Sue Goo Rhee

The predominant enzymes responsible for elimination of hydrogen peroxide (H₂O₂) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H₂O₂-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr⁹⁰ by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I–IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H₂O₂ by mechanisms involving the SH3 domains of the kinases and the Pro²⁹³PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr²³¹ and Tyr³⁸⁶. Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.Antioxid. Redox Signal. 7, 619–626.

Phospholipase C-γ1 (PLC-γ1) contains two tandem Src homology 2 (SH2) domains. The NH₂-terminal SH2 domain has been known to mediate the binding of PLC-γ1 to receptor protein tyrosine kinases, which then activate PLC-γ1 via phosphorylation at Y783. We now show that the phosphorylated Y783 residue (pY783) associates with the COOH-terminal SH2 domain [SH2(C)] within the same molecule of PLC-γ1. The specificity of this intramolecular interaction is demonstrated in several ways. The mutation of SH2(C), but not of the NH₂-terminal SH2 domain, exposes pY783 and makes it available for binding by anti-pY783 antibodies, for intermolecular association with a GST fusion protein containing the tandem SH2 domains of PLC-γ1 and for dephosphorylation by phosphatases. The intramolecular interaction between pY783 and SH2(C) induces a rearrangement of surface charge such that PLC-γ1 molecules phosphorylated at Y783 are retained more strongly by heparin resins than are unphosphorylated molecules. Finally, the intramolecular interaction of pY783 with SH2(C) results in activation of phospholipase activity. Our results thus clarify the molecular mechanism of PLC-γ1 activation, revealing the specific function of pY783 and the distinct roles of the two SH2 domains in this process.

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 .

Phospholipase C-γ2 (PLC-γ2) plays an important role in B-cell signaling. Phosphorylation of various tyrosine residues of PLC-γ2 has been implicated in regulation of its lipase activity. With the use of antibodies specific for each of the putative phosphorylation sites, we have now shown that PLC-γ2 is phosphorylated on Y753, Y759, and Y1217 in response to engagement of the B-cell receptor in Ramos cells, as well as in murine splenic B cells. In cells stimulated maximally via this receptor, the extent of phosphorylation of Y1217 was three times that of Y753 or of Y759. Stimulation of Jurkat T cells or platelets via their immunoreceptors also elicited phosphorylation of Y753 and Y759 but not that of Y1217. A basal level of phosphorylation of Y753 was apparent in unstimulated lymphocytes. The extent of phosphorylation of Y753 and Y759, but not that of Y1217, correlated with the lipase activity of PLC-γ2. Examination of the effects of various pharmacological inhibitors and of RNA interference in Ramos cells suggested that Btk is largely, but not completely, responsible for phosphorylation of Y753 and Y759, whereas phosphorylation of Y1217 is independent of Btk. Finally, phosphorylation of Y1217 and that of Y753 and Y759 occurred on different PLC-γ2 molecules.