Furanoterpenes,new types of protein tyrosine phosphatase 1B inhibitors,from two Indonesian marine sponges,Ircinia and Spongia spp.

Protein tyrosine phosphatase (PTP) 1B negatively regulates the insulin and leptin signaling pathways, and, thus, the clinical application of PTP1B inhibitors to the prevention and treatment of type 2 diabetes and obesity is expected. During our studies on PTP1B inhibitors, two furanosesterterpenes and a C21 furanoterpene were obtained as new types of PTP1B inhibitors from two Indonesian marine sponges. (7E, 12E, 20Z, 18S)-Variabilin (1) and (12E, 20Z, 18S)-8-hydroxyvariabilin (2) from Ircinia sp. and furospongin-1 (3) from Spongia sp. inhibited PTP1B activity with IC₅₀ values of 1.5, 7.1, and 9.9 μM, respectively. The inhibitory activity of compound 1 against T-cell PTP (TCPTP) was approximately 2-fold that against PTP1B, whereas the vaccinia H-1-related phosphatase (VHR) inhibitory effects of 1 were 4-fold weaker than that of its PTP1B inhibitory activity. Compounds 1–3 at 50 μM did not show cytotoxicity against two human cancer cell lines, hepatoma Huh-7 and bladder carcinoma EJ-1. Compound 1 did not enhance the phosphorylation level of Akt, a key downstream effector of the cascade, in Huh-7 cells.     

Structure-based design of a low molecular weight, nonphosphorus, nonpeptide, and highly selective inhibitor of protein-tyrosine phosphatase 1B

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.     

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations.     

Protein Tyrosine Phosphatase 1B and Insulin Resistance: Role of Endoplasmic Reticulum Stress/Reactive Oxygen Species/Nuclear Factor Kappa B Axis

Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [³H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH₂-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.     

Inhibition of protein tyrosine phosphatase 1B by diterpenoids isolated from Acanthopanax koreanum

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been proposed as a therapy to treat type 2 diabetes and obesity. In our preliminary screening study on the PTP1B inhibitory activity, a CH2Cl2-soluble extract of the roots of Acanthopanax koreanum (Araliaceae) was found to inhibit PTP1B activity at 30 microg/ml. Eight diterpenoids were isolated from the active fraction and were evaluated for their inhibitory effect on PTP1B. A kaurane-type diterpene, 16alphaH,17-isovaleryloxy-ent-kauran-19-oic acid (7), inhibited PTP1B with an IC50 value of 7.1+/-0.9 microM in a non-competitive manner. Acanthoic acid (2) and ent-kaur-16-en-19-oic acid (5) also inhibited PTP1B in dose-dependent manners. Either introduction of a hydroxyl group or reduction of a carboxyl group at C-19 in pimarane-type to alcohol abolished the inhibitory effects toward PTP1B.     

蛋白质酪氨酸磷酸酶1B（PTP1B）与2型糖尿病及肥胖的关系

蛋白质酪氨酸磷酸酶1B（PTP1B）是一种在体内广泛表达的胞内蛋白质酪氨酸磷酸酶,在调节胰岛素敏感性和能量代谢的过程中起着重要作用。通过抑制PTP1B可增加胰岛素和瘦蛋白（leptin）的活性, 为寻找2型糖尿病、肥胖的治疗提供了光明前景。     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Modulation of protein tyrosine phosphatase 1B by erythropoietin in UT-7 cell line.

BACKGROUND/ AIMS: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. METHODS: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. RESULTS: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). CONCLUSION: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.     

Reduction of protein-tyrosine phosphatase-1B increases insulin signaling in FAO hepatoma cells

Protein-tyrosine phosphatase-1B (PTP1B) has been implicated as a negative regulator of insulin signaling. PTP1B dephosphorylates the insulin receptor and insulin receptor substrates (IRS-1/2), inhibiting the insulin-signaling pathway. PTP1B has been reported to be elevated in diabetes and insulin-resistant states. Conversely, PTP1B null mice have increased insulin sensitivity. To further investigate the effect of PTP1B reduction on insulin signaling, FAO rat hepatoma cells were transfected, by electroporation, with a specific PTP1B antisense oligonucleotide (ASO), or a control oligonucleotide. The PTP1B ASO caused a 50-70% reduction in PTP1B protein expression as measured by Western blot analysis. Upon insulin stimulation, an increase in the phosphorylation of the insulin receptor and insulin receptor substrates was observed, without any change in protein expression levels. Reduction of PTP1B expression in FAO cells also caused an increase in insulin-stimulated phosphorylation of PKB and GSK3, without any change in protein expression. These results demonstrate that reduction of PTP1B can modulate key insulin signaling events downstream of the insulin receptor.     

