Parkin-catalyzed ubiquitin-ester transfer is triggered by PINK1-dependent phosphorylation

Abstract

PINK1 and PARKIN are causal genes for autosomal recessive familial Parkinsonism. PINK1 is a mitochondrial Ser/Thr kinase, whereas Parkin functions as an E3 ubiquitin ligase. Under steady-state conditions, Parkin localizes to the cytoplasm where its E3 activity is repressed. A decrease in mitochondrial membrane potential triggers Parkin E3 activity and recruits it to depolarized mitochondria for ubiquitylation of mitochondrial substrates. The molecular basis for how the E3 activity of Parkin is re-established by mitochondrial damage has yet to be determined. Here we provide in vitro biochemical evidence for ubiquitin-thioester formation on Cys-431 of recombinant Parkin. We also report that Parkin forms a ubiquitin-ester following a decrease in mitochondrial membrane potential in cells, and that this event is essential for substrate ubiquitylation. Importantly, the Parkin RING2 domain acts as a transthiolation or acyl-transferring domain rather than an E2-recruiting domain. Furthermore, formation of the ubiquitin-ester depends on PINK1 phosphorylation of Parkin Ser-65. A phosphorylation-deficient mutation completely inhibited formation of the Parkin ubiquitin-ester intermediate, whereas phosphorylation mimics, such as Ser to Glu substitution, enabled partial formation of the intermediate irrespective of Ser-65 phosphorylation. We propose that PINK1-dependent phosphorylation of Parkin leads to the ubiquitin-ester transfer reaction of the RING2 domain, and that this is an essential step in Parkin activation.

Keywords: Mitochondria; Parkin; Pink1; RING Finger; Ubiquitin; Ubiquitin Ligase.

FIGURE 1.

A, a higher molecular mass population (indicated by the red asterisk) was specifically observed in the Parkin C431S mutant following CCCP treatment in HeLa cell lysates. B, immunoblotting of the Parkin C431S mutant was repeated in the absence or presence of Myc₁-ubiquitin (Ub) co-expression. The slower migrating band resolved as a doublet because of the endogenous-ubiquitin adduct (indicated by a red arrow) and the Myc₁-ubiquitin adduct (black arrow). C, HeLa cell lysates co-expressing HA-Parkin and Myc₆-ubiquitin were immunoprecipitated with an anti-HA antibody, followed by immunoblotting with the indicated antibodies. The anti-Myc antibody specifically detected the modified Parkin(C431S) mutant. Red asterisk shows Parkin with the endogenous-ubiquitin adduct; blue asterisk shows Parkin with the exogenous Myc₆-ubiquitin adduct; black asterisk indicates the cross-reacting band. D and E, Cys-431 of Parkin is important for substrate ubiquitylation. Both ubiquitylation of a pseudo-substrate (D) and a genuine substrate Mfn2 (E) were inhibited in the transfected cells by C431A and C431S mutations of Parkin. The red asterisks indicate the oxyester-linked ubiquitin and black asterisks indicate ordinary substrate ubiquitylation. F, HeLa cells expressing HA-Parkin mutants harboring the C431S mutation and one of the disease-relevant mutations (K211N, C352G, and T415N) were subjected to immunoblotting following CCCP treatment. The red asterisk indicates the ubiquitin-oxyester band. G, Parkin co-localization with mitochondria was analyzed in >100 cells per Cys-431 mutation. Example figures indicative of robust colocalization (counted as 1) and the absence of colocalization (counted as 0) are shown on the right (bars, 10 μm). Error bars represent the mean ± S.D. values of three experiments. Statistical significance was calculated using Welch's t test; NS, not significant. H, the ubiquitylated form of C431S Parkin (marked by a red asterisk) is sensitive to NaOH treatment, confirming the presence of the oxyester adduct.

FIGURE 2.

Reconstitution of ubiquitin-oxyester formation using recombinant MBP-Parkin protein in vitro. A, schematic depiction of the domain structure of Parkin and the deletion mutant (IBR-RING2). B, in vitro E3 activity of Parkin C431 mutants. MBP-Parkin with the C431A or C431S mutation was purified from E. coli and reconstituted with ATP, ubiquitin, E1, and E2. Ub indicates ubiquitin, the red asterisk indicates the oxyester-linked ubiquitin and the black asterisk indicates conventional isopeptide-linked ubiquitylation, unless otherwise specified. C, E3 activity of the Parkin IBR-RING2 domain ± C431S mutation. D, observed variances in the MBP-Parkin C431S and MBP-IBR-RING2 C431S mutants are the result of ubiquitylation. In vitro ubiquitylation was performed with recombinant HA-ubiquitin and followed by immunoblotting with the indicated antibodies. The anti-HA antibody specifically detected the modified Parkin(C431S) mutants. E, ubiquitin-oxyester formation of MBP-Parkin and MBP-IBR-RING2 with the C431S mutation in the absence of ubiquitin, E1 or E2, or in the presence of all three components. F, ubiquitin-oxyester formation of MBP-IBR-RING2 (C431S) was repeated as E, except at neutral pH conditions (pH 7.0).

FIGURE 3.

A, reaction scheme for in vitro labeling experiments performed in B to E using the active site-directed probe Ub-VS. B, Ub-VS conjugates to Parkin and IBR-RING2 proteins. C and D, the Ub-VS adduct was inhibited by preincubation of IBR-RING2 with NEM (C) or the C431S mutation (D). E, C431S is the lone free cysteine mutation to specifically inhibit Ub-VS conjugation. F, E3 activity of Parkin lacking the Ubl and RING1 domains in cells. HeLa cells expressing GFP-Parkin or GFP-IBR-RING2 with the C431A or T415N mutation were treated with CCCP and subjected to immunoblotting. G, GFP-IBR-RING2 catalyzes autoubiquitylation in cells irrespective of a decrease in ΔΨm. H, cytosolic localization of GFP-IBR-RING2 following CCCP treatment. The mitochondrial localization of GFP-Parkin following CCCP treatment is shown as a control.

