Regulation and functions of protein kinase B in DNA damage signaling

Conserved from primitive metazoans to humans, protein kinase B (PKB or Akt) is a serine/threonine kinase with well-characterized functions in a number of essential cellular processes such as transcription, proliferation and survival. Its activity is regulated via phosphorylation of the conserved residues, Thr308 in the activation loop, and Ser473 in the hydrophobic motif. This results in a 10x and 1000x increase in PKB kinase activity, respectively. Clearly, deregulation of its activity can contribute to development of pathogenic states such as diabetes or cancer. Hence much recent PKB research has focused on the identification and characterization of the PKB hydrophobic motif Ser473 kinase.
We investigated the specific contributions of three PI3-kinase-like family members (PIKKs) in PKB Ser473 phosphorylation. These included DNA-dependent protein kinase (DNA-PK), mammalian target of rapamycin complex 2 (mTORC2) and ataxia-telangiectasia-mutated (ATM), as work from our and other laboratories proposed these kinases as PKB regulators. Stable inducible RNAi system was employed to specifically deplete TRex293 cells of respective kinases. We found that in growing cells (cycling cells cultured in fully supplemented medium), the integrity of mTORC2 is necessary for PKB Ser473 phosphorylation. When cells were starved (cultured in serum-free medium), and subsequently mitogen stimulated, both mTOR and DNA-PK contributed to the activation of PKB, which was reflected by PKB downstream signaling. Finally, DNA-PK was required for ?-IR-induced PKB phosphorylation/activation. We found that ablation of ATM by RNAi has no effect on PKB activity in any of the conditions tested. We concluded that mTOR and DNA-PK have overlapping and distinct physiological roles in regulating PKB activity, these being determined by the incitement or challenge to the cells.
Further, we investigated in more detail PKB activation following DNA damage. This required 3-phosphoinositide-dependent kinase 1 (PDK1) and DNA-dependent protein kinase (DNA-PK). Active PKB localized in the nucleus of ?-irradiated cells adjacent to DNA double-strand breaks, where it co-localized and interacted with DNA-PK. Levels of active PKB inversely correlated with DNA damage-induced apoptosis. Accordingly, a significant portion of p53- and DNA damage-regulated genes were misregulated in cells lacking PKB?. Lastly, PKB? knock-out mice showed impaired DNA damage-dependent induction of p21 and increased tissue apoptosis after single dose total body irradiation.
Our findings place PKB downstream of DNA-PK in the DNA damage response signaling cascade, where it provides a pro-survival signal, in particular by affecting transcriptional p21 regulation. Furthermore, this function is apparently restricted to the PKB? isoform.
Together, our results reveal a complex pattern of PKB regulation in response to various stimuli. This is achieved by PKB Ser473 phosphorylation being carried out by (at least) two different enzymes. Furthermore, this also possibly enables a context-dependent result of PKB activation, as we describe its specific roles in DNA damage signaling where active PKB promotes survival.