Redox regulation of PTPN22 affects the severity of T-cell-dependent autoimmune inflammation.

Chronic autoimmune diseases are associated with mutations in PTPN22, a modifier of T cell receptor (TCR) signaling. As with all protein tyrosine phosphatases, the activity of PTPN22 is redox regulated, but if or how such regulation can modulate inflammatory pathways in vivo is not known. To determine this, we created a mouse with a cysteine-to-serine mutation at position 129 in PTPN22 (C129S), a residue proposed to alter the redox regulatory properties of PTPN22 by forming a disulfide with the catalytic C227 residue. The C129S mutant mouse showed a stronger T-cell-dependent inflammatory response and development of T-cell-dependent autoimmune arthritis due to enhanced TCR signaling and activation of T cells, an effect neutralized by a mutation in Ncf1, a component of the NOX2 complex. Activity assays with purified proteins suggest that the functional results can be explained by an increased sensitivity to oxidation of the C129S mutated PTPN22 protein. We also observed that the disulfide of native PTPN22 can be directly reduced by the thioredoxin system, while the C129S mutant lacking this disulfide was less amenable to reductive reactivation. In conclusion, we show that PTPN22 functionally interacts with Ncf1 and is regulated by oxidation via the noncatalytic C129 residue and oxidation-prone PTPN22 leads to increased severity in the development of T-cell-dependent autoimmunity.

Qualitative Differences in Protection of PTP1B Activity by the Reductive Trx1 or TRP14 Enzyme Systems upon Oxidative Challenges with Polysulfides or H2O2 Together with Bicarbonate.

Protein tyrosine phosphatases (PTPs) can be regulated by several redox-dependent mechanisms and control growth factor-activated receptor tyrosine kinase phosphorylation cascades. Reversible oxidation of PTPs is counteracted by reductive enzymes, including thioredoxin (Trx) and Trx-related protein of 14 kDa (TRP14), keeping PTPs in their reduced active states. Different modes of oxidative inactivation of PTPs concomitant with assessment of activating reduction have been little studied in direct comparative analyses. Determining PTP1B activities, we here compared the potency of inactivation by bicarbonate-assisted oxidation using H2O2 with that of polysulfide-mediated inactivation. Inactivation of pure PTP1B was about three times more efficient with polysulfides as compared to the combination of bicarbonate and H2O2. Bicarbonate alone had no effect on PTP1B, neither with nor without a combination with polysulfides, thus strengthening the notion that bicarbonate-assisted H2O2-mediated inactivation of PTP1B involves formation of peroxymonocarbonate. Furthermore, PTP1B was potently protected from polysulfide-mediated inactivation by either TRP14 or Trx1, in contrast to the inactivation by bicarbonate and H2O2. Comparing reductive activation of polysulfide-inactivated PTP1B with that of bicarbonate- and H2O2-treated enzyme, we found Trx1 to be more potent in reactivation than TRP14. Altogether we conclude that inactivation of PTP1B by polysulfides displays striking qualitative differences compared to that by H2O2 together with bicarbonate, also with regard to maintenance of PTP1B activity by either Trx1 or TRP14.

Bicarbonate is essential for protein-tyrosine phosphatase 1B (PTP1B) oxidation and cellular signaling through EGF-triggered phosphorylation cascades.

Protein-tyrosine phosphatases (PTPs) counteract protein tyrosine phosphorylation and cooperate with receptor-tyrosine kinases in the regulation of cell signaling. PTPs need to undergo oxidative inhibition for activation of cellular cascades of protein-tyrosine kinase phosphorylation following growth factor stimulation. It has remained enigmatic how such oxidation can occur in the presence of potent cellular reducing systems. Here, using in vitro biochemical assays with purified, recombinant protein, along with experiments in the adenocarcinoma cell line A431, we discovered that bicarbonate, which reacts with H2O2 to form the more reactive peroxymonocarbonate, potently facilitates H2O2-mediated PTP1B inactivation in the presence of thioredoxin reductase 1 (TrxR1), thioredoxin 1 (Trx1), and peroxiredoxin 2 (Prx2) together with NADPH. The cellular experiments revealed that intracellular bicarbonate proportionally dictates total protein phosphotyrosine levels obtained after stimulation with epidermal growth factor (EGF) and that bicarbonate levels directly correlate with the extent of PTP1B oxidation. In fact, EGF-induced cellular oxidation of PTP1B was completely dependent on the presence of bicarbonate. These results provide a plausible mechanism for PTP inactivation during cell signaling and explain long-standing observations that growth factor responses and protein phosphorylation cascades are intimately linked to the cellular acid-base balance.

