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Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity*

  • Author Footnotes
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
    Su Jin Ham
    Footnotes
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
    Affiliations
    From the Interdisciplinary Graduate Program in Genetic Engineering,

    National Creative Research Initiatives Center for Energy Homeostasis Regulation,

    Institute of Molecular Biology and Genetics, and
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  • Soo Young Lee
    Affiliations
    National Creative Research Initiatives Center for Energy Homeostasis Regulation,

    Institute of Molecular Biology and Genetics, and
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  • Saera Song
    Affiliations
    National Creative Research Initiatives Center for Energy Homeostasis Regulation,

    Institute of Molecular Biology and Genetics, and
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  • Author Footnotes
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
    Ju-Ryung Chung
    Footnotes
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
    Affiliations
    School of Biological Sciences, Seoul National University, Seoul 51-742, Republic of Korea
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  • Sekyu Choi
    Affiliations
    National Creative Research Initiatives Center for Energy Homeostasis Regulation,

    Institute of Molecular Biology and Genetics, and
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  • Author Footnotes
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
    Jongkyeong Chung
    Correspondence
    To whom correspondence should be addressed: School of Biological Sciences, Seoul National University, 1 Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Republic of Korea. Tel.: 82-2-880-4399; Fax: 82-2-876-4401;.
    Footnotes
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
    Affiliations
    From the Interdisciplinary Graduate Program in Genetic Engineering,

    National Creative Research Initiatives Center for Energy Homeostasis Regulation,

    Institute of Molecular Biology and Genetics, and

    School of Biological Sciences, Seoul National University, Seoul 51-742, Republic of Korea
    Search for articles by this author
  • Author Footnotes
    * Supported by the National Creative Research Initiatives grant through the National Research Foundation of Korea (NRF) funded by Ministry of Science, ICT, and Future Planning (MSIP), the Republic of Korea (2010-0018291). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Supported by BK21 Plus Program from Ministry of Education, Republic of Korea.
Open AccessPublished:December 02, 2015DOI:https://doi.org/10.1074/jbc.M115.687319
      Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.

      Introduction

      Parkinson disease (PD)
      The abbreviations used are: PD, Parkinson disease; UBL, ubiquitin-like; PINK1, PTEN-induced putative kinase 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazine; RING1, really interesting new gene 1; IBR, in-between-ring; R2, RING2, really interesting new gene 2; Mfn, mitofusin; Drp1, dynamin-related protein 1; VDAC1, voltage-dependent anion channel protein 1; TOM20, translocase of outer membrane 20; USP, ubiquitin-specific protease; FL, full-length; co-IP, co-immunoprecipitation.
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      • Sixma T.K.
      The DUSP-Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange.
      ). Heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1) is an E3 ligase that contains the UBL domain. The UBL domain of HOIL-1 interacts with the 26S proteasome to promote degradation of its substrates by the ubiquitin-proteasome system (
      • Beasley S.A.
      • Safadi S.S.
      • Barber K.R.
      • Shaw G.S.
      Solution structure of the E3 ligase HOIL-1 Ubl domain.
      ). The deletion of the UBL domain in Parkin also enhances Parkin autoubiquitination activity (
      • Chaugule V.K.
      • Burchell L.
      • Barber K.R.
      • Sidhu A.
      • Leslie S.J.
      • Shaw G.S.
      • Walden H.
      Autoregulation of Parkin activity through its ubiquitin-like domain.
      ). Furthermore, an x-ray crystal structure of Parkin revealed that the UBL domain of Parkin binds to its C-terminal catalytic region to block association with E2 (
      • Trempe J.F.
      • Sauvé V.
      • Grenier K.
      • Seirafi M.
      • Tang M.Y.
      • Ménade M.
      • Al-Abdul-Wahid S.
      • Krett J.
      • Wong K.
      • Kozlov G.
      • Nagar B.
      • Fon E.A.
      • Gehring K.
      Structure of parkin reveals mechanisms for ubiquitin ligase activation.
      ). These findings raised the possibility that the UBL domain is critical for regulation of Parkin activation.
      Here, we investigate the molecular mechanism that underlies the function of the R1 and UBL domains of Parkin. We found that the UBL domain of Parkin suppresses Parkin autoubiquitination, substrate ubiquitination, mitochondria translocation, and mitophagy via interaction with the R1 domain of the E3 ligase. We also showed that the interaction between the R1 domain and the UBL domain is diminished when the UBL domain was phosphorylated at Ser-65 by PINK1. Furthermore, we showed that Parkin activity is regulated by competitive interaction of the R1 domain with the UBL domain and Parkin substrates such as VDAC1. Consistent with these in vitro data, Parkin mutations in the UBL domain of PD patients affected the interaction between the R1 domain and the UBL domain. Together, these results suggest that the interaction between the R1 domain and the UBL domain is critical for proper regulation of Parkin functions both in vitro and in vivo.

      Experimental Procedures

      Antibodies and Reagents

      Rabbit anti-HA (Cell Signaling Technology), mouse anti-GST (Upstate Biotechnology), rabbit anti-GFP (Santa Cruz), mouse or rabbit anti-Myc (Cell Signaling Technology), rabbit anti-ubiquitin (Cell Signaling Technology), mouse anti-FLAG (Medical and Biological Laboratories), mouse anti-MFN2 (Millipore), mouse anti-MFN1 (Abcam), mouse anti-VDAC1 (Santa Cruz), mouse anti-NDUFS3 (Abcam), rabbit anti-COIV4 (Abcam), mouse anti-TIM23 (BD Biosciences), and mouse anti-β-tubulin (Developmental Studies Hybridoma Bank) antibody were used for immunoblot analyses. Rabbit anti-TOM20 (Santa Cruz) mouse anti-GST (Upstate Biotechnology) antibody, rabbit anti-SQSTM1/p62 (Cell Signaling), and rabbit anti-LC3B antibody (Cell Signaling) were used for immunocytochemistry. CCCP was purchased from Calbiochem. Glutathione-Sepharose 4B beads (GE Healthcare) were used for GST pulldown assays.

      Plasmids

      The N-terminal GST-tagged pEBG vector was used to generate truncated Parkin constructs. The N-terminal GFP-tagged pGFPC1 vector was used to generate the Parkin UBL WT domain and its point mutant constructs (S65A and S65D). For site-directed mutagenesis, the QuikChangeTM kit (Stratagene) was used. Human PINK1 WT 3×Myc, kinase-dead human PINK1 K219A, D362A, and D384A 3×Myc, N-terminal HA-tagged human VDAC1, and N-terminal FLAG-tagged human Parkin were generated using the pcDNA3.1 zeo (+) vector. The pRK5 vector was used to express N-terminal HA-tagged human ubiquitin.

      Cell Culture and Transfection

      HEK293T and HeLa cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. HeLa cells stably expressing GFP-Parkin (
      • Okatsu K.
      • Uno M.
      • Koyano F.
      • Go E.
      • Kimura M.
      • Oka T.
      • Tanaka K.
      • Matsuda N.
      A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment.
      ) were cultured in advanced DMEM (Invitrogen) with 10% fetal bovine serum (Invitrogen), 5 μg/ml puromycin (Invitrogen), 200 mm l-glutamine (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. Expression plasmids were transfected using Lipofectamine Plus Reagent (Invitrogen) or polyethyleneimine (Sigma) according to the manufacturer's instructions.

      Cell Lysis and Immunoblotting

      Cells were prepared in lysis buffer A (25 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 50 mm NaF, 1 mm sodium vanadate, 2 mm DTT, 1 mm PMSF, 10 g/ml leupeptin, 1 g/ml pepstatin A, and 0.1% Nonidet P-40) for GST pulldown assays. Mitochondrial protein immunoblot analyses were performed using radioimmune precipitation assay buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 2 mm DTT, 1 mm PMSF, 10 g/ml leupeptin, and 1 g/ml pepstatin A). Lysis buffers were supplemented with the protease inhibitor mixture (Roche Applied Science). Total protein was quantified using the BCA protein assay kit (Pierce). Lysates were subjected to SDS-PAGE analysis followed by immunoblotting according to standard procedures. The blots were developed and visualized using LAS-4000 (Fujifilm).

