(Patho‐)physiological relevance of PINK 1‐dependent ubiquitin phosphorylation
2015; Springer Nature; Volume: 16; Issue: 9 Linguagem: Inglês
10.15252/embr.201540514
ISSN1469-3178
AutoresFabienne C. Fiesel, Maya Ando, Roman Hudec, Anneliese R. Hill, Monica Castanedes‐Casey, Thomas R. Caulfield, Elisabeth L. Moussaud-Lamodière, Jeannette N. Stankowski, Peter Bauer, Oswaldo Lorenzo‐Betancor, Isidró Ferrer, José Matías Arbelo, Joanna Siuda, Li Chen, Valina L. Dawson, Ted M. Dawson, Zbigniew K. Wszołek, Owen A. Ross, Dennis W. Dickson, Wolfdieter Springer,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoArticle14 July 2015free access (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation Fabienne C Fiesel Fabienne C Fiesel Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Maya Ando Maya Ando Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Roman Hudec Roman Hudec Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Anneliese R Hill Anneliese R Hill Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Monica Castanedes-Casey Monica Castanedes-Casey Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Thomas R Caulfield Thomas R Caulfield Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Elisabeth L Moussaud-Lamodière Elisabeth L Moussaud-Lamodière Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Jeannette N Stankowski Jeannette N Stankowski Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Peter O Bauer Peter O Bauer Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Oswaldo Lorenzo-Betancor Oswaldo Lorenzo-Betancor Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Isidre Ferrer Isidre Ferrer Institut de Neuropatologia, Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, Hospitalet del Llobregat, Spain CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author José M Arbelo José M Arbelo Department of Neurology, Parkinson's and Movement Disorders Unit, Hospital Universitario Insular de Gran Canaria, Las Palmas de Gran Canaria, Spain Search for more papers by this author Joanna Siuda Joanna Siuda Department of Neurology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland Search for more papers by this author Li Chen Li Chen Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Valina L Dawson Valina L Dawson Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA, USA Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA Search for more papers by this author Ted M Dawson Ted M Dawson Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA, USA Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Zbigniew K Wszolek Zbigniew K Wszolek Department of Neurology, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Owen A Ross Owen A Ross Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA Search for more papers by this author Dennis W Dickson Dennis W Dickson Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA Search for more papers by this author Wolfdieter Springer Corresponding Author Wolfdieter Springer Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA Search for more papers by this author Fabienne C Fiesel Fabienne C Fiesel Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Maya Ando Maya Ando Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Roman Hudec Roman Hudec Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Anneliese R Hill Anneliese R Hill Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Monica Castanedes-Casey Monica Castanedes-Casey Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Thomas R Caulfield Thomas R Caulfield Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Elisabeth L Moussaud-Lamodière Elisabeth L Moussaud-Lamodière Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Jeannette N Stankowski Jeannette N Stankowski Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Peter O Bauer Peter O Bauer Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Oswaldo Lorenzo-Betancor Oswaldo Lorenzo-Betancor Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Isidre Ferrer Isidre Ferrer Institut de Neuropatologia, Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, Hospitalet del Llobregat, Spain CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Barcelona, Spain Search for more papers by this author José M Arbelo José M Arbelo Department of Neurology, Parkinson's and Movement Disorders Unit, Hospital Universitario Insular de Gran Canaria, Las Palmas de Gran Canaria, Spain Search for more papers by this author Joanna Siuda Joanna Siuda Department of Neurology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland Search for more papers by this author Li Chen Li Chen Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Valina L Dawson Valina L Dawson Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA, USA Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA Search for more papers by this author Ted M Dawson Ted M