PTPase Activity Research Articles

Inhibitory effects of green tea catechins on protein tyrosine phosphatase in Prevotella intermedia.

Members of the Prevotella intermedia group possess protein tyrosine phosphatase (PTPase). The purpose of this study was to investigate the effects of catechin derivatives from Japanese green tea on the activity of PTPase in P. intermedia and related organisms. Multilocus enzyme electrophoresis of alkaline phosphatase derived from P. intermedia, Prevotella nigrescens, Prevotella pallens and Porphyromonas gingivalis revealed a species-specific migration pattern. Among the tea catechin derivatives tested, (-)-epigallocatechin gallate (EGCg), similar to orthovanadate, a specific inhibitor for PTPase, was effective in inhibiting the PTPase activity in P. intermedia at 0.5 microm, and related species at 5 microm. The results suggested that the inhibitory effect observed is due to the presence of galloyl moiety in the structure. In contrast, neither the green tea catechins nor orthovanadate inhibited the phosphatase activity in P. gingivalis, suggesting that this organism possessed a different family of alkaline phosphatase.

Chemical dissection of the effects of tyrosine phosphorylation of SHP-2.

The regulation of the protein tyrosine phosphatase (PTPase) SHP-2 by tyrosine phosphorylation has been difficult to elucidate because of the intrinsic instability of the phosphoprotein. In the past, expressed protein ligation has been used to site-specifically incorporate the phosphotyrosine mimic Pmp (phosphonomethylene phenylalanine) into the two tyrosine phosphorylation sites (542, 580) of SHP-2 one at a time to analyze the effects on catalytic behavior. In this study, we have incorporated two Pmps into the phosphorylation sites simultaneously and examined the effects of double SHP-2 tyrosine phosphorylation. We have found that the Pmp groups show close to additive effects on PTPase stimulation, suggesting dual SH2 domain occupancy. The relative effects of the phosphotyrosine analogue difluoromethylene phosphonophenylalanine (F(2)Pmp) compared to those of Pmp were also examined. It was found that the F(2)Pmp analogue showed slightly enhanced PTPase stimulation compared with the Pmp analogue, consistent with its higher affinity for SH2 domains. Taken together with the bis-Pmp studies, these data suggest that double phosphorylation of the SHP-2 C-terminus could give rise to a 9-fold overall PTPase activation, 30-50% of the value associated with deletion of the SH2 domains. Catalytically inactive forms of phosphorylated SHP-2 proteins were also produced by expressed protein ligation. This allowed for a systematic analysis of intermolecular autodephosphorylation of SHP-2, which revealed how conformational plasticity can modulate phosphotyrosine stability.

Membrane-bound acid phosphatase (MAP) from Entamoeba histolytica has phosphotyrosine phosphatase activity and disrupts the actin cytoskeleton of host cells.

Protein tyrosine phosphatases (PTPases) have been described as virulence factors in different pathogenic microorganisms. The pathogenic process by Enatamoeba histolytica is a multifactorial phenomenon that occurs in 3 steps: adhesion, cytolytic and cytotoxic effect, and phagocytosis. Lytic enzymes may participate during the second part of this process. In this work, we determined that purified membrane-bound acid phosphatase (MAP) from E. histolytica trophozoites has PTPase activity. The enzyme specifically dephosphorylated O-phospho-L-tyrosine at optimum pH of 5.0, with little activity towards O-phospho-L-serine, O-phospho-L-threonine, and ATP. It was inhibited by ammonium molybdate and sodium tungstate, and trifluoperazine did not show any effect. A monoclonal antibody against the catalytic domain of the human placental PTPase 1B, cross-reacted with a 55 kDa molecule present in the solubilized fraction. The interaction of the amoebic PTPase with HeLa cells resulted in the alteration of the cell actin cytoskeleton by disruption of the actin stress fibres.

