Fig. 6

PINK1 p.I368N is a kinase dead mutant that fails to activate PARKIN and the mitochondrial quality control. a HeLa cells were simultaneously transfected with PINK1 siRNA and V5 empty vector, PINK1 WT or p.I368N mutant. Cells were left untreated or incubated with 10 μM CCCP for 4 h. Cell lysates were analyzed by WB, probed with anti-V5, PINK1 and p-Ser65-Ub antibodies. GAPDH was used as a loading control. Auto-phosphorylated PINK1 (anti-V5) (asterisk) was detected on phos-tag gels only in lysates from PINK1-V5 WT transfected, CCCP treated cells. In PINK1-V5 p.I368N transfected cells, CCCP-induced phosphorylation of Ub was largely abolished. b IP of PINK1-V5 using V5 antibody followed by in vitro kinase assay. HeLa cells were transfected with the V5 empty vector, PINK1-V5 WT or p.I368N and treated with or without CCCP for 2 h. Washed immunoprecipitates were incubated with N-terminally biotinylated mono-Ub and 500 μM ATP in phosphorylation buffer at 37 °C for 24 h. Total and phosphorylated Ub were analyzed by WB using streptavidin-HRP and p-Ser65-Ub antibody, respectively. c PARKIN translocation to damaged mitochondria measured by HCI. HeLa cells stably overexpressing GFP-tagged PARKIN were simultaneously transfected with PINK1 (or control) siRNA and with V5 empty vector, PINK1-V5 WT or p.I368N mutant, as indicated, and with mCherry as a selection marker. Cells were left untreated or treated with CCCP for 2 h and GFP-PARKIN translocation was measured in mCherry-positive cells. Data represents the mean of two independent experiments run with each six replicate wells. Statistical significance was assessed by two-way ANOVA with Tukey’s post hoc; **, p < 0.005; ***, p < 0.0005). d Ub-charging of PARKIN C431S as a measure of its enzymatic activation. HeLa cells stably expressing 3xFLAG-tagged PARKIN C431S were pretreated with control or PINK1 siRNA and then transfected with V5 empty vector, PINK1-V5 WT or p.I368N. After treatment with CCCP for 4 h, cell lysates were analyzed by WB with the indicated antibodies. PARKIN C431S traps Ub in an oxyester-bond (resulting in an 8 kDa band shift) that is cleavable by NaOH treatment. e Densitometric analysis of Ub-charged 3xFLAG-PARKIN that was normalized to total PARKIN levels. Error bars indicate mean ± SEM from three independent experiments (one-way ANOVA with Tukey’s post hoc; ***, p < 0.0005; ns, not significant)