Dawson Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA, USA Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Zbigniew K Wszolek Zbigniew K Wszolek Department of Neurology, Mayo Clinic, Jacksonville, FL, USA Search for more papers by this author Owen A Ross Owen A Ross Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA Search for more papers by this author Dennis W Dickson Dennis W Dickson Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA Search for more papers by this author Wolfdieter Springer Corresponding Author Wolfdieter Springer Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA Search for more papers by this author Author Information Fabienne C Fiesel1, Maya Ando1, Roman Hudec1, Anneliese R Hill1, Monica Castanedes-Casey1, Thomas R Caulfield1, Elisabeth L Moussaud-Lamodière1, Jeannette N Stankowski1, Peter O Bauer1, Oswaldo Lorenzo-Betancor1, Isidre Ferrer2,3, José M Arbelo4, Joanna Siuda5, Li Chen6,7, Valina L Dawson6,7,8,9,10, Ted M Dawson6,7,9,10,11,12, Zbigniew K Wszolek13, Owen A Ross1,14, Dennis W Dickson1,14 and Wolfdieter Springer 1,14 1Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA 2Institut de Neuropatologia, Servei d'Anatomia Patològica, Hospital Universitari de Bellvitge, Hospitalet del Llobregat, Spain 3CIBERNED, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, Barcelona, Spain 4Department of Neurology, Parkinson's and Movement Disorders Unit, Hospital Universitario Insular de Gran Canaria, Las Palmas de Gran Canaria, Spain 5Department of Neurology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland 6Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA 7Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 8Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA 9Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA, USA 10Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA 11Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA 12Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA 13Department of Neurology, Mayo Clinic, Jacksonville, FL, USA 14Neurobiology of Disease, Mayo Graduate School, Jacksonville, FL, USA *Corresponding author. Tel: +1 904 953 6129; Fax: +1 904 953 7117; E-mail: [email protected] EMBO Reports (2015)16:1114-1130https://doi.org/10.15252/embr.201540514 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development. Synopsis In this study, two newly generated antibodies are used to detect endogenous phospho-ubiquitin in cells and human brain samples. pS65-Ub is shown to be reversible, respond to mitochondrial stress and accumulate during aging and in Parkinson's disease (PD). pS65-Ub is amplified by the concerted action of PINK1 kinase and Parkin E3 ubiquitin ligase in response to mitochondrial stress. pS65-Ub specifically labels severely damaged mitochondria destined for degradation. pS65-Ub is a novel biomarker of mitochondrial quality control and could also serve as a potential therapeutic target for PD. Introduction Mutations in PINK1 and PARKIN are the most common cause of recessive early-onset Parkinson's disease (PD). Together, they coordinate a mitochondrial quality control pathway that ensures safe disposal of defective (mitophagy) and maintenance of healthy mitochondria 1. This stress-induced pathway is tightly controlled and underlies complex regulation at multiple steps of a sequential process 2. Upon mitochondrial damage, the protein kinase PINK1 is stabilized on the outer membrane and recruits the E3 ubiquitin (Ub) ligase Parkin from the cytosol 3. PINK1 has been shown to phosphorylate Parkin 456 in its N-terminal Ub-like (UBL) domain, which is required for Parkin's structural 7 and functional activation 8. Parkin is "charged" with Ub by E2 co-enzymes that modulate its mitochondrial translocation and enzymatic functions, both of which are linked 910. Parkin then labels mitochondrial substrate proteins with poly-Ub chains of distinct topologies to mediate their sequestration and/or degradation. Parkin and generated Ub conjugates are also subject to regulation by specific de-ubiquitinating enzymes (DUBs) 11. Removal of individual Ub moieties or chains from substrates modulates downstream functions that are decoded by Ub-binding adaptors. PINK1 has just recently been identified to phosphorylate Ub, in addition to the Ub ligase Parkin, at a conserved serine 65 (S65) residue 121314. Both phosphorylation events are required for full activation of Parkin by feed-forward mechanisms during mitophagy 151617. While phosphorylation of the modifier protein further increases complexity, it also provides more selectivity and specificity for a seemingly universal ubiquitination process. In addition to activation of Parkin, consequences of pS65-Ub on structure, chain assembly, hydrolysis, and recognition have been reported in vitro 18. During preparation of this manuscript, another study suggested pS65-Ub as the Parkin receptor on damaged mitochondria 19. However, the (patho-)physiological