      Ubiquitination Assay

      Cells were lysed with Lysis buffer B (2% SDS, 150 mm NaCl, and 10 mm Tris, pH 8.0) with the protease inhibitor mixture (Roche Applied Science). Transfected cells were harvested and boiled for 10 min at 95 °C. Lysis buffer A and lysis buffer B were mixed at a 10:1 ratio. Samples were incubated at 4 °C for 1 h and centrifuged at 16,000 × g for 20 min. Glutathione-Sepharose 4B beads were added to the supernatant of cell lysates and incubated with rotation at 4 °C for 60 min. Beads were washed four times with lysis buffer A without Nonidet P-40. Laemmli 2× sample buffer was added, and samples were boiled for 10 min at 95 °C and subjected to SDS-PAGE analysis.

      Immunofluorescence

      HeLa cells were subcultured on coverslips in a 12-well tissue culture plate. Cells were treated with 20 μm CCCP or DMSO for 4 h, washed once with PBS, fixed in 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 in PBS for 5 min. For SQSTM1/p62 and LC3B staining, cells were permeabilized with ice-cold 100% methanol for 20 min. Then the cells were washed with 0.1% Triton X-100 in PBS (PBS-T) and incubated in blocking solution (4% BSA and 1% normal goat serum in PBS-T) for 1 h. Primary antibodies were added to the blocking solution, and the cells were incubated overnight at 4 °C. After washing with PBS-T four times, cells were incubated with appropriate secondary antibodies in blocking solution for 1 h at room temperature. The antibody-labeled cells were washed with PBS-T six times and mounted with mounting solution (100 mg/ml 1,4-diazabicyclo[2.2.2] octane (DABCO) in 90% glycerol). The slides were observed with a LSM710 laser scanning confocal microscope (Carl Zeiss). All of the immunostaining experiments with HeLa cells were conducted at least three times.

      Statistical Analysis

      The statistical analyses were performed using analysis of variance Tukey's tests or unpaired t tests. The p values were calculated from three independent experiments.

      Results

      Parkin UBL Domain Interacts with the R1 Domain

      Recently, the UBL domain of Parkin has been linked to the autoinhibitory function of Parkin (
      • Chaugule V.K.
      • Burchell L.
      • Barber K.R.
      • Sidhu A.
      • Leslie S.J.
      • Shaw G.S.
      • Walden H.
      Autoregulation of Parkin activity through its ubiquitin-like domain.
      ). However, the mechanism of how the UBL domain and other domains regulate Parkin activation is not known. To identify the regions of Parkin that contribute to the autoregulatory activity of Parkin, we generated GST-tagged truncation mutants of Parkin (Fig. 1A), overexpressed the constructs in HEK293T cells expressing HA-tagged human ubiquitin, and tested their function in vitro using an autoubiquitination assay. Compared with full-length (FL) Parkin, autoubiquitination activity was increased by Parkin mutants that lack the UBL domain, namely by the UBL domain deletion mutant (ΔUBL) or by the C-terminal RING motif, R1-IBR-R2 (C) (Fig. 1B). To determine whether the UBL domain can suppress the function of Parkin, we tested whether overexpression of the UBL domain with the truncation mutants could suppress their autoubiquitination activity. Strikingly, the autoubiquitination activity of ΔUBL or C was noticeably lower with overexpression of the UBL domain (Fig. 1C), indicating that Parkin autoubiquitination is negatively regulated by the UBL domain, in agreement with a previous report (
      • Chaugule V.K.
      • Burchell L.
      • Barber K.R.
      • Sidhu A.
      • Leslie S.J.
      • Shaw G.S.
      • Walden H.
      Autoregulation of Parkin activity through its ubiquitin-like domain.
      ).
      Figure thumbnail gr1
      FIGURE 1.Parkin UBL domain interacts with the R1 domain. A, schematic representation of the domains of human Parkin protein. FL Parkin and truncated Parkin constructs used for this study are shown, including ΔUBL (deletion of the UBL domain), C (R1-IBR-R2), UBL, Linker, R1, IBR-R2, and R2. All Parkin plasmids were generated in the pEBG vector with an N-terminal GST tag. B and C, autoubiquitination assays of Parkin in HEK293T cells. B, lysates from cells transfected with GST-tagged Parkin FL, ΔUBL, or C constructs and HA-tagged ubiquitin (Ub) were prepared. Samples from GST pulldown were analyzed by immunoblot with anti-HA antibody (top panel) or anti-GST antibody (middle panel), and whole cell lysates (WCL) were detected with anti-HA antibody (bottom panel). C, GFP-tagged UBL domain was co-expressed with GST-tagged Parkin FL, ΔUBL, or C constructs and HA-tagged Ub in HEK293T cells. Samples were subjected to GST pulldown as in B and analyzed by immunoblot with anti-Ub antibody (first panel) or anti-GST antibody (second panel), and whole cell lysates were detected with anti-GFP antibody (third panel) and anti-HA antibody (fourth panel). D and E, interaction between the UBL domain and various Parkin deletion mutants by GST pulldown assay. D, GST-tagged Parkin constructs and GFP-tagged UBL construct were co-expressed in HEK293T cells. Cell lysates were subjected to GST pulldown. GST pulldown samples (top and middle panels) and whole cell lysates samples (bottom panel) were immunoblotted with anti-GFP antibody and anti-GST antibody. E, GST-tagged R1 and GFP-tagged UBL were transfected in HEK293T cells. GST pulldown samples were analyzed by immunoblot with anti-GFP antibody (top panel) or anti-GST antibody (middle panel) as indicated. Whole cell lysates were used to detect the same level expression of GFP-tagged UBL domain (bottom panel). F, interaction between the R1 domain and the UBL domain was shown by GST pulldown assay. GST-tagged WT or C238S mutant R1 domain and GFP-tagged UBL domain were expressed in HEK 293T cells. GST pulldown samples were analyzed by immunoblot with anti-GFP antibody (top panel) or anti-GST antibody (middle panel) as indicated. WCL were used to detect the expression levels of GFP-tagged UBL domain (bottom panel).
      We hypothesized that the autoubiquitination activity of Parkin could be suppressed by direct interaction of the UBL domain with the domains in the C terminus of Parkin. To test this hypothesis, we performed co-immunoprecipitation (co-IP) of the GFP-tagged UBL domain with the GST-fused C-terminal regions of Parkin. We confirmed a direct interaction between the UBL domain and C (Fig. 1D) and, moreover, found that the R1 domain is sufficient for this interaction (Fig. 1E). From these data, we concluded that the UBL domain binds to the R1 domain, through which E2 enzymes interact with Parkin substrates (
      • Riley B.E.
      • Lougheed J.C.
      • Callaway K.
      • Velasquez M.
      • Brecht E.
      • Nguyen L.
      • Shaler T.
      • Walker D.
      • Yang Y.
      • Regnstrom K.
      • Diep L.
      • Zhang Z.
      • Chiou S.
      • Bova M.
      • Artis D.R.
      • Yao N.
      • Baker J.
      • Yednock T.
      • Johnston J.A.
      Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.
      ,
      • Trempe J.F.
      • Sauvé V.
      • Grenier K.
      • Seirafi M.
      • Tang M.Y.
      • Ménade M.
      • Al-Abdul-Wahid S.
      • Krett J.
      • Wong K.
      • Kozlov G.
      • Nagar B.
      • Fon E.A.
      • Gehring K.
      Structure of parkin reveals mechanisms for ubiquitin ligase activation.
      ) to negatively regulate Parkin activity.
      Conserved cysteine residues in the R1 domain, including Cys-238, are important for maintaining the structure of the RING-finger motif of the R1 domain (
      • Trempe J.F.
      • Sauvé V.
      • Grenier K.
      • Seirafi M.
      • Tang M.Y.
      • Ménade M.
      • Al-Abdul-Wahid S.
      • Krett J.
      • Wong K.
      • Kozlov G.
      • Nagar B.
      • Fon E.A.
      • Gehring K.
      Structure of parkin reveals mechanisms for ubiquitin ligase activation.
      ,
      • Ozawa K.
      • Komatsubara A.T.
      • Nishimura Y.
      • Sawada T.
      • Kawafune H.
      • Tsumoto H.
      • Tsuji Y.
      • Zhao J.
      • Kyotani Y.
      • Tanaka T.
      • Takahashi R.
      • Yoshizumi M.
      S-nitrosylation regulates mitochondrial quality control via activation of parkin.
      ). Mutation of cysteine 238 to serine (C238S) prevented binding of the UBL domain to the R1 domain (Fig. 1F). This result indicated that the proper folding of the R1 domain is critical for the interaction between the R1 domain and the UBL domain.

