The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and apparently sporadic Parkinson disease. LRRK2 is a multidomain protein kinase with autophosphorylation activity. It has previously been shown that the kinase activity of LRRK2 is required for neuronal toxicity, suggesting that understanding the mechanism of kinase activation and regulation may be important for the development of specific kinase inhibitors for Parkinson disease treatment. Here, we show that LRRK2 predominantly exists as a dimer under native conditions, a state that appears to be stabilized by multiple domain-domain interactions. Furthermore, an intact C terminus, but not N terminus, is required for autophosphorylation activity. We identify two residues in the activation loop that contribute to the regulation of LRRK2 autophosphorylation. Finally, we demonstrate that LRRK2 undergoes intramolecular autophosphorylation. Together, these results provide insight into the mechanism and regulation of LRRK2 kinase activity.

FIGURE 1.

LRRK2 self-interacts. A, schematic of the yeast two-hybrid results obtained using a central part of LRRK2 as bait (upper illustration). Interacting fragments of LRRK2 are shown by double-headed arrows (lower illustration). ANK = ankyrin domain. B, mapping interactions of the ROC domain against Myc-LRRK2 fragments expressed in 293FT cells. The left panel shows inputs, the central panel represents GST-ROC pulldowns, and the right panel represents GST pulldowns. Membranes were probed with Myc (9e10) antibody. Kin, kinase. C, Coomassie Blue staining of GST and GST-ROC proteins used for pulldown experiments. D, mapping interactions of the ROC domain by co-immunoprecipitation (IP). Myc-ROC or vector control plasmids were co-expressed together with FLAG-ROC and FLAG-WD40 in 293FT cells, and proteins were co-immunoprecipitated. The left panels show inputs (Myc-ROC on lanes 1-2; FLAG-ROC and FLAG-WD40 on lanes 3-4), and the right panels show co-immunoprecipitations by Myc-ROC (lanes 5-6) or by vector control (lanes 7-8). Membranes were probed with anti-Myc (9e10; lanes 1-2) or anti-FLAG (M2; lanes 3-8) antibodies. IB, immunoblot. E, GST pulldowns of ROC-COR-kinase with GFP-full-length LRRK2 expressed in 293FT cells. Recombinant ROC-COR-kinase was expressed in E. coli as GST fusion protein and purified by glutathione affinity chromatography. The upper panel indicates inputs, and the lower panel indicates pulldowns. Blots were probed with anti-GFP monoclonal antibody. F, full-length LRRK2 interaction. Co-immunoprecipitation of GFP-LRRK2 and LRRK2-V5 overexpressed in 293FT cells. Immunoprecipitations were performed using anti-GFP antibodies. Inputs (upper panels) and immunoprecipitates (lower panels) were probed with either GFP or V5 antibodies. The arrows indicate position of LRRK2, and the markers on the right of the blots are in kilodaltons.

FIGURE 2.

Truncated forms of LRRK2 show decreased autophosphorylation. A, schematic of deletion constructs analyzed for their ability to undergo autophosphorylation. ANK = ankyrin domain. B, the left panel represents autophosphorylation of immunoprecipitated proteins, and the right panel shows total protein loading by immunoblot with Myc (9e10) antibody. Full-length proteins (lane 1) exhibit autophosphorylation as expected; ΔN terminus (lane 2) displayed high levels of autophosphorylation; ROC-COR-kinase (lane 3) and kinase (lane 4) were incompetent to undergo autophosphorylation, although the amounts immunoprecipitated were comparable with active proteins (Western blot (WB) with anti-Myc, right panel, lanes 5-8). C, quantitation indicates that ΔN terminus (ΔN term; amino acids 1328-2527) incorporates approximately three times more ³²P than full-length. Both ROC-COR-kinase and kinase domains incorporated almost undetectable amounts of ³²P. All experiments were carried out in triplicate.

FIGURE 3.

