Reevaluation of phosphorylation sites in the Parkinson disease-associated leucine-rich repeat kinase 2 - PubMed (original) (raw)

Reevaluation of phosphorylation sites in the Parkinson disease-associated leucine-rich repeat kinase 2

Xiaojie Li et al. J Biol Chem. 2010.

Abstract

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an important cause of late-onset, autosomal dominant familial Parkinson disease and contribute to sporadic Parkinson disease. LRRK2 is a large complex protein with multiple functional domains, including a Roc-GTPase, protein kinase, and multiple protein-protein interaction domains. Previous studies have suggested an important role for kinase activity in LRRK2-induced neuronal toxicity and inclusion body formation. Disease-associated mutations in LRRK2 also tend to increase kinase activity. Thus, enhanced kinase activity may therefore underlie LRRK2-linked disease. Similar to the closely related mixed-lineage kinases, LRRK2 can undergo autophosphorylation in vitro. Three putative autophosphorylation sites (Thr-2031, Ser-2032, and Thr-2035) have been identified within the activation segment of the LRRK2 kinase domain based on sequence homology to mixed-lineage kinases. Phosphorylation at one or more of these sites is critical for the kinase activity of LRRK2. Sensitive phospho-specific antibodies to each of these three sites have been developed and validated by ELISA, dot-blot, and Western blot analysis. Using these antibodies, we have found that all three putative sites are phosphorylated in LRRK2, and Ser-2032 and Thr-2035 are the two important sites that regulate LRRK2 kinase activity.

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Figures

FIGURE 1.

FIGURE 1.

Overview of the three possible phosphorylation sites within LRRK2 kinase activation loop and the peptides used for the production of phospho-LRRK2 specific antibodies. A, sequence alignment of the activation loop of LRRK2 and other homologous MAP kinases. The activation loop is defined as the region between two conserved motifs, DF/YG and APE. Three possible sites for the autophosphorylation of LRRK2, Thr-2031, Ser-2032, and Thr-2035, are denoted by a dark bar. B, peptides used in the production of LRRK2 phospho-specific antibodies. Phosphopeptides pT2031, pS2032, and pT2035 were used for the generation and purification of the antibodies. Nonphosphorylated peptide was used for the purification and characterization of the antibodies. C, schematic diagram depicting the purification procedure of LRRK2 phospho-specific antibodies.

FIGURE 2.

FIGURE 2.

Characterization of the purified LRRK2 phospho-specific antibodies using specific peptides. A, ELISA using antibody anti-pT2031 and phospho-LRRK2 peptide (pT2031) and nonphosphorylated peptide control. B, ELISA using antibody anti-pS2032 and phospho-LRRK2 peptide (pS2032) and nonphosphorylated peptide control. C, ELISA using antibody anti-pT2035 and phospho-LRRK2 peptide (pT2035) and nonphosphorylated peptide control. D, dot blot using specific phospho- and its corresponding nonphosphoantibody.

FIGURE 3.

FIGURE 3.

Characterization of the purified LRRK2 phospho-specific antibodies using overexpressed LRRK2 protein. Immunoprecipitated LRRK2 wild type (WT) protein and various phosphorylation mutant proteins, derived from transfected HEK-293 cells, were analyzed by SDS-PAGE and Western blotting using specific phosphoantibody and anti-myc antibody. Phosphorylation-deficient mutant proteins were T2031A (A), S2032A (B), and T2035A (C); phospho-mimic mutant proteins were T2031D and T2031E (A), S2032D and S2032E (B), and T2035D and T2035E (C).

FIGURE 4.

FIGURE 4.

