Cell Protein Tyrosine Phosphatase Research Articles

Inhibition of PTPN2 by PTP inhibitor V

Protein phosphorylation is closely linked to critical pathways in numerous human diseases, which is regulated by the coordinated activities of kinases and phosphatases. Protein phosphorylation occurs predominantly on serine, threonine, and tyrosine residues of proteins and plays important roles in the regulation of physiological processes including gene expression, proliferation, differentiation, cell cycle arrest, and apoptosis. About 30% of proteins are phosphorylated in the mammalian cell. In case of eukaryotic cells, tyrosine phosphorylation occupies only 0.01-0.05% of total protein phosphorylation, while most of protein phosphorylation occurs on serine or threonine residues. However, growth factor stimulation or tumorigenesis enhances the level of tyrosine phosphorylation up to 1-2% of total protein phosphorylation. Protein phosphorylation is a reversible process that is controlled by protein kinases and protein phosphatases. In particular, protein tyrosine phosphatases (PTPs) are involved in most signaling pathways and thus play important roles in eukaryotes. PTPs superfamily comprises 107 enzymes in human genome. Based on the amino acid sequences of their catalytic domains, the PTPs can be grouped into four main families. Since PTPs play critical roles for cell homeostasis and thus diseases, chemical inhibitors that regulate PTPs have been extensively investigated to be used as therapeutic reagents. PTP inhibitor V, also known as phenyl hydrazono pyrazolone sulfate 1 (PHPS1), is 4-{N'-[3-(4-nitrophenyl)-5-oxo-1phenyl-1,5-dihydro-pyrazol-(4Z)-ylidene]-hydrazino}-benzenesulfonic acid (Fig. 1(a)). PTP inhibitor V was originally identified as a potent cell-permeable inhibitor that acts as active-site targeting, reversible, competitive inhibitor of SHP2 with the IC50 value of 2.1 μM. 5 PTP inhibitor V inhibits ECPTP, PTP1B and SHP-1 (IC50 = 5.4, 19, 30 μM, respectively). To investigate the effect of PTP inhibitor V on other PTPs, we performed in vitro phosphatase assays with purified recombinant PTPs (Table 1). We found that protein tyrosine phosphatase non-receptor type 2 (PTPN2) was inhibited by PTP inhibitor V while other PTPs were not. PTPN2 that is also called T cell protein tyrosine phosphatase (TC-PTP) belongs to class I Cys-based PTPs as the intracellular non-receptor PTPs with a high degree of sequence and structural homology within the catalytic domain. The mRNA levels of PTPN2 increase at G1 phase and return to basal level after G1 phase during cell cycle progression. 8

Spermidine Stimulates T Cell Protein-tyrosine Phosphatase-mediated Protection of Intestinal Epithelial Barrier Function

The gene locus encoding protein-tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with inflammatory bowel disease. Expression of the PTPN2 gene product, T cell protein-tyrosine phosphatase (TCPTP), in intestinal epithelial cells has been shown to play an important role in the protection of epithelial barrier function during periods of inflammation by acting as a negative regulator of the proinflammatory cytokine IFN-γ. Therefore, agents that increase the activity of TCPTP are of general interest as modifiers of inflammatory signaling events. A previous study demonstrated that the small molecule spermidine is a selective activator of TCPTP in vitro. The aim of this study was to investigate whether activation of TCPTP by spermidine was capable of alleviating IFN-γ-induced, proinflammatory signaling and barrier dysfunction in human intestinal epithelial cells. Studies revealed that treatment of T84 and HT29/cl.19A colonocytes with spermidine increased both TCPTP protein levels and enzymatic activity, correlating with a decrease in the phosphorylation of the signal transducers and activators of transcription 1 and 3, downstream mediators of IFN-γ signaling, upon coadministration of spermidine to IFN-γ-treated cells. On a functional level, spermidine protected barrier function in the setting of inflammation, restricting the decrease in transepithelial electrical resistance and the increase in epithelial permeability induced by IFN-γ in coincubation experiments. These data implicate spermidine as a potential therapeutic agent to treat conditions associated with elevated IFN-γ signaling and a faulty mucosal barrier.

