DNA-dependent protein kinase regulates lysosomal AMP-dependent protein kinase activation and autophagy

Abstract

Macroautophagy/autophagy is a central component of the cytoprotective cellular stress response. To enlighten stress-induced autophagy signaling, we screened a human kinome siRNA library for regulators of autophagic flux in MCF7 human breast carcinoma cells and identified the catalytic subunit of DNAdependent protein kinase PRKDC/DNA-PKcs as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed PRKDC upstream of the AMPdependent protein kinase (AMPK) complex and ULK1 kinase. In normal culture conditions, PRKDC interacted with the AMPK complex and phosphorylated its nucleotide-sensing γ1 subunit PRKAG1/AMPKγ1 at Ser192 and Thr284, both events being significantly reduced upon the activation of the AMPK complex. Alanine substitutions of PRKDC phosphorylation sites in PRKAG1 reduced AMPK complex activation without affecting its nucleotide sensing capacity. Instead, the disturbance of PRKDC-mediated phosphorylation of PRKAG1 inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with STK11 (serine/threonine kinase 11). Taken together, our data suggest that PRKDC-mediated phosphorylation of PRKAG1 primes AMPK complex to the lysosomal activation by STK11 in cancer cells thereby linking DNA damage response to autophagy and cellular metabolism.

DNA-PKcs: A Multi-Faceted Player in DNA Damage Response

“…The treatment of M059K cells with DNA-PKcs antisense oligonucleotides caused radiation-induced autophagy and radiosensitized the cells. Similar observations were also obtained by Puustinen et al (2020) study. Inactivation or depression of DNA-PKcs increases ionizing radiation-induced cell autophagy as well as occurrence of cell apoptosis.…”

Cell Metabolism and DNA Repair Pathways: Implications for Cancer Therapy

“…This finding suggests that DNA-PK is essential for activating AMPK under low energy levels as a result of glucose deprivation in mammalian cells. Similarly, another study confirmed that DNA-PKcs is a positive regulator of AMPK activity and was found to phosphorylate two residues on AMPKγ (S192 and T284) (Puustinen et al, 2020). Conversely, another study showed that an aging-related increase in DNA-PKcs activity led to decreased AMPK activity, via phosphorylation-mediated inhibition of Hsp90 chaperone activity toward AMPKαα2 (Park et al, 2017).…”

Cell Signaling and Translational Developmental Therapeutics

“… 117 , 118 , 119 The AMPK is itself regulated by phosphorylation, with the most notable regulators being Liver kinase B1 (LKB1) and ataxia-telangiectasia mutated (ATM). 120 , 121 , 122 , 123 , 124 , 125 LKB1 is often mutated in tumor cells, leading to dysregulation of energy sensing and autophagy regulation. In the nucleus ATM senses DNA damage and cytosolic ATM senses the levels of reactive oxygen species; ATM at both cellular locations phosphorylates and activates the AMPK.…”