      PINK1 Negatively Regulates the Interaction between the UBL and the R1 Domain

      PINK1 is an upstream kinase of Parkin that enhances Parkin activity by phosphorylation of the UBL domain (
      • Kondapalli C.
      • Kazlauskaite A.
      • Zhang N.
      • Woodroof H.I.
      • Campbell D.G.
      • Gourlay R.
      • Burchell L.
      • Walden H.
      • Macartney T.J.
      • Deak M.
      • Knebel A.
      • Alessi D.R.
      • Muqit M.M.
      PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.
      ,
      • Shiba-Fukushima K.
      • Imai Y.
      • Yoshida S.
      • Ishihama Y.
      • Kanao T.
      • Sato S.
      • Hattori N.
      PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy.
      • Kazlauskaite A.
      • Kelly V.
      • Johnson C.
      • Baillie C.
      • Hastie C.J.
      • Peggie M.
      • Macartney T.
      • Woodroof H.I.
      • Alessi D.R.
      • Pedrioli P.G.
      • Muqit M.M.
      Phosphorylation of Parkin at serine 65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity.
      ). We wanted to determine whether PINK1 regulates the binding between the UBL domain and the R1 domain of Parkin. GST pulldown assays were performed in HEK293T cells overexpressing the GST-tagged Parkin R1 domain and C with the GFP-tagged UBL domain. In CCCP-treated cells (20 μm), the interaction between the UBL domain and C and between the UBL domain and the R1 domain were reduced (Fig. 2, A and B, respectively). To test whether the effect of CCCP treatment is PINK1-dependent, we performed co-IP of the R1 domain and the UBL domain in HEK293T cells overexpressing WT PINK1 or a kinase-dead PINK1 mutant (K219A/D362A/D384A). With CCCP treatment, the UBL domain showed a strong interaction with the R1 domain in cells expressing PINK1 kinase-dead mutant at a level comparable with that in cells with no exogenous PINK1 control, whereas the interaction was weak in WT PINK1-expressing cells (Fig. 2C). Together, these results suggested that PINK1 interferes with the interaction between the UBL domain and the R1 domain of Parkin.
      Figure thumbnail gr2
      FIGURE 2.Interaction between the R1 domain and the UBL domain is regulated in a PINK1-dependent manner. A–E, HEK293T cells were transfected with GST-tagged truncated Parkin constructs and GFP-tagged UBL constructs as indicated. Cell lysates were subjected to GST pulldown as described under “Experimental Procedures.” A and B, GST-tagged R1 domain or GST-tagged C (R1-IBR-R2) construct was co-transfected with GFP-tagged UBL construct in HEK293T. Cells were treated with 20 μm CCCP for 4 h. Cell lysates were subjected to GST pulldown and immunoblotted with anti-GFP antibody (top panel) or anti-GST antibody (middle panel). Whole cell lysates (WCL) were immunoblotted with anti-GFP antibody (bottom panel). C, the C terminus 3×Myc-tagged PINK1 WT or kinase dead mutant (K219A/D362A/D384A (3KD)) was co-expressed with GST-tagged R1 and GFP-tagged UBL domain in HEK293T as indicated. GST pulldown samples and whole cell lysates were analyzed by immunoblot with anti-GFP antibody (first and third panels), anti-GST antibody (second panel), or anti-Myc antibody (fourth panel). D, GFP-tagged UBL WT, S65A, or S65D domain was co-expressed with GST-tagged R1 domain. GST pulldown samples and whole cell lysates were immunoblotted with anti-GFP antibody (top and bottom panels) and anti-GST antibody (middle panel). E, GFP-tagged S65A mutant UBL domain was co-expressed with GST-tagged R1 domain. Where indicated, cells were treated with 20 μm CCCP for 4 h. GST pulldown samples were immunoblotted anti-GFP antibody (top panel) and anti-GST antibody (middle panel). Whole cell lysates were immunoblotted with anti-GFP antibody (bottom panel).
      Because PINK1 activates Parkin by phosphorylation at Ser-65 of the UBL domain (
      • Kondapalli C.
      • Kazlauskaite A.
      • Zhang N.
      • Woodroof H.I.
      • Campbell D.G.
      • Gourlay R.
      • Burchell L.
      • Walden H.
      • Macartney T.J.
      • Deak M.
      • Knebel A.
      • Alessi D.R.
      • Muqit M.M.
      PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.
      ), we tested whether the phosphorylation contributes to the binding between the UBL domain and the R1 domain. To do this we performed co-IP of the R1 domain with the WT UBL domain or derivatives of the UBL domain in which Ser-65 was mutated to alanine or glutamate.
      The interaction between the UBL phosphomimetic S65D mutant and the R1 domain was very weak, whereas the interaction between the nonphosphorylatable S65A mutant and the R1 domain was at a level comparable with to WT (Fig. 2D). Furthermore, the interaction between the R1 domain and the UBL S65A mutant did not show significant differences upon treatment with CCCP (Fig. 2E). Thus, we concluded that the interaction between the R1 domain and the UBL domain is regulated by phosphorylation of the UBL domain at Ser-65.

