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 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-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β 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.
Oncogenic RAS (H-RASV12) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We ...investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RASV12-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RASV12-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RASV12-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNAs) and thus miRNA-mediated gene silencing, which counteracted the function of H-RASV12-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RASV12-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing.
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•PTP1B is reversibly oxidized by ROS in H-RASV12-transformed cells•Inactivation of PTP1B is required for H-RASV12-induced senescence•AGO2 phospho-Tyr 393 is a direct substrate of PTP1B•AGO2 phosphorylation at Tyr 393 inhibits its miRNA loading and gene silencing
Yang et al. demonstrate inactivation of protein tyrosine phosphatase PTP1B in response to oncogenic RAS-induced production of hydrogen peroxide. This led to enhanced tyrosine phosphorylation of argonaute 2, which impaired its ability to bind microRNA and its silencing of genes, such as p21, involved in onset of senescence.
Protein Tyrosine Phosphatase 1B (PTP1B) catalytic activity is negatively regulated by reversible oxidation. In vivo, PTP1B reversible inactivation by hydrogen peroxide allows phosphorylation of ...specific tyrosine residues of several receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). Hence PTP1B acts as a tumor suppressor by preventing excessive signaling from growth factors. We re-examined the redox cycle of PTP1B in order to find underlying molecular mechanisms that control its activation. For this purpose, we performed structural analyses of reduced and oxidized PTP1B (PTP1B-OX) and identified peptides that were uniquely exposed to the cytosol in PTP1B-OX (OX-Peptides). We used these OX-Peptides, fished for novel PTP1B interactors and we identified 14-3-3β as a specific binding partner for PTP1B-OX. As expected, the interaction between 14-3-3β and PTP1B was dependent on PTP1B oxidation downstream of EGFR in cells. 14-3-3s are phosphoserine binding proteins and inhibiting PKB activity and PTP1B Ser-50 phosphorylation prevented interaction between 14-3-3β- and PTP1B-OX. Interestingly, preventing PTP1B Ser-50 phosphorylation or inhibiting 14-3-3s using R18 also prevented PTP1B oxidation in cells, suggesting that 14-3-3β binding is necessary for PTP1B to become oxidized. Similarly, the interaction between 14-3-3 β and PTP1B-OX was compromised in cells following exposure to cell-permeable OX-Peptides. Preventing this protein-protein interaction directly perturbed the redox cycle of PTP1B: cell-permeable OX-peptides effectively prevented PTP1B reversible inactivation by reactive oxygen species. We propose a mechanism whereby 14-3-3β stabilizes the inactive form of PTP1B, transiently allowing EGFR signaling to occur. Destabilizing the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for drugs that activate protein tyrosine phosphatases.
The production of reactive oxygen species (ROS) exerts an additional tier of control over tyrosine phosphorylation-dependent signal transduction by transiently inhibiting the catalytic activity of ...specific protein tyrosine phosphatases (PTPs). Hence, the ability to detect reversible oxidation of PTPs in vivo is critical to understanding the complex biological role of ROS in the control of cellular signaling. Here, we describe an assay for identifying those PTPs that are reversibly oxidized in vivo, which utilizes the unique chemistry of the invariant catalytic Cys residue in labeling the active site with biotinylated small molecules under mildly acidic conditions. We have applied this cysteinyl-labeling assay to the study of platelet-derived growth factor (PDGF) receptor signaling in an angiomyolipoma cell model. Doing so has allowed us to detect reversible oxidation of several proteins in response to sustained PDGF stimulation. As in other cell systems, we have observed the reversible oxidation of the classical PTP SHP2 and the tumor suppressor phosphatase PTEN in response to PDGF stimulation. Furthermore, we detected reversible oxidation of members of two other subclasses of PTPs, the receptor PTP LAR and the dual-specificity phosphatase MKP1. These data demonstrate the broad selectivity of the assay, allowing us to detect representatives of all of the major subgroups of the PTP superfamily. We anticipate that this cysteinyl-labeling enrichment strategy can be applied broadly to study reversible oxidation as a mechanism of harnessing PTP catalytic activity in a variety of signaling pathways.
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