PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy - PubMed (original) (raw)

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PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy

Kahori Shiba-Fukushima et al. Sci Rep. 2012.

Abstract

Parkinson's disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (ΔΨm), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism - an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ΔΨm. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.

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Figures

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 mutation 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).

Figure 2. Ser65 in the Ubl domain of Parkin is phosphorylated upon depolarisation of ΔΨm.

(a) Phos-tag Western blotting detected phosphorylation of Ser65. HeLa cells were transiently transfected with Parkin WT and a series of alanine mutants for the candidate phospho-residues followed by treatment with or without 20 µM CCCP for 1 hr. Cell lysates were analysed by Phos-tag Western blotting. An asterisk indicates degraded Parkin. (b) Alignment of the amino acid sequences surrounding Ser65 (marked by a black dot) from a variety of animal species. The numbers on the left correspond to the residue numbers of Parkin proteins. (c) Introduction of the S65A mutation delayed Parkin translocation to the depolarised mitochondria in PINK1 WT/GFP-Parkin/PINK1−/− MEFs. Cells retrovirally introduced with GFP-Parkin WT or its phospho-mutants (S65A and S65E) were treated with or without 30 µM CCCP for the indicated periods of time. GFP-Parkin and mitochondria were visualised with anti-GFP (green) and anti-Tom20 (red), respectively. Parkin signals are also shown as monochrome images. Scale bar = 10 µm. (d) Mitochondrial translocation efficiency of Parkin mutants. PINK1 WT/PINK1−/− MEFs stably expressing GFP-Parkin WT, S65A or S65E were treated as in (c). Cells expressing GFP-Parkin perfectly overlapped (Complete, examples are shown on the right), partially overlapped (Partial) or non-overlapped (No) with the Tom20 signal were counted. The data represent means ± SE from three experiments (n = 99–143 cells in each). ** p < 0.01, * p < 0.05 vs. WT at each time point.

Figure 3. Pathogenic mutants of Parkin are subjected to Ser65 phosphorylation.

(a) Diagram of Parkin protein illustrating the pathogenic mutants used in this study. The Ser65 residue in the Ubl domain is shown as a yellow circle. RING, Ring-finger motif; IBR, in-between-Ring fingers domain. (b) Phos-tag Western blotting for Parkin and Western blotting for PINK1 were performed using Parkin WT and a series of pathogenic mutants as shown in Figure 2a. (c) Endogenous Parkin was also phosphorylated in SH-SY5Y cells after CCCP treatment. Post-nuclear cell lysates from SH-SY5Y cells treated with or without 10 µM CCCP for 30 and 60 min were fractionated into mitochondria-rich (Mito) and cytosolic (Cyto) fractions. These two fractions and their combination (Mito + Cyto) were subjected to Phos-tag or normal Western blotting analyses. Endogenous PINK1 was fractionated in the Mito fraction, as previously reported. Lactate dehydrogenase (LDH) and Tom20 were used as cytosolic and mitochondrial marker proteins, respectively. Asterisks: putative cleaved Parkin; dots: non-specific bands.

Figure 4. Ser65 phosphorylation affects the subsequent autophagy reaction.

(a) CCCP-dependent degradation of mitochondrial outer membrane proteins in PINK1 WT/PINK1−/− MEFs expressing WT or mutant forms of GFP-Parkin. Mfn1, VDAC1 and Tom20 were used as markers of mitochondrial outer membrane proteins. Actin: a loading control. Dots: ubiquitinated Mfn1. (b) Long-term time-course analysis of CCCP-dependent mitochondrial protein degradation. The degradation of outer membrane proteins was impaired in cells expressing GFP-Parkin S65A or S65E mutations. Hsp60 was used as a marker of mitochondrial matrix proteins. (c) S65A and S65E mutations do not affect proteasome recruitment to the mitochondria during mitophagy. PINK1 WT/PINK1−/− MEFs expressing WT or mutant forms of GFP-Parkin (green) were treated with 30 µM CCCP for 3 or 6 hr. Cells were stained with anti-proteasome subunit alpha type 7 (α7, red). α7-immunoreactivity was enriched in the nuclei of all three cell genotypes under normal conditions, as displayed in the representative image of S65E (CCCP 0 hr), and overlapped with the aggregated mitochondria (arrowheads) 6 hr after CCCP treatment irrespective of genotype. Similar results were obtained 3 hr after CCCP treatment. Scale bar = 10 µm. (d) Model for Parkin translocation and activation. The Parkin Ubl domain masks C-terminal RING-IBR-RING (RBR) domains for E3 activity. A Parkin phosphorylation event at Ser65 (P), combined with unknown factor(s) (?), stimulates the mitochondrial translocation of Parkin, releasing the RBR domains from autoinhibition by the Ubl domain.

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PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy - PubMed (original) (raw)