significance of this posttranslational modification in particular in neurons and in brain remains unclear. Here, we developed and carefully characterized two phospho-specific antibodies as tools to demonstrate the (patho-)physiological relevance of pS65-Ub. While one of the antibodies was specific to pS65-Ub, the other antibody recognized both pS65-Ub and pS65-Parkin. We confirmed that the obtained signals were: (i) specific to phosphorylated S65, (ii) induced by mitochondrial stress, (iii) dependent on PINK1 kinase, and (iv) reversible by and sensitive to phosphatase activity. For the first time, we corroborated the presence of pS65-Ub under endogenous conditions in stressed primary neurons and in vivo in human postmortem brains. Importantly, primary cells and brain tissue from PD patients carrying PINK1 mutations were largely devoid of pS65-Ub signal. Our findings suggest that pS65-Ub accumulates with stress, disease, or age, and highlight its significance and potential for future biomarker and/or therapeutic development. Results Validation of pS65-Ub antibodies in vitro We sought to develop antibodies specific to Ub phosphorylated at Ser65 (pS65-Ub) to investigate its significance in primary neurons, in human brain, and in PD patient samples. Affinity purification yielded two selective and sensitive rabbit polyclonal antibodies (hereafter referred to as pS65-Ub#1 and pS65-Ub#2) as shown by dot blot with the immunogenic and an unmodified control peptide (Fig 1A). Western blots (WBs) of synthetic or PINK1-phosphorylated pS65-Ub confirmed their selectivity for modified Ub only (Fig 1B and C). Similar to monomeric pS65-Ub, antibodies also detected poly-Ub chains that had been phosphorylated by PINK1 wild-type (WT), but not kinase-dead (KD) mutant (Figs 1D and EV1A). The slight preference for K48 over K63 linkage might be explained by the proximity of S65 to K63 (Fig 1E) and distinct topologies of the respective chains (Fig EV1B). K63 linkage could equally affect PINK1 phosphorylation of S65 or binding of the pS65-Ub antibodies. Click here to expand this figure. Figure EV1. anti-pS65-Ub antibodies detect PINK1-phosphorylated poly-Ub chains Untagged K48- and K63-linked poly-Ub chains (n = 2–7) were incubated with recombinant MBP-PINK1 WT or KD for the indicated times in vitro prior to WB. Both, pS65-Ub#1 and #2 similarly recognized phosphorylated K48-linked poly-Ub chains, whereas pS65-Ub#2 appeared to react stronger with phosphorylated K63-linked Ub conjugates. Blots were probed with MBP and total Ub antibodies to show equal loading. Axial and lateral views of a K48-linked or K63-linked (8-mers) poly-Ub chains show the different topologies. In the more closed and compacted structure of K48-linked poly-Ub chains, S65 residues of the individual Ub moieties are faced outwards. However, in the rather extended conformation of K63-linked chains with larger spacing between Ub moieties, S65 residues are positioned more toward the inside of the chain. Each Ub moiety and corresponding S65 is shown in a different color. pS65 is shown in VdW representation. K48-Gly76 and K63-Gly76 linkages are shown in licorice rendering colored by atom type. Download figure Download PowerPoint Figure 1. Novel antibodies selectively detect phosphorylated Ub monomers and poly-Ub chains in vitro Dot blots were performed with the immunogen (pSer65, amino acids 59–71 of Ub: YNIQKE[pS]TLHLVL) and a corresponding, non-phosphorylated peptide (Ser65) and probed with sera and affinity-purified antibodies pS65-Ub#1 and #2. Western blots (WBs) with increasing amounts of Ub and synthetic pS65-Ub (both N-terminally biotinylated, N-biotin) were probed with anti-pS65-Ub antibodies or streptavidin coupled to horseradish peroxidase (SA-HRP) as indicated. Monomeric N-biotin-Ub was incubated with recombinant MBP-tagged PINK1 kinase wild-type (WT) or kinase-dead (KD) mutant for the indicated times in vitro prior to WB. N-biotin-tagged K48- and K63-linked poly-Ub chains (n = 2–7) were incubated with or without MBP-tagged PINK1 WT in vitro prior to WB. Membranes were also probed with SA-HRP and K48 or K63 linkage-specific anti-Ub antibodies as controls, which did not discriminate between phosphorylated or non-phosphorylated Ub conjugates. Ribbon diagram of the pS65-Ub monomer. pSer65 is shown in VdW representation, while all seven lysine (Lys) residues are shown in stick/licorice, both colored by atom type. Download figure Download PowerPoint To determine a potential cross-reactivity of the pS65-Ub antibodies with Parkin, the other substrate of PINK1, we performed further experiments. Direct comparison of S65-phosphorylated Ub and Parkin peptides showed a minor detection of pS65-Parkin in addition to pS65-Ub, with one of two antibodies (pS65-Ub#2, Fig EV2A). In vitro phosphorylation of Parkin with PINK1 confirmed some cross-reactivity of pS65-Ub#2 with phosphorylated full-length