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. 2014 Apr 28;205(2):143-53.
doi: 10.1083/jcb.201402104. Epub 2014 Apr 21.

PINK1 Phosphorylates Ubiquitin to Activate Parkin E3 Ubiquitin Ligase Activity

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Free PMC article

PINK1 Phosphorylates Ubiquitin to Activate Parkin E3 Ubiquitin Ligase Activity

Lesley A Kane et al. J Cell Biol. .
Free PMC article

Abstract

PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.

Figures

Figure 1.
Figure 1.
Mutation of conserved serine/threonine residues of Parkin does not completely inhibit Parkin translocation or activity. (A) YFP-Parkin is normally cytosolic (left panels), but upon mitochondrial damage (10 µM CCCP for 2.5 h), YFP-Parkin translocates to mitochondria and causes the ubiquitination proteins (right panels). Cells were stained for Tom20 (mitochondria, blue) and Ub (red). (B) ParkinΔUBL, as well as alanine mutants of Ser/Thr residues previously reported to be phosphorylated, were all capable of translocating to damaged mitochondria (10 µM CCCP for 2.5 h). Fewer cells expressing ParkinS65A displayed mitochondrial translocation than any other mutant (see Table S1). Cells were stained for Parkin (green) and Tom20 (mitochondria, red). For quantification and references of observed phosphorylation, see Table S1. (C) CCCP-treated (10 µM CCCP for 3 h) PINK1 KO cells expressing the indicated Parkin mutants showed that Parkin translocation is PINK1 dependent. (D) Phos-tag and SDS-PAGE gels revealed a shift of WT Parkin on Phos-tag gels after CCCP treatment (arrow), indicating it is phosphorylated (lane 2 vs. lane 3), and this phosphorylation was removed by phosphatase (CIP, lane 1). ParkinΔUBL, S65A, and S65E displayed no observable shift. (E) Parkin in vitro ubiquitination assay revealed that Parkin mutants are capable of ubiquitinating Mfn1 (bottom arrow). Cytosolic extracts from cells expressing the indicated Parkin mutants were incubated with mitochondria from cells not expressing Parkin (±CCCP). Ubiquitination of Mfn1 was observed (arrow + Ub and polyUb) only in the presence of mitochondria from CCCP-treated cells. Bars: (A–C) 10 µm.
Figure 2.
Figure 2.
Proteomics search for PINK1 substrates revealed that PINK1 phosphorylates ubiquitin. (A) Workflow of the sample preparation for the PINK1 substrate search. (B) Identification of Ub in mitochondria from CCCP-treated PINK1 WT or KO cells. (C) Extracted ion chromatogram of the m/z 737.38 form of the Ub phosphopeptide TLSDYNIQKEpSTLHLVLR from endogenous PINK1 WT (red) and KO (blue) samples as prepared in A. (D) Extracted ion chromatograms of the Ub phosphopeptide TLSDYNIQKEpSTLHLVLR as observed with m/z 553.29, 737.38, and 1105.57 for the in vitro–phosphorylated His-Ub incubated with either CCCP-treated (red) or untreated (blue) mitochondria. Insets in C and D represent zoomed sections surrounding the phosphopeptide peak (asterisks). (E and F) Representative spectra for the Ub phosphopeptide TLSDYNIQKEpSTLHLVLR from in vitro–phosphorylated His-Ub from electron-transfer dissociation (panel E, ETD) and higher-energy collisional dissociation (panel F, HCD) fragmentation methods. ETD and HCD spectra obtained for this peptide from PINK1 WT mitochondria samples prepared as in A were almost identical to the spectra shown in E and F. (G) Alignment of Ub and the UBL domain of Parkin with the conserved S65 (bold) and unique Ub peptide observed by MS (red). Bold-type pS, phospho-Ser.
Figure 3.
Figure 3.
Phos-tag gels and 32P radiolabeling of in vitro phosphorylation of ubiquitin by PINK1. (A) Recombinant MBP-TcPINK1 WT, but not MBP-TcPINK1 KD, causes HA-Ub to shift on Phos-tag gels (top, lanes 7–10, phospho-Ub) but not SDS-PAGE (bottom). This shifted band is removed by phosphatase treatment (+CIP, lane 11). MBP-TcPINK1 WT is also shifted on Phos-tag gels, indicating auto-phosphorylation. (B) Radiolabeled phosphate was incorporated into recombinant Ub during incubation with γ-[32P]ATP and WT (but not KD) MBP-TcPINK1.
Figure 4.
Figure 4.
Phospho-ubiquitin activates Parkin. (A) YFP-ParkinWT translocates to damaged mitochondria in cells expressing high levels of WT HA-Ub (red, HA immunostaining, 10 µM CCCP for 1 h). Cells expressing high levels of HA-UbS65A have impaired YFP-Parkin translocation. Bars, 10 µm. (B) Quantification of A shown as averages ± SD from n = 3 experiments, 100 cells/experiment. (C) Immunoprecipitation of HA-Ub WT, S65D, S65E, and S65A from cells stably expressing YFP-Parkin revealed a stronger interaction between activated YFP-Parkin and HA-UbS65D and HA-UbS65E than WT or S65A. In the absence of HA-Ub, some YFP-Parkin bound to the HA IP beads (†), and this was consistent in all immunoprecipitations. The higher molecular weight form of YFP-Parkin was found only in the HA-Ub and was bound to a much larger extent to the HA-UbS65D and S65E. (D) Quantification of C shown as averages ± SD from n = 3 experiments. (E) ParkinC431S forms an oxyester-bound Ub intermediate upon activation with CCCP, which is cleaved by NaOH (lanes 1 and 2). The oxyester was not detected in cells without CCCP, with overexpressing HA-UbWT (lane 9), but was detected with overexpression of HA-UbS65D for both YFP-ParkinC431S and YFP-ParkinS65A/C431S (lanes 13 and 15). (F and G) HA-Ub was in vitro phosphorylated by (F) incubation with mitochondria from control or CCCP treated cells or (G) recombinant MBP-TcPINK1 WT or KD using the same protocol as used for the Phos-tag gels in Fig. 3 A. This HA-Ub was then added to an in vitro reaction (at 4 ng and 20 ng) with recombinant Parkin, E1, E2, untreated Ub (to a total of 1 µg), and ATP. In both cases the addition of phospho-Ub to the reaction caused an increase in Parkin activity (Ubn). (H) To ensure activation in F was due to S65 phospho-Ub, the experiment was repeated with WT, S65A, and no Ub (−). Activation was only observed with WT His-Ub (top). MBP-TcPINK1 was removed from the His-Ub phosphorylation reaction by binding to amylose beads (bottom). The “†” symbol in G and H represents a nonspecific band. *, P < 0.01; ***, P < 0.001.
Figure 5.
Figure 5.
A model of PINK1 phosphorylation of ubiquitin to activate Parkin and the conservation of S65 among UBL proteins and domains. (A) A model of the cyclical activation of Parkin by PINK1 phosphorylation and amplification of the cascade via PINK1 phosphorylation of ubiquitin. (B) Alignment of the ubiquitin-like domains of several small ubiquitin-like proteins shows several conserved serines at the sites homologous to ubiquitinS65. (C) Alignment of other UBL domain–containing proteins with Parkin’s UBL revealed that other proteins contain a homologous serine or threonine at the S65 position. Arrow indicates the position of Ub S65 in UBL proteins (B) and UBL domain–containing proteins (C).

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