2. Academic Validation
  3. Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics

Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics

  • Mol Cell. 2018 Apr 19;70(2):211-227.e8. doi: 10.1016/j.molcel.2018.03.012.
Alban Ordureau 1,  Joao A Paulo 1,  Wei Zhang 2,  Tim Ahfeldt 3,  Jiuchun Zhang 1,  Erin F Cohn 1,  Zhonggang Hou 1,  Jin-Mi Heo 1,  Lee L Rubin 3,  Sachdev S Sidhu 2,  Steven P Gygi 1,  J Wade Harper 4
Affiliations

Affiliations

  • 1 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
  • 2 Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, and Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto, ON M5S3E1, Canada.
  • 3 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02132, USA.
  • 4 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: [email protected].
PMID: 29656925 DOI: 10.1016/j.molcel.2018.03.012
Abstract

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin Ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to Mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and Mitophagy receptors via unphosphorylated chains.

Keywords

E3 ubiquitin ligase; PARKIN; PINK1; Parkinson’s disease; feed-forward mechanism; kinase; mitochondria; phosphorylation; proteomic; ubiquitylation.

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