Regulation of LRRK2 autophosphorylation activity by the three possible phosphorylation sites (Thr-2031, Ser-2032, and Thr-2035). A and C, immunoblot and autoradiogram of autophosphorylated LRRK2, as resolved by SDS-PAGE. LRRK2 protein levels were determined by Western blot analysis, using anti-myc antibody. B and D, normalization of incorporated 32P compared with LRRK2 protein content. Data represent three independent experiments, in arbitrary units, where wild type (WT)-LRRK2 kinase activity is defined as 100%. Control bar represents mean ± S.E. *, p < 0.05; **, p < 0.01 compared with wild type LRRK2 kinase activity, assessed by a two-tailed one-sample Student's t test.

FIGURE 5.

FIGURE 5.

Hydrogen peroxide (H2O2) activates LRRK2 kinase through the phosphorylation of all three phosphorylation sites (Thr-2031, Ser-2032, and Thr-2035). A, immunoblot and autoradiogram of autophosphorylated LRRK2, as resolved by SDS-PAGE. LRRK2 protein levels were determined by Western blot analysis, using anti-myc antibody. B, normalization of incorporated 32P compared with LRRK2 protein content. Data represent three independent experiments, in arbitrary units, where wild type (WT)-LRRK2 kinase activity is defined as 100%. Control bar represent mean ± S.E. *, p < 0.05; **, p < 0.01 compared with wild type LRRK2 kinase activity, assessed by a two-tailed one-sample Student's t test. C–E, Western blot using phospho-specific antibodies to detect the increasing phosphosignal of LRRK2 under the peroxide treatment. HEK-293 cells transfected with wild type or phospho-deficient mutant LRRK2 were treated with peroxide and immunoprecipitated by anti-myc antibody. Data represent five independent experiments, in arbitrary units, where wild type LRRK2 signal is defined as 100%. Control bar represent mean ± S.E. *, p < 0.05 compared with phosphorylation level of WT-LRRK2, assessed by a two-tailed one-sample Student's t test. n.s is nonsignificant, comparing the treated and untreated LRRK2 mutant samples, assessed by two tailed unpaired Students' t test.

FIGURE 6.

FIGURE 6.

Autophosphorylation of LRRK2 happens on all three putative sites (Thr-2031, Ser-2032 and Thr-2035. A, Western blotting using phospho-specific antibodies to detect the decreasing phosphosignal of LRRK2 under the treatment of kinase inhibitor staurosporine at 100 n

m

. Overexpressed human wild type LRRK2 protein was immunoprecipitated by anti-myc antibody and subjected to the standard LRRK2 kinase assay, with or without prior treatment of staurosporine (100 n

m

). B, Western blotting using phospho-specific antibodies to detect the increasing phosphosignal of LRRK2-G2019S mutant. HEK-293 cells transfected with wild type LRRK2 or G2019S mutant were treated with peroxide and immunoprecipitated (IP) by anti-myc antibody. WT+, wild type LRRK2 treated with H2O2; GS, G2019S; GS+, G2019S mutant LRRK2 treated with H2O2; n.s., not significant. Data represent three independent experiments, in arbitrary units, where WT-LRRK2 signal is defined as 100%. Control bar represents mean ± S.E. *, p < 0.05; **, p < 0.01, compared with untreated wild type LRRK2, assessed by a two-tailed one-sample Student's t test.

FIGURE 7.

FIGURE 7.

LRRK2-induced neuronal toxicity correlated with its kinase activity. A, mouse cortical neurons stained by TUNEL and an anti-GFP antibody after 48-h transfection (13 total days in vitro) with the indicated LRRK2 (wild type and mutant) constructs in a 10:1 molar ratio with EGFP. White arrows indicate neurons counted as nonviable EGFP- and TUNEL-positive neurons. B, quantitative data analysis of neuronal viability of LRRK2-transfected mouse cortical neurons. Data are representative of three independent experiments. Error bars represent mean ± S.E. +, p < 0.05; ++, p < 0.01 compared with control (EGFP only); *, p < 0.05 compared with wild type LRRK2-transfected neurons, assessed by one-way nonparametric analysis of variance with Dunnett's Multiple Comparison test.

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