Design and Synthesis of Imidazolidine‐2,4‐Dione Derivatives as Selective Inhibitors by Targeting Protein Tyrosine Phosphatase‐1B Over T‐Cell Protein Tyrosine Phosphatase

Owing to its special role as a negative regulator in both insulin and leptin signaling, protein tyrosine phosphatase-1B (PTP1B) has drawn considerable attention as a target for treating type 2 diabetes and obesity. It, however, is a great challenge to discover inhibitors specific to each PTP due to the highly homologous. In this study, a series of compounds were discovered to inhibit PTP1B based on imidazolidine-2,4-dione by means of 'core hopping'. A selective PTP1B inhibitor (comp#h) was identified, and molecular dynamics simulation and binding free energy calculation were carried out to propose the most likely binding mode of comp#h with PTP1B. The findings reported here may provide a new strategy in discovering selective and effective inhibitors for treating diabetes.

TRAF3 restrains regulatory T cell development by modulating IL-2 signaling (P1219)

Abstract The signaling protein TNF receptor associated factor 3 (TRAF3) has multiple diverse effects on signaling transduction. We recently produced a new strain of T cell specific TRAF3 deficient mice (CD4CreTRAF3flox/flox (T-T3-/-)), and found that the frequency and number of Foxp3+ regulatory T cells (Treg) are increased 2-3 times compared with litter mate control mice (LMC), though total CD4+ and CD8+ T cell numbers do not change. These Treg are Helios+ and the increase is cell intrinsic. Interestingly, thymic Treg emerge as early as day 1 after birth in T-T3-/- mice, compared to day 3 after birth in LMC. Notably, the percentage of Treg precursor cells is unchanged. However, following IL-2 stimulation, TRAF3-deficient Treg precursor cells convert into Foxp3+ Treg more efficiently than those of LMC, though expression levels of IL-2 receptor components are comparable. We further found that phosphorylation of STAT5 is remarkably enhanced in TRAF3-/- Treg precursors in response to IL-2 stimulation. We also noticed that T-T3-/- conventional CD4 T cells exhibit robustly enhanced p-STAT5, but not p-ERK and p-AKT, when stimulated with IL-2. Additionally, TRAF3 deficiency affects the plasma membrane localization of T Cell Protein Tyrosine Phosphatase (TCPTP), an enzyme that plays a negative role in IL-2 signaling. Taken together, our data show for the first time that TRAF3 is an important regulator of IL-2 signaling and affects Treg development.

Inhibition of T cell protein tyrosine phosphatase enhances IL-18-dependent hematopoietic stem cell expansion (P6268)

Abstract The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to augment the progenitor cell pool. We report that inhibition of T cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a 9-fold increase in the number of hematopoietic progenitors in murine bone marrow. This effect could be reproduced using a short (48 h) treatment with a pharmacological inhibitor of TC-PTP in murine bone marrow, as well as in human bone marrow peripheral blood and cord blood. We also demonstrate that the ex-vivo use of TC-PTP inhibitor only provides a temporary effect on stem cells and did not alter their capacity to reconstitute all hematopoietic components in vivo. We establish that one of the mechanisms whereby inhibition of TC-PTP mediates its effects involves the IL-18 signaling pathway, leading to increased production of IL-12 and IFN-gamma by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in contributing to the augmentation of the stem cell pool, and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound.

Click Synthesis of Triazolyl Phenylalaninyl and Tyrosinyl Derivatives as New Protein Tyrosine Phosphatase Inhibitors

The efficient construction of triazolyl peptidomimetics via the powerful click chemistry for the discovery of small molecule-based chemotherapeutic agents represents a promising strategy in drug development today. Herein, the synthesis of novel mono-triazolyl or bis-triazolyl amino acid derivatives was rapidly achieved via microwave-assisted Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). Subsequent in vitro enzymatic assay on several homologous protein tyrosine phosphatases (PTPs) identified the triazolyl dimers as new specific inhibitors of Cell Cycle Division 25B (CDC25B) phosphatase and Protein Tyrosine Phosphatase 1B (PTP1B).

