Hydrogen peroxide is an intracellular mediator of the mitogenic response. Thus, exposure of cells to nontoxic concentrations of H2O2 stimulates proliferation, whereas growth arrest induced by contact inhibition is associated with a decrease in the intracellular concentration of H2O2. Furthermore, cells transiently produce H2O2 in response to growth stimuli such as platelet-derived growth factor (PDGF), and inhibition of this effect blocks downstream signaling by such stimuli. Although H2O2 is implicated in intracellular signaling, molecules that are directly targeted by H2O2 and thereby propagate the signal remain to be identified. It is unlikely that protein targets bind H2O2 specifically. However, certain protein cysteine residues exist as thiolate anions at neutral pH as a result of interaction with nearby positively charged amino acids, and these residues are more susceptible to oxidation by H2O2 than are protonated cysteines. Proteins that contain an ionized cysteine include members of the protein tyrosine phosphatase family. PTEN is a member of this family and reverses the action of PI 3-kinase by catalyzing the removal of the phosphate attached to the 3'-hydroxyl group of the PI inositol ring. By negatively modulating the PI 3-kinase?Akt signaling pathway, PTEN functions as an important tumor suppressor. The essential cysteine-124 residue of human PTEN is surrounded by three basic amino acid residues in the active site pocket Exposure of cells to H2O2 or to PDGF also results in the generation of 3'-phosphorylated phosphoinositides (PIs), which are important regulators of cell survival and proliferation. The extent of 3'-phosphorylation of PIs in a cell reflects the balance between the action of PI 3-kinase and that of the 3'-phosphatase PTEN. Our results show that H2O2 induces reversible inactivation of PTEN by mediating oxidation of the essential cysteine-124 and the formation by this residue of a disulfide with cysteine-71. The oxidized enzyme was reactivated by thioredoxin. PTEN was also oxidized by H2O2 produced in cells in response to PDGF. Our data suggest that the reversible inactivation of PTEN by H2O2 produced in response to PDGF and other growth stimuli is important for the accumulation of 3'-phosphorylated PIs, and that the uncontrolled generation of H2O2 associated with certain pathological conditions might contribute to cell proliferation by inhibiting PTEN function. As exemplified by cyclic nucleotides, timely elimination of intracellular mediator after completion of their functions is critical for cellular signaling. Thus, elimination as well as production of second messengers are process highly controlled following the occupancy of receptors This would seem especially true for H2O2, which is readily converted to deleterious hydroxyl radicals. Enzymes that are capable of eliminating H2O2 includes catalase and glutathione peroxidase, and peroxiredoxin (Prx). There are no evidence that the activities of the two commonly known enzymes catalase and glutathione peroxidase are regulated In addition, catalase is exclusively localized in the peroxisosmes. Prx is a novel family of peroxidases that are present in organisms from all kindoms. All Prx enzymes contain a conserved cysteine residue at the amino-terminal region, which is the primary site of oxidation by H2O2. Mammalian Prx exists as at least six isoforms, which can be divided into three subgroups, namely 2-Cys, atypical 2-Cys and 1-Cys subgroups . The 2-Cys members, which include two cytosolic enzymes Prx I and Prx II. When overexpressed in various cells, Prx enzymes efficiently reduced the intracellular level of H2O2 produced in the cells stimulated with platelet-derived growth factor and tumor necrosis factor-alpha (TNF-alpha), inhibited NF-kappaB activation induced by TNF-alpha, and blocked the apoptosis induced by ceramide, indicating that Prx enzymes serve as component of signaling cascades by removing H2O2. However, there is no evidence that the activity of Prx enzymes is regulated in response to changes in cellular environments as in the case of many other intracellular messenger metabolizing enzymes. We noticed that the amino acid sequences of the 2-Cys Prx subgroup members from human, rat, and mouse all contain the consensus sequence for phosphorylation by cyclin-dependnet kinases (Cdks), (Ser/Thr)-Pro-X-(Lys/Arg), whereas Prx V and Prx VI do not contain the consensus sequence. The putative phosphorylation site Thr, which corresponds to Thr 90 of human Prx I, is located 38 amino acids down from the amino-terminal conserved Cys for all four 2-Cys Prx members. To test whether Cdk can phosphorylate 2-Cys Prx enzymes, purified Prx I, II, III, and IV were subjected to kinase reaction by recombinant Cdc2 kinase/cyclinB complex in. Prx I was the far best substrate. To facilitate the monitoring of Prx I phosphorylation in cells, antibody specific to Prx I phosphorylated at Thr 90 was prepared by immunizing rabbits with a 13-residue phosphopeptide that corresponds to the sequence between residues 83 and 95 of Thr90-phosphorylated Prx I Using this phosphorylation-specific antibodies, we demonstrated that purified Prx I is phosphorylated at Thr90 by cyclin-dependent kinases (Cdks) including the cell division cycle 2 (Cdc2) kinase. The phosphorylation of Thr90 resulted in substantial reduction in the peroxiase activity of Prx I. In HeLa, HepG2, NIH3T3 cells Prx I phosphorylation occurrs in cells in mitotic phase but not in interphase. These results suggest that Cdc2 kinase, the Cdk that is activated in mitotic phase, encounters and phosphorylates Prx I after the nuclear envelop breaks down The Cdc 2 kinase-dependent inactivation of Prx I and the resulting accumulation of H2O2 are likely important processes for the progression of cell cycle.
