The NADPH oxidase subunit p22^phox inhibits the function of the tumor suppressor protein tuberin

Mutations in the von Hippel-Lindau (VHL) gene give rise to renal cell carcinoma. Reactive oxygen species, generated by Nox oxidases, are involved in tumorigenesis. We have previously demonstrated that in VHL-deficient cells, p22^phox-dependent Nox1 and Nox4 oxidases maintain hypoxia inducible factor-2α (HIF-2α) protein expression through an Akt-dependent translational pathway. Phosphorylation of tuberin, by Akt, results in its inactivation. Here we show that diphenyleneiodonium chloride, an inhibitor of Nox oxidases, and small-interfering RNA-mediated down-regulation of p22 ^phox inhibit Akt-dependent phosphorylation of tuberin and stabilizes tuberin protein levels in VHL-deficient renal carcinoma cells. p22 ^phox-mediated inactivation of tuberin is associated with an increase in ribosomal protein S6 kinase 1 and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) phosphorylation as well as HIF-2α stabilization. Importantly, we find that marked up-regulation of p22^phox in human renal cell carcinoma correlates with increased tuberin phosphorylation, decreased tuberin protein levels, and increased phosphorylation of 4E-BP1. Our data provide the first evidence that p22^phox-based Nox oxidases maintain HIF-2α protein expression through inactivation of tuberin and downstream activation of ribosomal protein S6 kinase 1/4E-BP1 pathway.