The A to Z of modulated cell patterning by mammalian thioredoxin reductases.

Mammalian thioredoxin reductases (TrxRs) are selenocysteine-containing proteins (selenoproteins) that propel a large number of functions through reduction of several substrates including the active site disulfide of thioredoxins (Trxs). Well-known enzymatic systems that in turn are supported by Trxs and TrxRs include deoxyribonucleotide synthesis through ribonucleotide reductase, antioxidant defense through peroxiredoxins and methionine sulfoxide reductases, and redox modulation of a number of transcription factors. Although these functions may be essential for cells due to crucial roles in maintenance of cell viability and proliferation, findings during the last decade reveal that mammals have major redundancy in their cellular reductive systems. The synthesis of glutathione (GSH) and reductive functions of GSH-dependent pathways typically act in parallel with Trx-dependent pathways, with only one of these systems often being sufficient to support viability. Importantly, this does not imply that a modulation of the Trx system will remain without consequences, even when GSH-dependent pathways remain functional. As suggested by several recent findings, the Trx system in general and the TrxRs in particular, function as key regulators of signaling pathways. In this review article we will discuss findings that collectively suggest that modulation in mammalian systems of cytosolic TrxR1 (TXNRD1) or mitochondrial TrxR2 (TXNRD2) influence cell patterning and cellular stress responses. Effects of lower activities include increased adipogenesis, insulin responsiveness, glycogen accumulation, hyperproliferation, and distorted embryonic development, while increased activities correlate with decreased proliferation and extended lifespan, as well as worse cancer prognosis. The molecular mechanisms that underlie these diverse effects, involving regulation of protein phosphorylation cascades and of key transcription factors that guide cellular differentiation pathways, will be discussed. We conclude that the selenium-dependent oxidoreductases TrxR1 and TrxR2 should be considered as key components of signaling pathways that control cell differentiation and cellular stress responses.

Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H2O2 exposure.

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H2O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2O2 Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways.

Knockout of mitochondrial thioredoxin reductase stabilizes prolyl hydroxylase 2 and inhibits tumor growth and tumor-derived angiogenesis.

AIMS: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H2O2) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. RESULTS: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. INNOVATION: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. CONCLUSION: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.

Inhibition of protein tyrosine phosphatases enhances cerebral collateral growth in rats.

UNLABELLED: Arteriogenesis involves the rapid proliferation of preexisting arterioles to fully functional arteries as a compensatory mechanism to overcome circulatory deficits. Stimulation of arteriogenesis has therefore been considered a treatment concept in arterial occlusive disease. Here, we investigated the impact of inhibition of protein tyrosine phosphatases (PTPs) on cerebral arteriogenesis in rats. Arteriogenesis was induced by occlusion of one carotid and both vertebral arteries (three-vessel occlusion (3-VO)). Collateral growth and functional vessel perfusion was assessed 3-35 days following 3-VO. Furthermore, animals underwent 3-VO surgery and were treated with the pan-PTP inhibitor BMOV, the SHP-1 inhibitor sodium stibogluconate (SSG), or the PTP1B inhibitor AS279. Cerebral vessel diameters and cerebrovascular reserve capacity (CVRC) were determined, together with immunohistochemistry analyses and proximity ligation assays (PLA) for determination of tissue proliferation and phosphorylation patterns after 7 days. The most significant changes in vessel diameter increase were present in the ipsilateral posterior cerebral artery (PCA), with proliferative markers (PCNA) being time-dependently increased. The CVRC was lost in the early phase after 3-VO and partially recovered after 21 days. PTP inhibition resulted in a significant increase in the ipsilateral PCA diameter in BMOV-treated animals and rats subjected to PTP1B inhibition. Furthermore, CVRC was significantly elevated in AS279-treated rats compared to control animals, along with hyperphosphorylation of the platelet-derived growth factor-ß receptor in the vascular wall in vivo. In summary, our data indicate PTPs as hitherto unrecognized negative regulators in cerebral arteriogenesis. Further, PTP inhibition leading to enhanced collateral growth and blood perfusion suggests PTPs as novel targets in anti-ischemic treatment. KEY MESSAGES: PTPs exhibit negative regulatory function in cerebral collateral growth in rats. Inhibition of pan-PTP/PTP1B increases vessel PDGF-ß receptor phosphorylation. PTP1B inhibition enhances arteriogenesis and cerebrovascular reserve capacity.

