GAK and PRKCD kinases regulate basal mitophagy

Abstract

The removal of mitochondria in a programmed or stress-induced manner is essential for maintaining cellular homeostasis. To date, much research has focused upon stress-induced mitophagy that is largely regulated by the E3 ligase PRKN, with limited insight into the mechanisms regulating basal "housekeeping" mitophagy levels in different model organisms. Using iron chelation as an inducer of PRKN-independent mitophagy, we recently screened an siRNA library of lipid-binding proteins and determined that two kinases, GAK and PRKCD, act as positive regulators of PRKN-independent mitophagy. We demonstrate that PRKCD is localized to mitochondria and regulates recruitment of ULK1-ATG13 upon induction of mitophagy. GAK activity, by contrast, modifies the mitochondrial network and lysosomal morphology that compromise efficient transport of mitochondria for degradation. Impairment of either kinase in vivo blocks basal mitophagy, demonstrating the biological relevance of our findings.Abbreviations: CCCP: carbonyl cyanide-m-chlorophenyl hydrazone; DFP: deferiprone; GAK: cyclin G associated kinase; HIF1A: hypoxia inducible factor 1 subunit alpha; PRKC/PKC: protein kinase C; PRKCD: protein kinase C delta; PRKN: parkin RBR E3 ubiquitin protein ligase.

Keywords: Cyclin-G-associated kinase; GAK; PKC; PRKCD; PRKN; mitophagy; protein kinase C.

Figure 1.

The regulation of PRKN-independent mitophagy by GAK and PRKCD. Several subtypes of autophagy and mitophagy pathways exist, including PRKN-independent mitophagy (DFP-induced), PRKN-dependent mitophagy (CCCP-induced), and starvation-induced autophagy (EBSS-induced). PRKCD localizes to mitochondria independent of its C1 or C2 domains and becomes degraded upon induction of mitophagy pathways. Recruitment of ATG13-ULK1-RB1CC1/FIP200-ATG101 to phagophore initiation sites upon induction of PRKN-independent mitophagy is blocked by PRKCD siRNA (siPRKCD) or PRKC kinase inhibitors (PRKCi). Treatment with GAK siRNA (siGAK) or GAK kinase inhibitor (GAKi) causes mitochondrial clustering and an accumulation of swollen multi-lamellar lysosomes. In vivo examination of the hindbrain region of zebrafish (D. rerio) demonstrates basal mitophagy, which is reduced by dual knockout of prkcda and prkcdb (homologs of PRKCD). Similarly, RNAi-mediated depletion of gakh-1 (GAK homolog) in worms (C. elegans) reduces basal mitophagy levels in muscle cell walls.