequences and roles of other mutations reported in the literature are conflicting, I2020T causing an increase in kinase activity [13] or a decrease [14] Similarly, mutations in the GTPase domain have been demonstrated to increase kinase activity [8,15,16], whereas in other studies they have had no effect [14] Characterization of these mutations and understanding LRRK2’s pathogenic function has proven to be challenging, due to technical difficulties in expressing the protein The majority of studies have used immunoprecipitated LRRK2 from recombinant mammalian expression systems [8–10,13], and there is one report of Escherichia coli-expressed LRRK2 [17] These studies have investigated autophosphorylation, or phosphorylation of the surrogate substrate myelin basic protein, due to the lack of knowledge of physiological substrate(s); however, both of these are very weak events The recent identification of moesin as a putative physiological substrate for LRRK2 provided the first alternative for an in-depth investigation of LRRK2’s enzymatic properties [14]; however, its physiological relevance remains to be determined The only other proposed substrate of LRRK2 is eukaryotic initiation factor 4E-binding protein (4E-BP), identified in Drosophila [18], which may play a role in regulating protein translation, although the precise residue that is phosphorylated remains to be clarified In order to have a viable target for drug development, it is essential to know whether LRRK2 has appreciable activity towards its substrates In these studies, we have, for the first time, utilized highly purified LRRK2 produced from baculovirusinfected insect cells to generate significant quantities of active proteins for thorough enzymatic characterization Importantly, a truncated construct consisting of all the conserved functional domains of LRRK2 was found to behave similarly to the full-length protein, proving that results obtained with such constructs are valid We have investigated the detailed kinetics of wild-type LRRK2 in terms of measuring the rate con- LRRK2 characterization stants of autophosphorylation and phosphorylation of LRRKtide, a short peptide substrate derived from moesin [14] This characterization significantly extends the results from previous studies, which have been limited by protein supply [14], preventing the measurement of catalytic rate constants and other enzymatic parameters Furthermore, a time-resolved fluorescence resonance energy transfer (TR-FRET) methodology has been used to characterize LRRK2’s enzymological properties and assess the potency of small molecule, nonselective, kinase inhibitors Finally, we have assessed the effects of a number of common pathological mutations in LRRK2 on its enzymatic activity Overall, our studies provide a detailed enzymatic characterization of LRRK2’s kinase activity, and highlight its potential tractability as a drug target for PD Results LRRK2 proteins expressed by baculovirus Previous studies have primarily used LRRK2 constructs expressed in mammalian expression systems such as HEK-293 cells [8–10] Owing to the low yields obtained from such recombinant overexpression, alternatives are preferable for larger-scale expression and enzymological characterization Expression in E coli has been previously reported [17]; however, this study demonstrated difficulty in the production of large constructs consisting of more than just the COR-kinase domains, and the kinase activity associated with these domains was found to be relatively weak Therefore, in this study baculovirus-infected insect cells were used to express proteins Efficient expression of N-terminal glutathione S-transferase (GST) fusion proteins of LRRK2 residues 970– 2527, consisting of the Roc, COR, kinase, WD40 and entire C-terminal domains, produced significant amounts of protein for in-depth characterization Mutant forms of LRRK2 were also used, namely the pathogenic mutants G2019S, I1122V, I2020T, R1441C and Y1699C, along with the predicted kinase-dead D1994A mutant [10,15,19], in which the critical aspartate residue in the catalytic loop of the kinase domain is mutated Separation of the proteins (5 lg of each preparation) by SDS ⁄ PAGE followed by Coomassie Blue staining demonstrated that they are all of similar, high, purity (> 85%) and have similar banding patterns of minor contaminants (Fig 1A) Western blotting showed that the major band at 204 kDa for the wild-type protein is LRRK2 immunoreactive (Fig 1B) Samples were also characterized by MS, revealing the LRRK2 sequence as the dominant species; b-tubulin was also detected, but no other known kinases were detectable FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS 467 LRRK2 characterization V S Anand et al A LRRK2 exhibits weak autophosphorylation activity WT Y1699C R1441C I2020T I1122V ** CPM 1500 1000 500 WT C Time (min) D D1994A -LRRK2 0.24 10 15 20 30 B 250150- 32P 100- 50- B 2000 incorporation (pmol) 220160120- D1994A WT A G2019S We first assessed the kinase activity of baculovirusexpressed wild-type LRRK2 by autophosphorylation LRRK2 enzymes (50 nm) were incubated with 32 P-labeled ATP (200 lm) for 30 at 30 °C, and separated by SDS ⁄ PAGE The resultant autoradiograms showed that wild-type LRRK2 autophosphorylates, whereas the predicted kinase-dead, D1994A, mutant form [9] does not exhibit any autophosphorylation (Fig 2A) The only band appearing on the autoradiograms was at the size of the LRRK2 protein (204 kDa), indicating that