Figure 1.
LRRK2 and the hypothetical mechanism of its inhibitor-induced dephosphorylation.
(A) Schematic depiction of the domain structure of LRRK2. The phosphorylation hot spot is located amino-terminal to the LRR domain. The residues mutated in this study (Lys1906, Asp1994, Ala2016, Asp2017, Gly2019, Thr2031, Ser2032, and Thr2035) are also indicated. LRR: leucine-rich repeat, ROC: Ras of complex proteins, COR: carboxyl-terminal of ROC. (B) The hypothetical mechanism of the inhibitor-induced dephosphorylation. Dashed lines represent inhibited pathways.
Figure 2.
The basal phosphorylation of LRRK2 harboring kinase-modifying mutations.
The combined results of quantification of the levels of basal phosphorylation at Ser910 (A and D), Ser935 (B and E), or Ser955 (C and F) of LRRK2 harboring the kinase-inactive mutations (A–C) and the inhibitor-insensitive or hyperactive mutations (D–F). The corresponding immunoblots are shown in Figure 3 and 4. The data are given as the percentage of those observed in WT LRRK2 (n = 6, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Kruskal-Wallis test; comparison with WT LRRK2).
Figure 3.
Inhibitor-induced dephosphorylation of kinase-inactive LRRK2.
HEK293 cells transfected with wild-type, K1906A, K1906M, D1994A, D1994N, D2017A, S2032A or T2035A LRRK2 were treated with (A) 3 µM LRRK2-IN-1 or the solvent (0.1% DMSO) for 30 min, (B) 5 µM sunitinib or the solvent (0.1% DMSO) for 90 min, or (C) 30 µM H-1152 or the solvent (1% sterilized distilled water) for 90 min, and the phosphorylation of LRRK2 at Ser910, Ser935, or Ser955 was examined by immunoblotting. The levels of the phosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in solvent-treated WT LRRK2 (n = 3, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Two-way ANOVA test followed by Bonferroni’s test).
Figure 4.
Inhibitor-induced dephosphorylation of inhibitor-resistant or hyperactive LRRK2.
HEK293 cells transfected with wild-type, K1906M, A2016T, G2019S, T2031S LRRK2 were treated with (A) 3 µM LRRK2-IN-1 or the solvent (DMSO) for 30 min, (B) 5 µM sunitinib or the solvent (0.1% DMSO) for 90 min, or (C) 30 µM H-1152 or the solvent (1% sterilized distilled water) for 90 min, and the phosphorylation of LRRK2 at Ser910, Ser935, or Ser955 was examined by immunoblotting. Non-specific bands were marked with asterisks (*). The levels of the phosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in DMSO-treated WT LRRK2 (n = 3, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Two-way ANOVA followed by Bonferroni’s test). Asterisks with parentheses mean that the distribution of either sample did not follow a normal distribution (Shapiro-Wilk test).
Table 1.
Summary of the basal phosphorylation and inhibitor-induced dephosphorylation of kinase-inactive LRRK2.
Figure 5.
The kinase activity of LRRK2 harboring kinase-modifying mutations.
(A) Phosphorylation of biotin-LRRKtide was examined by ELISA using an antibody that specifically recognizes phosphorylated LRRKtide. The data are given as the amount of phosphorylation per minute (n = 3, mean ± standard error). The amount of immunoprecipitated LRRK2 was examined by immunoblotting with an anti-LRRK2 antibody (bottom panel). (B) Autophosphorylations of WT and mutant LRRK2 at Thr1357, Thr1491, and Thr1503 were examined by immunoblotting with an antibody specifically recognizing corresponding phosphorylated threonines. The levels of the autophosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in WT LRRK2 (n = 3, mean ± standard error). ***p<0.001 (One-way ANOVA followed by Bonferroni’s test).
Table 2.
Summary of the basal phosphorylation and inhibitor-induced dephosphorylation of inhibitor-resistant or hyperactive LRRK2.