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation.

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFßR phosphorylation in two independent cell systems demonstrated a decrease in PDGFßR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFßR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFßR phosphorylation, showing that mitochondrial ROS in general promote PDGFßR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.

Regulation of protein tyrosine phosphatase oxidation in cell adhesion and migration.

SIGNIFICANCE: Redox-regulated control of protein tyrosine phosphatases (PTPs) through inhibitory reversible oxidation of their active site is emerging as a novel and general mechanism for control of cell surface receptor-activated signaling. This mechanism allows for a previously unrecognized crosstalk between redox regulators and signaling pathways, governed by, for example, receptor tyrosine kinases and integrins, which control cell proliferation and migration. RECENT ADVANCES: A large number of different molecules, in addition to hydrogen peroxide, have been found to induce PTP inactivation, including lipid peroxides, reactive nitrogen species, and hydrogen sulfide. Characterization of oxidized PTPs has identified different types of oxidative modifications that are likely to display differential sensitivity to various reducing systems. Accumulating evidence demonstrates that PTP oxidation occurs in a temporally and spatially restricted manner. Studies in cell and animal models indicate altered PTP oxidation in models of common diseases, such as cancer and metabolic/cardiovascular disease. Novel methods have appeared that allow characterization of global PTP oxidation. CRITICAL ISSUES: As the understanding of the molecular and cellular biology of PTP oxidation is developing, it will be important to establish experimental procedures that allow analyses of PTP oxidation, and its regulation, in physiological and pathophysiological settings. Future studies should also aim to establish specific connections between various oxidants, specific PTPs, and defined signaling contexts. FUTURE DIRECTIONS: Modulation of PTP activity still appears as a valid strategy for correction or inhibition of dys-regulated cell signaling. Continued studies on PTP oxidation might present yet unrecognized means to exploit this regulatory mechanism for pharmacological purposes.

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-ß receptor tyrosine kinase signaling.

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-ß receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-ß receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.

Targeting density-enhanced phosphatase-1 (DEP-1) with antisense oligonucleotides improves the metabolic phenotype in high-fat diet-fed mice.

BACKGROUND: Insulin signaling is tightly controlled by tyrosine dephosphorylation of the insulin receptor through protein-tyrosine-phosphatases (PTPs). DEP-1 is a PTP dephosphorylating tyrosine residues in a variety of receptor tyrosine kinases. Here, we analyzed whether DEP-1 activity is differentially regulated in liver, skeletal muscle and adipose tissue under high-fat diet (HFD), examined the role of DEP-1 in insulin resistance in vivo, and its function in insulin signaling. RESULTS: Mice were fed an HFD for 10 weeks to induce obesity-associated insulin resistance. Thereafter, HFD mice were subjected to systemic administration of specific antisense oligonucleotides (ASOs), highly accumulating in hepatic tissue, against DEP-1 or control ASOs. Targeting DEP-1 led to improvement of insulin sensitivity, reduced basal glucose level, and significant reduction of body weight. This was accompanied by lower insulin and leptin serum levels. Suppression of DEP-1 in vivo also induced hyperphosphorylation in the insulin signaling cascade of the liver. Moreover, DEP-1 physically associated with the insulin receptor in situ, and recombinant DEP-1 dephosphorylated the insulin receptor in vitro. CONCLUSIONS: These results indicate that DEP-1 acts as an endogenous antagonist of the insulin receptor, and downregulation of DEP-1 results in an improvement of insulin sensitivity. DEP-1 may therefore represent a novel target for attenuation of metabolic diseases.

