Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease
Figure 5
PD-linked mutants affect S935 phosphorylation and 14-3-3 binding of LRRK2.
(A) FLAG-LRRK2 WT, S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected into HEK-293T cells. The LRRK2 variants were immunoprecipitated and analyzed by Western blot using anti-pS935 and anti-FLAG antibodies. Quantification of the signals was performed using LI-COR and Odyssey software and the data was analyzed by One-way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments. (B) FLAG-LRRK2-WT, G2019S, R1441G and K1906M was immunoprecipitated from the brain lysates of the corresponding BAC-transgenic mice. The quantification of the signals was performed as described above. The ratio of pS935 signal over total LRRK2 signal is shown. The data was analyzed with One-way ANOVA (* P<0.05, ** P<0.01). Data are presented as mean value (± SEM) from three mice. (C) Myc-14-3-3γ (or control Myc vector) was co-transfected with FLAG-LRRK2 Wt, S935A, R1441G or G2019S mutant plasmid into HEK-293T cells. Co-IP was performed by using anti-Myc antibody. The pulled down LRRK2 variant levels were analyzed by Western blot analysis with the indicated antibodies and quantified using LI-COR and Odyssey system. The data was analyzed by One-way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments. (D) FLAG-LRRK2-WT, S935A, R1441G, Y1699C, G2019S or K1906M mutant plasmid was transfected individually into HEK-293T cells. Sepharose 4B beads-conjugated GST-14-3-3γ protein was incubated with different transfected cell lysates containing LRRK2-Wt or various mutants. Western blot analysis was performed to determine the LRRK2 variant amount after the GST pull-down assay. The results were quantified as described above. The ratio of pulled down LRRK2 and input LRRK2 was measured and analyzed by One-Way ANOVA (** P<0.01). Data are presented as mean value (± SEM) from three independent experiments.