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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 DNA-dependent 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 AMP-dependent 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. Abbreviations: AXIN1: axin 1; 3-MA: 3-methyladenine; 5-FU: 5-fluorouracil; AA mutant: double alanine mutant (S192A, T284A) of PRKAG1; ACACA: acetyl-CoA carboxylase alpha; AICAR: 5-Aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ATM: ataxia telangiectasia mutated; ATR: ATM serine/threonine kinase; AV: autophagic vacuole; AVd: degradative autophagic vacuole; AVi: initial autophagic vacuole; BECN1: beclin 1; BSA: bovine serum albumin; CBS: cystathionine beta-synthase; CDK7: cyclin dependent kinase 7; CDKN1A: cyclin dependent kinase inhibitor 1A; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S transferase; H2AX/H2AFX: H2A.X variant histone; HBSS: Hanks balanced salt solution; IP: immunopurification; IR: ionizing radiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K9: mitogen-activated protein kinase kinase kinase 9; mRFP: monomeric red fluorescent protein; mCh: mCherry; MCM7: minichromosome maintenance complex component 7; MTORC1: mechanistic target of rapamycin kinase complex 1; NHEJ: non-homologous end joining; NRBP2: nuclear receptor binding protein 2; NTC: non-targeting control; NUAK1: NUAK family kinase 1; PBS: phosphate-buffered saline; PIK3AP1: phosphoinositide-3-kinase adaptor protein 1; PIK3CA: phosphatidylinositol-4,5-biphosphate 3-kinase catalytic subunit alpha; PIKK: phosphatidylinositol 3-kinase-related kinase; PRKAA: protein kinase AMP-activated catalytic subunit alpha; PRKAB: protein kinase AMP-activated non-catalytic subunit beta; PRKAG: protein kinase AMP-activated non-catalytic subunit gamma; PRKDC: protein kinase, DNA-activated, catalytic subunit; RLuc: Renilla luciferase; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TP53: tumor protein p53; TSKS: testis specific serine kinase substrate; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild type.

SUBMITTER: Puustinen P

PROVIDER: S-EPMC8386596 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na+,K+/ATPase ?1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the ?1 subunit was required for autophagy induction, since in ?1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (?1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition.IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the ?1 subunit of the Na+,K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in ?1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

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

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