High-resolution double vision of the allosteric phosphatase PTP1B.

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments, including diabetes, obesity and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High-resolution X-ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high-resolution structures is often complicated by differences between data sets, including non-isomorphism. Here, the highest resolution crystal structure of apo wild-type (WT) PTP1B to date is presented out of a total of ∼350 PTP1B structures in the PDB. This structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. The conformational differences between these chains are interrogated in the apo structure and between several recently reported high-resolution ligand-bound structures. Electron-density maps in a high-resolution structure of a recently reported activating double mutant are also examined, and unmodeled alternate conformations in the mutant structure are discovered that coincide with regions of enhanced conformational heterogeneity in the new WT structure. These results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, these new data and analysis provide a detailed view of the conformational ensemble of PTP1B and highlight the utility of high-resolution crystallography for elucidating conformational heterogeneity with potential relevance for function.

Measuring the Reversible Oxidation of Protein Tyrosine Phosphatases Using a Modified Cysteinyl-Labeling Assay.

The modified cysteinyl-labeling assay enables the labeling, enrichment, and detection of all members of the protein tyrosine phosphatase (PTP) superfamily that become reversibly oxidized in cells to facilitate phosphorylation-dependent signaling. In this chapter, we describe the method in detail and highlight the pitfalls of avoiding post-lysis oxidation of PTPs to measure the dynamic and transient oxidation and reduction of PTPs in cell signaling.

A role for cytoglobin in regulating intracellular hydrogen peroxide and redox signals in the vasculature.

The oxidant hydrogen peroxide serves as a signaling molecule that alters many aspects of cardiovascular functions. Recent studies suggest that cytoglobin - a hemoglobin expressed in the vasculature - may promote electron transfer reactions with proposed functions in hydrogen peroxide decomposition. Here, we determined the extent to which cytoglobin regulates intracellular hydrogen peroxide and established mechanisms. We found that cytoglobin decreased the hyperoxidation of peroxiredoxins and maintained the activity of peroxiredoxin 2 following challenge with exogenous hydrogen peroxide. Cytoglobin promoted a reduced intracellular environment and facilitated the reduction of the thiol-based hydrogen peroxide sensor Hyper7 after bolus addition of hydrogen peroxide. Cytoglobin also limited the inhibitory effect of hydrogen peroxide on glycolysis and reversed the oxidative inactivation of the glycolytic enzyme GAPDH. Our results indicate that cytoglobin in cells exists primarily as oxyferrous cytoglobin (CygbFe 2+ -O 2 ) with its cysteine residues in the reduced form. We found that the specific substitution of one of two cysteine residues on cytoglobin (C83A) inhibited the reductive activity of cytoglobin on Hyper7 and GAPDH. Carotid arteries from cytoglobin knockout mice were more sensitive to glycolytic inhibition by hydrogen peroxide than arteries from wildtype mice. Together, these results support a role for cytoglobin in regulating intracellular redox signals associated with hydrogen peroxide through oxidation of its cysteine residues, independent of hydrogen peroxide reaction at its heme center.

Cholesterol Chip for the Study of Cholesterol-Protein Interactions Using SPR.

Cholesterol, an important lipid in animal membranes, binds to hydrophobic pockets within many soluble proteins, transport proteins and membrane bound proteins. The study of cholesterol-protein interactions in aqueous solutions is complicated by cholesterol's low solubility and often requires organic co-solvents or surfactant additives. We report the synthesis of a biotinylated cholesterol and immobilization of this derivative on a streptavidin chip. Surface plasmon resonance (SPR) was then used to measure the kinetics of cholesterol interaction with cholesterol-binding proteins, hedgehog protein and tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B regulates miR-208b-argonaute 2 association and thyroid hormone responsiveness in cardiac hypertrophy.

Increased production of reactive oxygen species plays an essential role in the pathogenesis of several diseases, including cardiac hypertrophy. In our search to identify redox-sensitive targets that contribute to redox signaling, we found that protein tyrosine phosphatase 1B (PTP1B) was reversibly oxidized and inactivated in hearts undergoing hypertrophy. Cardiomyocyte-specific deletion of PTP1B in mice (PTP1B cKO mice) caused a hypertrophic phenotype that was exacerbated by pressure overload. Furthermore, we showed that argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, was a substrate of PTP1B in cardiomyocytes and in the heart. Our results revealed that phosphorylation at Tyr393 and inactivation of AGO2 in PTP1B cKO mice prevented miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1; also known as MED13) and contributed to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3) with propylthiouracil rescued pressure overload-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity is cardioprotective and that redox signaling is linked to thyroid hormone responsiveness and microRNA-mediated gene silencing in pathological hypertrophy.

