Redox Regulation of Akt Phosphorylation in PTEN-/- Mouse Embryonic Fibroblasts

Abstract

Over the years, studies have demonstrated the emerging roles of the superoxide anion (O2.-) as an essential signalling molecule. The involvement of O2.- in cell proliferation and cell growth and has been demonstrated in different systems. Moreover, there are accumulating evidence pointing to the anti-apoptotic role of O2.-. Our group has shown that an increase in intracellular O2.- endows tumour cells with a survival advantage against a variety of apoptotic triggers. In line with the pro-survival role of O2.-, our group recently demonstrated the role of O2.- in regulating the survival kinase Akt via an oxidative inhibition of PTEN by S-nitrosylation. During the course of this study, it was noticed that in mouse embryonic fibroblasts that do not express the tumour suppressor PTEN (MEFPTEN-/- cells), a decrease in the intracellular level of O2.- abrogated the hyperphosphorylation of Akt that was observed in these cells. Therefore, we hypothesize that O2.- may regulate the PI3K-Akt pathway not only through the inhibition of PTEN but also through a novel pathway that may be critical for the maintenance of the hyperphosphorylated Akt observed in MEFPTEN-/- cells.

In the current project, the PTEN-independent pathways involved in the regulation of Akt phosphorylation by O2.- in MEFPTEN-/- cells is investigated. Using diphenyleneiodonium chloride (DPI), the inhibitor for the O2.--producing NADPH oxidases and silencing of the Nox4 isoform by small interference RNA, we show that the reduction of intracellular level of O2.- in MEFPTEN-/- cells results in a decrease in the phosphorylation level of the otherwise hyperphosphorylated Akt kinase.

In investigating how O2.- regulates Akt phosphorylation level in MEFPTEN-/- cells, we provide evidence that the dephosphorylation of Akt is not dependent on any alterations in the level of PIP3, an important secondary messenger regulating Akt phosphorylation. Instead, the Akt molecules present in the cytosol are the primary target of this O2.- -mediated regulation, which is achieved via PP2A-dependent dephosphorylation. Furthermore, we also show that Akt oxidation status is inversely correlated with the level of intracellular O2.-. The proposed regulation of Akt phosphorylation by O2.- is possibly dependent on the shift between reduced-Akt and oxidized-Akt, which is associated with the susceptibility of Akt to the PP2A phosphatase.

In addition to the cytosolic regulation of Akt phosphorylation by O2.-, we have also reported NHE1 as a regulator of Akt phosphorylation at the membrane. We showed for the first time that NHE1 interacts directly with Akt. This interaction allows NHE1 to serve as an additional anchor point for Akt recruitment to the membrane. Moreover, complex formation between NHE1 and Akt is disrupted by a reduction in intracellular O2.- level, which further illustrates the importance of O2.- in regulating Akt phosphorylation.

Taken together, our data show that O2.- can regulate the level of Akt phosphorylation in various ways in MEFPTEN-/- cells. This suggests that in addition to be due to the elevated level of PIP3, hyperphosphorylation of Akt in PTEN-defective cells could be dependent on intracellular O2.- level as well.