Parkin. However, compared to pS65-Ub (Fig 1C), pS65-Parkin was detected only after longer kinase reactions (60 min) and longer exposures (Fig EV2B). As this could be consistent with the idea that Ub is the preferred substrate for PINK1 over Parkin, we generated equimolar amounts of both phosphorylated proteins (in a 2-day kinase reaction to ensure complete modification of all Ub and Parkin molecules). In this setting, pS65-Ub#2 showed a stronger signal for pS65-Parkin compared to pS65-Ub (Fig EV2C). Click here to expand this figure. Figure EV2. pS65-Ub#1 is highly specific, whereas pS65-Ub#2 recognizes both PINK1-modified substrates, pS65-Ub and pS65-Parkin Dot blots were performed with the immunogen (pSer65-Ub, amino acids 59–71 of Ub: YNIQKE[pS]TLHLVL) and a phosphorylated Parkin peptide (pSer65-Parkin, amino acids 60–71 of Parkin: DLDQQ[pS]IVHIVQ) and probed with affinity-purified antibodies pS65-Ub#1 and #2. While pS65-Ub#1 was specific to the phosphorylated Ub peptide, pS65-Ub#2 showed reactivity toward Ub and Parkin phospho-peptides. Recombinant untagged Parkin was incubated with MBP-tagged PINK1 WT or KD for the indicated times in vitro. PINK1-phosphorylated Parkin is detected by pS65-Ub#2, but not #1, although at much lower level than pS65-Ub (compared to Fig 1C). Equimolar amounts of recombinant N-biotin-Ub or untagged Parkin were incubated with MBP-PINK1 for 2 days to achieve complete phosphorylation as shown by phos-tag gel electrophoresis. Under these conditions, pS65-Ub#1 specifically detected pS65-Ub, while pS65-Ub#2 cross-reacted somewhat stronger with pS65-Parkin. Both antibodies did not recognize unphosphorylated Ub or Parkin, respectively. Download figure Download PowerPoint Cellular pS65-Ub signal is induced by stress and amplified by functional Parkin Next, we tested pS65-Ub antibodies on samples from human HeLa cells. Parental cells, which lack detectable amounts of endogenous Parkin, or cells stably overexpressing native, untagged Parkin, were treated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (Fig 2A). WB of lysates revealed almost no signal in untreated cells, but a robust increase in pS65-Ub signal with mitochondrial damage over time. Presence of functional Parkin WT amplified the pS65-Ub levels, likely through enhanced formation of poly-Ub chains that in turn serve as substrates for PINK1. While pS65-Ub signal steadily increased over longer times CCCP treatment in cells without Parkin, it never reached levels observed in the presence of functional Parkin. Here, a peak was reached already around 4 h upon CCCP treatment, after which pS65-Ub levels decreased possibly due to substrate degradation. In addition to the prominent high molecular weight (HMW) smear, we also detected monomers and dimers of pS65-modified Ub. Interestingly, monomeric pS65-Ub accumulated more in HeLa cells lacking Parkin and appeared to be utilized in Parkin overexpressing cells for the formation of HMW conjugates. Immunofluorescence (IF) staining of HeLa cells expressing GFP-Parkin corroborated lack of pS65-Ub signal at basal conditions and robust induction upon CCCP treatment. Moreover, IF revealed an exclusive co-localization of pS65-Ub with Parkin on mitochondria as expected (Fig 2B and C). Figure 2. Cellular pS65-Ub signal is stress-induced and amplified by functional Parkin on mitochondria A. Parental HeLa cells and cells stably overexpressing untagged, native human Parkin WT were treated with CCCP for the indicated times, and lysates were analyzed by WB as indicated. HeLa cells without or with functional Parkin both showed an increase in pS65-Ub signal over time of CCCP treatment; however, the signal was strongly amplified in the presence of Parkin. Inlay WB (16%) for the stronger pS65-Ub#2 antibody shows phosphorylated mono- and di-Ub (as indicated by one or two black circles). Note the increase of both species over time in parental cells. In the presence of Parkin, these appear to be utilized for conjugation of higher molecular weight (HMW) phosphorylated poly-Ub chains as indicated by enhanced overall total Ub levels. B, C. HeLa cells stably overexpressing GFP-tagged Parkin (green) were treated with CCCP as indicated, fixed and stained with anti-TOM20 (mitochondria, cyan) and anti-pS65-Ub#1 (B) or anti-pS65-Ub#2 (C) (red) as well as Hoechst (nucleus, blue). Upon CCCP treatment, pS65-Ub signal forms on mitochondria in the presence of functional Parkin. Scale bars, 10 μm. Download figure Download PowerPoint pS65-Ub#2 recognizes both PINK1 kinase products, pS65-Ub and pS65-Parkin To further investigate the PINK1- and Parkin-dependent amplification of pS65-Ub signal, we stably expressed a catalytically dead Parkin C431S mutant in HeLa cells. Similar to parental cells that lack Parkin, expression of this inactive variant did not result in amplification of pS65-Ub signal and appearance of HMW species as compared