In Vitro Activity Assays to Quantitatively Assess the Endogenous Reversible Oxidation State of Protein Tyrosine Phosphatases in Cells.

The reversible oxidation of protein tyrosine phosphatases (PTPs) impairs their ability to dephosphorylate substrates in vivo. This transient inactivation of PTPs occurs as their conserved catalytic cysteine residue reacts with cellular oxidants thereby abolishing the ability of this reactive cysteine to attack the phosphate of the target substrate. Hence, in vivo, the inhibition of specific PTPs in response to regulated and localized rises in cellular oxidants enables phospho-dependent signaling. We present assays that measure the endogenous activity of specific PTPs that become transiently inactivated in cells exposed to growth factors. Here, we describe the methods and highlight the pitfalls to avoid post-lysis oxidation of PTPs in order to assess the inactivation and the reactivation of PTPs targeted by cellular oxidants in signal transduction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Cell transfection (optional) Support Protocol: Preparation of degassed lysis buffers Basic Protocol 2: Cellular extraction in anaerobic conditions Basic Protocol 3: Enrichment and activity assay of specific PTPs Alternate Protocol: Measurement of active PTPs via direct cysteinyl labeling.

Regulation of PTP1B activation through disruption of redox-complex formation.

We have identified a molecular interaction between the reversibly oxidized form of protein tyrosine phosphatase 1B (PTP1B) and 14-3-3ζ that regulates PTP1B activity. Destabilizing the transient interaction between 14-3-3ζ and PTP1B prevented PTP1B inactivation by reactive oxygen species and decreased epidermal growth factor receptor phosphorylation. Our data suggest that destabilizing the interaction between 14-3-3ζ and the reversibly oxidized and inactive form of PTP1B may establish a path to PTP1B activation in cells.

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.

Reversible oxidation controls the activity and oligomeric state of the mammalian phosphoglycolate phosphatase AUM.

Redox-dependent switches of enzyme activity are emerging as important fine-tuning mechanisms in cell signaling. For example, protein tyrosine phosphatases employ a conserved cysteine residue for catalysis, which also renders them highly susceptible to reversible inactivation by oxidation. In contrast, haloacid dehalogenase (HAD)-type phosphatases perform catalysis via a phosphoaspartyltransferase reaction. The potential regulation of HAD phosphatases by reversible oxidation has not yet been explored. Here, we investigate the redox-sensitivity of the HAD-type phosphoglycolate phosphatase PGP, also known as AUM or glycerol-3-phosphate phosphatase. We show that recombinant, purified murine PGP is inhibited by oxidation and re-activated by reduction. We identify three reactive cysteine residues in the catalytic core domain of PGP (Cys35, Cys104 and Cys243) that mediate the reversible inhibition of PGP activity and the associated, redox-dependent conformational changes. Structural analysis suggests that Cys35 oxidation weakens van-der-Waals interactions with Thr67, a conserved catalytic residue required for substrate coordination. Cys104 and Cys243 form a redox-dependent disulfide bridge between the PGP catalytic core and cap domains, which may impair the open/close-dynamics of the catalytic cycle. In addition, we demonstrate that Cys297 in the PGP cap domain is essential for redox-dependent PGP oligomerization, and that PGP oxidation/oligomerization occurs in response to stimulation of cells with EGF. Finally, employing a modified cysteinyl-labeling assay, we show that cysteines of cellular PGP are transiently oxidized to sulfenic acids. Taken together, our findings establish that PGP, an aspartate-dependent HAD phosphatase, is transiently inactivated by reversible oxidation in cells.

Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-ß1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats.

PTP1B: mediating ROS signaling to silence genes.

Numerous studies have shown that normal cells often respond to the activation of oncogenes by undergoing reactive oxygen species-dependent induction of senescence. Here, we discuss our recent publication identifying protein tyrosine phosphatase PTP1B as an important redox-controlled checkpoint for senescence downstream of oncogenic RAS.