      The UBL Domain Negatively Regulates Parkin Activity

      To determine whether the weakened interaction between the UBL domain and the R1 domain by phosphorylation at Ser-65 has an effect on Parkin activation, we performed an autoubiquitination assay using FL WT Parkin or FL Parkin with S65A or S65D mutation. FL Parkin with S65D mutation showed stronger autoubiquitination activity compared with FL WT Parkin or FL Parkin with S65A mutation (Fig. 3A), indicating that phosphorylation of Parkin at Ser-65 promotes its E3 ligase activity, in agreement with previous studies (
      • Kondapalli C.
      • Kazlauskaite A.
      • Zhang N.
      • Woodroof H.I.
      • Campbell D.G.
      • Gourlay R.
      • Burchell L.
      • Walden H.
      • Macartney T.J.
      • Deak M.
      • Knebel A.
      • Alessi D.R.
      • Muqit M.M.
      PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65.
      ,
      • Shiba-Fukushima K.
      • Imai Y.
      • Yoshida S.
      • Ishihama Y.
      • Kanao T.
      • Sato S.
      • Hattori N.
      PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy.
      ). Furthermore, overexpression of the UBL domain strongly suppressed autoubiquitination of FL WT Parkin or FL Parkin with S65A or S65D mutation (Fig. 3A). These data suggested that exogenous UBL domain blocks Parkin autoubiquitination activity by an intermolecular interaction.
      Figure thumbnail gr3
      FIGURE 3.Parkin activity is negatively regulated by its UBL domain. A, autoubiquitination assays for Parkin Ser-65 mutants. GST-tagged FL WT, S65A, or S65D Parkin was co-expressed with GFP-tagged UBL WT, S65A, or S65D domain and HA-tagged human Ub in HEK293T cells. GST pulldown samples were analyzed with anti-HA antibody (first panel) or anti-GST antibody (second panel) as indicated. Whole cell lysates (WCL) were blotted with anti-GFP antibody (third panel) and anti-HA antibody (fourth panel) to detect the same level expression of proteins. B, FLAG-tagged FL Parkin and GFP-tagged WT, S65A, or S65D UBL domain constructs were co-expressed in HeLa cells as indicated. Transfected cells were treated with 20 μm CCCP for 4 h as indicated. Whole cell lysates were prepared and analyzed for immunoblot with anti-Ub antibody (first panel), anti-FLAG antibody (second panel), and anti-GFP antibody (third panel). Immunoblot analyses with anti-β-tubulin antibody were used as loading controls (fourth panel). C, HeLa cells stably expressing GFP-tagged Parkin were transfected with HA-tagged ubiquitin and GFP-tagged UBL WT, S65A, or S65D domain and were treated with 10 μm CCCP for 4 h. Whole cell lysates were quantified and immunoblotted for endogenous mitochondrial proteins using anti-Mfn1, anti-Mfn2, anti-VDAC1, and anti-β-tubulin antibody. Anti-GFP antibody was used to detect the UBL domain and Parkin proteins. D, HeLa cells stably expressing GFP-tagged Parkin were transfected with GFP-tagged UBL WT, S65A, or S65D domain and treated with 10 μm CCCP for 12 h as indicated. Cell lysates were subjected to immunoblot analyses for endogenous mitochondrial proteins using anti-Mfn1, anti-Mfn2, anti-VDAC1, anti-COXIV, anti-TIM23, or anti-NDUFS3 antibody. Immunoblot analyses with anti-β-tubulin antibody were used as loading controls. Anti-GFP immunoblot was used to detect the UBL domain and FL Parkin proteins. E–G, confocal images of HeLa cells transfected as indicated and treated with 20 μm CCCP for 12 h. Cells expressing both GST-tagged FL Parkin and GFP-tagged UBL were marked by dotted circles. Bars, 20 μm. E, GST-tagged FL Parkin proteins were immunolabeled with anti-GST antibody (red), and the mitochondria were labeled with anti-TOM20 antibody (blue). The WT, S65A, and S65D UBL domain were GFP tagged (green). F and G, GST-tagged FL Parkin protein was immunolabeled with anti-GST antibody (red). To detect mitophagy, cells were stained with anti-p62/SQSTM1 (p62) antibody (gray, F) or anti-LC3B antibody (gray, G). The WT, S65A, and S65D UBL domain were GFP-tagged (green).
      We also co-expressed FL WT Parkin with the WT UBL domain or the UBL domain with S65A or S65D mutation in HeLa cells, which have little or no endogenous Parkin expression, and treated the cells with 20 μm CCCP to induce Parkin autoubiquitination. As expected, the polyubiquitination level in cells expressing FL Parkin alone was comparable with control, and with CCCP treatment the polyubiquitination levels were significantly increased (Fig. 3B). The CCCP-induced increase in polyubiquitination was noticeably reduced with co-expression of the WT UBL domain or the UBL domain with S65A mutation but less with co-expression of the S65D mutant (Fig. 3B).
      We next sought to determine whether the UBL domain affects Parkin E3 ligase activity by checking the ubiquitination levels of Parkin substrates, VDAC1, Mfn1, and Mfn2. We used a HeLa cell line stably expressing GFP-tagged Parkin and transfected with the GFP-tagged UBL domain with WT, S65A, or S65D mutation. We treated the cells with 10 μm CCCP to measure the polyubiquitination levels of endogenous substrate proteins. As in the case of Parkin autoubiquitination, expression of the UBL domain inhibited the CCCP-induced polyubiquitination of Parkin substrates, VDAC1, Mfn1, and Mfn2 (Fig. 3C). We also observed that the polyubiquitination of substrates was reduced with expression of the UBL domain with WT Parkin or Parkin with S65A mutation; however, expression of Parkin with S65D mutation did not affect the polyubiquitination level of Parkin substrates (Fig. 3C).
      Activated Parkin induces mitophagy (
      • Geisler S.
      • Holmström K.M.
      • Skujat D.
      • Fiesel F.C.
      • Rothfuss O.C.
      • Kahle P.J.
      • Springer W.
      PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.
      ,
      • Matsuda N.
      • Sato S.
      • Shiba K.
      • Okatsu K.
      • Saisho K.
      • Gautier C.A.
      • Sou Y.S.
      • Saiki S.
      • Kawajiri S.
      • Sato F.
      • Kimura M.
      • Komatsu M.
      • Hattori N.
      • Tanaka K.
      PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy.
      ,
      • Vives-Bauza C.
      • Zhou C.
      • Huang Y.
      • Cui M.
      • de Vries R.L.
      • Kim J.
      • May J.
      • Tocilescu M.A.
      • Liu W.
      • Ko H.S.
      • Magrané J.
      • Moore D.J.
      • Dawson V.L.
      • Grailhe R.
      • Dawson T.M.
      • Li C.
      • Tieu K.
      • Przedborski S.
      PINK1-dependent recruitment of Parkin to mitochondria in mitophagy.
      ,
      • Kawajiri S.
      • Saiki S.
      • Sato S.
      • Sato F.
      • Hatano T.
      • Eguchi H.
      • Hattori N.
      PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy.
      ). We investigated whether expression of the UBL domain may inhibit this process. The WT UBL domain, the UBL domain with S65A mutation, or the UBL domain with S65D mutation was overexpressed in a HeLa cell line stably expressing GFP-Parkin, and the levels of endogenous mitochondrial proteins were analyzed after treatment with or without 10 μm CCCP for 12 h. With CCCP treatment, the protein levels of Mfn1, Mfn2, VDAC1, COXIV, TIM23, and NDUFS3 were reduced compared with controls that were not treated with CCCP. Overexpression of the WT UBL domain or the UBL domain with S65A mutation partially prevented the reduction of the protein levels. However, overexpression of the UBL domain with S65D mutation was unable to prevent the CCCP-induced reduction of mitochondrial protein levels (Fig. 3D). Next, we observed mitochondria undergoing CCCP-induced mitophagy by using the mitochondrial marker TOM20 (
      • Geisler S.
      • Holmström K.M.
      • Skujat D.
      • Fiesel F.C.
      • Rothfuss O.C.
      • Kahle P.J.
      • Springer W.
      PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.
      ,
      • Bingol B.
      • Tea J.S.
      • Phu L.
      • Reichelt M.
      • Bakalarski C.E.
      • Song Q.
      • Foreman O.
      • Kirkpatrick D.S.
      • Sheng M.
      The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.
      ). Levels of TOM20 staining were markedly decreased in HeLa cells expressing GST-tagged FL Parkin with CCCP treatment for 12 h (Fig. 3E). In cells co-expressing FL Parkin and GFP-tagged WT or S65A UBL domain, TOM20 staining was not decreased with treatment of CCCP compared with control cells expressing FL Parkin alone; however, in cells co-expressing FL Parkin and S65D UBL domain, TOM20 staining levels were significantly weaker with CCCP treatment compared with control (Fig. 3E). Previous studies have shown that after CCCP treatment Parkin translocates to the mitochondria and ubiquitinates various substrates in the mitochondria and that an adaptor protein containing the LC3B-interaction region, such as p62/SQSTM1, is recruited to the mitochondria followed by recruitment of LC3B (
      • Geisler S.
      • Holmström K.M.
      • Skujat D.
      • Fiesel F.C.
      • Rothfuss O.C.
      • Kahle P.J.
      • Springer W.
      PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.
      ,
      • Okatsu K.
      • Saisho K.
      • Shimanuki M.
      • Nakada K.
      • Shitara H.
      • Sou Y.S.
      • Kimura M.
      • Sato S.
      • Hattori N.
      • Komatsu M.
      • Tanaka K.
      • Matsuda N.
      p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria.
      ,
      • Huang C.
      • Andres A.M.
      • Ratliff E.P.
      • Hernandez G.
      • Lee P.
      • Gottlieb R.A.
      Preconditioning involves selective mitophagy mediated by Parkin and p62/SQSTM1.
      ,
      • Gao J.
      • Qin S.
      • Jiang C.
      Parkin-induced ubiquitination of Mff promotes its association with p62/SQSTM1 during mitochondrial depolarization.
      ). This sequence of mitophagy events can be visualized by immunostaining of p62/SQSTM1 and LC3B (
      • Geisler S.
      • Holmström K.M.
      • Skujat D.
      • Fiesel F.C.
      • Rothfuss O.C.
      • Kahle P.J.
      • Springer W.
      PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.
      ). We further characterized the role of WT, S65A, or S65D UBL domain in the steps leading to Parkin-mediated mitophagy. In HeLa cells overexpressing FL Parkin, we observed co-localization of Parkin and p62/SQSTM1 in the mitochondria with CCCP treatment for 12 h (Fig. 3F). However, when FL Parkin and the WT or S65A UBL domain were co-expressed, p62/SQSTM1 failed to be recruited to the mitochondria (Fig. 3F). In contrast, we observed co-localization of Parkin and p62/SQSTM1 in cells co-expressing the UBL domain with S65D mutation and FL Parkin (Fig. 3F). Similar results were observed when CCCP-treated cells were stained with antibody against LC3B; lower levels of LC3B were detected in the mitochondria with expression of the WT UBL domain or the UBL domain with S65A mutation, whereas LC3B were detected in the mitochondria in cells co-expressing the S65D UBL domain at similar levels compared with cells expressing FL Parkin alone (Fig. 3G). These results indicated that the UBL domain can inhibit activation of Parkin E3 ligase activity and Parkin-induced mitophagy and that this inhibitory function of the UBL domain is negatively regulated by PINK1-dependent phosphorylation at Ser-65.