Non-structural 3 protein expression is associated with T cell protein tyrosine phosphatase and viral RNA levels in chronic hepatitis C patients

The hepatitis C virus (HCV) non-structural 3 (NS3) protein plays key roles in both the viral life cycle and in the modulation of intrahepatic signaling and immunity. We recently showed that NS3 cleaves the T cell protein tyrosine phosphatase (TCPTP).To better understand the inactivation of TCPTP in HCV-infected humans, we investigated whether there is an association between TCPTP cleavage, NS3 protein levels and clinical parameters in hepatitis C patients.Liver biopsies were obtained from 69 HCV RNA positive patients with confirmed chronic HCV infection and 16 control patients. Hepatic NS3 and TCPTP protein levels were determined and correlated to viral load or clinical parameters for the severity of liver disease.We found a positive correlation between the viral load and the intrahepatic NS3 protein levels in patients infected with HCV. HCV-infected patients had significantly lower intrahepatic TCPTP levels than non-infected control patients. In HCV-infected patients both intrahepatic NS3 expression and the viral load were inversely correlated with the intrahepatic TCPTP protein levels. Detection of NS3 did not associate with any other clinical parameters such as liver damage, the grade of liver inflammation or fibrosis stage.This is the first study reporting a detailed analysis of HCV NS3 and TCPTP protein levels in the liver. It demonstrates a clear link between HCV viral load, NS3 expression in the liver and intrahepatic TCPTP levels. Thus, the association between TCPTP cleavage and viral replication may have important consequences for the HCV life cycle and HCV-induced liver diseases.

HPN, a Synthetic Analogue of Bromophenol from Red Alga Rhodomela confervoides: Synthesis and Anti-Diabetic Effects in C57BL/KsJ-db/db Mice

3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(isopropoxymethyl)benzyl)benzene-1,2-diol (HPN) is a synthetic analogue of 3,4-dibromo-5-(2-bromo-3,4-dihydroxy-6-(ethoxymethyl)benzyl)benzene-1,2-diol (BPN), which is isolated from marine red alga Rhodomela confervoides with potent protein tyrosine phosphatase 1B (PTP1B) inhibition (IC50 = 0.84 μmol/L). The in vitro assay showed that HPN exhibited enhanced inhibitory activity against PTP1B with IC50 0.63 μmol/L and high selectivity against other PTPs (T cell protein tyrosine phosphatase (TCPTP), leucocyte antigen-related tyrosine phosphatase (LAR), Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2). The results of antihyperglycemic activity using db/db mouse model demonstrated that HPN significantly decreased plasma glucose (P < 0.01) after eight weeks treatment period. HPN lowered serum triglycerides and total cholesterol concentration in a dose-dependent manner. Besides, both of the high and medium dose groups of HPN remarkably decreased HbA1c levels (P < 0.05). HPN in the high dose group markedly lowered the insulin level compared to the model group (P < 0.05), whereas the effects were less potent than the positive drug rosiglitazone. Western blotting results showed that HPN decreased PTP1B levels in pancreatic tissue. Last but not least, the results of an intraperitoneal glucose tolerance test in Sprague–Dawley rats indicate that HPN have a similar antihyperglycemic activity as rosiglitazone. HPN therefore have potential for development as treatments for Type 2 diabetes.

Endothelial cell PTP1B regulates leukocyte recruitment during allergic inflammation

Pulmonary eosinophilia is a consistent hallmark of allergic lung inflammation. Infiltration of eosinophils into ovalbumin (OVA)-challenged lungs is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. Ligation of VCAM-1 activates endothelial cell protein tyrosine phosphatase 1B (PTP1B), which is required for VCAM-1-dependent leukocyte migration in vitro. To examine whether nonhematopoietic PTP1B modulates eosinophil recruitment in vivo, mice deficient in PTP1B were irradiated and received wild-type hematopoietic cells to generate chimeric PTP1B-/- mice. In response to OVA challenge, the chimeric PTP1B-/- mice had reduced eosinophilia in the lung tissue and bronchoalveolar lavage, indicating a role for PTP1B in nonhematopoietic cells during leukocyte recruitment. To determine whether endothelial cell PTP1B modulates eosinophil recruitment, mice with an inducible endothelial cell-specific PTP1B deletion (iePTP1B mice) were generated and the PTP1B deletion was induced after antigen sensitization before antigen challenge. In response to OVA challenge, the iePTP1B mice with the endothelial cell PTP1B deletion had an increased accumulation of eosinophils bound to the luminal surface of the endothelium in the lung vasculature and had a decrease in leukocyte recruitment into the lung tissue. In the iePTP1B mice, expression of adhesion molecules, cytokines, or chemokines that regulate leukocyte recruitment during inflammation was not altered, consistent with other studies that deletion of endothelial adhesion molecule signals does not alter lung cytokines and chemokines. In summary, these data suggest that VCAM-1 activation of PTP1B in the endothelium is necessary for eosinophil recruitment during allergic inflammation. Moreover, these studies provide a basis for targeting VCAM-1-dependent signaling pathways in allergy therapies.

