Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities

Abstract

The leucine-rich repeat kinase 2 (LRRK2) protein has both guanosine triphosphatase (GTPase) and kinase activities, and mutation in either enzymatic domain can cause late-onset Parkinson disease. Nucleotide binding in the GTPase domain may be required for kinase activity, and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation, and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. Parkinson-disease-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase domain mutation resulted in a multiplicative increase (∼7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activities. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occur in an ATP- and autophosphorylation-independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.

Fig. 1. Autophosphorylation profile of G2019S mutant and WT-LRRK2

A) In vitro kinase assays were conducted with the indicated LRRK2 (Δ1–970) enzyme at 100 nM concentration and 100 μM ATP at 30°C and reactions resolved by SDS-PAGE. After exposure to autoradiography film, bands were excised from dried gels. B) Incorporation of ³²P into LRRK2 protein bands in A was measured by liquid scintillation with correction for counting efficiency and normalized to a standard curve, and average phosphates incorporated per LRRK2 at each time point were determined. Error bars represent ±S.E.M. No significant additional incorporation was noted after 30 minutes. C) Recombinant LRRK2 Δ1–970 and full-length (FL) proteins were treated with 100 μM LRRKtide peptide for the indicated interval in kinase assays. ³²P incorporation into peptides was determined via liquid scintillation. Activity during the denoted intervals was normalized to the activity in the first 20 min interval for each condition. ‘Free’ denotes the condition where LRRK2 was included in soluble form; ‘Solid Surface’ denotes reactions where LRRK2 protein was bound to agarose beads. Error bars represent ±S.E.M. D) Kinase reactions were conducted over the indicated time and analyzed by silver stained SDS-PAGE gels and native-PAGE Blue gels.

Fig. 2. LRRK2 autophosphorylation specificity in the ROCO domain is unchanged by the pathogenic G2019S mutation

LRRK2 in vitro kinase assays were conducted for 30 min with 100 nM G2019S (kinase-overactive), WT or D1994A (kinase-dead) LRRK2 protein (Δ1–970) in the presence of 100 μM ATP. A) Reactions were resolved by SDS-PAGE, and Pro-Q diamond staining highlights phosphorylation of LRRK2 protein after a 30 min in vitro kinase assay. Fluorescence was detected using a Typhoon Trio scanner. B) ATP treated and naïve LRRK2 proteins were run on the same gel and phosphorylation at threonine residues measured C–H) Kinase reactions were resolved by SDS-PAGE, and coomassie stained bands were digested with LysC, or chymotrypsin, or trypsin. Minimum spectra required to make an assignment for a phosphorylated residue are represented. In most cases multiple high quality peptides (Table 1) corroborate the assignment. Blue text highlights matched values from expected. Identical phosphorylated residues were detected in both G2019S and WT-LRRK2 protein, but no phosphorylated residues were detected in D1994A-LRRK2 protein. C) Representative CID spectra from 1343 phosphorylated peptide. D–H) Representative ETD spectra from other identified LRRK2 phosphorylated peptides.

Fig. 3. LRRK2 autophosphorylation occurs primarily in the GTP binding pocket

A–C) Cartoon models of the LRRK2 GTPase domain with autophosphorylation at detected sites. N=Blue, O=Red, P=Orange and the bound GDP (Purple) is shown in stick format and the Mg²⁺ ion (Green) shown as a sphere. Pictures were created with PyMOL with domain structure 2ZEJ (Protein Data Bank) D) Linear representation of the LRRK2 GTPase domain illustrates the location of phosphorylations (yellow flags) and residues lining the binding pocket (colored in red) and those unresolved in the crystal structure (gray).

Fig. 4. Autophosphorylation-specific LRRK2 antibodies identify pT1503 in LRRK2 protein derived from cell lines and tissue

A) Recombinant LRRK2 Δ1–970 derived from SF-9 cells was probed for phosphorylated T1503, with or without ATP treatment. Specificity of the pT1503 antibody is demonstrated through lack of reactivity with kinase dead (KD, D1994) and naïve LRRK2. B and C) Full-length LRRK2 protein purified from HEK-293FT cells was probed for total LRRK2 protein and phosphorylated T1503. Specificity of the pT1503 antibody is demonstrated through minimal reactivity with kinase dead (KD, D1994), phosphomimetic T1503D and unphosphorylatable T1503A LRRK2. D) In vitro kinase assays were conducted with LRRK2 recombinant protein prior to treatment with PPase1. Treatment of autophosphorylated LRRK2 with PPase1 (2.5 U, where 0.1 units removes 0.5 nmol serine/threonine phosphates from 10 μM phospho-MBP) and sunitinib (500nM) after in vitro kinase assays. (++) denotes that LRRK2 was treated with sunitinib both during the kinase assay and phosphatase treatment. E) Western blots for LRRK2 protein derived from BAC LRRK2-Flag G2019S mouse tissue. Western blots for pT1503 were conducted before and after treating the membrane with λ-phosphatase. Total LRRK2 was determined with anti-Flag. Quantifications depicted are from 3 independent experiments.

Fig. 5. Effect of variation in the ROC domain on LRRK2 enzymatic activity

A and C) Full length LRRK2 protein conjugated to agarose beads was incubated in kinase reaction buffer containing 100 μM LRRKtide substrate for 30 min. Beads were removed from the reaction, LRRK2 removed from the beads, denatured and resolved by SDS-PAGE, with LRRK2 protein highlighted by coomassie stain and accompanying autoradiography exposure. B and D) Kinase reaction supernatant from A and C containing phosphorylated LRRKtide was spotted on P-81 cellulose paper and ³²P incorporation was measured by liquid scintillation. Results from three experiments are given, and error bars represent ±S.E.M., n.s. is not significant and * indicates p<.001 compared to WT-LRRK2, determined by one-way ANOVA with Tukey’s post hoc test. E) Lysate from HEK-293FT cells transiently transfected with full length LRRK2 were combined with γ-S-GTP sepharose beads. Input fractions and GTP pull-down fractions were resolved by SDS-PAGE and western blotting. F) Raw luminescence values were determined on an Alpha-Innotech Flurochem HD, background signal removed and values normalized to T1348N LRRK2 across experiments after correction for total protein levels (Input fraction) in 4 independent experiments. Error bars are ±S.E.M. and * indicates p<.05 and *** indicates p<.001 relative to WT, determined by one-way ANOVA and Tukey’s post hoc test.

Fig. 6. Identification of LRRK2 sites of phosphorylation on MBP

A) In vitro kinase assay with recombinant dephosphorylated MBP with WT or kinase dead (D1994A) LRRK2 (Δ1–970) analyzed by SDS-PAGE and protein bands resolved with coomassie stain. Exposure to autoradiography film indicates comparative phosphorylation of substrate proteins. B) Web logo identified no significant trends in peptide sequence using the 13mer peptide sequence around the 12 phosphorylated residues identified by mass spectrometry in this study. C–H) In vitro kinase assays composed of 50 nM LRRK2 (Δ1–970) and 1 μM dephosphorylated MBP were resolved by SDS-PAGE, stained with coomassie and the predominate MBP isoform excised and digested with LysC, trypsin or chymotrypsin. Detected peptides and corresponding spectra required to make an assignment are given along with the identity of the phosphorylated residue. In all cases multiple high quality peptides (not shown) corroborate the assignment.