      The UBL Domain Regulates Parkin Translocation to the Mitochondria

      Parkin is cytoplasmic but translocates to the mitochondria upon activation by PINK1 (
      • Kim Y.
      • Park J.
      • Kim S.
      • Song S.
      • Kwon S.K.
      • Lee S.H.
      • Kitada T.
      • Kim J.M.
      • Chung J.
      PINK1 controls mitochondrial localization of Parkin through direct phosphorylation.
      ,
      • Song S.
      • Jang S.
      • Park J.
      • Bang S.
      • Choi S.
      • Kwon K.Y.
      • Zhuang X.
      • Kim E.
      • Chung J.
      Characterization of PINK1 (PTEN-induced putative kinase 1) mutations associated with Parkinson disease in mammalian cells and Drosophila.
      ). We sought to determine whether the UBL domain regulates the mitochondrial translocation of Parkin. We expressed FL Parkin in HeLa cells, treated the cells with 20 μm CCCP, and observed Parkin translocation to the mitochondria by confocal microscopy. FL Parkin was localized in the cytoplasm but translocated to the mitochondria with CCCP treatment as expected (Fig. 4A). Surprisingly, when the WT UBL domain was co-expressed with FL Parkin, Parkin showed less localization to the mitochondria. However, when the UBL S65D mutant was expressed with FL Parkin, Parkin highly localized to the mitochondria (Fig. 4B). Quantitative analyses revealed that of 200 cells that were counted, ∼20% of cells showed reduced translocation of FL Parkin to the mitochondria when the WT UBL domain was co-expressed; however, co-expression of the UBL domain S65D mutant showed no difference in the mitochondrial translocation of Parkin compared with control cells that did not express exogenous UBL domain (Fig. 4C). Therefore, we concluded that the UBL domain of Parkin negatively regulates CCCP-induced mitochondrial translocation of Parkin.
      Figure thumbnail gr4
      FIGURE 4.The UBL domain delays Parkin translocation to the mitochondria. A, confocal images of Parkin translocation to the mitochondria in HeLa cells. HeLa cells transfected with GST-tagged FL Parkin or GFP-tagged UBL domain constructs (green) were treated with DMSO (top panel) or 20 μm CCCP (bottom panel) for 4 h. GST-tagged Parkin protein was immunolabeled with anti-GST antibody (green), and the mitochondria were labeled with anti-TOM20 antibody (red). Bars, 20 μm. B, HeLa cells were transfected with plasmids expressing GST-tagged FL Parkin and GFP-tagged UBL domain with WT or S65D mutation. The cells were treated with 10 μm CCCP for 4 h and measured for the subcellular localization of GFP-tagged UBL WT or S65D (green). The same cells were also immunostained for GST-tagged FL Parkin with anti-GST antibody (red) and for mitochondria with anti-TOM20 antibody (blue). Bars, 20 μm. C, quantification of the percentage of Parkin localized to the mitochondria. n = 200. Error bars: S.D. *, p < 0.05 by analysis of variance Tukey's test.

      The UBL Domain Regulates Interaction between Parkin and Its Substrate VDAC1

      The Parkin R1 domain is necessary for Parkin binding to the E2 enzyme or its substrates (
      • Shimura H.
      • Hattori N.
      • Kubo S.i.
      • Mizuno Y.
      • Asakawa S.
      • Minoshima S.
      • Shimizu N.
      • Iwai K.
      • Chiba T.
      • Tanaka K.
      • Suzuki T.
      Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.
      ). We found above that polyubiquitination or mitophagy of Parkin substrates, Mfn1, Mfn2, and VDAC are regulated by the direct interaction of the Parkin UBL domain and R1 domain (Fig. 3). This suggested that the interaction may affect Parkin binding to its substrates. Before testing this possibility, we first sought to identify Parkin substrates that directly bind to Parkin among previously reported substrates that showed direct binding, namely Mfn1, Mfn2, VDAC1, and Drp1, by co-IP with FL Parkin (
      • Chen Y.
      • Dorn 2nd., G.W.
      PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria.
      ,
      • Wang H.
      • Song P.
      • Du L.
      • Tian W.
      • Yue W.
      • Liu M.
      • Li D.
      • Wang B.
      • Zhu Y.
      • Cao C.
      • Zhou J.
      • Chen Q.
      Parkin ubiquitinates Drp1 for proteasome-dependent degradation: implication of dysregulated mitochondrial dynamics in Parkinson disease.
      ,
      • Tanaka A.
      • Cleland M.M.
      • Xu S.
      • Narendra D.P.
      • Suen D.F.
      • Karbowski M.
      • Youle R.J.
      Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin.
      ,
      • Sun Y.
      • Vashisht A.A.
      • Tchieu J.
      • Wohlschlegel J.A.
      • Dreier L.
      Voltage-dependent anion channels (VDACs) recruit Parkin to defective mitochondria to promote mitochondrial autophagy.
      ). We observed that only VDAC1 directly interacts with Parkin (Fig. 5A). Next, to identify which region of Parkin interacts with VDAC1, we examined the ability of Parkin deletion mutants to bind to VDAC1. We found that C (Fig. 5B) and, more specifically, the R1 domain of Parkin, are sufficient for Parkin interaction with VDAC1 (Fig. 5C).
      Figure thumbnail gr5
      FIGURE 5.The UBL domain inhibits the interaction between Parkin and VDAC1. GST-tagged FL Parkin, C, R1, or IBR-R2 Parkin constructs was co-expressed with HA-tagged VDAC1 and/or GFP-tagged UBL WT, S65A, and S65D in HEK293T cells as indicated. Whole cell lysates (WCL) were subjected to GST pulldown or immunoprecipitation with anti-HA antibody followed by SDS-PAGE and immunoblot analyses with the indicated antibodies.
      To determine whether this inhibition occurs by interfering with the interaction between the R1 domain and VDAC1, we performed co-IP to measure the interaction between the R1 domain and VDAC1 when the WT UBL domain or the UBL S65A or S65D mutant was co-expressed. With co-expression of the WT UBL domain or the UBL domain with S65A mutation, the interaction between the R1 domain and VDAC1 was noticeably reduced, but co-expression of the UBL domain with S65D mutation showed little or no difference in the ability to interact (Fig. 5D). Thus, we concluded that the UBL domain and VDAC1 competitively binds to the R1 domain of Parkin. Furthermore, these results suggested that the UBL domain of Parkin simultaneously regulates the activation of Parkin E3 ligase activity and the interaction between Parkin and its substrates.

