Figure 1. PINK1-dependent phosphorylation of Parkin in vivo.

(a) PINK1-FLAG WT or KD/HA-Parkin/PINK1^−/− MEFs were labelled with [³²P] orthophosphate and treated with 30 µM CCCP for 1.5 hr. Phosphorylated Parkin was detected by autoradiography (³²P). Immunoprecipitated HA-Parkin was detected by Western blotting (WB) with anti-Parkin. (b) PINK1-FLAG WT or KD/HA-Parkin/PINK1^−/− MEFs were treated with or without 30 µM CCCP for the indicated periods of time. Cell lysate was subsequently separated on a Phos-tag gel, followed by WB with anti-PINK1 or anti-Parkin antibodies (Phos-tag WB). Phosphorylated bands of Parkin and PINK1 were confirmed by their disappearance with lambda protein phosphatase (λPP) treatment. Mitochondrial Hsp60 was used as a loading control. (c) Suppression of endogenous PINK1 expression inhibits Parkin phosphorylation. HeLa cells stably expressing non-tagged Parkin were treated with the indicated concentrations of stealth siRNA duplex against PINK1 (Invitrogen) with or without 10 µM CCCP for 1 hr. Long- (LE) and short-exposure (SE) blot signals for PINK1 were shown. Actin was used as a loading control. (d) Truncated PINK1 mutants used in this study. Putative mitochondria-targeting sequence, 1–34 aa; transmembrane domain, 94–110 aa; kinase domain, 156–509 aa. (e) Full-length PINK1 is required for Parkin phosphorylation. PINK1^−/− MEFs stably expressing non-tagged Parkin were transfected with various PINK1 constructs with C-terminal FLAG-tags. PINK1 expression was confirmed with anti-FLAG-HRP. (f) Human fibroblasts from a normal control and a PARK6 case with a homozygous C388R mutation⁴⁴ were transfected with Parkin and were treated with or without 30 µM CCCP for 1 hr. (g) Cells treated with CCCP up to 60 min as in (b) were further incubated with fresh culture medium without CCCP for the indicated periods of time (Washout).