Vanadate enhances leptin-induced activation of JAK/STAT pathway in CHO cells

Leptin, the product of the ob gene, is an adipocyte-derived hormone that plays a key role in the control of food intake and energy expenditure. Leptin acts through receptors that belong to a member of the class I cytokine receptor family. It has been demonstrated that the SH2 domain-containing tyrosine phosphatase 2 (SHP-2) negatively regulates STAT3-mediated transcriptional activation through long form leptin receptor (OBRb). Vanadate has been shown to be a potent and selective inhibitor of PTPase activity in vitro. In this study, we have demonstrated that vanadate increases leptin-induced JAK2 and STAT3 phosphorylation in CHO cells expressing OBRb. The increased leptin-dependent luciferase activity of SOCS3 gene was also seen in vanadate-treated cell. Furthermore, vanadate reversed the inhibitory effects of SOCS3 on leptin-induced STAT3 phosphorylation. The present findings suggest that PTP inhibitors including vanadate and vanadate-derived compounds could be used as a therapeutic agent in the treatment of obesity.

Dipeptidyl peptidase IV on activated T cells as a target molecule for therapy of rheumatoid arthritis.

The extracellular domain of the T cell co-stimulatory molecule CD26 possesses dipeptidyl peptidase IV (DP IV) enzyme activity. Activated T cells are known to increase expression of cell surface DP IV and some specific inhibitors of this enzyme have been reported to suppress T cell function. Previously we have identified a DP IV inhibitor, designated TMC-2, found in culture supernatant of Aspergillus oryzae. Administration of TMC-2 to rats with adjuvant arthritis caused marked suppression of paw swelling. To elucidate the mechanism of TMC-2 antiarthritic activity, we have studied its effects on T cell function. Here we show that TMC-2 inhibited DP IV activity of CD26 immunoprecipitated from T cell lysates, and also inhibited proliferative responses of T cells to specific antigen or anti-CD3 antibody. Suppression of IL-2 production was demonstrated at both the mRNA and protein levels. TMC-2 did not alter the PTPase activity of pure CD45, but when this molecule was co-precipitated from T cell lysates together with associated CD26, its PTPase was virtually completely abolished by TMC-2. These results suggest that modulation of CD45 PTPase activity might be responsible for functional suppression of T cells by TMC-2. Because the effects of TMC-2 on T cells were reversible and it was not toxic at the concentrations used, TMC-2 may be a candidate novel therapeutic agent for rheumatoid arthritis.

Chicken Anemia Virus VP2 Is a Novel Dual Specificity Protein Phosphatase

The function of viral protein 2 (VP2) of the immunosuppressive circovirus chicken anemia virus (CAV) has not yet been established. We show that the CAV VP2 amino acid sequence has some similarity to a number of eukaryotic, receptor, protein-tyrosine phosphatase (PTPase) alpha proteins as well as to a cluster of human TT viruses within the Sanban group. To investigate if CAV VP2 functions as a PTPase, purified glutathione S-transferase (GST)-VP2 fusion protein was assayed for PTPase activity using the generalized peptide substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine), with free phosphate detected using the malachite green colorimetric assay. CAV GST-VP2 was shown to catalyze dephosphorylation of both substrates. CAV GST-VP2 PTPase activity for the ENDpYINASL substrate had a V(max) of 14,925 units/mg.min and a K(m) of 18.88 microm. Optimal activity was observed between pH 6 and 7, and activity was specifically inhibited by 0.01 mm orthovanadate. We also show that the ORF2 sequence of the CAV-related human virus TT-like minivirus (TLMV) possessed PTPase activity and steady state kinetics equivalent to CAV GST-VP2 when expressed as a GST fusion protein. To establish whether these viral proteins were dual specificity protein phosphatases, the CAV GST-VP2 and TLMV GST-ORF2 fusion proteins were also assayed for serine/threonine phosphatase (S/T PPase) activity using the generalized peptide substrate RRApTVA, with free phosphate detected using the malachite green colorimetric assay. Both CAV GST-VP2 and TLMV GST-ORF2 fusion proteins possessed S/T PPase activity, which was specifically inhibited by 50 mm sodium fluoride. CAV GST-VP2 exhibited S/T PPase activity with a V(max) of 28,600 units/mg.min and a K(m) of 76 microm. Mutagenesis of residue Cys(95) to serine in CAV GST-VP2 abrogated both PTPase and S/T PPase activity, identifying it as the catalytic cysteine within the proposed signature motif. These studies thus show that the circoviruses CAV and TLMV encode dual specificity protein phosphatases (DSP) with an unusual signature motif that may play a role in intracellular signaling during viral replication. This is the first DSP gene to be identified in a small viral genome.