The CREB/KMT5A complex regulates PTP1B to modulate high glucose-induced endothelial inflammatory factor levels in diabetic nephropathy

Diabetic nephropathy (DN) is the primary microvascular complication of diabetes mellitus and may result in end-stage renal disease. The overproduction of various inflammatory factors is involved in the pathogenesis of DN. Protein tyrosine phosphatase 1B (PTP1B) modulates the expression of a series of cytokines and nuclear factor kappa B (NF-κB) activity. cAMP response element-binding protein (CREB) and lysine methyltransferase 5A (KMT5A) have been reported to participate in the maintenance of a healthy endothelium. In the present study, we hypothesise that CREB associates with KMT5A to modulate PTP1B expression, thus contributing to high glucose-mediated glomerular endothelial inflammation. Our analyses revealed that plasma inflammatory factor levels, glomerular endothelial p65 phosphorylation and PTP1B expression were increased in DN patients and rats. In vitro, high glucose increased endothelial inflammatory factor levels and p65 phosphorylation by augmenting PTP1B expression in human umbilical vein endothelial cells (HUVECs). Moreover, high glucose decreased CREB and KMT5A expression. CREB overexpression and KMT5A overexpression both inhibited high glucose-induced PTP1B expression, p65 phosphorylation and endothelial inflammatory factor levels. si-CREB- and sh-KMT5A-induced p65 phosphorylation and endothelial inflammatory factor levels were reversed by si-PTP1B. Furthermore, CREB was associated with KMT5A. Mechanistic research indicated that CREB and histone H4 lysine 20 methylation (H4K20me1, a downstream target of KMT5A) occupy the PTP1B promoter region. sh-KMT5A augmented PTP1B promoter activity and activated the positive effect of si-CREB on PTP1B promoter activity. Our in vivo study demonstrated that CREB and KMT5A were downregulated in glomerular endothelial cells of DN patients and rats. In conclusion, CREB associates with KMT5A to promote PTP1B expression in vascular endothelial cells, thus contributing to hyperglycemia-induced inflammatory factor levels in DN patients and rats.Subject terms: Diabetes complications, Type 2 diabetes     

The reciprocal role of Egr-1 and Sp family proteins in regulation of the PTP1B promoter in response to the p210 Bcr-Abl oncoprotein-tyrosine kinase

Protein-tyrosine phosphatase 1B (PTP1B) is an important regulator of protein-tyrosine kinase-dependent signaling pathways. Changes in expression and activity of PTP1B have been associated with various human diseases; however, the mechanisms by which PTP1B expression is regulated have yet to be characterized. Previously, we have shown that the expression of PTP1B is enhanced by p210 Bcr-Abl and that PTP1B is a specific antagonist of transformation induced by this oncoprotein protein-tyrosine kinase. Here we have characterized the PTP1B promoter and demonstrate that a motif with features of a stress-response element acts as a p210 Bcr-Abl-responsive sequence, termed PRS. We have shown that three C(2)H(2) zinc finger proteins, namely Sp1, Sp3, and Egr-1, bind to PRS. Whereas binding of either Sp1 or Sp3 induced promoter function, Egr-1 repressed Sp3-mediated PTP1B promoter activation. The binding of Egr-1 to PRS is suppressed by p210 Bcr-Abl due to the inhibition of Egr-1 expression, resulting in the enhancement of PTP1B promoter activity. Our data indicate that Egr-1 and Sp family proteins play a reciprocal role in the control of expression from the PTP1B promoter.     

PTP1B regulates leptin signal transduction in vivo

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.     

