Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization - PubMed (original) (raw)

Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization

Nicolas Dzamko et al. Biochem J. 2010.

Abstract

LRRK2 (leucine-rich repeat protein kinase 2) is mutated in a significant number of Parkinson's disease patients. Since a common mutation that replaces Gly2019 with a serine residue enhances kinase catalytic activity, small-molecule LRRK2 inhibitors might have utility in treating Parkinson's disease. However, the effectiveness of inhibitors is difficult to assess, as no physiological substrates or downstream effectors have been identified that could be exploited to develop a robust cell-based assay. We recently established that LRRK2 bound 14-3-3 protein isoforms via its phosphorylation of Ser910 and Ser935. In the present study we show that treatment of Swiss 3T3 cells or lymphoblastoid cells derived from control or a Parkinson's disease patient harbouring a homozygous LRRK2(G2019S) mutation with two structurally unrelated inhibitors of LRRK2 (H-1152 or sunitinib) induced dephosphorylation of endogenous LRRK2 at Ser910 and Ser935, thereby disrupting 14-3-3 interaction. Our results suggest that H-1152 and sunitinib induce dephosphorylation of Ser910 and Ser935 by inhibiting LRRK2 kinase activity, as these compounds failed to induce significant dephosphorylation of a drug-resistant LRRK2(A2016T) mutant. Moreover, consistent with the finding that non-14-3-3-binding mutants of LRRK2 accumulated within discrete cytoplasmic pools resembling inclusion bodies, we observed that H-1152 causes LRRK2 to accumulate within inclusion bodies. These findings indicate that dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and/or monitoring LRRK2 cytoplasmic localization can be used as an assay to assess the relative activity of LRRK2 inhibitors in vivo. These results will aid the elaboration and evaluation of LRRK2 inhibitors. They will also stimulate further research to understand how phosphorylation of Ser910 and Ser935 is controlled by LRRK2, and establish any relationship to development of Parkinson's disease.

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Figures

Figure 1. H-1152 and sunitinib treatment leads to dephosphorylation of Ser910 and Ser935 and disruption of 14-3-3 interaction

(A) Endogenous LRRK2 was immunoprecipitated (IP) with anti-LRRK2-(100–500) (S348C) antibody from Swiss 3T3 cells treated with DMSO vehicle control or the indicated concentrations of H-1152 for 90 min. Immunoprecipitates were subjected to immunoblot analysis with the indicated antibody as well as 14-3-3 overlay far-Western analysis. The immunoblot analysis was quantified by Odyssey® LI-COR analysis with the histogram at the top of each panel representing the ratio of Ser910 phosphorylation to total LRRK2, the lower histogram represents the ratio of Ser935 phosphorylation to total LRRK2. (B) Endogenous LRRK2 immunoprecipitates were analysed as in (A), except that cells were treated with 30 μM H-1152 for the indicated time prior to cell lysis. (Cand D) Endogenous LRRK2 immunoprecipitates were analysed as in (A andB) respectively, except that sunitinib was employed rather than H-1152. Results are means±S.E.M. and are representative of at least two separate experiments performed in duplicate. AU, arbitrary units.

Figure 2. Evidence that LRRK2 kinase activity controls Ser910 and Ser935 phosphorylation, as well as 14-3-3 binding

(A and B) HEK-293 cells transiently expressing the indicated forms of FLAG–LRRK2 were treated with DMSO vehicle control or indicated concentrations of H-1152 or sunitinib for 90 min. Cells were lysed in lysis buffer supplemented with 0.5% NP-40 and 150 mM NaCl and subjected to anti-FLAG immunoprecipitation. Immunoprecipitates were resolved by SDS/PAGE (4–12% Novex gels) and subjected to immunoblotting with anti-FLAG (total LRRK2), anti-phospho-Ser910 (αpS910), anti-phospho-Ser935 (αpS935) antibodies, as well as a 14-3-3 overlay assay, as indicated. Similar results were obtained in two separate experiments.

