- Top of page
- Experimental procedures
Mutations in leucine-rich repeat kinase 2 (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 (LanthaScreen™) 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 μm) than with the G2019S mutant (Km,app of 134 μm). 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 2 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 . 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  consisting of a number of putative protein–protein 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) . 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 , 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  or a decrease . 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 . 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 . 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 ; 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 , 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 baculovirus-infected 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 constants of autophosphorylation and phosphorylation of LRRKtide, a short peptide substrate derived from moesin . This characterization significantly extends the results from previous studies, which have been limited by protein supply , 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.
- Top of page
- Experimental procedures
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 LanthaScreen™ 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 provides a protein and assay system that can be utilized for significantly higher throughput than has previously been possible.
Our studies have, for the first time, used baculovirus-expressed 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 , 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. 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 β-tubulin copurified with LRRK2; a recent study has also identified an interaction between the proteins .
Recent data indicate that LRRK2 predominantly exists as a dimer and undergoes cis-mediated intramolecular autophosphorylation . Many protein kinases require phosphorylation of residues within their kinase domains to be activated , 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 ∼ 8 min . 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 ∼ 3 min) and 4.2 min−1 (complete reaction in ∼ 3 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 min. 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  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 . Their functional roles, with respect to involvement with the cytoskeleton, fit with observations of modulation of LRRK2 expression affecting neuronal morphology . In these studies, we have used the short peptide LRRKtide, based around the putative phosphorylation site of moesin , to further characterize the enzymological activity of LRRK2. The homology of LRRK2, although low, has placed it in the TKL family of protein kinases , 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 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 pmol·min−1·μg−1 for wild-type LRRK2 and 138 pmol·min−1·μg−1 for G2019S LRRK2 for such a large, 204 kDa protein, this activity is respectable, in line with the findings of others . We have shown that 4E-BP  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 . 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 has been shown to modulate kinase activity [15,16,28,29]. The I2020T mutation has previously been shown to increase [13,15], decrease  or have no effect on kinase activity . 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 . 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 do 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, LanthaScreen™, to assess the activity of LRRK2; this can be used as a high-throughput assay to screen for inhibitory compounds. The LanthaScreen™ 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 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.