to cells expressing Parkin WT (Fig EV3A). The Parkin C431S mutant, which traps the Ub moiety on the catalytic center in a stable oxyester, but not WT, was recognized by the antibody pS65-Ub#2 as a discrete doublet band upon CCCP treatment. The upper band of these was indeed sensitive to NaOH cleavage, suggesting the accumulation of Ub-charged pS65-Parkin C431S over time (Fig EV3B). Of note, pS65-Ub#2 did not recognize the Parkin S65A mutant, suggesting its specificity for the S65-phosphorylated form of Parkin (Fig EV3C). To exclude a major contribution of pS65-Parkin detection to the overall cellular signal observed with pS65-Ub#2, we studied GFP-Parkin WT and C431S HeLa cells. We confirmed that the pS65-Ub signal is amplified only in the presence of functional Parkin (Appendix Fig S1). Yet, after longer times of CCCP incubation (8 h), we noticed a very weak signal in the presence of non-functional C431S Parkin with the antibody pS65-Ub#1 and, to a stronger extent, with pS65-Ub#2. However, Parkin C431S did not translocate to damaged mitochondria and remained evenly distributed throughout the cell (Appendix Fig S1). The signal obtained with pS65-Ub#2 rather resembled the intracellular localization of mitochondria. Click here to expand this figure. Figure EV3. Cross-reactivity of the pS65-Ub#2 antibody with cellular pS65-Parkin Parental HeLa cells and cells stably expressing 3× FLAG-tagged Parkin WT or C431S mutants were treated with CCCP for the indicated times, lysed and analyzed by WB. In comparison with parental HeLa cells and with cells overexpressing the ligase-dead Parkin mutant C431S, cells overexpressing functional Parkin show strongly enhanced pS65-Ub signal. Similarly, enhanced overall ubiquitination is present in WT overexpressing cells as reflected by total Ub signal. In addition to pS65-Ub, pS65-Ub#2 also recognizes Parkin as observed with the inactive Parkin mutant C431S, where it labels two discrete bands, which likely correspond to pS65-Parkin (open arrowhead) and pS65-Parkin charged with Ub or pS65-Ub (filled arrowhead). HeLa cells stably expressing 3× FLAG-tagged Parkin C431S were incubated with 4 or 10 μM CCCP for 2 h, lysed and left untreated or treated with NaOH to strip off Ub from "Ub-charged" Parkin. Chemical cleavage of the Ub moiety from C431S by NaOH results in the collapse of the upper band (Ub-charged pS65-Parkin, filled arrowhead) into the lower band (pS65-Parkin, open arrowhead) as shown by FLAG and pS65-Ub#2 antibodies. HeLa cells were transiently transfected with FLAG-Parkin C431S or a C431S+S65A double mutant as an additional specificity control. Cells were challenged with CCCP, lysed, and left untreated or treated with NaOH. Of note, pS65-Ub#2 does only recognize a discrete band where Parkin WT but not the phospho-dead variant Parkin S65A was expressed. Download figure Download PowerPoint To finally confirm the nature of the cellular signal obtained with the pS65-Ub antibodies, we performed immunoprecipitation (IP) of denatured lysates. Both pS65-Ub antibodies pulled down increasing amounts of Ub conjugates over time following CCCP treatment from HeLa cells overexpressing Parkin WT (Appendix Fig S2A). Yet, Parkin was not detectable from either pS65-Ub IP (not shown). Reciprocal anti-FLAG IP, this time under non-denaturing, but stringent RIPA buffer conditions, pulled down substantial amounts of phosphorylated poly-Ub conjugates with 3× FLAG-tagged Parkin WT, but not C431S mutant (Appendix Fig S2B). Using an anti-FLAG antibody, Parkin protein did not show a major shift into HMW species (Appendix Fig S2B). This suggests that Parkin strongly binds to pS65-Ub chains rather than it is overtly modified by them. pS65-Ub is specific to mitochondrial damage in all cells Given that our novel tools allowed detection of a mitochondrial stress-induced signal, we aimed to corroborate the mitochondrial co-localization of pS65-Ub with Parkin. Subcellular fractionations from HeLa cells stably expressing 3× FLAG-Parkin WT or C431S identified stabilized PINK1 protein and functional Parkin in mitochondrial samples upon CCCP treatment (Fig 3A). In contrast to WT, Parkin C431S remained in the cytosol and was detected only with pS65-Ub#2 as the characteristic doublet band. In both cells, polymeric HMW pS65-Ub species accumulated in the mitochondrial fraction, at much higher levels in the presence of functional Parkin as seen with a total Ub antibody. However, monomeric pS65-Ub and dimeric pS65-Ub were exclusively detected in the cytoplasmic fraction and were much more abundant in the absence of functional Parkin. Figure 3. Levels of cellular pS65-Ub species are dependent on PINK1 kinase activity HeLa cells stably expressing 3× FLAG-tagged Parkin WT or C431S were treated for 2.5 h with CCCP and subjected to subcellular fractionation. Post-nuclear supernata
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