Our data reveal that LRRK2 tolerates a wider range of amino acids in its substrates compared with some other protein kinases that have strong requirements for specific amino acids within the substrates that they phosphorylate ( and ). Significant substrate specificity preferences are the −5 (tryptophan and arginine), −2 (phenylalanine, tyrosine and histidine), −1 (tyrosine, arginine and tryptophan), +2 (arginine) and +3 (arginine) positions. Importantly, our data suggest that LRRK2 has a strong preference for phosphorylating threonine, as mutation of the phosphorylated threonine residue in Nictide to serine abolished phosphorylation of the peptide by LRRK2 (). Positional scanning peptide library analysis also suggested that LRRK2 poorly tolerated acidic glutamic acid or aspartic acid residues at all positions surrounding the phosphorylation site (). LRRKtide only possesses a single acidic residue (Asp−4), and the only mutation we tested that improved LRRK2 phosphorylation, was mutation of this residue to alanine (). It is possible that this knowledge of substrate specificity of LRRK2 may aid in the identification of LRRK2 substrates and/or potential phosphorylation sites within identified substrates. This analysis has also enabled us to generate the Nictide peptide, a much improved substrate compared with LRRKtide peptide that is widely deployed to assay LRRK2. A key advantage of Nictide is that it can be used at much lower concentrations in kinase assays. We have been able to assay the activity of endogenous LRRK2 using Nictide, with virtually no background activity observed in the control immunoprecipitate (). To our knowledge, this is the first time that activity of endogenous LRRK2 has been assessed. When trying to assay activity of endogenous LRRK2 employing LRRKtide at concentrations of 300 μM (Km value), we observed significant background activity in the pre-immune immunoprecipitation (N. Dzamko, unpublished work). Assessment of activity of endogenous LRRK2 is important, as it paves the way to study LRRK2 activity in cells/tissues derived from PD patients. It will enable evaluation of whether LRRK2 protein kinase activity is controlled by extracellular agonists and may also help in the screening for inhibitors for LRRK2. We also observed that fusing Nictide to GST, yielded a highly expressed protein in Escherichia coli (4 mg/l) which was efficiently phosphorylated by LRRK2 in vitro at a greater initial rate than GST–ezrin-(505–586) or GST–LRRKtide (). GST–Nictide would serve as a good positive control when evaluating efficiency of phosphorylation of LRRK2 substrates that are identified in future studies.
Our analysis reveals that the substrate specificity of LRRK2 is quite distinct from that of ROCK2. LRRK2 does not phosphorylate MYPT, and ROCK2 poorly phosphorylates ezrin. Moreover, mutations in LRRKtide affected phosphorylation by LRRK2 and ROCK2 in different ways. For example, mutation of the +1 position of the LRRKtide peptide from a leucine residue to alanine abolished ROCK phosphorylation, without affecting LRRK2 phosphorylation. Many LRRKtide mutations enhanced phosphorylation by ROCK, but inhibited phosphorylation by LRRK2. Consistent with ROCK2 phosphorylating ezrin poorly in vitro
, we also found that in vivo
various ROCK inhibitors failed to inhibit ERM phosphorylation under conditions which they suppressed MYPT phosphorylation. This is consistent with other studies where the Y-27632 ROCK inhibitor was found not to suppress ERM phosphorylation [32
]. Taken together, these data cast doubt on earlier suggestions that ERM proteins are physiologically phosphorylated by ROCK isoforms.
The finding that the H-1152, Y-27632 and sunitinib failed to suppress ERM phosphorylation indicates that either LRRK2 does not phosphorylate ERM in HEK-293 cells or that LRRK2 is not the sole kinase that phosphorylates ERM proteins. As we were unable to detect significant levels of endogenous LRRK2 in HEK-293 cells (), we overexpressed LRRK2 and LRRK2[G2019S] in HEK-293 cells, but this also failed to induce phosphorylation of ERM proteins (R.J. Nichols, unpublished work). Taken together, this suggests that, although ERM proteins are efficiently phosphorylated by LRRK2 in vitro
, there is no strong evidence that ERM proteins comprise physiological substrates for LRRK2. Recent studies in Drosophila
] and primary mouse lymphocytes [36
] have suggested that the SLK/LOK STE20 protein kinase might be a key player in controlling ERM phosphorylation. Consistent with this, ERM phosphorylation is reduced but not abolished in lymphocytes derived from SLK/LOK-knockout mice [36
]. It was shown in 1994 that SLK has an optimal motif of R-R/K-F-G-S/T-L-R-R-F/I [37
], resembling the ERM site D-K-Y-K-T-L-R-Q-I and is also remarkably similar to the optimal substrate specificity of LRRK2, W-R-F-Y-T-L-R-R-A. It would also be interesting to test whether residual ERM phosphorylation observed in the SLK/LOK-knockout cells was reduced further by treatment with sunitinib and Y-27632 LRRK2 inhibitors. Another recent study has indicated that another STE20 family kinase termed Mst4 kinase can phosphorylate ezrin in polarized epithelial cells in a pathway controlled by the LKB1 tumour suppressor [38
]. MST4 was not inhibited by any compound used in the present study ().