      Mutations of the UBL Domain Identified in PD Patients Affect Parkin Activity

      To determine whether the UBL domain plays a role in the pathogenesis of Parkinson disease, we utilized five mutants of the UBL domain (K27N, R33Q, R42P, A46P, and K48A) in Parkin that were previously reported in PD patients (
      • Shimura H.
      • Hattori N.
      • Kubo S.i.
      • Mizuno Y.
      • Asakawa S.
      • Minoshima S.
      • Shimizu N.
      • Iwai K.
      • Chiba T.
      • Tanaka K.
      • Suzuki T.
      Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.
      ,
      • Oliveira S.A.
      • Scott W.K.
      • Martin E.R.
      • Nance M.A.
      • Watts R.L.
      • Hubble J.P.
      • Koller W.C.
      • Pahwa R.
      • Stern M.B.
      • Hiner B.C.
      • Ondo W.G.
      • Allen Jr., F.H.
      • Scott B.L.
      • Goetz C.G.
      • Small G.W.
      • Mastaglia F.
      • Stajich J.M.
      • Zhang F.
      • Booze M.W.
      • Winn M.P.
      • Middleton L.T.
      • Haines J.L.
      • Pericak-Vance M.A.
      • Vance J.M.
      Parkin mutations and susceptibility alleles in late-onset Parkinson's disease.
      • Terreni L.
      • Calabrese E.
      • Calella A.M.
      • Forloni G.
      • Mariani C.
      New mutation (R42P) of the parkin gene in the ubiquitinlike domain associated with parkinsonism.
      ,
      • Dev K.K.
      • van der Putten H.
      • Sommer B.
      • Rovelli G.
      Part I: parkin-associated proteins and Parkinson's disease.
      ,
      • Henn I.H.
      • Gostner J.M.
      • Lackner P.
      • Tatzelt J.
      • Winklhofer K.F.
      Pathogenic mutations inactivate parkin by distinct mechanisms.
      • Tan E.K.
      • Skipper L.M.
      Pathogenic mutations in Parkinson disease.
      ).
      We observed the intracellular localization of Parkin in HeLa cells expressing FL WT Parkin or FL Parkin with the pathogenic mutations in the UBL domain. As a negative control, we utilized Parkin with T240R mutation in the R1 domain or C431S mutation in the R2 domain, which are known to hinder interaction with the E2 enzyme or prevent activation of Parkin, respectively (
      • Riley B.E.
      • Lougheed J.C.
      • Callaway K.
      • Velasquez M.
      • Brecht E.
      • Nguyen L.
      • Shaler T.
      • Walker D.
      • Yang Y.
      • Regnstrom K.
      • Diep L.
      • Zhang Z.
      • Chiou S.
      • Bova M.
      • Artis D.R.
      • Yao N.
      • Baker J.
      • Yednock T.
      • Johnston J.A.
      Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases.
      ,
      • Shimura H.
      • Hattori N.
      • Kubo S.i.
      • Mizuno Y.
      • Asakawa S.
      • Minoshima S.
      • Shimizu N.
      • Iwai K.
      • Chiba T.
      • Tanaka K.
      • Suzuki T.
      Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase.
      ). When treated with CCCP, WT, R42P, and K48A FL Parkin localized to the mitochondria, but K27N, R33Q, and A46P FL Parkin mutants distributed throughout the cytosol (Fig. 6A). Also, in agreement with previous reports (
      • Lazarou M.
      • Narendra D.P.
      • Jin S.M.
      • Tekle E.
      • Banerjee S.
      • Youle R.J.
      PINK1 drives Parkin self-association and HECT-like E3 activity upstream of mitochondrial binding.
      ,
      • Geisler S.
      • Holmström K.M.
      • Skujat D.
      • Fiesel F.C.
      • Rothfuss O.C.
      • Kahle P.J.
      • Springer W.
      PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.
      ,
      • Geisler S.
      • Vollmer S.
      • Golombek S.
      • Kahle P.J.
      The ubiquitin-conjugating enzymes UBE2N, UBE2L3, and UBE2D2/3 are essential for Parkin-dependent mitophagy.
      ,
      • Zhang C.
      • Lee S.
      • Peng Y.
      • Bunker E.
      • Giaime E.
      • Shen J.
      • Zhou Z.
      • Liu X.
      PINK1 triggers autocatalytic activation of Parkin to specify cell fate decisions.
      ), the T240R or C431S FL Parkin mutant did not translocate to the mitochondria (Fig. 6A). Quantitative analysis revealed that ∼84% of cells showed translocation of WT FL Parkin to the mitochondria with CCCP treatment; however, in cells expressing K27N, R33Q, or A46P mutant, Parkin localization to the mitochondria was rare or not detected (Fig. 6B). In cells expressing FL Parkin with R42P or K48A mutation, Parkin was localized to the mitochondria in 67% or 78% of the cells, respectively (Fig. 6B).
      Figure thumbnail gr6
      FIGURE 6.PD pathogenic mutations in the UBL domain affect mitochondrial translocation of Parkin. A, confocal microscopy images of WT Parkin and mutant Parkin with pathogenic mutations in the UBL domain and catalytic sites, such as K27N, R33Q, R42P, A46P, K48A, T240R, and C431S. HeLa cells transfected with GST-tagged FL Parkin were treated with DMSO or 20 μm CCCP for 2 h. Cells were immunostained for GST-tagged FL Parkin constructs with anti-GST antibody (green) and for mitochondria with anti-TOM20 antibody (red). Bars, 20 μm. B, quantification of the percentage of Parkin localized to the mitochondria. n = 200. Error bars: S.D. Different letters indicate the statistical significance by analysis of variance Tukey's test. Statistical significance is p < 0.05.
      To test whether the defect in mitochondrial localization of the pathogenic Parkin mutants is a result of altered interaction between the UBL domain and the R1 domain, we performed an autoubiquitination assay to test the inhibitory function of the UBL domain. The autoubiquitination activity of FL Parkin with R42P or K48A mutation was higher than that of FL WT Parkin without CCCP treatment, indicating that the UBL domain with R42P or K48A mutation loses the ability to tightly regulate Parkin activation (Fig. 7A). We also observed that the autoubiquitination activity of FL WT Parkin and FL Parkin with R42P or K48A mutation was induced by CCCP treatment compared with that in untreated controls (Fig. 7A). However, the autoubiquitination activity of FL Parkin with K27N, R33Q, or A46P mutation was low or barely detectable even with CCCP treatment (Fig. 7A). These results indicated that K27N, R33Q, or A46P mutation in the UBL domain blocks Parkin activation upon CCCP treatment.
      Figure thumbnail gr7
      FIGURE 7.PD pathogenic mutations in the UBL domain disrupt Parkin autoubiquitination. A, autoubiquitination assays for Parkin pathogenic mutants in the UBL domain (K27N, R33Q, R42P, A46P, and K48A). HEK293T cells were transfected with GST-tagged FL Parkin mutants in the UBL domain and HA-tagged Ub as indicated. Whole cell lysates (WCL) were subjected to GST pulldown and immunoblotted with anti-HA antibody (top and bottom panel) and anti-GST antibody (middle panel). HEK293T cells were non-treated or treated with 20 μm CCCP for 2 h. B, autoubiquitination assays for negative controls (T240R and C431S) and Parkin pathogenic mutants in the UBL domain (K27N, R33Q, and A46P). HEK293T cells were transfected with GST-tagged FL Parkin mutants and HA-tagged Ub as indicated. Whole cell lysates were subjected to GST pulldown and immunoblotted with anti-HA antibody (top and bottom panel) and anti-GST antibody (middle panel). HEK293T cells were non-treated or treated with 20 μm CCCP for 2 h. C, GFP-tagged UBL constructs with K27N, R33Q, R42P, A46P, or K48A mutation and GST-tagged R1 construct were transfected in HEK293T cells. WCL were subjected to GST pulldown assay followed by immunoblot analyses with anti-GFP antibody (top panel) or anti-GST antibody (middle panel). WCL were immunoblotted with anti-GFP antibody (bottom panel) to detect the expression levels of GFP-tagged UBL proteins. HEK293T cells were non-treated (left) or treated with 20 μm of CCCP for 2 h (right).
      We tested whether autoubiquitination levels differ between cells expressing Parkin mutants that do not lead to an increased autoubiquitination, K27N, R33Q, or A46P, and Parkin T240R or C431S mutants with CCCP treatment. We found that autoubiquitination levels were comparable in cells expressing K27N, R33Q, or A46P Parkin mutant and the T240R or C431S mutant controls, even with CCCP treatment (Fig. 7B).
      To investigate whether the changes in the autoubiquitination level of the mutant Parkin proteins are due to changed interactions between the R1 domain and the UBL domain, we examined whether the ability of the UBL domain with K27N, R33Q, R42P, A46P, or K48A mutation to bind to the R1 domain is altered compared with the WT UBL domain. Interaction between the R1 domain and the UBL domain with R42P or K48A mutation was decreased compared with the WT UBL domain, as determined by co-IP (Fig. 7C, left). Strikingly, although the interaction between the R1 domain and the WT UBL domain or between the R1 domain and the UBL domain with R42P or K48A mutation was dramatically decreased upon CCCP treatment, the interaction between the R1 domain and the UBL domain with K27N, R33Q, or A46P mutation was not affected by CCCP treatment (Fig. 7C, right). Interestingly, this unaffected interaction between the R1 domain and the UBL domain by CCCP treatment correlated well with the decreased autoubiquitination and mitochondria localization levels of FL Parkin with K27N, R33Q, or A46P mutation (Figs. 7A and 6).
      Together, these results strongly suggested that the mechanism of regulating Parkin activation by the interaction between the UBL domain and the R1 domain is linked to PD pathogenesis and that the interaction can be a key target in developing effective treatments against Parkin-mediated pathogenesis.