Dephosphorylation of tyrosine 393 in argonaute 2 by protein tyrosine phosphatase 1B regulates gene silencing in oncogenic RAS-induced senescence.

Oncogenic RAS (H-RAS(V12)) 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-RAS(V12)-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RAS(V12)-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RAS(V12)-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-RAS(V12)-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RAS(V12)-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing.

Receptor protein-tyrosine phosphatase α regulates focal adhesion kinase phosphorylation and ErbB2 oncoprotein-mediated mammary epithelial cell motility.

We investigated the role of protein-tyrosine phosphatase α (PTPα) in regulating signaling by the ErbB2 oncoprotein in mammary epithelial cells. Using this model, we demonstrated that activation of ErbB2 led to the transient inactivation of PTPα, suggesting that attenuation of PTPα activity may contribute to enhanced ErbB2 signaling. Furthermore, RNAi-induced suppression of PTPα led to increased cell migration in an ErbB2-dependent manner. The ability of ErbB2 to increase cell motility in the absence of PTPα was characterized by prolonged interaction of GRB7 with ErbB2 and increased association of ErbB2 with a ß1-integrin-rich complex, which depended on GRB7-SH2 domain interactions. Finally, suppression of PTPα resulted in increased phosphorylation of focal adhesion kinase on Tyr-407, which induced the recruitment of vinculin and the formation of a novel focal adhesion kinase complex in response to ErbB2 activation in mammary epithelial cells. Collectively, these results reveal a new role for PTPα in the regulation of motility of mammary epithelial cells in response to ErbB2 activation.

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.

Characterization of hsp27 kinases activated by elevated aortic pressure in heart.

Chronic hemodynamic overload results in left ventricular hypertrophy, fibroblast proliferation, and interstitial fibrosis. The small heat shock protein hsp27 has been shown to be cardioprotective and this requires a phosphorylatable form of this protein. To further understand the regulation of hsp27 in heart in response to stress, we investigated the ability of elevated aortic pressure to activate hsp27-kinase activities. Isolated hearts were subjected to retrograde perfusion and then snap frozen. Hsp27-kinase activity was measured in vitro as hsp27 phosphorylation. Immune complex assays revealed that MK2 activity was low in non-perfused hearts and increased following crystalline perfusion at 60 or 120 mmHg. Hsp27-kinase activities were further studied following ion-exchange chromatography. Anion exchange chromatography on Mono Q revealed 2 peaks (b and c) of hsp27-kinase activity. A third peak a was detected upon chromatography of the Mono Q flow-through fractions on the cation exchange resin, Mono S. The hsp27-kinase activity underlying peaks a and c increased as perfusion pressure was increased from 40 to 120 mmHg. In contrast, peak b increased over pressures 60-100 mmHg but was decreased at 120 mmHg. Peaks a, b, and c contained MK2 immunoreactivity, whereas MK3 and MK5 immunoreactivity was detected in peak a. p38 MAPK and phospho-p38 MAPK were also detected in peaks b and c but absent from peak a. Hsp27-kinase activity in peaks b and c (120 mmHg) eluted from a Superose 12 gel filtration column with an apparent molecular mass of 50 kDa. Hence, peaks b and c were not a result of MK2 forming complexes. In-gel hsp27-kinase assays revealed a single 49-kDa renaturable hsp27-kinase activity in peaks b and c at 60 mmHg, whereas several hsp27-kinases (p43, p49, p54, p66) were detected in peaks b and c from hearts perfused at 120 mmHg. Thus, multiple hsp27-kinases were activated in response to elevated aortic pressure in isolated, perfused rat hearts and hence may be implicated in regulating the cardioprotective effects of hsp27 and thus may represent targets for cardioprotective therapy.

Targeting the reversibly oxidized protein tyrosine phosphatase superfamily.

Controlled production of reactive oxygen species leads to reversible oxidation of protein tyrosine phosphatases (PTPs) and has emerged as an important tier of regulation over phosphorylation-dependent signal transduction. We present a modified cysteinyl-labeling assay that detects reversible oxidation of members of each of the different PTP subclasses. Here, we describe the methods for enriching reversibly oxidized PTPs from complex protein extracts, illustrating the procedure in IMR90 fibroblasts.

