The tumor suppressor PTEN regulates cell migration, growth, and survival by removing the 3′-phosphate of phosphoinositides. Exposure of purified PTEN or of cells to H₂O₂ resulted in inactivation of PTEN in a time- and H₂O₂concentration-dependent manner. Analysis of various cysteine mutants, including mass spectrometry of tryptic peptides, indicated that the essential Cys¹²⁴ residue in the active site of PTEN specifically forms a disulfide with Cys⁷¹during oxidation by H₂O₂. The reduction of H₂O₂-oxidized PTEN in cells appears to be mediated predominantly by thioredoxin. Thus, thioredoxin was more efficient than glutaredoxin, glutathione, or a 14-kDa thioredoxin-like protein with regard to the reduction of oxidized PTEN in vitro. Thioredoxin co-immunoprecipitated with PTEN from cell lysates; and incubation of cells with 2,4-dinitro-1-chlorobenzene (an inhibitor of thioredoxin reductase) delayed the reduction of oxidized PTEN, whereas incubation with buthioninesulfoximine (an inhibitor of glutathione biosynthesis) did not. These results suggest that the reversible inactivation of PTEN by H₂O₂might be important for the accumulation of 3′-phosphorylated phosphoinositides and that the uncontrolled generation of H₂O₂ associated with certain pathological conditions might contribute to cell proliferation by inhibiting PTEN function.

Stimulation of cells with various peptide growth factors induces the production of phosphatidylinositol 3,4,5-trisphosphate (PIP ₃ ) through activation of phosphatidylinositol 3-kinase. The action of this enzyme is reversed by that of the tumor suppressor PTEN. With the use of cells overexpressing NADPH oxidase 1 or peroxiredoxin II, we have now shown that H ₂ O ₂ produced in response to stimulation of cells with epidermal growth factor or platelet-derived growth factor potentiates PIP ₃ generation and activation of the protein kinase Akt induced by these growth factors. We also show that a small fraction of PTEN molecules is transiently inactivated as a result of oxidation of the essential cysteine residue of this phosphatase in various cell types stimulated with epidermal growth factor, platelet-derived growth factor, or insulin. These results suggest that the activation of phosphatidylinositol 3-kinase by growth factors might not be sufficient to induce the accumulation of PIP ₃ because of the opposing activity of PTEN and that the concomitant local inactivation of PTEN by H ₂ O ₂ might be needed to increase the concentration of PIP ₃ sufficiently to trigger downstream signaling events. Furthermore, together with previous observations, our data indicate that peroxiredoxin likely participates in PIP ₃ signaling by modulating the local concentration of H ₂ O ₂ .

Mammalian cytosolic thioredoxin reductase (TrxR) has a redox center, consisting of Cys ⁵⁹ /Cys ⁶⁴ adjacent to the flavin ring of FAD and another center consisting of Cys ⁴⁹⁷ /selenocysteine (SeCys) ⁴⁹⁸ near the C terminus. We now show that the C-terminal Cys ⁴⁹⁷ -SH/SeCys ⁴⁹⁸ -Se ⁻ of NADPH-reduced enzyme, after anaerobic dialysis, was converted to a thioselenide on incubation with excess oxidized Trx (TrxS ₂ ) or H ₂ O ₂ . The Cys ⁵⁹ -SH/Cys ⁶⁴ -SH pair also was oxidized to a disulfide. At lower concentrations of TrxS ₂ , the Cys ⁵⁹ -SH/Cys ⁶⁴ -SH center was still converted to a disulfide, presumably by reduction of the thioselenide to Cys ⁴⁹⁷ -SH/SeCys ⁴⁹⁸ -Se ⁻ . Specific alkylation of SeCys ⁴⁹⁸ completely blocked the TrxS ₂ -induced oxidation of Cys ⁵⁹ -SH/Cys ⁶⁴ -SH, and the alkylated enzyme had negligible NADPH-disulfide oxidoreductase activity. The effect of replacing SeCys ⁴⁹⁸ with Cys was determined by using a mutant form of human placental TrxR1 expressed in Escherichia coli . The NADPH-disulfide oxidoreductase activity of the purified Cys ⁴⁹⁷ /Cys ⁴⁹⁸ mutant enzyme was 6% or 11% of that of wild-type rat liver TrxR1 with 5,5′-dithio bis (2-nitrobenzoic acid) or TrxS ₂ , respectively, as substrate. Disulfide formation induced by excess TrxS ₂ in the mutant form was 12% of that of the wild type. Thus, SeCys has a critical redox function during the catalytic cycle, which is performed poorly by Cys.

Production of reactive oxygen species, often by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, plays a role in the signaling responses of cells to many receptor stimuli. Here, we describe the function of the calcium-dependent, nonphagocytic NADPH oxidase Duox1 in primary human CD4⁺ T cells and cultured T cell lines. Duox1 bound to inositol 1,4,5-trisphosphate receptor 1 and was required for early T cell receptor (TCR)–stimulated production of hydrogen peroxide (H₂O₂) through a pathway that was dependent on TCR-proximal kinases. Transient or stable knockdown of Duox1 inhibited TCR signaling, especially phosphorylation of tyrosine-319 of ζ chain–associated protein kinase of 70 kilodaltons (ZAP-70), store-operated entry of calcium ions (Ca²⁺), and activation of extracellular signal–regulated kinase. The production of cytokines was also inhibited by knockdown of Duox1. Duox1-mediated inactivation of Src homology 2 domain–containing protein tyrosine phosphatase 2 promoted the phosphorylation of ZAP-70 and its association with the Src family tyrosine kinase Lck and the CD3ζ chain of the TCR complex. Thus, we suggest that activation of Duox1, downstream of proximal TCR signals, generates H₂O₂ that acts in a positive feedback loop to enhance and sustain further TCR signaling.