none of the other minor contaminant bands seen on Coomassie-stained gels (Fig 1A) are other active kinases, or substrates for LRRK2 On performing filter-binding assays, it was confirmed that D1994A there is significant, although low, incorporation of 32P into LRRK2 as compared to reactions in the presence of the D1994A kinase-dead LRRK2, or in the absence of enzyme (Fig 2B) These findings are consistent with previous studies that have used proteins expressed in mammalian cells [8–10,14] To extend these findings and further understand the kinetics of LRRK2 autophosphorylation, a time-course experiment was WT (data not shown) As previous studies have shown that the chaperone proteins Hsp90 and p50cdc37 can interact with LRRK2 in a recombinant expression system and mammalian cells [13,20], western blots for these proteins were performed, but immunoreactivity was not detected (data not shown) Additionally, the presence of Hsp60 and Hsp70, which are often found to interact with proteins expressed in insect cells, was investigated, but these were also not detected (data not shown) 75- 0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 10 15 20 25 Time (min) 50- 20- Fig Characterization of LRRK2 proteins (A) Coomassie Bluestained gel of lg of each GST–LRRK2 protein preparation separated by SDS ⁄ PAGE (B) Western blot of 0.5 lg of wild-type (WT) LRRK2 with antibody against LRRK2 indicates that the major protein is LRRK2 immunoreactive Data are representative of three independent experiments 468 Fig Autophosphorylation of wild-type (WT) LRRK2 (A) Autoradiogram of wild-type and kinase-dead (D1994A) LRRK2 proteins (50 nM) that have been incubated with 200 lM ATP for 30 at 30 °C (B) Wild-type and kinase-dead (D1994A) LRRK2 proteins (50 nM) were allowed to autophosphorylate in the presence of 200 lM ATP, and the incorporated 32P was quantitated using filterbinding assays (data from three independent experiments) Significant autophosphorylation was observed in the presence of LRRK2 as compared to kinase-dead (D1994A) LRRK2 and without LRRK2 (**P < 0.01) (C) The autoradiograph shows a time-dependent increase in LRRK2 autophosphorylation (D) The rate of autophosphorylation was determined using filter-binding assays and quenching reactions with 100 mM EDTA at varying reaction times Counts of incorporation were then plotted with respect to time and fitted to a linear equation to obtain the rate constant of autophosphorylation FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Anand et al LRRK2 characterization peptide based on its sequence that is sufficient for LRRK2 activity, named LRRKtide [14] To test whether wild-type LRRK2 was able to phosphorylate LRRKtide, LRRK2 (100 nm) was mixed with LRRKtide (300 lm), and reactions were initiated by adding ATP (200 lm) at 30 °C for 30 min, before loading reactions onto phosphocellulose filters There was significant incorporation of 32P into LRRKtide in comparison with autophosphorylation as detected by the filter-binding assay (Fig 3A) As LRRKtide contains both a threonine residue and a tyrosine residue as potential phosphorylation sites, we investigated which residue was targeted for phosphorylation by mutagenesis Mutation performed, demonstrating increased LRRK2 phosphorylation over time (Fig 2C) Expressing higher amounts of purified protein gives the advantage of being able to perform a detailed enzymatic characterization; hence, the rate of 32P incorporation into LRRK2 was quantified using filter-binding assays, and wild-type LRRK2 autophosphorylation was found to be very slow, with a rate constant of 0.006 ± 0.0005 pmolỈmin)1 (Fig 2D) LRRK2 activity on LRRKtide The identification of moesin as a potential substrate for LRRK2 led to the identification of a 15 amino acid B 10 000 A 1000 1000 CPM(x103) CPM(x103) *** 100 10 *** *** 100 10 + – +LRRK2 D 30 LRRKtide 32P incorporation (pmol·min–1·µg–1) LRRKtide 32P incorporation (pmol) C 25 20 15 10 No peptide + LRRKtide Y-F – LRRKtide T-A LRRKtide LRRKtide – LRRKtide T-A – LRRKtide Y-F + No peptide + LRRKtide 1 LRRK2 –LRRK2 2.5 2.0 1.5 1.0 0.5 0.0 10 20 Time (min) 30 100 200 300 400 500 Concentration LRRKtide (µM) 600 Fig LRRKtide phosphorylation by wild-type LRRK2 (A) LRRK2 (100 nM) was incubated with 200 lM ATP in kinase reaction buffer in the presence of 300 lM LRRKtide; filter-binding assays showed a significant incorporation of 32P into the substrate as compared to autophosphorylation (***P < 0.0001, column 1) or in the absence of enzyme (column 3) or with no LRRK2 or LRRKtide present (column 4) (data from three independent experiments) (B) LRRK2 phosphorylates LRRKtide at its threonine residue LRRK2 at 100 nM was incubated in the presence of 200 lM ATP with a series of peptides (400 lM); LRRKtide and a form of LRRKtide in which the tyrosine residue was mutated to phenylalanine (LRRKtide Y–F) showed significant and robust phosphorylation as compared to background (***P < 0.0001) A peptide in which the threonine of LRRKtide was mutated to alanine (LRRKtide T–A) showed no incorporation of 32P as compared to the control of LRRK2 alone Data from three independent experiments (C) The rate of LRRKtide phosphorylation was determined by incubating 50 nM LRRK2 with 300 lM LRRKtide in kinase reaction buffer containing 200 lM ATP Reactions were quenched with 100 mM EDTA after 1, 5, 10, 15, 20 and 30 min, before loading onto phosphocellulose filters and subsequent washing and counting Data from three independent experiments (D) The apparent Km of wild-type LRRK2 for LRRKtide was determined by incubating 50 nM LRRK2 with varying concentrations of LRRKtide in the presence of 200 lM ATP Data were fitted to a hyperbola to yield an apparent Km of 186 ± 77 lM Data from three independent experiments FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS 469 V S Anand et al 32P 30 G2019S WT 25 20 15 10 0.1 0.3 0.2 0.4 Concentration WT/G2019S(µg) B 140 (nmol·min–1·mg–1) 120 100 80 60 WT 40 20 0 200 400 600 800 1000 1200 Concentration ATP (µM) C 600 500 (nmol·min–1·mg–1) Having identified that LRRKtide is an efficient substrate for LRRK2, we assessed the catalytic efficiency of wild-type LRRK2 on this peptide Varying concentrations of LRRK2 were incubated with 300 lm LRRKtide, and the incorporation of 32P over the course of the reaction was measured, yielding a specific activity of 42 ± 1.5 pmolỈmin)1Ỉlg)1 (Fig 4A) In addition, to investigate the effect of the G2019S mutation on the catalytic activity of LRRK2, varying concentrations of G2019S LRRK2 were incubated with LRRKtide and ATP, and yielded a specific activity of 138 ± pmolỈmin)1Ỉlg)1 (Fig 4A), about three-fold greater than that determined for wild-type LRRK2 As G2019S LRRK2 showed greater activity than the wild-type, and as the mutation is located within the activation segment of the kinase domain, we investigated its influence on the affinity of ATP for LRRK2 Proteins were incubated with 400 lm LRRKtide in the presence of varying concentrations of ATP, and the incorporation of 32P into LRRKtide was assessed The apparent Km for ATP of wild-type LRRK2 was found to be 57 ± lm (Fig 4B), approximately three-fold lower than that of G2019S LRRK2, which had an apparent Km of 134 ± lm (P < 0.01; Fig 4C) 35 LRRKtide 32P incorporation Activity of wild-type and G2019S mutant forms of LRRK2 A LRRKtide 32P incorporation of the tyrosine to a phenylalanine (LRRKtide Y–F) did not significantly affect the phosphorylation of the peptide, whereas mutation of threonine to alanine (LRRKtide T–A) completely blocked the ability of the peptide to be phosphorylated (Fig 3B) Therefore, LRRK2 appears to act on LRRKtide as a serine ⁄ threonine kinase with no tyrosine kinase activity To assess the rate of phosphorylation of this peptide, LRRKtide was incubated with LRRK2 for various times The rate of phosphorylation was determined to be 0.7 ± 0.02 pmolỈmin)1, approximately 100-fold faster than the measured rate constant of autophosphorylation (Fig 3C) Furthermore, the apparent Km of LRRKtide was determined by performing reactions with LRRK2 at varying concentrations of LRRKtide, and was determined to be 186 ± 70 lm (Fig 3D); this is consistent with data obtained using proteins expressed in mammalian cells [14] incorporated into LRRKtide (pmol·min–1) LRRK2 characterization 400 300 200 G2019S 100 0 200 400 600 800 1000 1200 Concentration ATP (µM) A time-resolved fluorescent based assay for measuring LRRK2 activity Fig Activity of wild-type (WT) and G2019S LRRK2 on LRRKtide (A) The specific activity of G2019S LRRK2 ( ) is greater than that of wild-type LRRK2 (d) Proteins were incubated at varying concentrations with 300 lM LRRKtide and 200 lM ATP for 30 min, and the amount of 32P incorporated per minute was calculated Data from three independent experiments (B) Wild-type LRRK2 or G2019S LRRK2 at 100 nM was incubated with 400 lM LRRKtide in the presence of varying concentrations of ATP The apparent Km for ATP for wild-type LRRK2 is 57 ± lM and that for G2019S LRRK2 is 134 ± lM Because LRRK2 showed high activity with LRRKtide, we were able to convert the radioactive assay into a TR-FRET-based LanthaScreenTM format A fluores- cein-labeled LRRKtide is used as the substrate, and after a kinase reaction has occurred, a terbium-labeled antibody against phospho-LRRKtide is added for 470 FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Anand et al Potency of broad-spectrum kinase inhibitors on LRRK2 The LanthaScreenTM format allows the rapid and efficient screening of kinase inhibitors, and therefore allows further characterization of LRRK2 properties A set of approximately 120 known kinase inhibitors was screened against wild-type and G2019S LRRK2 (data not shown) A small panel of kinase inhibitors was then selected for further study (Table 1), and dose–response relationships were obtained using wild-type (Fig 6A) and G2019S (Fig 6B) LRRK2 with 200 nm LRRKtide and ATP at apparent Km values The inhibitors tested showed dose-dependent inhibition of the kinase activity (Fig 6A,B), with the most potent compound being staurosporine, with an IC50 of 1.8 ± 0.09 nm for G2019S LRRK2 (Table 1) There was no significant difference in the inhibitory efficacy of compounds between wild-type and G2019S LRRK2 (Table 1) Effect of additional clinical mutations on LRRK2 kinase activity As we had seen significant differences in activity between wild-type and G2019S LRRK2 using the LRRKtide peptide, further analysis of other mutant forms of LRRK2 was performed Previously, conflicting results have been reported for the effects of these mutations [8,13–15], although the majority of studies have investigated autophosphorylation, or other surrogate substrates that are weakly phosphorylated We therefore tested the effects of each mutation on both autophosphorylation and LRRKtide phosphorylation, to determine whether there are any differences Wildtype and mutant LRRK2 proteins, G2019S, D1994A, R1441C, Y1699C, I1122V, and I2020T, were