DNA-PKcs-mediated phosphorylation of AMPKγ1 regulates lysosomal AMPK activation by LKB1

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 (DNA-PKcs) as a positive regulator of basal and DNA damage-induced autophagy. Analysis of autophagy-regulating signaling cascades placed DNA-PKcs upstream of the AMP-dependent protein kinase (AMPK) and ULK1 kinase. In normal culture conditions, DNA-PKcs interacted with AMPK and phosphorylated its nucleotide-sensing {gamma}{gamma}1 subunit at Ser-192 and Thr-284, both events being significantly reduced upon AMPK activation. Alanine substitutions of DNA-PKcs phosphorylation sites in AMPK{gamma}{gamma}1 reduced AMPK activation without affecting its nucleotide sensing capacity. Instead, the disturbance of DNA-PKcs-mediated phosphorylation of AMPK{gamma}{gamma} inhibited the lysosomal localization of the AMPK complex and its starvation-induced association with LKB1. Taken together, our data suggest that DNA-PKcs-mediated phosphorylation of AMPK{gamma}{gamma} primes AMPK complex to the lysosomal activation by LKB1 thereby linking DNA damage response to autophagy and cellular metabolism.