Figure 3. Analysis of endogenous LRRK2 Ser910 and Ser935 responsiveness to LRRK2 inhibition in Parkinson's disease patient-derived samples

(A) LRRK2 kinase activity was assessed in EBV-immortalized lymphoblastoid cells from a patient homozygous for the LRRK2(G2019S) mutation and diagnosed with Parkinson's disease and an individual without the G2019S mutation. Kinase assays were performed in triplicate and results are means±S.E.M. (B–E) These cells were then treated with increasing doses of (B and C) H-1152 or (D andE) increasing doses of sunitinib for 60 min prior to lysis. LRRK2 was immunoprecipitated (IP) and the phosphorylation status of Ser910 and Ser935was assessed following immunoblot and LI-COR quantification analysis. The upper histogram in each panel represents the ratio of Ser910 phosphorylation to total LRRK2, the lower histogram represents the ratio of Ser935 phosphorylation to total LRRK2. Results are means±S.E.M. for a duplicate analysis. Similar results were obtained in two separate experiments.

Figure 4. Disruption of 14-3-3 binding induces accumulation of LRRK2 within cytoplasmic aggregates

(A) Stable-inducible T-REx cells lines harbouring the indicated forms of LRRK2 were incubated for 24 h with 0.1 μg/ml doxycycline to induce expression of GFP–LRRK2. The indicated cell lines were treated in the absence or presence of the indicated dose of H-1152 for 90 min prior to fixation. Representative fluorescent micrographs of GFP–LRRK2 localization are shown. Cytoplasmic aggregates of GFP–LRRK2 are indicated with white arrows. (B) Fluorescent micrographs representative of cultures of the indicated forms of GFP–LRRK2 are shown. Cytoplasmic aggregates of GFP–LRRK2 are indicated with white arrows. Localization analyses were performed in duplicate, and similar results were observed in two independent experiments. (C) Cells were treated as in (A) except that slides were co-stained with an anti-tubulin antibody to detect microtubules (red). GFP–LRRK2 is shown in green and co-localization is shown in yellow.

Figure 5. Evidence that Ser910/Ser935 phosphorylation is not mediated by autophosphorylation

Endogenous LRRK2 was immunoprecipitated from Swiss 3T3 cells treated with DMSO, 30 μM H-1152 or 10 μM sunitinib for 2 h to induce dephosphorylation of Ser910 and Ser935. Immunoprecipitates were washed multiple times with lysis buffer containing 0.5 M NaCl to remove inhibitor and were then incubated in kinase buffer containing 20 μM Nictide in the presence or absence of magnesium ATP (Mg:ATP) for 30 min. Following incubation, immunoprecipitates were centrifuged at 6000 g for 0.5 min and the supernatant was spotted on to P81 paper for measurement of LRRK2 kinase activity. Sample buffer was added to the pelleted beads and LRRK2 Ser910 and Ser935 phosphorylation was quantified following immunoblot analysis with the indicated antibodies. A membrane was also subjected to autoradiography to assess LRRK2 autophosphorylation (32P). The minor effect that H-1152 had on LRRK2 kinase assay is not significant.

Figure 6. Proposed model of how LRRK2 controls Ser910 and Ser935 phosphorylation leading to 14-3-3 binding

Our data suggest that LRRK2 functions as an upstream component of a signal transduction pathway that either directly or indirectly stimulates the activity of a protein kinase or inhibits the activity of a protein phosphatase that acts on Ser910 and Ser935. This enables LRRK2 to interact with 14-3-3 isoforms and stabilizes diffuse cytoplasmic localization of LRRK2. Treatment of cells with LRRK2 inhibitors thus leads to dephosphorylation of Ser910 and Ser935 and dissociation of 14-3-3. Our findings indicate that LRRK2 phosphorylation of Ser910 and Ser935 as well as 14-3-3 binding could be employed as a biomarker to benchmark efficacy of LRRK2 inhibitors that are being developed.

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Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization - PubMed (original) (raw)