The finding that the widely utilized ROCK inhibitor Y-27632 (used in > 1400 papers) as well as H-1152 and hydroxyfasudil inhibit recombinant as well as endogenous LRRK2 with a similar potency to that which they target ROCK2 was unexpected, as LRRK2 and ROCK2 are not closely related kinases. LRRK2 lies within the tyrosine-like kinases of the human kinome, whereas ROCK2 belongs to the distinct AGC branch [39
]. It is therefore possible that some of the physiological effects observed with these ROCK inhibitors could result from inhibition of LRRK2 rather than ROCK isoforms. Identification of GSK429286A is significant, in that not only is it more selective than Y-27632 as assessed on our kinase-specificity panel, but also it does not significantly inhibit LRRK2 even at doses as high as 30 μ
M (500-fold higher than IC50
of inhibition of ROCK2). GSK429286A will be a useful reagent to use in addition to Y-27632 to assess cellular roles of ROCK isoforms. The finding that the LRRK2[G2019S] mutant was 2–4-fold more sensitive H-1152, Y-27632 and sunitinib than the wild-type LRRK2 also indicates that it may be possible to develop compounds that have greater potency towards the PD mutant. It has also been reported that the LRRK2[G2019S] and LRRK2[I202T] mutants that possess elevated activity were also moderately more sensitive to a panel of non-selective kinase inhibitors [40
]. If compounds that specifically inhibited PD mutant forms of LRRK2 could be elaborated, they might have lower side effects and not suppress the normal functions of wild-type LRRK2. In drug discovery screens being undertaken to identify LRRK2 inhibitors, it could be beneficial to screen compounds against both mutant and wild-type forms of LRRK2.
Molecular modelling of the kinase domain of LRRK2 and comparing it with the structures of other kinases revealed a model of how LRRK2 might interact with H-1152. Several residues in the active site of ROCK that are key for binding to H-1152 are also conserved in LRRK2. These include Ala2016
, the equivalent of Ala215
in ROCK2 that plays an important role in mediating binding to the inhibitor [30
]. Mutation of Ala2016
in LRRK2 to a threonine residue, equivalent to Thr182
in PKA that is weakly inhibited by H-1152, did not affect the basal LRRK2 activity, but markedly suppressed inhibition of LRRK2 by H-1152 and other ROCK inhibitors. The inhibitor-resistant LRRK2[A2016T] mutant might aid in exploring the physiological roles of LRRK2. The wild-type and the LRRK2[A2016T] mutant could be overexpressed in cells and phosphorylation of any target should be less sensitive to inhibition by H-1152, Y-27632 or sunitinib in the cells overexpressing the drug-resistant mutant.
Our findings also provide a pharmacological strategy in which phosphorylation of identified LRRK2 substrates could be validated. We suggest that phosphorylation of a LRRK2 substrate should be suppressed by Y-27632 and H-1152 (dual ROCK and LRRK2 inhibitors) as well as sunitinib (inhibits LRRK2, but not ROCK), but not be affected by GSK429286A (inhibits ROCK, but not LRRK2). In contrast, ROCK-mediated processes should be sensitive to GSK429286A in addition to Y-27632 and H-1152, but should not be inhibited by sunitinib. Consistent with this, sunitinib does not inhibit the phosphorylation of MYPT at Thr850 under conditions where this phosphorylation is inhibited by GSK429286A, Y-27632 and H-1152 ().
In conclusion, we have undertaken basic analysis of the LRRK2 substrate specificity and developed improved assays to isolate and assess its activity. This will aid in assessing how LRRK2 is regulated and might also facilitate identification of LRRK2 inhibitors that might have potential for treatment of PD. We have also developed a strategy making use of Y-276332 or H-1152, sunitinib and GSK429286A to explore the roles of the LRRK2 kinase. Finally, we recommend that effects prescribed to ROCK based mainly on the use of Y-276332 and/or H-1152 be re-evaluated with the more selective GSK429286A inhibitor to ensure that these are indeed mediated by ROCK and not LRRK2.