Abstract

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.

Highlights

  • The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive

  • We focused on sotrastaurin for further PKC family inhibitors (PKCi) experiments as our other data indicated there was an important role for the kinase activity beyond the decrease in BNIP3L seen with enzastaurin

  • As our data indicate a requirement of G-associated kinase (GAK) kinase activity for efficient mitophagy in mammalian cells, we examined the effect of targeting gakh-1 upon basal mitophagy in a C. elegans reporter line expressing mtRosella GFP-DsRed in body-wall muscle cells, following the concept of the IMLS reporter cell lines

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Summary

Introduction

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Elucidation of the molecular mechanisms of mitophagy has largely focused upon hereditary forms of PD and the role of the genes PINK1 and Parkin (PRKN) in mediating mitophagy in response to mitochondrial depolarisation[3] Such studies have shown that selective recognition of damaged mitochondria involves PINK1-mediated phosphorylation of ubiquitin and PRKN, leading to further ubiquitination of outer mitochondrial membrane proteins that are recognised by specific autophagy receptors that interact with LC3 and GABARAP proteins in the autophagy membrane to facilitate mitophagosome formation. Further characterisation of the mechanisms involved in PRKNindependent basal mitophagy pathways is needed to understand their role in normal physiology and disease development One such pathway involves HIF1α/hypoxia-dependent upregulation of the mitophagy receptor BNIP3 that has been well characterised for its role in the clearance of red blood cell mitochondria[10]. These kinases represent targets for the study and regulation of basal mitophagy

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