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Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox

Significance Although the three Akt isoforms share mechanisms of activation and exhibit overlaps in their downstream signaling pathways, significant differences are now being uncovered. Here, we show that among Akt isoforms, Akt3 preferably phosphorylates p47phox and activates NADPH oxidase, resulting in robust induction of reactive oxygen species (ROS). Akt3-induced ROS activate the DNA damage response and upregulate p53 expression. Consequently, the proliferation rate of Akt3-expressing cells is reduced, an effect reversed by p53 loss. In cancer, Akt3 expression correlates with the abundance of p53; however, tumors can adapt to high Akt3 activity by inactivating the DNA damage response. These findings reveal differences in the regulation of ROS by Akt isoforms, which may be exploited therapeutically for Akt3-driven cancers. Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53−/− and INK4a−/−/Arf−/− mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47phox−/− MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox. In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.