GAK and PRKCD are positive regulators of PRKN-independent mitophagy

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.

DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion

“…A clear contrast between our results and the published literature on iron depletion is timing, as most studies have focused predominantly on signalling events following 18–24 h of chelation treatment (Oexle et al , 1999 ; Allen et al , 2013 ; Hara et al , 2020 ; Zhao et al , 2020 ; Munson et al , 2021 ). Specifically, temporal metabolomics revealed that iron depletion is a potent signal that induces alterations in cellular lipid recomposition within minutes.…”

“…Lysosomes appeared to retain their overall acidity upon DGAT1/2 inhibition, but whether fusion dynamics, trafficking or autophagosome‐lysosome reformation might be affected is unclear. In agreement with our discovery that lipid homeostasis modifies mitophagy, a recent study reported a role for two lipid‐binding kinases (GAK and PRKCD) in Parkin‐independent mitophagy, and their inactivation induced endolysosomal dysfunction independently of effects on acidity (Munson et al , 2021 ). Of interest, the accumulation of mitochondrial DAG is hypothesised to play a role here, which is related to oxidative stress (Cowell et al, 2019 ).…”

“…This raises several fundamental questions regarding the regulatory interplay between mitophagy and metabolism and the control of metabolism by iron homeostasis. For instance, most signalling studies investigating DFP‐induced mitophagy have focussed on timepoints corresponding to the peak of mitochondrial turnover (typically 24 h) (Allen et al , 2013 ; Hara et al , 2020 ; Zhao et al , 2020 ; Munson et al , 2021 ). Consequently, the metabolic events upon immediate iron depletion leading to mitophagy remain unexplored.…”

“…Basal mitophagy may also provide an additional means to supplement lysosomes with iron, thereby facilitating functional acidity (Yambire et al , 2019 ; Weber et al , 2020 ). Therapeutic iron chelators such as deferiprone (DFP) are potent inducers of mitophagy (Allen et al , 2013 ; Hara et al , 2020 ; Zhao et al , 2020 ; Munson et al , 2021 ), and the therapeutic induction of mitophagy using clinically approved chelators may also prove more tractable than targeting endogenous mitophagy receptors (Rosignol et al , 2020 ). Iron depletion induces mitophagy independently of PINK1/Parkin (Allen et al , 2013 ) and involves induction of mitochondrial ferritin (Hara et al , 2020 ), yet several key questions remain.…”

GAK and PRKCD kinases regulate basal mitophagy

“…Given the membrane dependence of the autophagy pathway, we focused specifically on proteins that contain domains known to interact with lipids (e.g., FYVE, PX, C1, C2 domains, etc.). Carrying out an image-based siRNA screen in U2OS cells expressing a mitochondrial matrix localized EGFP-mCherry reporter, we determined that the kinases GAK (cyclin G associated kinase) and PRKCD (protein kinase C delta) act as positive regulators of DFP-induced mitophagy [ 1 ]. We validated their effect on mitophagy through several means, including biochemical activity assays, Western blot, and proteomic-based approaches.…”