Gene expression during long-term culture of mesenchymal stem cells obtained from patients with amyotrophic lateral sclerosis

Ex-vivo expansion of bone marrow (BM)-derived mesenchymal stem cells (MSCs) before clinical use for amyotrophic lateral sclerosis (ALS) patients is essential but occasionally results in cellular senescence as well as decrease of differentiation potential. In this study, we analyzed the genes that are up- or down-regulated by the long-term culture of MSCs from the BM of ALS patients using microarray and real-time RT-PCR. Among the analyzed genes, 878 (cluster 11) including RUNX2, SMO, IHH, GPNMB, HGF, COL10A1 and WNT3 were continually downregulated during long-term culture, whereas 732 (cluster 14) including PCGF5, HHIP and SERPINB2 were continually up-regulated. A gene ontology (GO) analysis revealed that genes related to cell differentiation (GO:0030154), growth factor activity (GO:0008201) and ossification (GO:0001503) belonged to cluster 11. Genes related to cell morphogenesis (GO:0000902) and protein tyrosine phosphatase activity (GO:0004725) belonged to cluster 14. When we also analyzed DNA methylation in RUNX2 using methylation-specific PCR, the −3.8 kb and −3.0 kb CpG island shores adjacent to the promoter of RUNX2 DNA became hypermethylated upon successive subculture. These results suggest that the decrease in RUNX2 mRNA observed during the long-term culture of MSCs may be at least partially related to the hypermethylation of the RUNX2 CpG island shores.

The tyrosine phosphatase TC48 interacts with and inactivates the oncogenic fusion protein BCR-Abl but not cellular Abl

The chimeric oncoprotein BCR-Abl exhibits deregulated protein tyrosine kinase activity and is responsible for the pathogenesis of certain human leukemias, such as chronic myelogenous leukemia. The activities of cellular Abl (c-Abl) and BCR-Abl are stringently regulated and the cellular mechanisms involved in their inactivation are poorly understood. Protein tyrosine phosphatases can negatively regulate Abl mediated signaling by dephosphorylating the kinase and/or its substrates. This study investigated the ability of the intracellular T cell protein tyrosine phosphatase (TCPTP/PTPN2) to dephosphorylate and regulate the functions of BCR-Abl and c-Abl. TCPTP is expressed as two alternately spliced isoforms — TC48 and TC45, which differ in their C-termini and localize to the cytoplasm and nucleus respectively. We show that TC48 dephosphorylates BCR-Abl but not c-Abl and inhibits its activity towards its substrate, CrkII. Y1127 and Y1294 residues whose phosphorylation corresponds with BCR-Abl activation status were the primary sites targeted by TC48. Co-localization and immunoprecipitation experiments showed that TC48 interacted with BCR-Abl but not with c-Abl, and BCR domain was sufficient for interaction. TC48 expression resulted in the stabilization of Bcr-Abl protein dependent on its phosphatase activity. Inactivation of cellular TC48 in K562 cells by stable expression of a dominant negative catalytically inactive mutant TC48, enhanced proliferation. TC48 expressing K562 clones showed reduced proliferation and enhanced sensitivity to STI571 compared to control clones suggesting that TC48 can repress the growth of CML cells. This study identifies a novel cellular regulator that specifically inhibits the activity of oncogenic BCR-Abl but not that of the cellular Abl kinase.

