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

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.

Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy

“…Hence, ubiquitination on a conserved lysine on the periphery of the hydrophobic groove of BAK significantly impaired the ability of BAK to permeabilise liposomes and mediate cytochrome c release. The non‐degradative ubiquitination of BAK was supported by the predominantly K11‐linked ubiquitin chains, consistent with the profile of atypical linkages catalysed by Parkin during mitophagy (Cunningham et al , ) and also a recent study reporting that endogenous Parkin does not promote significant proteasomal turnover of mitochondrial substrates (Ordureau et al , ). Ubiquitination of BAK by the E3 ligase HERC1 was shown to promote its proteasomal degradation following irradiation of papillomavirus‐infected cells (Holloway et al , ).…”

“…However, this was in the absence of detectable BAX ubiquitination either prior to or after induction of mitochondrial depolarisation, even under conditions of proteasomal inhibition. Although we cannot exclude degradation of BAX by alternative mechanisms, our data suggest that ubiquitination of VDAC2, an early and selective substrate of Parkin (Ordureau et al , ), may also contribute to the defect in BAX mitochondrial association. Furthermore, Parkin limited cytochrome c release in the absence of BAX, suggesting that the inactivation of BAK is also a key function of Parkin.…”

“…BAX interacts with voltage‐dependent anion channel (VDAC) 2 at mitochondria and VDAC2 promotes BAX apoptotic activity (Yamagata et al , ; Ma et al , ). VDAC2 is one of the most prominent substrates of Parkin (Ordureau et al , ), suggesting that its ubiquitination could be responsible for the inability of BAX to mediate cytochrome c release. Consistent with this, the deubiquitination of VDAC2 observed following MG132 treatment correlated with the partial rescue of BAX localisation and cytochrome c release (Fig D).…”

“…Consistent with this, the deubiquitination of VDAC2 observed following MG132 treatment correlated with the partial rescue of BAX localisation and cytochrome c release (Fig D). It is important to note that Parkin‐conjugated ubiquitin chains are commonly non‐degradative (Ordureau et al , ) and so BAX inhibition may not be due to VDAC2 degradation, but rather steric hindrance of the interaction between BAX and ubiquitinated VDAC2. Nevertheless, BAX‐mediated apoptotic activity is suppressed by Parkin in the absence of significant BAX ubiquitination.…”

“…A recent study by Ordureau et al revealed that endogenous Parkin has a hierarchy of substrates based on the abundancy of their ubiquitinated forms in response to mitochondrial damage (Ordureau et al , ). To confirm that BAK was a substrate of endogenous Parkin, we analysed the SH‐SY5Y neuroblastoma cell line (Jiang et al , ; Vives‐Bauza et al , ).…”