Effects of troglitazone on skeletal muscle and liver protein tyrosine phosphatase activity in insulin-resistant otsuka long-evans tokushima fatty rats

Background: Protein tyrosine phosphatases (PTPases) appear to play an essential role in the regulation of steady-state phosphorylation of the insulin receptor and other proteins in the insulin-signaling pathway. Objectives: This study determined the role of PTPases in skeletal muscle and liver in an insulin-resistant model and the in vivo effect of troglitazone on PTPases. Methods: We used 26-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats as models of insulin resistance. PTPase activity was assessed with reduced carboxyamido-methylated and -maleylated lysozyme as substrates. The protein levels of 3 major candidate PTPases—leukocyte common antigen-related PTPase (LAR), PTPase 1B (PTP1B), and src homology 2-containing PTPase (SH-PTP2)—in skeletal muscle and liver of OLETF rats were analyzed using Western blot. The activities and amounts of PTPases in skeletal muscle and liver of OLETF rats treated with troglitazone for 7 days also were examined. Results: Particulate and cytosolic PTPase activities in skeletal muscle of OLETF rats were increased compared with those in control rats. Similar changes were observed in the liver. The protein levels of LAR, PTP1B, and SH-PTP2 in particulate fractions of skeletal muscle of OLETF rats were increased, but levels of PTP1B and SH-PTP2 in cytosolic fractions were unchanged. Similar findings were observed in the liver. Troglitazone normalized the increased PTPase activity and each protein level except cytosolic fraction of the liver. Conclusions: The findings suggest that troglitazone decreases elevated activities and levels of PTPases in the particulate fraction of the skeletal muscle and liver of OLETF rats, and that PTPase plays important roles through regulating the insulin-signaling system in the pathogenesis of insulin resistance in OLETF rats.

Antiproliferative activity induced by the somatostatin analogue, TT-232, in human pancreatic cancer cells

Somatostatin analogues have been developed as antiproliferative agents, but their administration as general antitumour agents is limited, mainly because of the wide distribution of somatostatin receptors throughout the human body. TT-232, a new somatostatin structural analogue, was reported to have tumour-selective antiproliferative activity without an antisecretory action. We examined whether TT-232 had antiproliferative activity in human pancreatic cancer cell lines, and compared its antiproliferative activity with that of RC-160 and other TT-232 derivatives. TT-232 inhibited the growth of all of the cell lines used in this study and induced apoptotic cell death. RC-160 showed no such growth inhibition. TT-232 also inhibited tumour formation in a xenograft model. A competitive binding assay was performed using the cell membrane fraction and 111In-DTPA-TT-232 in order to show the existence of a specific binding site on the cells. A specific binding site was detected in MIAPaCa-2 cells. It has been shown that the activation of protein tyrosine phosphatase (PTPase) is one of the main intracellular pathways responsible for somatostatinergic inhibition of cell growth. We found a significant PTPase stimulation after TT-232 administration using an immunoblot analysis assessing the level of protein tyrosine phosphorylation, and also a direct measurement of the PTPase activity. We also demonstrated that PTPase stimulation by TT-232 was involved in its antiproliferative activity as this activity was reversed by the addition of sodium orthovanadate, a PTPase inhibitor. Our results indicate that TT-232 could be a potentially useful therapeutic agent if these data are translated into clinical practice.

Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities.

Protein-tyrosine phosphatases (PTPases) have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible) fraction of the endogenous PTPase pool.

Entamoeba histolytica-induced dephosphorylation in host cells.

Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation.

EGFR-independent activation of ERK1/2 mediates growth inhibition by a PTPase antagonizing K-vitamin analog.