FIGURE 4.

A and B, PINK1^−/− MEFs co-expressing C431S Parkin mutant and various pathogenic (A) or autophosphorylation-relevant (B) PINK1 mutants were subjected to immunoblotting using an anti-Parkin antibody for detection of ubiquitin-oxyester formation (upper panel) or an anti-PINK1 antibody to confirm expression of the PINK1 mutants (lower panel). The red asterisks indicate the ubiquitin-oxyester band. FL, full-length PINK1; Δ1 and Δ2, the amino-terminal processed forms as reported in Ref. .

FIGURE 5.

A, exogenous or endogenous Parkin underwent phosphorylation following CCCP treatment. The cell lysate of exogenous Parkin-expressing HeLa cells or intact HEK293T cells ± CCCP treatment were subjected to Phos-tag PAGE. Note that mobility does not reflect the molecular weight of proteins in Phos-tag PAGE (58) and thus molecular weight markers are not shown. The blue asterisks indicate phosphorylated Parkin, whereas the arrows indicate the cross-reacting band unless otherwise specified. B, protein phosphatase treatment in cell lysates caused the high-molecular shift of endogenous Parkin to disappear. C, phosphorylation of Parkin following CCCP treatment in cells depends on PINK1. Parkin was not phosphorylated in PINK1^−/− MEFs following CCCP treatment, whereas exogenous PINK1 complemented the aforementioned defect. D, phosphorylation of Parkin was severely compromised by various pathogenic mutations of PINK1. Parkin C431S mutant-expressing PINK1^−/− MEFs complemented by disease-relevant PINK1 were treated with CCCP and subjected to immunoblotting. FL, full-length PINK1; Δ1 and Δ2, the amino-terminal processed form as described in the legend to Fig. 4A. E, intact HEK293T cells ± CCCP treatment were subjected to fractionation experiments. Cyt and Mt indicate the cytosol-rich supernatant and the mitochondria-rich membrane pellet, respectively. Arrowheads indicate endogenous Parkin.

FIGURE 6.

Parkin is phosphorylated at Ser-65 following CCCP treatment. A, Ser to Asp substitution at various putative phosphorylation sites did not alter the phosphorylation pattern of Parkin in HeLa cells upon dissipation of ΔΨm. Blue asterisks indicate phosphorylated Parkin unless otherwise specified. Note that mobility does not reflect the molecular weight in Phos-tag PAGE (58) and thus molecular weight markers are not shown. B, the S65A, S65D, and S65E mutations dramatically decreased the phosphorylation of Parkin in cells. C, mass spectrometric analysis of the in cell phosphorylation site of Parkin. GST-Parkin purified from cells ± CCCP treatment was subjected to LC-MS/MS analysis. A phosphorylated peptide equivalent to amino acids 52–75 was detected only in CCCP-treated cells. D, the MS/MS data suggest that phosphorylation occurs at Ser-65.

FIGURE 7.

A, the S65A/S65D/S65E mutations decreased the phosphorylation of ubiquitin ester-stabilized Parkin C431S mutants in cells. Blue asterisks indicate phosphorylated Parkin, unless otherwise specified. B, straight immunoblotting of HeLa cell lysates expressing Parkin harboring double mutations (i.e. C431S with the S65A, S65D, or S65E mutation). The red asterisks indicate ubiquitin-oxyester formation unless otherwise specified. The S65A/C431S Parkin mutant disturbed the ubiquitin-ester formation completely, whereas the S65D/C431S and S65E/C431S mutants partially complemented ubiquitin-ester formation. C, immunoprecipitation with an anti-HA antibody followed by immunoblotting with the indicated antibodies confirmed that Myc₆-ubiquitin is conjugated to the Parkin C431S, S65D/C431S, and S65E/C431S mutants. Red asterisks show Parkin with the Myc₆-ubiquitin adduct. D and E, the Parkin S65T or S65T/C431S mutants underwent phosphorylation (D) and exerted ubiquitin-oxyester formation (E) equivalent to WT or C431S mutant in cells. F, the number of HeLa cells with Parkin localized to the mitochondria per C431S/S65X mutation following CCCP treatment for 1 h was counted in >100 cells as Fig. 1G. Error bars represent the mean ± S.D. values and statistical significance was calculated using Welch's t test.

FIGURE 8.

A, mitochondria collected from cells ± CCCP pretreatment were incubated at 30 °C with cytosol expressing GFP-Parkin collected from cells with intact mitochondria. In this cell-free ubiquitylation assay, CCCP-treated mitochondria stimulate autoubiquitylation of GFP-Parkin and substrate ubiquitylation toward Mfn2. B, activation of GFP-Parkin by CCCP-pretreated mitochondria depends on PINK1. Mitochondria collected from PINK1^−/− MEFs following CCCP treatment do not activate GFP-Parkin in the cell-free ubiquitylation assay, whereas exogenous PINK1 complements the aforementioned defect. C, formation of an apparent ubiquitin-oxyester adduct on the Parkin C431S mutant is dependent on the presence of CCCP-pretreated mitochondria in the cell-free assay. The red asterisks indicate ubiquitin-oxyester formation unless otherwise specified. D, ubiquitin-oxyester formation of Parkin harboring C431S, C431S/S65A, or C431S/S65E mutations in the cell-free ubiquitylation experiments. The S65A mutation disrupted ubiquitin-ester formation completely, whereas the C431S/S65E mutant has weak but detectable ubiquitin-oxyester formation. E, a model for Parkin activation on damaged mitochondria. See text for details.