incubated 1.2 [WT] [G2019S] Emission ratio (520/495) detection Fluorescence resonance energy transfer (FRET) occurs from the terbium-labeled antibody to the fluorescein dye on the phosphorylated peptide Reactions were performed with varying concentrations of wild-type LRRK2 in the presence of 400 nm fluorescein–LRRKtide and mm ATP for h at room temperature The reaction was stopped by addition of 10 mm EDTA, and phosphorylation was detected by the terbium-labeled antibody against phospho-LRRKtide FRET was measured by the emission ratio at 520 ⁄ 495 nm The EC50 for wild-type LRRK2 was found to be 2728 ± 884 ngỈmL)1, whereas G2019S LRRK2 showed approximately two-fold greater activity, with an EC50 of 1276 ± 505 ngỈmL)1 (Fig 5), comparable to the differences seen in specific activity in radiometric assays (Fig 4A) LRRK2 characterization 1.0 0.8 0.6 0.4 0.2 0.0 –1 Log concentration WT/G2019S LRRK2 (ng·mL–1) Fig A time-resolved fluorescence-based LanthaScreenTM assay effectively measures LRRK2 kinase activity Titration of wild-type and G2019S LRRK2 demonstrates concentration-dependent phosphorylation of LRRKtide, with the mutant protein being more active Varying concentrations of each protein were incubated with 400 nM fluorescein–LRRKtide and mM ATP in kinase reaction buffer for h at room temperature The reaction was stopped, and phosphorylation of LRRKtide was detected by addition of terbium-labeled antibody against LRRKtide FRET was measured by excitation at 495 nm, and the emission ratio between 525 nm and 495 nm was calculated to measure the phosphorylation of the substrate Data are illustrated as a single experiment representative of at least three independent runs Table IC50 values of nonspecific kinase inhibitors on wild-type and G2019S LRRK2 Inhibitors were tested using the LanthaScreenTM format, mean IC50 ± SD, data from three independent experiments for each compound Compound Wild-type IC50 (nM) G2019S IC50 (nM) JAK3 inhibitor VI K252A Staurosporine Su-11248 Ro-31-8220 22 3.6 2.0 15 2671 40 2.8 1.8 26 1922 ± ± ± ± ± 2.5 0.2 0.1 1.3 895 ± ± ± ± ± 0.1 0.09 1.7 665 with 32P-labeled ATP for 30 at 30 °C, the reactions were stopped, proteins were separated by SDS ⁄ PAGE, and the resultant gel was exposed to a phosphoimaging screen (Fig 7A, top) G2019S LRRK2 showed significantly greater autophosphorylation than wild-type LRRK2 The other mutant LRRK2 proteins were not significantly different in activity with respect to the wild-type (Fig 7A) As the G2019S mutation provides a serine residue, and hence a potential extra phosphorylation site, we sought to confirm that the increased autophosphorylation observed was not due to phosphorylation at this site Analysis by MS failed to detect signal at this residue; FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS 471 LRRK2 characterization A V S Anand et al Furthermore, the mutant forms of LRRK2 were tested in the LanthaScreenTM assay G2019S LRRK2 showed the greatest activity as compared to the wildtype, and I2020T LRRK2 the least activity (Fig 7C), comparable to the data obtained from the radiometric assay (Fig 7B) 0.9 Emission ratio (520/495) 0.8 0.7 0.6 0.5 0.4 0.3 Full-length LRRK2 displays comparable properties to the truncated form JAK3 inhibitorVI K252A Staurosporine Su-11248 Ro-31-8220 0.2 0.1 –4 –3 –2 –1 5 Log concentration inhibitor (nM) B Emission ratio (520/495) 0.7 0.6 0.5 0.4 0.3 JAK3 inhibitorVI K252A Staurosporine Su-11248 Ro-31-8220 0.2 0.1 –4 –3 –2 –1 Log concentration inhibitor (nM) Fig Inhibition of wild-type and G2019S LRRK2 by nonspecific kinase inhibitors (A) The indicated inhibitors demonstrate dosedependent inhibition of wild-type LRRK2 activity Reactions were performed in the presence of 3.4 lgỈmL)1 LRRK2, 400 nM fluorescein–LRRKtide, 57 lM ATP, and varying doses of inhibitors (B) The same inhibitors were also tested against 1.0 lgỈmL)1 G2019S LRRK2 in a similar manner, except with 134 lM ATP however, as G2019S LRRK2 also displays increased activity on LRRKtide peptide (Fig 4A), it is evident that this mutation leads to a protein with greater activity than the wild-type The specific activities of mutant LRRK2 enzymes with respect to 32P incorporation into LRRKtide were measured by incubating varying concentrations of each enzyme in the presence of 300 lm LRRKtide for various times G2019S LRRK2 demonstrated the greatest activity of all the mutants, although R1441C LRRK2 also showed significantly greater specific activity than the wild-type Interestingly I2020T LRRK2 showed significantly lower activity As would be predicted, the D1994A kinase-dead mutant showed significantly less activity on LRRKtide than the wild-type (Fig 7B) 472 Our previous studies were all performed using a truncated construct consisting of all conserved structural domains of LRRK2, comprising amino acids 970–2527, as this could be obtained in reasonable quantities for performing enzymological characterization To further validate our findings, we wanted to ensure that full-length LRRK2 (LRRK2-FL) behaved similarly We were able to purify very low quantities of LRRK2-FL, as a FLAG-tagged construct, with activity in a dimer fraction being separated using size exclusion chromatography Autophosphorylation experiments showed that the full-length and truncated forms of LRRK2 had comparable activities (Fig 8A) Using the TR-FRET LanthaScreenTM format, the EC50 of LRRK2-FL was determined to be 2375 ± 536 ngỈmL)1 (Fig 8B), which is very similar to that obtained for