Stimulation of various cells with growth factors results in a transient increase in the intracellular concentration of H 2 O 2 that is required for growth factor-induced protein tyrosine phosphorylation. The effect of H 2 O 2 produced in response to epidermal growth factor (EGF) on the activity of protein-tyrosine phosphatase 1B (PTP1B) was investigated in A431 human epidermoid carcinoma cells. H 2 O 2 inactivated recombinant PTP1B in vitro by oxidizing its catalytic site cysteine, most likely to sulfenic acid. The oxidized enzyme was reactivated more effectively by thioredoxin than by glutaredoxin or glutathione at their physiological concentrations. Oxidation by H 2 O 2 prevented modification of the catalytic cysteine of PTP1B by iodoacetic acid, suggesting that it should be possible to monitor the oxidation state of PTP1B in cells by measuring the incorporation of radioactivity into the enzyme after lysis of the cells in the presence of radiolabeled iodoacetic acid. The amount of such radioactivity associated with PTP1B immunoprecipitated from A431 cells that had been stimulated with EGF for 10 min was 27% less than that associated with PTP1B from unstimulated cells. The amount of iodoacetic acid-derived radioactivity associated with PTP1B reached a minimum 10 min after stimulation of cells with EGF and returned to base line values by 40 min, suggesting that the oxidation of PTP1B is reversible in cells. These results indicate that the activation of a receptor tyrosine kinase by binding of the corresponding growth factor may not be sufficient to increase the steady state level of protein tyrosine phosphorylation in cells and that concurrent inhibition of protein-tyrosine phosphatases by H 2 O 2 might also be required.

Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), a substrate for both enzymes, is converted to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P 3 activates purified PLC-␥ isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-␥ isozymes indirectly, in the absence of PLC-␥ tyrosine phosphorylation, through the generation of PI(3,4,5)P 3 .Activation of both PLC 1 and PI 3-kinase often occurs in response to stimulation of cells by a variety of agonists. PLC catalyzes the hydrolysis of PI(4,5)P 2 to generate the second messengers inositol 1,4,5-trisphosphate (I(1,4,5)P 3 ) and diacylglycerol (1-3). PI 3-kinase phosphorylates the D-3 position of PI(4,5)P 2 to produce PI(3,4,5)P 3 , which is then sequentially dephosphorylated to PI(3,4)P 2 and phosphatidylinositol 3-phosphate (4 -7). The activation of each of these two enzymes has been implicated in such diverse cellular processes as mitogenesis, chemotaxis, secretion, and cytoskeletal assembly (4 -7).The phosphoinositides PI(3,4)P 2 and PI(3,4,5)P 3 are not substrates of any known PLC (8) and are normally absent from resting cells; however, they appear within seconds to minutes of stimulation of cells with various growth factors or other cellular activators. In contrast, the concentration of phosphatidylinositol 3-phosphate does not change substantially in response to cell stimulation (4 -7). It has thus been suggested that PI(3,4)P 2 and PI(3,4,5)P 3 might function as intracellular messengers (4 -7). With regard to potential targets of these D-3-phosphorylated lipids, they have been shown to activate Ca 2ϩ -independent isoforms of protein kinase C (9, 10) as well as to bind the pleckstrin homology (PH) domain of the protein serine-threonine kinase Akt, thereby activating its kinase activity (11-13), and to the SH2 domains of the 85-kDa (p85) subunit of PI 3-kinase, thereby preventing its binding to tyrosine-phosphorylated proteins (14).The 10 mammalian PLC isozymes identified to date are single polypeptides and can be divided into three types: PLC-␤, PLC-␥, and PLC-␦ (1). All contain a PH domain in their NH 2 -terminal region. The ␥ type isozymes differ from the other two types in that they contain two SH2 domains, one SH3 domain, and an additional PH domain that is split by the SH domains; these domains are arranged in the order PH(N)-SH2-SH2-SH3-PH(C), where N and C in parentheses denote NH 2 -and COOHterminal locations, respectively. Upon st...

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH 2 -terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 g/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 g/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H 2 O 2 produced by the mitochondrial respiratory chain.

The thiol (-SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (-SO 2 H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (-SO 3 H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to the H 2 O 2 concentrations administered to the yeast cells. We identified two species of hyperoxidized Tsa1p: one can be reduced back (reversible) with sulfiredoxin, and the other cannot (irreversible). Irreversibly hyperoxidized Tsa1p was identified as containing the active-site cysteine sulfonic acid (Tsa1p-SO 3 H) by mass spectrometry. Tsa1p-SO 3 H was not an autoxidation product of Tsa1p-SO 2 H and was maintained in yeast cells even after two doubling cycles. Tsa1p-SO 3 H self-assembled into a ring-shaped multimeric form was shown by electron microscopy. Although the Tsa1p-SO 3 H multimer lost its peroxidase activity, it gained ϳ4-fold higher chaperone activity compared with Tsa1p-SH. In this study, we identify an irreversibly hyperoxidized Prx, Tsa1p-SO 3 H, with enhanced molecular chaperone activity and suggest that Tsa1p-SO 3 H is a marker of cumulative oxidative stress in cells.

Vitamin D 3 up-regulated protein 1 (VDUP1) is a stressresponse gene that is up-regulated by 1,25(OH) 2 D 3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1 À/À fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27 kip1 , a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27 kip1 and to decrease the stability of p27 kip1 . VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27 kip1 stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27 kip1 from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27 kip1 stability via regulating JAB1. (Cancer Res 2005; 65(11): 4485-9)