Regulation of protein kinase B (PKB/Akt) by DNA-dependent protein kinase (DNA-PK) under physiological conditions

The serine/threonine protein kinase B (PKB/Akt) is a downstream effector of phosphatidylinositol 3-kinase (PI3K) and a major regulator of a variety of cellular processes, including metabolism, transcription, survival, proliferation, and growth. PKB is activated by several stimuli, including hormones, growth factors, cytokines and, as recently reported, also by DNA damage. Activation of PKB requires phosphorylation at two key regulatory sites: Thr308 and Ser473 (of PKBa). Phosphorylation by 3-phosphoinositide-dependent kinase-1 (PDK1) occurs on Thr308 in the activation loop of PKB. The phosphorylation on Ser473 within a C-terminal hydrophobic motif leads to full activation of PKB and mediated by two members of the PI3K-related kinase (PIKK) family, mTOR/rictor complex (mTORC2) or DNA-dependent protein kinase (DNA-PK) in a stimulus specific manner. Insulin or growth factor induced PKB Ser473 phosphorylation is regulated by mTORC2. In contrast, DNA damage-induced phosphorylation of PKB Ser473 is mediated by DNA-PK.
The present study made use of genetically modified mouse models to investigate PKB regulation by DNA-PK, as phosphorylation of Ser473 may be stimulus-, signalling pathway- and/or cell type-specific. In this study, we investigated the role of DNA-PK in basal, insulin-induced, and DNA damage-induced phosphorylation of PKB Ser473 under physiological conditions. We report that DNA-PK phosphorylated PKB on Ser473 upon DNA damage induced by γ-irradiation in vivo. In contrast, DNA-PK was dispensable for insulin and growth factor-induced PKB activation. Interestingly, analysis of basal PKB Ser473 phosphorylation in DNA-PKcs−/− mice showed tissue-specific deregulation of the PKB/FoxO pathway. In particular, we provide evidence that persistent PKB hyperactivity in the thymus apparently contributes to spontaneous tumourigenesis in DNA-PKcs−/− mice. Lymphomagenesis could be prevented by the deletion of PKBa and implies deregulation of PKB in DNA-PKcs−/− thymi.
Deregulation of PKB is implicated in various types of cancer and PI3K/PKB pathway is one of the most deregulated pathways in human malignancies. Therefore PI3K/PKB pathway is a major focus of current efforts for the treatment of cancer. In the second part of the study we made use of differential activation of PKB by upstream kinases in response to specific stimuli as a tool to dissect the mode of action of a small molecule inhibitor BBD130.