the truncated wild-type LRRK2 construct (Fig 5) Furthermore, staurosporine, the most potent kinase inhibitor of the truncated variant of LRRK2, was determined to have an IC50 of 8.2 ± 0.8 nm (Fig 8C), which is similar to that obtained for truncated wild-type LRRK2 (Fig 6) The low yields of LRRK2-FL obtained precluded a more thorough characterization of its enzymological properties; however, these data suggest that the truncated construct behaves very similarly to the full-length protein in terms of its kinase activity Discussion In the present study, we have investigated the kinase activity of a new protein source of LRRK2, with respect to autophosphorylation and LRRKtide phosphorylation These studies demonstrate LRRK2 to be an effective kinase whose activity is dependent upon its substrate, and how mutations in LRRK2 that have been clinically linked to PD may affect its function Finally, a novel fluorescence-based assay system using LanthaScreenTM technology, which robustly measures LRRK2 kinase activity, and is amenable for testing the efficacy of small molecule kinase inhibitors, has been evaluated Overall, this adds to the enzymological characterization of LRRK2 and FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Anand et al LRRK2 characterization provides a protein and assay system that can be utilized for significantly higher throughput than has previously been possible A B 400 300 200 100 I2020T I1122V 1699C G2019S WT R1441C *** D1994A 32 P incorporation (% WT) *** 160 *** Specific activity (pmol·min–1·µg–1) 140 120 100 80 * 60 40 *** 20 *** WT G2019S D1994A R1441C Y1699C I1122V I2020T C 1.2 [D1994A] [WT] [G2019S] [I2020T] [I1122V] [R1441C] [Y1699C] 1.0 Emission ratio (520/495) Our studies have, for the first time, used baculovirusexpressed proteins The majority of previous studies on LRRK2 have used proteins expressed in mammalian cells [8,9,13,14] that can only be produced with low yields and low purity One study has reported the use of E coli-expressed proteins [17], but only short constructs, consisting of either the kinase or COR-kinase domains of LRRK2 Baculovirus-mediated production of proteins gives the advantage of being able to produce large amounts of post-translationally modified LRRK2 protein with all of its enzymatic domains In line with other studies, the generation of full-length LRRK2 has been difficult; however, we have been able to express and purify the full-length protein in Sf21 cells on a small scale These studies allowed us to demonstrate that a shorter construct lacking the N-terminal region behaves the same as full-length LRRK2 with respect to its kinase activity This region contains no conserved structural features, and no clinically relevant mutations have been characterized within it Therefore, characterization of the kinase activity of a construct lacking this domain gives valuable data about LRRK2 Our findings significantly extend the characterization of LRRK2’s enzymological properties, our results being consistent with the only previously published parameter, Km of LRRKtide, obtained from proteins expressed in mammalian cells, and adding the characterization of specific activity and Km for ATP Unlike studies using other expression systems [13,17], we have shown that LRRK2, when expressed in insect cells, does not copurify with chaperone proteins 0.8 0.6 0.4 0.2 0.0 –1 Log concentration protein (ng·mL–1) Fig Kinase activity of pathogenic LRRK2 mutants (A) Autophosphorylation of LRRK2 mutants G2019S, D1994A, I1122V, R1441C, Y1699C and I2020T was assessed by autoradiography Kinase reactions were performed with 50 nM each mutant in the presence of 200 lM ATP, and reactions were allowed to proceed for 30 at 30 °C, before being stopped by separation using SDS ⁄ PAGE The gels were exposed to a Phosphoimager screen and autoradiograms were developed; the results are representative of experiments performed on three independent occasions The autophosphorylated variant LRRK2 bands were quantitated and normalized with respect to wild-type (WT) LRRK2 Autophosphorylation of G2019S LRRK2 was significantly higher than that of wild-type LRRK2 (***P < 0.0001) (B) Specific activities of mutant LRRK2 proteins with respect to 32P incorporation into LRRKtide were assessed using filter-binding assays G2019S LRRK2 (***P < 0.0001), and R1441C LRRK2 (*P < 0.05) activities were significantly higher than that of wild-type LRRK2 D1994A LRRK2 and I2020T LRRK2 activities were found to be significantly lower than that of wild-type LRRK2 (***P < 0.0001) Data from three independent experiments (C) The activities of mutants were additionally assessed using the LanthaScreenTM format Experiments were performed as described previously; data are representative of at least three independent experiments FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS 473 Emission ratio (520/495) LRRK2 A B V S Anand et al LRRK2-FL LRRK2 characterization 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Log concentration WT LRRK2-FL (ng·mL–1) Emission ratio (520/495) C 0.5 0.4 0.3 0.2 0.1 0.0 0.01 0.1 10 100 1000 10 000 Log concentration staurosporine (nM) Fig Full-length LRRK2 is active (A) Autoradiogram of autophosphorylation of full-length and truncated wild-type LRRK2 constructs Full-length and truncated wild-type LRRK2 (100 nM) were incubated with 200 lM 32P-labeled ATP for 30 at 30 °C to allow autophosphorylation to proceed Reactions were subjected to SDS ⁄ PAGE, gels were exposed to a Phosphoimager