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Cited by 806Parkinson's Disease Mechanisms and TreatmentsUbiquitin and proteasome pathwaysAutophagy in Disease and Therapy

Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

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Kazlauskaite, Agne, Kondapalli, Chandana, Gourlay, Robert, Campbell, David G., Ritorto, Maria Stella, Hofmann, Kay, Alessi, Dario R., Knebel, Axel +2 more·2014·Biochemical Journal
DOI 10.1042/bj20140334PMC PMC4000136
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Abstract

We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease.

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  • Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane and phosphorylates ubiquitin and PRKN at Ser65.

    “Upon mitochondrial depolarization, PINK1 stabilizes on the outer mitochondrial membrane, where it phosphorylates ubiquitin (Ub) and PRKN at Ser65 [5–10].”

    From miRNA family miR-29 inhibits PINK1-PRKN signaling via ATG9A (2026) · cites this paper as [6]

    The Introduction says PINK1 becomes stabilized at mitochondria after membrane depolarization, and the Discussion’s Figure 9 model says activated PINK1 phosphorylates both Parkin (PRKN) and ubiquitin at Ser65. The copy does not specify that PINK1 stabilizes on the outer mitochondrial membrane, so that location is more specific than the cited paper supports. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Phosphorylation of ubiquitin and PRKN at Ser65 by PINK1 is required for activation of the PINK1-PRKN pathway.

    “Both events are required for the activation of the pathway.”

    From miRNA family miR-29 inhibits PINK1-PRKN signaling via ATG9A (2026) · cites this paper as [6]

    The Results section on the dual requirement (page not provided) says phosphorylation of both Parkin and ubiquitin at Ser65 is required for optimal Parkin E3 ligase activation. It also reports that only mutation of both sites completely abolishes activity. The claim omits the 'optimal' qualifier and overstates the evidence by saying both events are required for activation at all. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

  • PINK1 also phosphorylates ubiquitin at Ser65 (pUbS65) and PINK1-dependent phosphorylation of ubiquitin is required for translocation and activation of Parkin to initiate mitophagy.

    “Recently, several groups discovered that PINK1 also phosphorylates ubiquitin at Ser65 (pUbS65) and PINK1-dependent phosphorylation of ubiquitin is required for translocation and activation of Parkin to initiate mitophagy [16–18].”

    From miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1) (2016) · cites this paper as [17]

    The Introduction and Discussion show that PINK1 phosphorylates ubiquitin at Ser65 and that phosphorylated ubiquitin activates Parkin E3 ligase. The Introduction reports that PINK1 is required for Parkin recruitment to mitochondria, but our copy does not establish that ubiquitin phosphorylation is required for that translocation or for initiating mitophagy. No page numbers are available in the indexed passages. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

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Authors
Kazlauskaite, A., Kondapalli, C., Gourlay, R., Campbell, D. G., Ritorto, M. S., Hofmann, K., Alessi, D. R., Knebel, A., Trost, M., & Muqit, M. M. K.
Affiliations
  • University of Dundee (GB)
  • University of Cologne (DE)
Corresponding author
  • Kazlauskaite, Agne
  • Kondapalli, Chandana
  • Gourlay, Robert
  • Knebel, Axel
  • Trost, Matthias
ORCID
Venue
Biochemical Journal
Publisher
Portland Press Ltd.
Published
2014-04-25
Volume
460
Issue
1
Pages
127-141
ISSN
  • 0264-6021 (ISSN-L)
  • 1470-8728
DOI
10.1042/bj20140334
PMC
PMC4000136
OpenAlex
W2087592289
Cited by
806
References
4
Citations by year
2026: 38 · 2025: 40 · 2024: 72 · 2023: 46 · 2022: 68 · 2021: 83 · 2020: 75 · 2019: 54 · 2018: 73 · 2017: 65 · 2016: 81 · 2015: 75 · 2014: 35
FWCI
55.61
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemical Journal (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Kölner Universitäts PublikationsServer (Universität zu Köln) (publishedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
  • Newcastle University ePrints (Newcastle Univesity)
Funders
  • Michael J. Fox Foundation for Parkinson's Research
  • Pfizer
  • AstraZeneca
  • GlaxoSmithKline
  • University of Dundee
  • Merck KGaA
  • Wellcome Trust (101022, 089698, 101022/Z/13/Z)
  • Parkinson's UK (H-0901, H-1101)
  • University College London
  • Medical Research Council (MC_G1000735)
Topics
  • Health Sciences › Medicine › Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
Keywords
PINK1, Parkin, ubiquitin phosphorylation, mitochondrial depolarization, mitochondrial quality control, Parkinson's disease, ubiquitin-like domain, CCCP, phosphoproteomics
MeSH terms
PTEN-Induced Putative Kinase, Amino Acid Sequence, Humans, Molecular Sequence Data, Phosphorylation, Protein Kinases, Serine, Ubiquitin, Ubiquitin-Protein Ligases, HEK293 Cells

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