      Discussion

      In this study we presented evidence that the R1 domain and the UBL domain of Parkin interact and that this interaction leads to reduced Parkin E3 ligase activity. We also showed that the R1 domain directly binds to VDAC1 and that competitive binding of the UBL domain to the R1 domain prevents Parkin translocation to the mitochondria. Moreover, we demonstrated that PINK1-dependent phosphorylation of Parkin at Ser-65 negatively regulates the interaction between the R1 domain and the UBL domain, suggesting a novel mechanism of how PINK1 regulates Parkin activation (Fig. 8).
      Figure thumbnail gr8
      FIGURE 8.The open and closed conformations of Parkin are regulated by the interaction between the R1 and the UBL domain. An intramolecular regulation of Parkin by the UBL domain. Parkin activity is inhibited by interaction of the catalytic R1 domain and the UBL domain. When the UBL domain binds to the R1 domain, Parkin rests in an autoinhibited, or “closed” state (top panel). Phosphorylation of the UBL domain by PINK1 at Ser-65 leads to decreased binding between the UBL domain and the R1 domain (middle panel, left). The “open” C-terminal catalytic site of Parkin can promote substrate ubiquitination. The R1 domain of Parkin also interacts with VDAC1, and Parkin can translocate to the mitochondria. Parkin localized to the mitochondria can ubiquitinate various mitochondrial proteins, including Mfn1, Mfn2, and VDAC1 (bottom panel). Some pathogenic Parkin mutants with missense mutations in the UBL domain (K27N, R33Q, and A46P) do not respond to CCCP treatment and maintain the closed state (middle panel, right).
      Various proteins and functional domains, including ubiquitin-associated proteins (UBAs), ubiquitin-binding proteins (UBPs), ubiquitin binding domains (UBDs), and UBLs share structural similarities with ubiquitin and consist of a Leu-8–Ile-44–Val-70 hydrophobic patch that interacts with ubiquitin or other target proteins (
      • Beasley S.A.
      • Safadi S.S.
      • Barber K.R.
      • Shaw G.S.
      Solution structure of the E3 ligase HOIL-1 Ubl domain.
      ,
      • Chaugule V.K.
      • Burchell L.
      • Barber K.R.
      • Sidhu A.
      • Leslie S.J.
      • Shaw G.S.
      • Walden H.
      Autoregulation of Parkin activity through its ubiquitin-like domain.
      ,
      • Lowe E.D.
      • Hasan N.
      • Trempe J.F.
      • Fonso L.
      • Noble M.E.
      • Endicott J.A.
      • Johnson L.N.
      • Brown N.R.
      Structures of the Dsk2 UBL and UBA domains and their complex.
      ,
      • Dieckmann T.
      • Withers-Ward E.S.
      • Jarosinski M.A.
      • Liu C.F.
      • Chen I.S.
      • Feigon J.
      Structure of a human DNA repair protein UBA domain that interacts with HIV-1 Vpr.
      • Walters K.J.
      • Kleijnen M.F.
      • Goh A.M.
      • Wagner G.
      • Howley P.M.
      Structural studies of the interaction between ubiquitin family proteins and proteasome subunit S5a.
      ). The UBL domain of Parkin harbors a hydrophobic patch with a similar structure with conserved Ile-44 and Val-70 residues. Recently, the determination of the structure of rat FL Parkin by low resolution x-ray crystallography raised the possibility that the R1 domain may interact with the UBL domain via the Ile-44 residue of the hydrophobic patch and the surrounding hydrophobic surface (
      • Trempe J.F.
      • Sauvé V.
      • Grenier K.
      • Seirafi M.
      • Tang M.Y.
      • Ménade M.
      • Al-Abdul-Wahid S.
      • Krett J.
      • Wong K.
      • Kozlov G.
      • Nagar B.
      • Fon E.A.
      • Gehring K.
      Structure of parkin reveals mechanisms for ubiquitin ligase activation.
      ). We tested whether the hydrophobic patch in the UBL domain is required for binding between the R1 domain and the UBL domain by generating UBL domain mutants where the Ile-44 or Val-70 residue was mutated to an alanine. Using co-IP and autoubiquitination assays, we showed that there was no effect on interaction between the R1 domain and the UBL domain with I44A or V70A mutation and that there was no difference in autoubiquitination levels of the UBL domain mutants compared with WT. From these data we deduced that additional amino acids in addition to the hydrophobic patch are required for binding between the R1 domain and the UBL domain.
      To further identify amino acid residues that are required for binding between the R1 domain and the UBL domain of Parkin, we generated two Parkin UBL domain derivatives, in which one consisted of amino acid residues 1–35 and the other, of the remaining amino acids, residues 36–76. By co-IP assays, we showed that residues 1–35 of the UBL domain are necessary for binding of the UBL domain to the R1 domain. Three-dimensional structural analysis of the Parkin UBL domain obtained from Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank revealed that four amino acid residues of the UBL domain, Phe-4, Arg-6, Pro-14, and Glu-16, are externally exposed and lie in the same plane (
      • Tomoo K.
      • Mukai Y.
      • In Y.
      • Miyagawa H.
      • Kitamura K.
      • Yamano A.
      • Shindo H.
      • Ishida T.
      Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin.
      ,
      • Tashiro M.
      • Okubo S.
      • Shimotakahara S.
      • Hatanaka H.
      • Yasuda H.
      • Kainosho M.
      • Yokoyama S.
      • Shindo H.
      NMR structure of ubiquitin-like domain in PARKIN: gene product of familial Parkinson's disease.
      ). Furthermore, through sequence analysis, we found that the four amino acid residues are conserved in humans and Drosophila. To investigate whether these residues are required for binding of the UBL domain to the R1 domain, we mutated Phe-4, Arg-6, Pro-14, and Glu-16 into alanines and observed that the interaction between the R1 domain and the UBL domain was abolished. From this we concluded that the N terminus of the UBL domain mediates binding to the R1 domain.
      As phosphorylation of the Parkin UBL domain at Ser-65 leads to a decreased interaction between the R1 domain and the UBL domain, we investigated whether Ser-65 phosphorylation affects the amino acid residues that participate in binding of the two domains. To do so we measured the distance between the residues in a three-dimensional configuration. Interestingly, Ser-65 was located not far from the Phe-4 and Arg-6 residues of Parkin, suggesting the possibility that phosphorylation at Ser-65 may affect the interaction between the R1 domain and the UBL domain by interfering these two amino acid residues.
      Recently, it has been reported that phosphorylation of Parkin Ser-65 forms a cleft in the three-dimensional structure formed between the UBL domain and C and that the cleft is filled with an increased number of water molecules (
      • Caulfield T.R.
      • Fiesel F.C.
      • Moussaud-Lamodière E.L.
      • Dourado D.F.
      • Flores S.C.
      • Springer W.
      Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.
      ). With the conformational change, the distance between the UBL domain and the R1 domain increased from 20 Å to >50 Å, and the binding site of ubiquitin-conjugated E2 increased within the R1 domain is revealed, leading to docking of the UbcH5a/UBE2D1 E2 enzyme to the R1 domain. This promotes Parkin ubiquitination at Cys-431 and its E3 ligase activity (
      • Caulfield T.R.
      • Fiesel F.C.
      • Moussaud-Lamodière E.L.
      • Dourado D.F.
      • Flores S.C.
      • Springer W.
      Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.
      ).
      In agreement with these structural analyses, we found that phosphorylation of Parkin UBL domain at Ser-65 by PINK1 leads to a decreased interaction between the R1 domain and the UBL domain (Fig. 2D). Consistent with this finding, the UBL domain with S65D phosphomimetic mutation failed to suppress polyubiquitination of Parkin endogenous substrates, in contrast to wild-type or the UBL domain with S65A mutation (Fig. 3C). Moreover, we further demonstrated that interaction between Parkin R1 domain and Parkin substrate VDAC1 is regulated by Ser-65 phosphorylation of the UBL domain. Collectively, we provided evidence that PINK1 phosphorylation of Parkin at Ser-65, by preventing binding between the UBL domain and the R1 catalytic region, exposes the R1 domain and, consequently, promotes Parkin E3 ligase activity and translocation to the mitochondria (Fig. 8).
      The PINK1 phosphorylation site in the UBL domain is conserved in ubiquitin; ubiquitin is also phosphorylated by PINK1 at Ser-65 (
      • Koyano F.
      • Okatsu K.
      • Kosako H.
      • Tamura Y.
      • Go E.
      • Kimura M.
      • Kimura Y.
      • Tsuchiya H.
      • Yoshihara H.
      • Hirokawa T.
      • Endo T.
      • Fon E.A.
      • Trempe J.F.
      • Saeki Y.
      • Tanaka K.
      • Matsuda N.
      Ubiquitin is phosphorylated by PINK1 to activate parkin.
      ,
      • Wauer T.
      • Swatek K.N.
      • Wagstaff J.L.
      • Gladkova C.
      • Pruneda J.N.
      • Michel M.A.
      • Gersch M.
      • Johnson C.M.
      • Freund S.M.
      • Komander D.
      Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly, and hydrolysis.
      ). Phosphorylation of ubiquitin at Ser-65 leads to increased activation of FL Parkin (
      • Koyano F.
      • Okatsu K.
      • Kosako H.
      • Tamura Y.
      • Go E.
      • Kimura M.
      • Kimura Y.
      • Tsuchiya H.
      • Yoshihara H.
      • Hirokawa T.
      • Endo T.
      • Fon E.A.
      • Trempe J.F.
      • Saeki Y.
      • Tanaka K.
      • Matsuda N.
      Ubiquitin is phosphorylated by PINK1 to activate parkin.
      ,
      • Wauer T.
      • Swatek K.N.
      • Wagstaff J.L.
      • Gladkova C.
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      Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly, and hydrolysis.
      ). Furthermore, Parkin activation is increased in an in vitro ubiquitin assay when phospho-ubiquitin is coexpressed with WT or S65A mutant of FL Parkin (
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      Ubiquitin is phosphorylated by PINK1 to activate parkin.
      ,
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      • Michel M.A.
      • Gersch M.
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      • Freund S.M.
      • Komander D.
      Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly, and hydrolysis.
      ). For Parkin to be fully activated, phosphorylation at the Ser-65 residue of both ubiquitin and Parkin UBL domain is required (
      • Kazlauskaite A.
      • Kondapalli C.
      • Gourlay R.
      • Campbell D.G.
      • Ritorto M.S.
      • Hofmann K.
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      Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser-65.
      • Kane L.A.
      • Lazarou M.
      • Fogel A.I.
      • Li Y.
      • Yamano K.
      • Sarraf S.A.
      • Banerjee S.
      • Youle R.J.
      PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.
      ,
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      How phosphoubiquitin activates Parkin.
      ,
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      • Lilov A.
      • Seirafi M.
      • Vranas M.
      • Rasool S.
      • Kozlov G.
      • Sprules T.
      • Wang J.
      • Trempe J.F.
      • Gehring K.
      A Ubl/ubiquitin switch in the activation of Parkin.
      • Kumar A.
      • Aguirre J.D.
      • Condos T.E.
      • Martinez-Torres R.J.
      • Chaugule V.K.
      • Toth R.
      • Sundaramoorthy R.
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      • Knebel A.
      • Spratt D.E.
      • Barber K.R.
      • Shaw G.S.
      • Walden H.
      Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.
      ). In a recent study the crystal structure of phospho-ubiquitin and a UBL domain-deleted mutant Parkin (amino acids 140–461) of Pediculus humanus corporis was reported (
      • Wauer T.
      • Simicek M.
      • Schubert A.
      • Komander D.
      Mechanism of phospho-ubiquitin-induced PARKIN activation.
      ). This report suggested the possibility that Parkin activation may be regulated not only by interaction between the R1 domain and the UBL domain but also by interaction between the R1 domain and phospho-ubiquitin (
      • Wauer T.
      • Simicek M.
      • Schubert A.
      • Komander D.
      Mechanism of phospho-ubiquitin-induced PARKIN activation.
      ). In this study we showed that binding to the R1 domain is decreased when the UBL domain is phosphorylated at Ser-65 (Fig. 2) and that activation of FL Parkin is suppressed when co-expressed with the WT UBL domain or the UBL domain with S65A mutation, but not efficiently (Fig. 3). This may be explained by the fact that phospho-ubiquitin also regulates Parkin activation. Further study is needed to address whether phosphorylation of Ser-65 in ubiquitin, in addition to phosphorylation of Ser-65 in the UBL domain, regulates the interaction between the R1 domain and the UBL domain of Parkin.
      We found that the interaction between the R1 domain and the UBL domain with K27N, R33Q, or A46P mutation was not dissociated by CCCP treatment (Fig. 7C). The tight interaction between the R1 domain and the UBL domain with K27N, R33Q, or A46P mutation in the presence of CCCP prevents Parkin translocation to the mitochondria and inhibits Parkin autoubiquitination (FIGURE 6., FIGURE 7.A). These data suggested that, of the mutations found in PD patients, K27N, R33Q, or A46P mutation in the UBL domain leads to a continuous interaction of the UBL domain with the R1 domain and that the UBL domain interferes with interaction of the R1 domain with its substrates or E2 enzymes to prevent Parkin activation. From this we suggest a new mechanism of PD pathogenesis in which failure to regulate the interaction between the R1 domain and the UBL domain to regulate Parkin activation leads to the development of PD.
      In summary, our findings indicated that the interaction between the R1 domain and the UBL domain is critical for proper regulation of Parkin function and that interruption of this regulation may lead to PD pathogenesis. Given the importance of modulating the binding between two domains in Parkin, development of a new drug that can regulate this interaction may offer exciting possibilities of hindering PD pathogenesis by regulation of Parkin activity and of controlling various cellular processes, such as mitochondrial homeostasis, mitochondrial dynamics, and mitophagy.

      Author Contributions

      S. J. H., S. S., and J. C. conceived and designed the experiment. S. J. H. and S. S. performed the experiments. S. J. H., S. S., S. Y. L., S. C., and J. C. analyzed the data. S. S. and S. C. contributed reagents/materials/analysis tools. S. J. H., S. Y. L., J.-R. C., and J. C. wrote the paper.

      Acknowledgments

      We thank Dr. Kei Okatsu (Tokyo Metropolitan Institute of Medical Science) for the HeLa cells stably expressing GFP-Parkin.

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