screen, and an autoradiogram was developed The result is representative of three independent experiments (B) Varying concentrations of full-length and truncated LRRK2 proteins were incubated with 100 nM fluorescein– LRRKtide and 50 lM ATP in kinase reaction buffer for h at room temperature The reaction was stopped, and phosphorylation of LRRKtide was detected by addition of nM terbium-labeled antibody against LRRKtide FRET was measured by excitation at 495 nm, and the emission ratio between 525 nm and 495 nm was calculated to measure the phosphorylation of the substrate Data are illustrated as a single experiment representative of three independent runs (C) Dose-dependent staurosporine inhibition of fulllength LRRK2 LanthaScreenTM assays were performed with 3.6 lgỈmL)1 full-length LRRK2, 100 nM fluorescein–LRRKtide, 50 lM ATP and varying concentrations of staurosporine in kinase reaction buffer for h at room temperature 474 However, these proteins are highly active, indicating that, although chaperone-mediated folding may be important for LRRK2, its maintained interaction is not a prerequisite for kinase activity Interestingly, by MS, we found that b-tubulin copurified with LRRK2; a recent study has also identified an interaction between the proteins [21] Recent data indicate that LRRK2 predominantly exists as a dimer and undergoes cis-mediated intramolecular autophosphorylation [22] Many protein kinases require phosphorylation of residues within their kinase domains to be activated [23], and therefore can act as substrates of kinases, including themselves The S6 ⁄ H4 serine ⁄ threonine kinase, for example, has been found to autophosphorylate at an exponential rate constant of 0.91 min)1, with the reaction going to completion in  [24] Both the fibroblast growth factor and RET receptor tyrosine kinases have been found to autophosphorylate, with rate constants of 0.05 s)1 (complete reaction in  min) and 4.2 min)1 (complete reaction in  min), respectively [25,26] LRRK2 is also able to undergo autophosphorylation, which is indicative of such a process, and the majority of studies on LRRK2 to date have used this property to assay its activity In our studies, we have demonstrated that LRRK2 can autophosphorylate, but this process is inefficient, with data displaying linear kinetics, indicating that the reaction does not go to completion within 30 This suggests that LRRK2 is a relatively poor substrate for itself, in comparison to other well-characterized kinases, and that this may not be its major functional role in a physiological environment The search for more relevant substrates of LRRK2 has led to the identification of members of the ezrin– radixin–moesin family of proteins [14] as potential substrates Although the physiological relevance of these proteins as substrates has not been proven, they clearly provide an in vitro substrate that LRRK2 can phosphorylate with higher efficiency [14] Their functional roles, with respect to involvement with the cytoskeleton, fit with observations of modulation of LRRK2 expression affecting neuronal morphology [11] In these studies, we have used the short peptide LRRKtide, based around the putative phosphorylation site of moesin [14], to further characterize the enzymological activity of LRRK2 The homology of LRRK2, although low, has placed it in the TKL family of protein kinases [27], whose members have both serine ⁄ threonine and tyrosine kinase activity With respect to LRRKtide, we have demonstrated that LRRK2 acts purely as a serine ⁄ threonine kinase, and phosphorylates LRRKtide significantly more efficiently than it phosphorylates itself Potentially, LRRK2 could autophosphorylate in FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Anand et al the cells that it is being prepared in, and hence be already highly phosphorylated at this site, therefore preventing much additional phosphorylation from taking place Our results indicate that additional phosphorylation can still take place, showing linear kinetics over the course of the reactions performed; therefore, phosphorylation at this site is not saturated Nonetheless, these findings give rise to the notion that caution must be used when interpreting results based purely on autophosphorylation, as this is a relatively weak activity Even though the physiological relevance of moesin in relation to LRRK2 is still unclear, the findings indicate that LRRK2 has the ability to be a kinase with significant activity and high substrate turnover With specific activities of 42 pmolỈmin)1Ỉlg)1 for wild-type LRRK2 and 138 pmolỈmin)1Ỉlg)1 for G2019S LRRK2 for such a large, 204 kDa protein, this activity is respectable, in line with the findings of others [14] We have shown that 4E-BP [18] is also phosphorylated by our LRRK2 proteins (data not shown), but the precise site of phosphorylation and the physiological relevance are unclear It will be interesting to assess the activity on other physiological substrates as they are identified The linkage of mutations in LRRK2 to the development of PD has led to the interest in this protein In this study, we initially investigated the role of the most prevalent mutation found in humans, G2019S This mutation significantly increases the specific activity of LRRK2, and also alters its apparent Km for ATP The G2019S residue lies within the activation loop of the kinase, and potentially leads to the introduction of an extra residue that can be phosphorylated, placing LRRK2 in a