The K-vitamin analog Cpd 5 or [2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone] is a potent cell growth inhibitor in vitro and in vivo, likely due to arylation of enzymes containing a catalytic cysteine. This results in inhibition of protein tyrosine phosphatase (PTPase) activity with resultant hyperphosphorylation of EGF receptors (EGFR) and ERK1/2 protein kinases, which are downstream to EGFR in the MAPK pathway. We used NR6 fibroblast cells, which lack endogenous EGFR and its variant cells transfected with different EGFR mutants to assess the contribution of the EGFR-mediated signaling pathway to Cpd 5-mediated ERK activation and cell growth inhibition. Cpd 5 treatment resulted in enhanced phosphorylation of EGFR at carboxyl-terminal tyrosines. This phosphorylation and activation of EGFR were found to be necessary neither for growth inhibition nor for the activation of the downstream kinases ERK1/2, since both occurred in EGFR-devoid mutant cells. U0126 and PD 098059, specific inhibitors of MEK1/2, the ERK1/2 kinases, antagonized both cell growth inhibition and ERK1/2 phosphorylation mediated by Cpd5. Cpd 5 was also found to inhibit ERK1/2 phosphatase(s) activity in lysates from all the cells tested, irrespective of their EGFR status. These results show that EGFR-independent ERK1/2 phosphorylation was involved in the mechanism of Cpd5 mediated growth inhibition. This is likely due to the observed antagonism of ERK phosphatase activity. A candidate PTPase was found to be Cdc25A, a recently identified ERK phosphatase.

Protein tyrosine phosphatase activity in insulin-resistant rodent Psammomys obesus.

Phosphotyrosine phosphatase (PTPase) activity and its regulation by overnight food deprivation were studied in Psammomys obesus (sand rat), a gerbil model of insulin resistance and nutritionally induced diabetes mellitus. PTPase activity was measured using a phosphopeptide substrate containing a sequence identical to that of the major site of insulin receptor (IR) β-subunit autophosphorylation. The PTPase activity in membrane fractions was 3.5-, 8.3-, and 5.9-fold lower in liver, fat, and skeletal muscle, respectively, compared with corresponding tissues of albino rat.Western blotting of tissue membrane fractions in Psammomys showed lower PTPase and IR than in albino rats. The density of PTPase transmembrane protein band was 5.5-fold lower in liver and 12-fold lower in adipose tissue. Leukocyte antigen receptor (LAR) and IR were determined by specific immunoblotting and protein bands densitometry and were also found to be 6.3-fold lower in the liver and 22-fold lower in the adipose tissue in the hepatic membrane fractions. Liver cytosolic PTPase activity after an overnight food deprivation in the nondiabetic Psammomys rose 3.7-fold compared with postprandial PTPase activity, but it did not change significantly in diabetic fasted animals. Similar fasting-related changes were detected in the activity of PTPase derived from membrane fraction. In conclusion, the above data demonstrate that despite the insulin resistance, Psammomys is characterized by low level of PTPase activities in membrane and cytosolic fractions in all 3 major insulin responsive tissues, as well as in liver. PTPase activity does not rise in activity as a result of insulin resistance and nutritionally induced diabetes.

Troglitazone ameliorates abnormal activity of protein tyrosine phosphatase in adipose tissues of Otsuka Long-Evans Tokushima Fatty rats.

Protein tyrosine phosphatases (PTPases) play an essential role in the regulation of steady-state phosphorylation of the insulin receptor and other proteins in the insulin signaling pathway. To determine the role of PTPases in adipose tissue in the development into an insulin-resistant state, we examined PTPase activities and protein levels of three major candidate PTPases in adipose tissues of 26-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Particulate PTPase activities in visceral and epididymal adipose tissues of OLETF rats were increased compared to those in Long-Evans Tokushima Otsuka (LETO) rats, non-insulin-resistant controls. Cytosolic PTPase activities in these tissues were conversely decreased in OLETF rats. In subcutaneous adipose tissues, those changes were not observed. Western blot analysis showed that the amounts of leukocyte antigen-related PTPase (LAR), PTPase 1B (PTP1B), and src homology 2-containing PTPase (SH-PTP2) were increased in particulate fractions of visceral and epididymal fat of OLETF rats. On the other hand, those in the cytosolic fractions were slightly decreased. Troglitazone was administered to OLETF rats to examine the effect of the drug on the changes in PTPase activity and distribution. Troglitazone treatment restored those alterations in PTPase activity in the particulate fraction and the amounts of LAR, PTP1B and SH-PTP2 in both fractions of visceral and epididymal adipose tissues of OLETF rats. Although it remains unknown whether such effects of troglitazone are mediated by peroxisome proliferator-activated receptor y, these data provide useful information for understanding the significance of PTPase in insulin-resistant rats and the molecular mechanism of troglitazone action.

