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Oxidative stress initiates signaling pathways, which protect from stress-induced cellular damage, initiate apoptosis, or drive cells into senescence or into tumorigenesis. Oxidative stress regulates the activity of the cell survival factor PKB, through the regulation of PtdIns(3,4,5)P₃ synthesis. Whether oxidative stress regulates other phosphoinositides to control PKB activation is not clear. Here we show that PtdIns5P is a redox-regulated second messenger. In response to hydrogen peroxide (H₂O₂), we measured an increase in PtdIns5P in cells derived from human osteosarcoma, U2OS (5-fold); breast tumors, MDA-MB-468 (2-fold); and fibrosarcoma, HT1080 (3-fold); and in p53-null murine embryonic fibroblasts (8-fold). In U2OS cells, the increase in H₂O₂-dependent PtdIns5P did not require mTOR, PDK1, PKB, ERK, and p38 signaling or PIKfyve, a lipid kinase that increases PtdIns5P in response to osmotic and oncogenic signaling. A reduction in H₂O₂-induced PtdIns5P levels by the overexpression of PIP4K revealed its role in PKB activation. Suppression of H₂O₂-induced PtdIns5P generation reduced PKB activation and, surprisingly, reduced cell sensitivity to growth inhibition by H₂O₂. These data suggest that inhibition of PIP4K signaling might be useful as a novel strategy to increase the susceptibility of tumor cells to therapeutics that function through increased oxidative stress.

Oxidative signaling and oxidative stress contribute to aging, cancer, and diseases resulting from neurodegeneration. Pin1 is a proline isomerase that recognizes phosphorylated substrates and regulates the localization and conformation of its targets. Pin1(-/-) mice show phenotypes associated with premature aging, yet mouse embryonic fibroblasts (MEFs) from these mice are resistant to hydrogen peroxide (H(2)O(2))-induced cell death. We found that the abundance of phosphatidylinositol-5-phosphate (PtdIns5P) was increased in response to H(2)O(2), an effect that was enhanced in Pin1(-/-) MEFs. Reduction of H(2)O(2)-induced PtdIns5P compromised cell viability in response to oxidative stress, suggesting that PtdIns5P contributed to the enhanced cell viability of Pin1(-/-) MEFs exposed to oxidative stress. The increased PtdIns5P in the Pin1(-/-) MEFs stimulated the expression of genes involved in defense against oxidative stress and reduced the accumulation of reactive oxygen species. Pin1 and PtdIns5P 4-kinases (PIP4Ks), enzymes that phosphorylate and thereby reduce the amount of PtdIns5P, interacted in a manner dependent on the phosphorylation of PIP4K. Although reintroduction of Pin1 into the Pin1(-/-) MEFs reduced the amount of PtdIns5P produced in response to H(2)O(2), in vitro assays indicated that the isomerase activity of Pin1 inhibited PIP4K activity. Whether this isomerise-mediated inhibition of PIP4K occurs in cells remains an open question, but the data suggest that the regulation of PIP4K by Pin1 may be complex.