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ.

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by protein-tyrosine phosphatases (PTPs). We recently identified the PTP DEP-1/CD148/PTPRJ as a novel negative regulator of FLT3. This study addressed the role of DEP-1 for regulation of the acute myeloid leukemia (AML)-related mutant FLT3 internal tandem duplication (ITD) protein. Our experiments revealed that DEP-1 was expressed but dysfunctional in cells transformed by FLT3 ITD. This was caused by enzymatic inactivation of DEP-1 through oxidation of the DEP-1 catalytic cysteine. In intact cells, including primary AML cells, FLT3 ITD kinase inhibition reactivated DEP-1. DEP-1 reactivation was also achieved by counteracting the high levels of reactive oxygen species (ROS) production detected in FLT3 ITD-expressing cell lines by inhibition of reduced NAD phosphate (NADPH)-oxidases, or by overexpression of catalase or peroxiredoxin-1 (Prx-1). Interference with ROS production in 32D cells inhibited cell transformation by FLT3 ITD in a DEP-1-dependent manner, because RNAi-mediated depletion of DEP-1 partially abrogated the inhibitory effect of ROS quenching. Reactivation of DEP-1 by stable overexpression of Prx-1 extended survival of mice in the 32D cell/C3H/HeJ mouse model of FLT3 ITD-driven myeloproliferative disease. The study thus uncovered DEP-1 oxidation as a novel event contributing to cell transformation by FLT3 ITD.

Hypoxia followed by re-oxygenation induces oxidation of tyrosine phosphatases.

Hypoxia and hypoxia/reoxygenation (H/R) are components of tissue ischemia and reperfusion implicated in myocardial infarction, organ transplantation, and tumor perfusion. H/R enhances production of reactive oxygen species (ROS). Candidate molecular targets of ROS are the catalytic site cysteine of protein tyrosine phosphatases (PTPs), which are major regulators of tyrosine kinase signaling. This study aimed at analyzing potential effects of H/R on PTP-oxidation in cultured cells and in heart tissue. Exposure of mouse NIH3T3 fibroblasts to H/R increased the oxidation of the PTPs SHP-2- and DEP-1. The catalytic pan-PTP- and SHP-2-activity after H/R were also decreased in rat cardiomyoblasts. In vivo dephosphorylation of the Platelet-derived Growth Factor (PDGF)-receptor in NIH3T3 fibroblasts was delayed following H/R. Erk1/2 displayed an antioxidant-sensitive increase in H/R. Furthermore, increased PDGF-induced cytoskeleton re-arrangements were evident following H/R and could be prevented by antioxidant pretreatment. Finally, decreased pan-PTP- and SHP-2 activity was demonstrated in tissue extracts from an ex vivo Langendorff-model of rat heart ischemia-reperfusion. This study thus demonstrates PTP-oxidation as a previously unrecognized molecular component of the cellular response to H/R in cells and tissues. The study additionally provides the first demonstration of increased PTP-oxidation in tissues under patho-physiological settings.

Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases.

RATIONALE: Platelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated with pulmonary hypoxia. OBJECTIVES: To investigate whether hypoxia alters PDGF ß receptor (ßPDGFR) signaling in the pulmonary vasculature. METHODS: The impact of chronic hypoxia on signal transduction by the ßPDGFR was measured in human pulmonary arterial smooth muscle cells (hPASMC) in vitro, and in mice with hypoxia-induced PH in vivo. MEASUREMENTS AND MAIN RESULTS: Chronic hypoxia significantly enhanced PDGF-BB-dependent proliferation and chemotaxis of hPASMC. Pharmacologic inhibition of PI3 kinase (PI3K) and PLCγ abrogated these events under both normoxia and hypoxia. Although hypoxia did not affect ßPDGFR expression, it increased the ligand-induced tyrosine phosphorylation of the receptor, particularly at binding sites for PI3K (Y751) and PLCγ (Y1021). The activated ßPDGFR is dephosphorylated by protein tyrosine phosphatases (PTPs). Interestingly, hypoxia decreased expression of numerous PTPs (T cell PTP, density-enhanced phosphatase-1, PTP1B, and SH2 domain-containing phosphatase-2), resulting in reduced PTP activity. Hypoxia-inducible factor (HIF)-1α is involved in this regulation of gene expression, because hypoxia-induced ßPDGFR hyperphosphorylation and PTP down-regulation were abolished by HIF-1α siRNA and by the HIF-1α inhibitor 2-methoxyestradiol. ßPDGFR hyperphosphorylation and PTP down-regulation were also present in vivo in mice with chronic hypoxia-induced PH. CONCLUSIONS: Hypoxia reduces expression and activity of ßPDGFR-antagonizing PTPs in a HIF-1α-dependent manner, thereby enhancing receptor activation and proliferation and chemotaxis of hPASMC. Because hyperphosphorylation of the ßPDGFR and down-regulation of PTPs occur in vivo, this mechanism likely has significant impact on the development and progression of PH and other hypoxia-associated diseases.