Rhododendric acid A, a new ursane-type PTP1B inhibitor from the endangered plant Rhododendron brachycarpum G. Don

In spite of the critical role of the natural products in drug discovery, surprising little attention has been placed on endangered and rare plant species that could play a pivotal role in pharmaceutical and fiber development. Protein tyrosine phosphatase-1B (PTP1B), which blocks insulin signaling, has been gaining interest to be a promising therapeutic target for the treatment of type 2 diabetes mellitus. Bioassay-guided fractionation on the leaves of Rhododendron brachycarpum G. Don (Ericaceae) yielded seven PTP1B inhibitory triterpenoids, including a new triterpene, rhododendric acid A (1). Their PTP1B inhibitory potency and their lipophilicity were investigated to provide a feasible scaffold that may overcome the innate limitation of the previously reported PTP1B inhibitors.     

Galpha(i2) enhances insulin signaling via suppression of protein-tyrosine phosphatase 1B

Suppression of the expression of the heterotrimeric G-protein Galpha(i2) in vivo has been shown to provoke insulin resistance, whereas enhanced insulin signaling is observed when Galpha(i2) is overexpressed in vivo. The basis for Galpha(i2) regulation of insulin signaling was explored in transgenic mice with targeted expression of the GTPase-deficient, constitutively active Q205L Galpha(i2) in fat and skeletal muscle. Phosphorylation of insulin receptor and IRS-1 in response to insulin challenge in vivo was markedly amplified in fat and skeletal muscle expressing Q205L Galpha(i2). The expression and activity of the protein-tyrosine phosphatase 1B (PTP1B), but not protein-tyrosine phosphatases SHP-1, SHP-2, and LAR, were constitutively decreased in tissues expressing the Q205L Galpha(i2), providing a direct linkage between insulin signaling and Galpha(i2). The loss of PTP1B expression may explain, in part, the loss of PTP1B activity in the iQ205L transgenic mice. Activation of Galpha(i2) in mouse adipocytes with lysophosphatidic acid was shown to decrease PTP1B activity, whereas pertussis toxin inactivates Galpha(i2), blocks lysophosphatidic acid-stimulated inhibition of PTP1B activity, and blocks tonic suppression of PTP1B activity by Galpha(i2). Elevation of intracellular cAMP in fat cells is shown to increase PTP1B activity, whereas either depression of cAMP levels or direct activation of Galpha(i2) suppresses PTP1B. These data provide the first molecular basis for the interplay between Galpha(i2) and insulin signaling, i.e. activation of Galpha(i2) can suppress both the expression and activity of PTP1B in insulin-sensitive tissues.     

Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.     

Crystal structure of PTP1B complexed with a potent and selective bidentate inhibitor

Protein-tyrosine phosphatase 1B (PTP1B) has been implicated as an important regulator in several signaling pathways including those initiated by insulin and leptin. Potent and specific PTP1B inhibitors could serve as useful tools in elucidating the physiological functions of PTP1B and may constitute valuable therapeutics in the treatment of several human diseases. We have determined the crystal structure of PTP1B in complex with compound 2, the most potent and selective PTP1B inhibitor reported to date. The structure at 2.15-A resolution reveals that compound 2 simultaneously binds to the active site and a unique proximal noncatalytic site formed by Lys-41, Arg-47, and Asp-48. The structural data are further corroborated by results from kinetic analyses of the interactions of PTP1B and its site-directed mutants with compound 2 and several of its variants. Although many of the residues important for interactions between PTP1B and compound 2 are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which provide a structural basis for potent and selective PTP1B inhibition. Our data further suggest that potent, yet highly selective, PTP1B inhibitory agents can be acquired by targeting the area defined by residues Lys-41, Arg-47, and Asp-48, in addition to the previously identified second aryl phosphate-binding pocket.     

Protein tyrosine phosphatase 1B deficiency potentiates PERK/eIF2α signaling in brown adipocytes

Background

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and body mass, and has been implicated in endoplasmic reticulum (ER) stress. Herein, we assess the role of PTP1B in ER stress in brown adipocytes, which are key regulators of thermogenesis and metabolic response.