Immunohistochemical Analysis of IA-2 Family of Protein Tyrosine Phosphatases in Rat Gastrointestinal Endocrine Cells

Islet-associated protein-2 (IA-2) and IA-2β (also known as phogrin) are unique neuroendocrine-specific protein tyrosine phosphatases (PTPs). The IA-2 family of PTPs was originally identified from insulinoma cells and discovered to be major autoantigens in type 1 diabetes. Despite its expression in the neural and canonical endocrine tissues, data on expression of the IA-2 family of PTPs in gastrointestinal endocrine cells (GECs) are limited. Therefore, we immunohistochemically investigated the expression of the IA-2 family of PTPs in the rat gastrointestinal tract. In the stomach, IA-2 and IA-2β were expressed in GECs that secrete serotonin, somatostatin, and cholecystokinin/gastrin-1. In addition to these hormones, secretin, gastric inhibitory polypeptide (also known as the glucose-dependent insulinotropic peptide), glucagon-like peptide-1, and glucagon, but not ghrelin were coexpressed with IA-2 or IA-2β in duodenal GECs. Pancreatic islet cells that secrete gut hormones expressed the IA-2 family of PTPs. The expression patterns of IA-2 and IA-2β were comparable. These results reveal that the IA-2 family of PTPs is expressed in a cell type-specific manner in rat GECs. The extensive expression of the IA-2 family of PTPs in pancreo-gastrointestinal endocrine cells and in the enteric plexus suggests their systemic contribution to nutritional control through a neuroendocrine signaling network.

Inhibition of T cell Protein Tyrosine Phosphatase (TC-PTP) Enhances IL-18-dependent Hematopoietic Progenitor Expansion (111.57)

Abstract The clinical application of hematopoietic progenitor cell-based therapies for the treatment of hematological diseases is hindered by current protocols, which are cumbersome and have limited efficacy to expand the progenitor cell pool. We report that inhibition of T cell protein tyrosine phosphatase (TC-PTP), an enzyme involved in the regulation of cytokine signaling, through gene knockout results in a 9-fold expansion of functional hematopoietic progenitors in murine bone marrow (BM). This effect could be reproduced using a short (48 h) treatment with a pharmacological inhibitor of TC-PTP in murine BM, as well as in human BM, peripheral blood and cord blood. We also demonstrate that BM stem cells treated ex-vivo with a TC-PTP inhibitor can differentiate into functional progeny in vivo. We establish that the mechanism whereby inhibition of TC-PTP mediates its effects involves the IL-18 signaling pathway, leading to increased production of IL-12 and IFN-γ by progenitor cells. Together, our results reveal a previously unrecognized role for IL-18 in regulating progenitor cell expansion, and provide a novel and simple method to rapidly expand progenitor cells from a variety of sources using a pharmacological compound.

T cell protein tyrosine phosphatase deficiency results in spontaneous synovitis and subchondral bone resorption in mice

T cell protein tyrosine phosphatase (TC-PTP) is an important regulator of hematopoiesis and cytokine signaling. Recently, several genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in the locus of TC-PTP that are associated with rheumatoid arthritis and juvenile idiopathic arthritis, among other autoimmune diseases. The aim of this study was to evaluate the effect of TC-PTP deficiency on the bone and joint environment using a knockout mouse model. Radiographic and micro-computed tomography analyses were performed on femurs of 3-week-old mice. In addition, the femorotibial joints were assessed by histology, flow cytometry, and cytokine detection. Deficiency of TC-PTP resulted in decreased bone volume as well as an increase in osteoclast density within the mouse femurs. In addition, synovitis, characterized by infiltration of mixed inflammatory cell types and proinflammatory cytokines, developed in the knee joints of TC-PTP(-/-) mice. These findings demonstrate that loss of TC-PTP expression results in synovitis with several hallmarks of inflammatory arthritis. The inflammatory environment observed in the knee joints of TC-PTP(-/-) mice differs from the systemic inflammation previously described in these mice and merits further research into the role of TC-PTP in the synovium. Furthermore, the results support recently described associations between SNPs in the TC-PTP locus and arthritis incidence.

T cell protein tyrosine phosphatase (TCPTP) deficiency in muscle does not alter insulin signalling and glucose homeostasis in mice.

