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. 2010 Sep 7;107(38):16691–16696. doi: 10.1073/pnas.1006083107

Fig. 4.

Fig. 4.

Parkin is tyrosine-phosphorylated after dopaminergic stress. (A) SH-SY5Y cells were transiently transfected with V5-parkin and treated with MPP+ (250 μM) or dopamine (DA) (250 μM) for 24 h in the presence or absence of STI-571 (10 μM). Cell lysates were immunoprecipitated with an anti-V5 antibody and immunoblotted with anti-phosphotyrosine to show phosphorylated parkin, and anti-parkin to show equivalent IP. Inputs were immunoblotted with anti-phospho-c-Abl to show activated c-Abl, and with anti-c-Abl, anti-AIMP2, or anti-actin antibodies. Data were repeated with similar results. (B) SH-SY5Y cells were transiently transfected with V5-parkin and HA-ubiquitin and treated with MPP+ (250 μM) or DA (250 μM) for 24 h in the presence or absence of STI-571 (10 μM). Cell lysates were immunoprecipitated with an anti-V5 antibody and immunoblotted with anti-HA to monitor ubiquitination of parkin, anti-phosphotyrosine to show phosphorylated parkin, and anti-V5 to show equivalent levels of immunoprecipitated parkin. Inputs were immunoblotted with anti-phospho-c-Abl to show activated c-Abl, and with anti-c-Abl or anti-HA to show equivalent expression. Data were repeated with similar results. (C) PC12-AIMP2-inducible cells were transfected with V5-WT-parkin and V5-Y143F-parkin in the presence or absence of MPP+ (200 μM) for 24 h. Some samples were incubated with 10 μM STI-571. Cell death was assessed by trypan blue exclusion. Both STI-571 and transfection with phosphorylation-resistant Y143F-parkin protect against AIMP2-induced cell death and after MPP+ treatment. Doxycycline (Dox) (+) prevents the induction of AIMP2. Dox withdrawal (−) induces the expression of AIMP2 (Fig. S3). Data are mean ± SEM for three separate experiments performed in duplicate. Statistical significance was determined by one-way ANOVA and Tukey's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001; ***1: DMSO versus MPP+–Dox (+); **2: MPP+ versus STI+MPP+–Dox (+); ***3: DMSO–Dox (+) versus AIMP2–Dox (−); ***4: DMSO/AIMP2–Dox (−) versus AIMP2–Dox (−) + WT-parkin; *5: DMSO/AIMP2-Dox (−) versus MPP+/AIMP2–Dox (−) + WT-parkin; ***6: MPP++/AIMP2–Dox (−) + WT-parkin versus STI + MPP+/AIMP2–Dox (−) + WT-parkin; **7: DMSO/AIMP2–Dox (−) versus AIMP2–Dox (−) + Y143F parkin; ***8: MPP+/AIMP2–Dox (−) versus MPP+/AIMP2–Dox (−) + Y143F parkin. (D and E) The protective effect of STI-571 requires parkin. (D) SH-SY5Y cells were transfected with either parkin-shRNA or GFP-shRNA in the presence or absence of MPP+ (250 μM) for 24 h. Some samples were incubated with 10 μM STI-571. Cell lysates were subjected to immunoprecipitation with an anti-parkin antibody. The parkin immunoprecipitates were immunoblotted with anti-phosphotyrosine to monitor tyrosine phosphorylation of parkin and anti-parkin to monitor the levels of immunoprecipitated parkin. Total lysates were immunoblotted with anti-phospho-c-Abl to show tyrosine-phosphorylated c-Abl, anti-actin as a loading control, and with an AIMP2 antibody to monitor the levels of AIMP2 and an anti-parkin antibody to confirm the parkin knockdown by parkin-shRNA. Data were repeated with similar results. (E) Trypan blue cell death assessments in SH-SY5Y neuroblastoma cells that were transiently transfected with parkin-shRNA or GFP-shRNA and treated with MPP+ (200 μM). Some samples were incubated with 10 μM STI-571 as indicated. Data are mean ± SEM for three separate experiments performed in duplicate. Statistical significance was determined by one-way ANOVA and Tukey's post hoc test. ***P < 0.001.