A role of DNA-dependent protein kinase for the activation of AMP-activated protein kinase in response to glucose deprivation

Abstract

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an essential role in double-strand break repair by initially recognizing and binding to DNA breaks. Here, we show that DNA-PKcs interacts with the regulatory γ1 subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that has been proposed to function as a "fuel gauge" to monitor changes in the energy status of cells and is controlled by the upstream kinases LKB1 and Ca²⁺/calmodulin-dependent kinase kinase (CaMKK). In co-immunoprecipitation analyses, DNA-PKcs and AMPKγ1 interacted physically in DNA-PKcs-proficient M059K cells but not in DNA-PKcs-deficient M059J cells. Glucose deprivation-stimulated phosphorylation of AMPKα on Thr172 and of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, is substantially reduced in M059J cells compared with M059K cells. The inhibition or down-regulation of DNA-PKcs by the DNA-PKcs inhibitors, wortmannin and Nu7441, or by DNA-PKcs siRNA caused a marked reduction in AMPK phosphorylation, AMPK activity, and ACC phosphorylation in response to glucose depletion in M059K, WI38, and IMR90 cells. In addition, DNA-DNA-PKcs(-/-) mouse embryonic fibroblasts (MEFs) exhibited decreased AMPK activation in response to glucose-free conditions. Furthermore, the knockdown of DNA-PKcs led to the suppression of AMPK (Thr172) phosphorylation in LKB1-deficient HeLa cells under glucose deprivation. Taken together, these findings support the positive regulation of AMPK activation by DNA-PKcs under glucose-deprived conditions in mammalian cells.

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

“…Supporting our findings, the association between DNA-PKcs and AMPK has been identified by two previous studies. Amatya and coworkers have demonstrated an interaction between DNA-PKcs and AMPKg using yeast 2-hybrid method and co-immunoprecipitation from mammalian cells (Amatya, Kim et al, 2012), while Lu and coworkers have verified this interaction in mammalian cells by co-immunoprecipitation and immunocytochemistry (Lu, Tang et al, 2016). In line with our data, these studies have also identified DNA-PKcs as a positive regulator of AMPK activity, however, without addressing the underlying mechanism.…”

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…response to glucose-free conditions (59). However, although CRY1 is a substrate for AMPK, this is unlikely to be the mechanism in this case, because DNA-PK activates AMPK (rather than inhibiting its activity) and because the Ser-588 site on CRY1 is part of an SQ motif, which is not predicted to be a phosphorylation site for AMPK.…”

Beyond DNA Repair: DNA-PK Function in Cancer

“…AMP-activated protein kinase (AMPK) is an energy sensor that acts as a metabolic master switch influencing numerous processes [81]. DNA-PKcs interacts with the regulatory λ1 subunit of AMPK, and glucose-deprivation stimulated phosphorylation of AMPK is decreased in DNA-PKcs deficient cells [82]. Further, DNA-PKcs inhibition or depletion resulted in reduced AMPK phosphorylation and activation in response to glucose deprivation, suggesting that DNA-PKcs positively regulates AMPK phosphorylation and activation in glucose-deprived conditions.…”