Insulin activates insulin receptor protein tyrosine kinase and downstream phosphatidylinositol-3-kinase (PI3K)/Akt signalling in muscle to promote glucose uptake. The insulin receptor can serve as a substrate for the protein tyrosine phosphatase (PTP) 1B and T cell protein tyrosine phosphatase (TCPTP), which share a striking 74% sequence identity in their catalytic domains. PTP1B is a validated therapeutic target for the alleviation of insulin resistance in type 2 diabetes. PTP1B dephosphorylates the insulin receptor in liver and muscle to regulate glucose homeostasis, whereas TCPTP regulates insulin receptor signalling and gluconeogenesis in the liver. In this study we assessed for the first time the role of TCPTP in the regulation of insulin receptor signalling in muscle. We generated muscle-specific TCPTP-deficient (Mck-Cre;Ptpn2(lox/lox)) mice (Mck, also known as Ckm) and assessed the impact on glucose homeostasis and muscle insulin receptor signalling in chow-fed versus high-fat-fed mice. Blood glucose and insulin levels, insulin and glucose tolerance, and insulin-induced muscle insulin receptor activation and downstream PI3K/Akt signalling remained unaltered in chow-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. In addition, body weight, adiposity, energy expenditure, insulin sensitivity and glucose homeostasis were not altered in high-fat-fed Mck-Cre;Ptpn2(lox/lox) versus Ptpn2(lox/lox) mice. These results indicate that TCPTP deficiency in muscle has no effect on insulin signalling and glucose homeostasis, and does not prevent high-fat diet-induced insulin resistance. Thus, despite their high degree of sequence identity, PTP1B and TCPTP contribute differentially to insulin receptor regulation in muscle. Our results are consistent with the notion that these two highly related PTPs make distinct contributions to insulin receptor regulation in different tissues.

Unraveling the functional implications of GWAS: how T cell protein tyrosine phosphatase drives autoimmune disease

Genome-wide association studies (GWAS) have identified a large number of SNPs that are linked to human autoimmune diseases. However, the functional consequences of most of these genetic variations remain undefined. T cell protein tyrosine phosphatase (TCPTP, which is encoded by PTPN2) is a JAK/STAT and growth factor receptor phosphatase that has been linked to the pathogenesis of type 1 diabetes, rheumatoid arthritis, and Crohn's disease by GWAS. In this issue of the JCI, Wiede and colleagues have generated a T cell-specific deletion of TCPTP and identified a novel role for this phosphatase as a negative regulator of TCR signaling. These data provide new insight as to how noncoding PTPN2 SNPs identified in GWAS could drive human autoimmune diseases.

T cell protein tyrosine phosphatase attenuates T cell signaling to maintain tolerance in mice.

Many autoimmune diseases exhibit familial aggregation, indicating that they have genetic determinants. Single nucleotide polymorphisms in PTPN2, which encodes T cell protein tyrosine phosphatase (TCPTP), have been linked with the development of several autoimmune diseases, including type 1 diabetes and Crohn's disease. In this study, we have identified TCPTP as a key negative regulator of TCR signaling, which might explain the association of PTPN2 SNPs with autoimmune disease. We found that TCPTP dephosphorylates and inactivates Src family kinases to regulate T cell responses. Using T cell-specific TCPTP-deficient mice, we established that TCPTP attenuates T cell activation and proliferation in vitro and blunts antigen-induced responses in vivo. TCPTP deficiency lowered the in vivo threshold for TCR-dependent CD8(+) T cell proliferation. Consistent with this, T cell-specific TCPTP-deficient mice developed widespread inflammation and autoimmunity that was transferable to wild-type recipient mice by CD8(+) T cells alone. This autoimmunity was associated with increased serum levels of proinflammatory cytokines and anti-nuclear antibodies, T cell infiltrates in non-lymphoid tissues, and liver disease. These data indicate that TCPTP is a critical negative regulator of TCR signaling that sets the threshold for TCR-induced naive T cell responses to prevent autoimmune and inflammatory disorders arising.