Active LRRK2 forms dimers. A, autophosphorylation activity of immunoprecipitated Myc-tagged wild-type, kinase dead (KD), and K1347A LRRK2. Blots are representative of n = 3 independent experiments, and corrected activity for total loading (with anti-Myc antibody) are shown in a bar graph. B, native PAGE reveals that Myc-tagged wild-type (WT)-LRRK2, but not KD, and that K1347A is mainly present in a complex (∼550 kDa) around the dimer size. A faint band is detected at ∼270 kDa, corresponding to monomeric LRRK2. WB, Western blot. C, representative Western blot of 0.25-ml gel filtration fractions of wild-type, KD, and K1347A LRRK2 cell lysates. The column void volume (V₀) and the standards ferritin (440 kDa) and catalase (232 kDa) are indicated. Blots were probed either with anti-Myc (9e10) or with rabbit anti-LRRK2. D, graph illustrates the elution profile of Myc-tagged wild-type LRRK2 plotted as the average percentage of protein in each fraction from four independent extracts. The total signal in each fraction measured by densitometry was divided by the total signal of the protein in all the fractions to determine the percentages shown in the graph. LRRK2 exists mainly as a ∼600-kDa species. A portion of the protein is present in higher molecular weight complexes. Markers on the right of all blots are in kilodaltons.

FIGURE 4.

Ser-2032 and Thr-2035 regulate LRRK2 autophosphorylation activity but are not sufficient to mediate dimerization. A, the structural model of kinase domain of LRRK2 constructed using the serine/threonine kinase domain of TβR-I (PDB entry 1B6C) as a template highlights the position of potential regulatory sites Thr-2031, Ser-2032, and Thr-2035. The loop in red is the catalytic loop (it also chelates the secondary Mg²⁺ ion). The loop in orange contains the highly conserved ASP that chelates the primary activating Mg²⁺ ion. B, native PAGE followed by Western blots (WB) of the Myc-LRRK2 proteins where these sites are mutated to alanine or glutamate. All variants form dimers and a higher molecular weight smear with a minor band corresponding to monomeric LRRK2. C, WT, KD, and Ala/Glu mutants were cloned in fusion with an N terminus 2x-Myc tag and expressed in 293FT cells. Total lysates were blotted for Myc (9e10) and normalized with β-actin (upper panels). The same set of constructs was immunoprecipitated (IP) with anti-Myc polyclonal antibodies and subjected to in vitro kinase assays. Autophosphorylation activity is shown by autoradiography, and total proteins after immunoprecipitation are shown by Myc Western blot (middle panels). Activity against the generic Ser/Thr kinase substrate MBP was also assessed (lower two panels) using autoradiography, with MBP loading assessed using Ponceau staining. Markers on the right of the blots are in kilodaltons. D-F, quantitation indicates that and Thr-2035 and Ser-2032 mutations were fully stable (D) and had decreased autophosphorylation activity (E) but only minor effects on MBP phosphorylation (F). D-F were carried out in quadruplicate and analyzed by one-way analysis of variance with Tukey's multiple comparison test. ^*, p < 0.05; ^**, p < 0.01.

FIGURE 5.

Autophosphorylation of full-length LRRK2 is an intramolecular process. A, trans-phosphorylation assays using GFP-KD as substrate and Myc-WT as active LRRK2. Proteins were purified by immunoprecipitation, and concentrations were estimated using bovine serum albumin standard on Coomassie Brilliant Blue (CBB). Constant amounts of GFP-KD (∼100 ng) and increasing amounts of Myc-WT (from ∼50 ng to ∼1 μg) were loaded in 5% SDS-PAGE gels. Phosphorylation is shown by autoradiography (upper panel), and total protein loading is shown by Coomassie Brilliant Blue staining (lower panel). The experiment is representative of n = 3 replicates. B, trans-phosphorylation assays using Myc-LRRK2 WT, S2032A, S2032E, T2035A, T2035E, and KD and active GFP-WT. The upper panel represents autoradiograms, and the lower panel shows total protein loading by immunoblot (with monoclonal anti-GFP and Myc antibodies). WB, Western blot. EV, empty vector. C, trans-phosphorylation assays using the same set of Myc-tagged LRRK2 protein as A and GFP-KD as inactive substrate. As above, the upper panel represents autoradiograms, and the lower panel shows total protein loading by immunoblot. D, schematic showing that autophosphorylation (indicated by circled P) of dimeric LRRK2 is likely to be an intramolecular event.