12/15-lipoxygenase-derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H(2)O(2), as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF beta-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX-derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H(2)O(2) in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.

FBXW7/hCDC4 is a general tumor suppressor in human cancer.

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of approximately 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.

Oxidation sensitivity of the catalytic cysteine of the protein-tyrosine phosphatases SHP-1 and SHP-2.

Reversible oxidation of the catalytic cysteine of protein-tyrosine phosphatases (PTPs) has emerged as a putative mechanism of activity regulation by physiological cell stimulation with growth factors, and by cell treatments with adverse agents such as UV irradiation. We compared SHP-1 and SHP-2, two structurally related cytoplasmic protein-tyrosine phosphatases with different cellular functions and cell-specific expression patterns, for their intrinsic susceptibility to oxidation by H(2)O(2). The extent of oxidation was monitored by detecting the modification of the PTP catalytic cysteine by three different methods, including a modified in-gel PTP assay, alkylation with a biotinylated iodoacetic acid derivative, and an antibody against oxidized PTPs. Dose-response curves for oxidation of the catalytic domains of SHP-1 and SHP-2 were similar. SHP-1 and -2 require relatively high H(2)O(2) concentrations for oxidation (half-maximal oxidation at 0.1-0.5 mM). For SHP-1, the SH2 domains had a significant protective function with respect to oxidation. In EOL-1 cells, SHP oxidation by exogenous H(2)O(2) in general and SHP-2 oxidation in particular was strongly diminished compared to HEK293 cells, at least partially related to a generally lower oxidant sensitivity of the EOL-1 cells. The data suggest that the differential cell functions of SHP-1 and SHP-2 are not related to differences in oxidation sensitivity. The modulating effects of SH2 domains for oxidation of these PTPs are in support of an enhanced oxidation susceptibility of activated SHPs.

Interferon alpha-induced apoptosis in tumor cells is mediated through the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway.

Interferon (IFN) alpha induces a caspase-dependent apoptosis that is associated with activation of the proapoptotic Bak and Bax, loss of mitochondrial membrane potential, and release of cytochrome c. In addition to the onset of the classical Jak-STAT pathway, IFNalpha also induced phosphoinositide 3-kinase (PI3K) activity. Pharmacological inhibition of PI3K activity by Ly294002 disrupted IFN-induced apoptosis upstream of mitochondria. Inhibition of mTOR by rapamycin or by overexpression of a kinase dead mutant of mTOR, efficiently blocked IFNalpha-induced apoptosis. A PI3K and mTOR-dependent phosphorylation of p70S6 kinase and 4E-BP1 repressor was induced by IFNalpha treatment of cells and was strongly inhibited by Ly294002 or rapamycin. The activation of Jak-STAT signaling upon IFNalpha stimulation was not affected by abrogating PI3K/mTOR pathway. Neither was the expression of several IFNalpha target genes affected, nor the ability of IFNalpha to protect against virus-induced cell death affected by inhibition of the PI3K/mTOR pathway. These data demonstrate that an intact PI3K/mTOR pathway is necessary for the ability of IFNalpha to induce apoptosis, whereas activation of the Jak-STAT pathway alone appears to be insufficient for this specific IFNalpha-induced effect.