Assays for protein-tyrosine phosphatases

Many studies have implicated protein-tyrosine phosphatases (PTPases) as important regulators of cellular functions and as potential drug targets. This has led to an increased interest in the development of precise assays for detailed mechanistic studies of PTPases, rapid assays for high-throughput PTPase inhibitor screening, and more biologically relevant assays using physiological protein substrates. The first assays aimed specifically at PTPases utilized tyrosine-phosphorylated proteins such as myosin light chain LC 20 , insulin receptor, epidermal growth factor receptor, RCM-lysozyme, or myelin basic protein as substrates while 32 P i release was monitored as the readout of PTPase activity. The tyrosine-phosphorylated forms of other proteins and peptides such as Raytide, angiotensin II, glutamine synthase, and angiotensin II peptides have also been used as PTPase substrates. Studies involving phosphorylated, physiological substrates have also been carried out in which phosphoamino acid analysis, phosphopeptide mapping, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with phosphoimaging, were used to monitor PTPase activity.

Depot-Specific Variation in Protein-Tyrosine Phosphatase Activities in Human Omental and Subcutaneous Adipose Tissue: A Potential Contribution to Differential Insulin Sensitivity

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42–71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor β-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

Depot-specific variation in protein-tyrosine phosphatase activities in human omental and subcutaneous adipose tissue: a potential contribution to differential insulin sensitivity.

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42-71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor beta-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

Acquisition of a Specific and Potent PTP1B Inhibitor from a Novel Combinatorial Library and Screening Procedure

Protein-tyrosine phosphatases (PTPases) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTPase activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases including cancers and diabetes. For example, recent gene knockout studies in mice identify PTP1B as a promising target for anti-diabetes/obesity drug discovery. Thus, there is intense interest in obtaining specific and potent PTPase inhibitors for biological studies and pharmacological development. However, given the highly conserved nature of the PTPase active site, it is unclear whether selectivity in PTPase inhibition can be achieved. We describe a combinatorial approach that is designed to target both the active site and a unique peripheral site in PTP1B. Compounds that can simultaneously associate with both sites are expected to exhibit enhanced affinity and specificity. We also describe a novel affinity-based high-throughput assay procedure that can be used for PTPase inhibitor screening. The combinatorial library/high-throughput screen protocols furnished a small molecule PTP1B inhibitor that is both potent (K(i) = 2.4 nm) and selective (little or no activity against a panel of phosphatases including Yersinia PTPase, SHP1, SHP2, LAR, HePTP, PTPalpha, CD45, VHR, MKP3, Cdc25A, Stp1, and PP2C). These results demonstrate that it is possible to acquire potent, yet highly selective inhibitors for individual members of the large PTPase family of enzymes.

TU-572, a Potent and Selective CD45 Inhibitor, Suppresses IgE-Mediated Anaphylaxis and Murine Contact Hypersensitivity Reactions

Background: CD45, receptor-type protein tyrosine phosphatases (PTPases) are essential components of signaling through both the T cell receptor and the B cell antigen receptor. However, the functional significance of CD45 in the signaling pathway through the high-affinity immunoglobulin (Ig) E receptor has not yet been established. In this study, we demonstrate that the potent CD45 inhibitor negatively regulates IgE-dependent anaphylaxis and contact hypersensitivity reactions. Method: We have previously found that TU-572, 2-[(4-methylthiopyridin-2-yl)methylsulfinyl]-5-isopropoxybenzimidazole, had a potent and selective inhibitory effect against PTPase activity of CD45. Using a CD45 inhibitor, we examined in vitro and in vivo IgE-mediated responses. Results: TU-572 potently inhibited histamine release from rat peritoneal mast cells and mouse systemic anaphylaxis reaction using monoclonal anti-dinitrophenyl (DNP) IgE and DNP-BSA. TU-572 also suppressed the immediate-type hypersensitivity response induced by repeated epicutaneous application of trinitrochlorobenzene in BALB/c mice. Conclusion: These findings revealed that the PTPase activity of CD45 played a critical role in signal transduction of IgE-mediated anaphylaxis in vitro and in vivo. PTPase inhibitors such as TU-572 are useful in the treatment of allergic diseases.

Site-Specific Incorporation of a Phosphotyrosine Mimetic Reveals a Role for Tyrosine Phosphorylation of SHP-2 in Cell Signaling

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using “expressed protein ligation”, nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.