more active conformation [17] Our findings confirm that the G2019S mutation increases kinase activity, with respect not only to autophosphorylation, but also to the peptidic substrate LRRKtide The increased activity seen with respect to LRRKtide implies that differences seen are due to increased activity of the protein and not just to the presence of an extra phosphorylation site The mutation also affects the apparent Km of ATP, indicating that it modifies the active site of the enzyme to alter the affinity for ATP The R1441C and Y1699C mutations within the Roc and COR regions, despite having been linked to PD, did not, in our hands, increase kinase activity with respect to autophosphorylation, but resulted in small increases in activity with respect to LRRKtide phosphorylation Mutations within the Roc and COR regions have previously been demonstrated to increase, decrease or not affect kinase activity [14,15,17] The differences in the results may be due to different expression systems, construct lengths, or levels of GTP, as the Roc region forms a GTPase domain that LRRK2 characterization has been shown to modulate kinase activity [15,16,28,29] The I2020T mutation has previously been shown to increase [13,15], decrease [14] or have no effect on kinase activity [17] Our studies indicate that the I2020T mutation causes a decrease in the kinase activity of LRRK2 in the context of LRRKtide; this is possibly due to the critical role of this residue in the activation loop of the kinase domain, causing it to be in a less active state, but this is not observed with respect to autophosphorylation, which is a much weaker event It remains to be determined whether other substrates will be found to be affected differently by these mutant forms of LRRK2 Nonetheless, the differences in the results seen in multiple studies between different mutant forms of LRRK2 suggest that LRRK2 may either have multiple roles or act at multiple points in pathways relevant for PD Furthermore, different mutations in LRRK2 lead to PD with pleiomorphic pathology and symptoms For example, patients with mutations in the GTPase domains have been shown to have different combinations of tauopathies and synucleinopathies, in addition to the hallmark neuronal degeneration in the substantia nigra [2] Additionally, the occurrence of the other mutations in LRRK2 is not as common as that of G2019S [30–32] With our findings that different mutations differentially affect the kinase activity of LRRK2, yet all lead to PD, albeit with somewhat different symptoms, it appears that LRRK2 is a central protein in processes underlying the disease The mutations that not affect kinase activity may affect the localization of LRRK2, or other properties that modulate its roles in a critical pathway that underlies the disorder The prevalence, penetrance and functional significance of the G2019S mutation make the kinase activity of LRRK2 of major interest in developing therapeutic strategies for PD We have therefore taken advantage of a time-resolved fluorescence based assay, LanthaScreenTM, to assess the activity of LRRK2; this can be used as a high-throughput assay to screen for inhibitory compounds The LanthaScreenTM format with the LRRKtide peptide is comparable to radiometric assays, and has been effectively used to demonstrate that a number of nonselective kinase inhibitors display inhibitory activity on LRRK2 These studies have demonstrated that LRRK2 acts as a serine ⁄ threonine kinase with appreciable activity in relation to a peptidic substrate, as compared to its autophosphorylation, which is too weak and inefficient a process for thorough and high-throughput assays This study has been enabled by the generation of a baculovirus-expressed protein that contains all of the conserved structural domains of LRRK2 and that FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS 475 LRRK2 characterization V S Anand et al behaves in the same way as full-length LRRK2 with respect to its kinase properties With respect to LRRKtide, we have demonstrated that kinase inhibitors can be evaluated and the biochemical characteristics of LRRK2 efficiently assessed In addition, we have shown that clinical mutations in LRRK2 that are linked to PD affect its kinase activity differentially These studies increase our understanding of LRRK2 as an enzyme, and additionally provide tools that can be used in compound screening to identify novel LRRK2 inhibitors Clearly, LRRK2 plays a key role in critical pathways implicated in PD, and understanding its properties and functions will aid in our understanding of disease pathology and progression Experimental procedures Chemicals and reagents All compounds and proteins were provided by or purchased from Invitrogen (Madison, WI, USA), unless otherwise stated Ro-31-8220 and JAK3 Inhibitor VI were from EMD Biosciences (San Diego, CA, USA) Su-11248 was from American Custom Chemicals (San Diego, CA, USA) Antibodies against LRRK2, Hsp60 and Hsp90 were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against Hsp70 and cdc37 were purchased from Invitrogen [32P]ATP[cP] was purchased from GE Healthcare (Piscataway, NJ, USA) or Perkin Elmer (Waltham, MA, USA) The TR-FRET LanthaScreenTM assay system was developed by Invitrogen (Madison, WI, USA) Synthetic peptides were purchased from AnaSpec, Inc (San Jose, CA, USA) SDS ⁄ PAGE and western blotting For western blots, proteins were separated on 4–12% NuPAGE gels (Invitrogen) For purity