Analysis of the redox regulation of protein tyrosine phosphatase superfamily members utilizing a cysteinyl-labeling assay.

The catalytic activity of protein tyrosine phosphatase (PTP) superfamily members is regulated by the reversible oxidation of their invariant catalytic Cys residue in vivo. Transient and specific regulation of PTP activity by reactive oxygen species (ROS) attenuates dephosphorylation and, thereby, promotes phosphorylation, hence facilitating signal transduction. We have recently developed a modified cysteinyl-labeling assay [Boivin, B., Zhang, S., Arbiser, J. L., Zhang, Z. Y., and Tonks, N. K. (2008). Proc. Natl. Acad. Sci. USA105, 9959-9964.] that showed broad selectivity in detecting reversible oxidation of members from different PTP subclasses in platelet-derived growth factor (PDGF)-BB overexpressing cells. Herein, we applied this assay, which utilizes the unique chemistry of the invariant catalytic Cys residue to enrich and identify PTPs that are reversibly oxidized upon acute growth factor stimulation. Performing the cysteinyl-labeling assay with Rat-1 fibroblasts enabled us to capture both PTEN and SHP-2 as a consequence to acute PDGF-BB stimulation. Given the ability of this assay to detect reversible oxidation of a broad array of members of the PTP family, we anticipate that it should permit profiling of the entire ROS-regulated PTPome in a wide array of signaling paradigms.

Reactive oxygen species enhance insulin sensitivity.

Chronic reactive oxygen species (ROS) production by mitochondria may contribute to the development of insulin resistance, a primary feature of type 2 diabetes. In recent years it has become apparent that ROS generation in response to physiological stimuli such as insulin may also facilitate signaling by reversibly oxidizing and inhibiting protein tyrosine phosphatases (PTPs). Here we report that mice lacking one of the key enzymes involved in the elimination of physiological ROS, glutathione peroxidase 1 (Gpx1), were protected from high-fat-diet-induced insulin resistance. The increased insulin sensitivity in Gpx1(-/-) mice was attributed to insulin-induced phosphatidylinositol-3-kinase/Akt signaling and glucose uptake in muscle and could be reversed by the antioxidant N-acetylcysteine. Increased insulin signaling correlated with enhanced oxidation of the PTP family member PTEN, which terminates signals generated by phosphatidylinositol-3-kinase. These studies provide causal evidence for the enhancement of insulin signaling by ROS in vivo.

A modified cysteinyl-labeling assay reveals reversible oxidation of protein tyrosine phosphatases in angiomyolipoma cells.

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.

Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated oxidation and inactivation of phosphatases in growth factor signaling.

Superoxide dismutase 1 (SOD1) is an abundant copper/zinc enzyme found in the cytoplasm that converts superoxide into hydrogen peroxide and molecular oxygen. Tetrathiomolybdate (ATN-224) has been recently identified as an inhibitor of SOD1 that attenuates FGF-2- and VEGF-mediated phosphorylation of ERK1/2 in endothelial cells. However, the mechanism for this inhibition was not elucidated. Growth factor (GF) signaling elicits an increase in reactive oxygen species (ROS), which inactivates protein tyrosine phosphatases (PTP) by oxidizing an essential cysteine residue in the active site. ATN-224-mediated inhibition of SOD1 in tumor and endothelial cells prevents the formation of sufficiently high levels of H(2)O(2), resulting in the protection of PTPs from H(2)O(2)-mediated oxidation. This, in turn, leads to the inhibition of EGF-, IGF-1-, and FGF-2-mediated phosphorylation of ERK1/2. Pretreatment with exogenous H(2)O(2) or with the phosphatase inhibitor vanadate abrogates the inhibition of ERK1/2 phosphorylation induced by ATN-224 or SOD1 siRNA treatments. Furthermore, ATN-224-mediated SOD1 inhibition causes the down-regulation of the PDGF receptor. SOD1 inhibition also increases the steady-state levels of superoxide, which induces protein oxidation in A431 cells but, surprisingly, does not oxidize phosphatases. Thus, SOD1 inhibition in A431 tumor cells results in both prooxidant effects caused by the increase in the levels of superoxide and antioxidant effects caused by lowering the levels of H(2)O(2). These results identify SOD1 as a master regulator of GF signaling and as a therapeutic target for the inhibition of angiogenesis and tumor growth.