Methodology/Principal Findings

To determine the role of PTP1B in ER stress, we utilized brown adipose tissue (BAT) from mice with adipose-specific PTP1B deletion, and brown adipocytes deficient in PTP1B and reconstituted with PTP1B wild type (WT) or the substrate-trapping PTP1B D181A (D/A) mutant. PTP1B deficiency led to upregulation of PERK-eIF2α phosphorylation and IRE1α-XBP1 sub-arms of the unfolded protein response. In addition, PTP1B deficiency sensitized differentiated brown adipocytes to chemical-induced ER stress. Moreover, PERK activation and tyrosine phosphorylation were increased in BAT and adipocytes lacking PTP1B. Increased PERK activity resulted in the induction of eIF2α phosphorylation at Ser51 and better translatability of ATF4 mRNA in response to ER stress. At the molecular level, we demonstrate direct interaction between PTP1B and PERK and identify PERK Tyr615 as a mediator of this association.

Conclusions

Collectively, the data demonstrate that PTP1B is a physiologically-relevant modulator of ER stress in brown adipocytes and that PTP1B deficiency modulates PERK-eIF2α phosphorylation and protein synthesis.     

Protein-tyrosine phosphatase-1B (PTP1B) mediates the anti-migratory actions of Sprouty

Mammalian Sprouty proteins have been shown to inhibit the proliferation and migration of cells in response to growth factors and serum. In this communication, using HeLa cells, we have examined the possibility that human Sprouty 2 (hSPRY2) mediates its anti-migratory actions by modulating the activity or intracellular localization of protein-tyrosine phosphatases. In HeLa cells, overexpression of hSPRY2 resulted in an increase in protein-tyrosine phosphatase (PTP1B) amount and activity in the soluble (100,000 x g) fraction of cells without an increase in total amount of cellular PTP1B. This increase in the soluble form of PTP1B was accompanied by a decrease in the amount of the enzyme in the particulate fraction. The amounts of PTP-PEST or PTP1D in the soluble fractions were not altered. Consistent with an increase in soluble PTP1B amount and activity, the tyrosine phosphorylation of cellular proteins and p130(Cas) was decreased in hSPRY2-expressing cells. In control cells, overexpression of wild-type (WT) PTP1B, but not its C215S catalytically inactive mutant mimicked the actions of hSPRY2 on tyrosine phosphorylation of cellular proteins and migration. On the other hand, in hSPRY2-expressing cells, the C215S mutant, but not WT PTP1B, increased tyrosine phosphorylation of cellular proteins and attenuated the anti-migratory actions of hSPRY2. Interestingly, neither WT nor C215S mutant forms of PTP1B modulated the anti-mitogenic actions of hSPRY2. Therefore, we conclude that an increase in soluble PTP1B activity contributes to the anti-migratory, but not anti-mitogenic, actions of hSPRY2.     

The role of protein-tyrosine phosphatase 1B in integrin signaling

Protein-tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling and a novel therapeutic target for the treatment of type 2 diabetes, obesity, and other associated metabolic syndromes. Because PTP1B regulates multiple signal pathways and it can both enhance and antagonize a cellular event, it is important to establish the physiological relevance of PTP1B in these processes. In this study, we utilize potent and selective PTP1B inhibitors to delineate the role of PTP1B in integrin signaling. We show that down-regulation of PTP1B activity with small molecule inhibitors suppresses cell spreading and migration to fibronectin, increases Tyr(527) phosphorylation in Src, and decreases phosphorylation of FAK, p130(Cas), and ERK1/2. In addition, PTP1B "substrate-trapping" mutants bind Tyr(527)-phosphorylated Src and protect it from dephosphorylation by endogenous PTP1B. These results establish that PTP1B promotes integrin-mediated responses in fibroblasts by dephosphorylating the inhibitory pTyr(527) and thereby activating the Src kinase. We also show that PTP1B forms a complex with Src and p130(Cas), and that the proline-rich motif PPRPPK (residues 309-314) in PTP1B is essential for the complex formation. We suggest that the specificity of PTP1B for Src pTyr(527) is mediated by protein-protein interactions involving the docking protein p130(Cas) with both Src and PTP1B in addition to the interactions between the PTP1B active site and the pTyr(527) motif.