Ethyl-3,4-dephostatin Inhibits PTPN2 and Induces ERK Activation

The mitogen-activated protein kinases (MAPKs) are key signaling enzymes that play critical roles in cellular signal transduction pathways such as cell survival, proliferation, differentiation, and apoptosis. The three major groups of MAPK that are expressed in mammals are p38, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK). Signaling by MAPKs is processed following their phosphorylation at conserved threonine and tyrosine residues within the activation loop of MAPKs mediated by their upstream kinases in activation. Protein tyrosine phosphatases (PTPs) are responsible for regulation of MAPK activity through dephosphorylation of phospho-threonine and phospho-tyrosine residues on MAPKs. The human genome encodes 107 PTPs. PTPs can be classified into four groups based on amino-acid sequences of catalytic domains. They are class I Cys-based PTPs, class II Cys-based PTPs, class III Cys-based PTPs, and Asp-based PTPs. Especially, protein tyrosine phosphatase non-receptor type 2 (PTPN2) that is also known as T cell protein tyrosine phosphatase (TC-PTP) belongs to class I Cys-based PTPs as the intracellular, non-receptor PTPs (NRPTPs) with a high degree of sequence and structural homology within the catalytic domain. PTPN2 has been reported to modulate cytokine receptor signaling, including IFN-γ signaling. PTPN2 dephosphorylates and inactivates Src tyrosine kinases to suppress downstream signaling through ERK and production of interleukin 6. In a way to study biological functions of PTPN2, we performed the regulatory mechanism of PTPN2 activity using a specific chemical compound, ethyl-3,4-dephostatin. Ethyl-3,4-dephostatin (Fig. 1) was originally identified as a potent PTP-1B and src homology-2-containing protein tyrosine phosphatase-1 (SHP-1) inhibitor and showed inhibitory effect on several PTPs in vitro. Recently, dual-specificity phosphatase 22 (DUSP22) was identified as an additional target of ethyl-3,4-dephostatin. To investigate whether ethyl-3,4-dephostatin was able to inhibit PTPN2 activity, we treated various concentrations of ethyl-3,4-dephostatin on purified active PTPN2. PTPN2 activity was decreased by ethyl-3,4-dephostatin in a dosedependent manner. The half maximal inhibitory concentration (IC50) of PTPN2 was found to be about 6.5 ± 0.54 μM by using the curve fitting program PRISM 3.0 (Fig. 2(a)). In addition, the Lineweaver-Burk plot shows that the Ki was 8.6 μM (Fig. 2(b)). This result indicates that ethyl-3,4dephostatin acts as a competitive inhibitor of PTPN2, suggesting that ethyl-3,4-dephostatin suppresses the activity of PTPN2 through binding to the catalytic site. We further examined whether ethyl-3,4-dephostatin influences PTPN2-mediated ERK inhibition in vivo. Since PTPN2 inactivates Src tyrosine kinases that are located upstream of ERK signaling, we overexpressed PTPN2 in HEK 293 cells and detected phosphorylation levels of endogenous ERK after treatment with ethyl-3,4-dephostatin. HEK 293 cells were transfected with FLAG-PTPN2 expression plasmid and

Dinuclear copper complexes of organic claw: Potent inhibition of protein tyrosine phosphatases

Three dinuclear copper complexes of organic claw ligands (2,2′,2″,2‴-(5-R-2-hydroxy-1,3-phenylene)bis(methylene)bis(azanetriyl)tetraacetic acid, R = methyl (H 5L1), chloro (H 5L2) and bromo (H 5L3)): [Cu 2NaL1(H 2O) 2] ( 1), [Cu 2HL2(H 2O) 2] ( 2), [Cu 2NaL3(H 2O) 2] ( 3), have been synthesized and characterized by elemental analyses, infrared spectra, thermo-gravimetric analyses, X-ray diffraction analysis, electrospray ionization mass spectra, pH-potentiometric titration, molar conductivity. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T cell protein tyrosine phosphatase (TCPTP), Megakaryocyte protein tyrosinephosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) are evaluated in vitro. The three copper complexes exhibit potent and almost same inhibition against PTP1B and SHP-1 with IC 50 values ranging from 0.15 to 0.31 μM, about 2-fold stronger inhibition than against PTP-MEG2, 10-fold stronger inhibition than against TCPTP, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Molecular docking analyses confirm the inhibition model. Fluorescence titration studies suggest that the complexes bond to PTP1B with the formation of a 1:1 complex. The results demonstrate that copper complexes that are potent PTPs inhibitors but have different inhibitory effects over different PTPs, may be explored as new practical inhibitors towards individual PTP with some specificity.

Expression of T Cell Protein Tyrosine Phosphatase (PTPN2) is Enhanced in Mucosal T Cells in IBD Patients

Expression of T Cell Protein Tyrosine Phosphatase (PTPN2) is Enhanced in Mucosal T Cells in IBD Patients