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Mol Cell DOI:10.1016/j.molcel.2018.03.012

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

Publication TypeJournal Article
Year of Publication2018
AuthorsOrdureau, A, Paulo, JA, Zhang, W, Ahfeldt, T, Zhang, J, Cohn, EF, Hou, Z, Heo, J-M, Rubin, LL, Sidhu, SS, Gygi, SP, J Harper, W
JournalMol Cell
Volume70
Issue2
Pages211-227.e8
Date Published2018 04 19
ISSN1097-4164
KeywordsEnzyme Activation, HeLa Cells, Human Embryonic Stem Cells, Humans, Kinetics, Mitochondrial Degradation, Mitochondrial Membranes, Neural Stem Cells, Neurogenesis, Neurons, Phenotype, Phosphorylation, Protein Kinases, Proteomics, Signal Transduction, Ubiquitin-Protein Ligases, Ubiquitination, Voltage-Dependent Anion Channel 1
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.

DOI10.1016/j.molcel.2018.03.012
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/29656925?dopt=Abstract

Alternate JournalMol. Cell
PubMed ID29656925
PubMed Central IDPMC5910199
Grant ListK01 DK098285 / DK / NIDDK NIH HHS / United States
R01 GM067945 / GM / NIGMS NIH HHS / United States
R01 GM095567 / GM / NIGMS NIH HHS / United States
R37 NS083524 / NS / NINDS NIH HHS / United States