GAK and PRKCD are positive regulators of PRKN-independent mitophagy

Abstract

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy in response to different stimuli 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 novel 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 is required for ULK1/ATG13 recruitment 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 PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy.

Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report

“…PRKCD, which localizes to mitochondria and is turned over by mitophagy, facilitates the recruitment of the autophagic markers ATG13 and ULK1 to mitochondria. In vivo data in zebrafish confirm the importance of PRKCD in mitophagy 44 . While the PRKCD targets that mediate this effect are unknown, Simonsen proposed that PRKCD may phosphorylate mitophagy receptors and recruit FIP200.…”

“…In vivo data in zebrafish confirm the importance of PRKCD in mitophagy. 44 While the PRKCD targets that mediate this effect are unknown, Simonsen proposed that PRKCD may phosphorylate mitophagy receptors and recruit FIP200. Alternatively, PRKCD may act in a manner analogous to PINK in PINK/Parkin-dependent mitophagy, which phosphorylates ubiquitinated outer mitochondrial proteins, leading to the recruitment of the autophagy machinery.…”

Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1

“…A recent study has reported that two protein kinases, cyclin G-associated kinase (GAK) and PRKCδ, are previously unknown regulators of Parkin-independent mitophagy and recruit ULK1 to autophagosome structures close to mitochondria ( 57 ). While we did not detect either GAK or PRKCδ, we did detect multiple kinases in our mitochondrial enriched fractions that were ubiquitylated including PRKCβ, PRKCε, and PRKCγ isoforms, and in future work, it will be important to determine whether these and other kinases recruited to mitochondria regulate AO-induced mitophagy in neurons (table S5).…”

Global ubiquitylation analysis of mitochondria in primary neurons identifies physiological Parkin targets following activation of PINK1

“…A recent study has reported that two protein kinases, Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCδ) are novel regulators of Parkin-independent mitophagy and recruit ULK1 to autophagosome structures close to mitochondria (Munson et al, 2020). Whilst we did not detect either GAK or PRKδ, we did detect multiple kinases in our mitochondrial enriched fractions that were ubiquitylated including PRKCβ, PRKCε and PRKC γ isoforms and in future work it will important to determine whether these and other kinases recruited to mitochondria regulate AO-induced mitophagy in neurons (Table S4).…”