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

Molecular cell | 2018

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.

Pubmed ID: 29656925 RIS Download

Mesh terms: Enzyme 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

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This is a list of tools and resources that we have found mentioned in this publication.


PCNA (PC10) antibody (antibody)

RRID:AB_628110

This monoclonal antibody targets PCNA (PC10)

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PINK1 Antibody (antibody)

RRID:AB_2237092

This monoclonal antibody targets PINK1

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HRP/Anti-M13 Monoclonal Conjugate antibody (antibody)

RRID:AB_2616587

This monoclonal antibody targets PII (M13 phage)

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Parkin Antibody (antibody)

RRID:AB_10693040

This polyclonal antibody targets Parkin

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Anti-OPTN antibody (antibody)

RRID:AB_1079527

This polyclonal antibody targets OPTN

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IRDye 680RD Goat anti-Mouse IgG (H + L), 0.5 mg antibody (antibody)

RRID:AB_10956588

This unknown targets IRDye 680RD Goat anti-Mouse IgG (H + L) 0.5 mg

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Mitofusin 2 antibody [NIAR164] (antibody)

RRID:AB_10999860

This monoclonal antibody targets Mitofusin 2 antibody [NIAR164]

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CALCOCO2 antibody (antibody)

RRID:AB_1640255

This polyclonal antibody targets CALCOCO2

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Parkin (PRK8) antibody (antibody)

RRID:AB_628104

This monoclonal antibody targets Parkin (PRK8)

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MAP2 Monoclonal Antibody (MT-08) (antibody)

RRID:AB_1076853

This monoclonal antibody targets MAP2

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CISD1 antibody (antibody)

RRID:AB_2080268

This polyclonal antibody targets CISD1

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Monoclonal ANTI-FLAG® M2 antibody (antibody)

RRID:AB_262044

This monoclonal antibody targets FLAG

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beta-Actin (AC-15) antibody (antibody)

RRID:AB_1119529

This monoclonal antibody targets beta-Actin (AC-15)

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Anti-Rabbit IgG (H+L), HRP Conjugate antibody (antibody)

RRID:AB_430833

This unknown targets Rabbit IgG (H+L) HRP Conjugate

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SQSTM1/p62 (D10E10) Rabbit mAb (IF Preferred) antibody (antibody)

RRID:AB_10949900

This monoclonal antibody targets SQSTM1/p62 (D10E10) Rabbit mAb (IF Preferred)

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Tom20 (FL-145) Antibody (antibody)

RRID:AB_2207533

This polyclonal antibody targets Tom20 (FL-145)

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Anti-Mouse IgG (H+L), HRP Conjugate antibody (antibody)

RRID:AB_430834

This unknown targets Mouse IgG (H+L) HRP Conjugate

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