analysis, proteins were separated on 4–20% Tris ⁄ glycine gels (Invitrogen), stained following standard methods with Coomassie Brilliant Blue, and imaged using a digital camera (Kodak, Rochester, NY, USA) For western blots, proteins were transferred from gels onto nitrocellulose membranes; the membranes were then blocked with buffer (Rockland Immunochemicals, Gilbertsville, PA, USA) and incubated with the indicated primary antibodies overnight at °C The blots were washed and incubated with AlexaFluor-conjugated secondary antibodies (LI-COR Biotechnology, Lincoln, NE, USA) and imaged using an Odyssey Infra Red Imaging System (LI-COR) Protein expression and purification Wild-type LRRK2 consisted of amino acids 970–2527 of human LRRK2 fused to an N-terminal GST tag, and was 476 provided by or purchased from Invitrogen Mutant forms of LRRK2 were generated by PCR-mediated mutagenesis, and expressed and purified in parallel with the wild-type protein by identical procedures (provided by Invitrogen) Full-length LRRK2 was C-terminally His-tagged and FLAG-tagged, purified on a FLAG immunoaffinity column (Sigma, St Louis, MO, USA), and subjected to separation by size exclusion chromatography (Tosoh Bioscience, Grove City, OH, USA) LRRK2 radiometric assays For autophosphorylation experiments, LRRK2 was incubated with [32P]ATP (200 lm) in kinase reaction buffer consisting of 20 mm Tris ⁄ HCl (pH 7.5), 10 mm MgCl, mm EGTA, mm Na3VO4, mm b-glycerolphosphate, 0.02% Triton X-100, and mm fresh dithiothreitol at 30 °C for 30 in a final reaction volume of 25 lL; reactions were terminated by the addition of LDS sample buffer (Invitrogen) LRRK2 autophosphorylation was detected by running samples on 4–12% SDS ⁄ PAGE gels and exposing the gel to a phosphor screen, scanned on a STORM 840 scanner (GE Healthcare, Piscataway, NJ, USA), and quantitated (when indicated) using imagequant software (GE Healthcare) For LRRKtide 32P incorporation, assays similar to those described above were performed along with LRRKtide (300 lm), unless otherwise noted Reactions were terminated by applying 30 lL of the reaction mixture to P81 phosphocellulose paper (Millipore, Billerica, MA, USA) 32P incorporation into LRRKtide was quantified by washing the phosphocellulose membranes in 50–75 mm phosphoric acid and liquid scintillation counting The kinetics of LRRK2 autophosphorylation were qualitatively assessed by autoradiography and quantitatively by phosphocellulose membrane filter-binding assay, as described above for LRRKtide 32P incorporation For experiments to determine the binding affinity of ATP for LRRK2, enzyme concentrations were 2.1 lgỈmL)1 for wild-type LRRK2 and 1.0 lgỈmL)1 for G2019S LRRK2; the LRRKtide concentration in the reactions was 400 lm The kinase reaction buffer consisted of 20 mm Tris ⁄ HCl (pH 8.5), 10 mm MgCl, mm EGTA, 0.01% Brij-35, and mm fresh dithiothreitol The specific activity was determined by performing the assay described above with varying concentrations of LRRK2 and fitting the kinetics to a linear equation using sigmaplot software (Jandel Scientific, Corte Madera, CA, USA) Statistical significance was determined using the t-test LanthaScreenTM assay Kinase reactions were performed in triplicate in a volume of 10 lL in 50 mm Tris ⁄ HCl (pH 8.5), 10 mm MgCl, mm EGTA, 0.01% Brij-35, mm fresh dithiothreitol, and 400 nm fluorescein–LRRKtide For kinase titrations, the FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS V S Anand et al ATP concentration was mm, whereas for inhibitor studies, the ATP concentration was equal to the apparent Km determined in radiometric format, which was 57 lm for wild-type LRRK2 and 134 lm for G2019S LRRK2 For inhibitor studies, the enzyme concentrations were 3.4 lgỈmL)1 for wild-type LRRK2 and 1.0 lgỈmL)1 for G2019S LRRK2; these are equal to EC80 levels from kinase titrations For inhibitor studies, all reactions contained 1% residual dimethylsulfoxide from compound dilutions After h of kinase reactions, a 10 lL solution of terbium-labeled anti-p-LRRKtide and EDTA (in 50 mm Tris, pH 7.5, and 0.01% Nonidet-P40) was added to each well, for a final concentration of 2.5 nm antibody and 10 mm EDTA After a h incubation, the plate was read on a BMG Pherastar plate reader using the LanthaScreenTM filter module (BMG Labtech, Inc Durham, NC, USA) with excitation at 340 nm The TR-FRET ratio was calculated as the intensity of the acceptor signal (520 nm) divided by the intensity of the donor signal (495 nm) Statistical significance was determined using the t-test Acknowledgements This work was funded in part by the Michael J Fox Foundation We would like to thank Marie Uphoff and 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of LRRK2 mutations and Parkinson’s disease in Brazil Neurosci Lett 433, 17–21 FEBS Journal 276 (2009) 466–478 ª 2008 The Authors Journal compilation ª 2008 FEBS ... IC50 (nM) G2019S IC50 (nM) JAK3 inhibitor VI K252A Staurosporine Su-1 124 8 Ro-31- 822 0 22 3.6 2. 0 15 26 71 40 2. 8 1.8 26 1 922 ± ± ± ± ± 2. 5 0 .2 0.1 1.3 895 ± ± ± ± ± 0.1 0.09 1.7 665 with 32P-labeled... that of G2019S LRRK2, which had an apparent Km of 134 ± lm (P < 0.01; Fig 4C) 35 LRRKtide 32P incorporation Activity of wild-type and G2019S mutant forms of LRRK2 A LRRKtide 32P incorporation of. .. compilation ª 20 08 FEBS 469 V S Anand et al 32P 30 G2019S WT 25 20 15 10 0.1 0.3 0 .2 0.4 Concentration WT/G2019S(µg) B 140 (nmol·min–1·mg–1) 120 100 80 60 WT 40 20 0 20 0 400 600 800 1000 120 0 Concentration