Investigation of protein-tyrosine phosphatase 1B function by quantitative proteomics

Because of their antagonistic catalytic functions, protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases act together to control phosphotyrosine-mediated signaling processes in mammalian cells. However, unlike for protein-tyrosine kinases, little is known about the cellular substrate specificity of many PTPs because of the lack of appropriate methods for the systematic and detailed analysis of cellular PTP function. Even for the most intensely studied, prototypic family member PTP1B many of its physiological functions cannot be explained by its known substrates. To gain better insights into cellular PTP1B function, we used quantitative MS to monitor alterations in the global tyrosine phosphorylation of PTP1B-deficient mouse embryonic fibroblasts in comparison with their wild-type counterparts. In total, we quantified 124 proteins containing 301 phosphotyrosine sites under basal, epidermal growth factor-, or platelet-derived growth factor-stimulated conditions. A subset of 18 proteins was found to harbor hyperphosphorylated phosphotyrosine sites in knock-out cells and was functionally linked to PTP1B. Among these proteins, regulators of cell motility and adhesion are overrepresented, such as cortactin, lipoma-preferred partner, ZO-1, or p120ctn. In addition, regulators of proliferation like p62DOK or p120RasGAP also showed increased cellular tyrosine phosphorylation. Physical interactions of these proteins with PTP1B were further demonstrated by using phosphatase-inactive substrate-trapping mutants in a parallel MS-based analysis. Our results correlate well with the described phenotype of PTP1B-deficient fibroblasts that is characterized by an increase in motility and reduced cell proliferation. The presented study provides a broad overview about phosphotyrosine signaling processes in mouse fibroblasts and, supported by the identification of various new potential substrate proteins, indicates a central role of PTP1B within cellular signaling networks. Importantly the MS-based strategies described here are entirely generic and can be used to address the poorly understood aspects of cellular PTP function.     

The tumor suppressor PTEN is necessary for human Sprouty 2-mediated inhibition of cell proliferation

Sprouty family proteins are novel regulators of growth factor actions. Human Sprouty 2 (hSPRY2) inhibits the proliferation of a number of different cell types. However, the mechanisms involved in the anti-proliferative actions of hSPRY2 remain to be elucidated. Here we have demonstrated that hSPRY2 increases the amount of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and decreases its phosphorylation. The resultant increase in PTEN activity is reflected in decreased activation of Akt by epidermal growth factor and serum. Consistent with increased PTEN activity, in hSPRY2-expressing cells, the progression of cells from the G1 to S phase is decreased. By using PTEN null primary mouse embryonic fibroblasts and their isogenic controls as well as small interfering RNA against PTEN, we demonstrated that PTEN is necessary for hSPRY2 to inhibit Akt activation by epidermal growth factor as well as cell proliferation. Overall, we concluded that hSPRY2 mediates its anti-proliferative actions by altering PTEN content and activity.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in l6 myocytes and Fao hepatoma cells

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.     

A novel role of Sprouty 2 in regulating cellular apoptosis

Sprouty (SPRY) proteins modulate receptor-tyrosine kinase signaling and, thereby, regulate cell migration and proliferation. Here, we have examined the role of endogenous human SPRY2 (hSPRY2) in the regulation of cellular apoptosis. Small inhibitory RNA-mediated silencing of hSPRY2 abolished the anti-apoptotic action of serum in adrenal cortex adenocarcinoma (SW13) cells. Silencing of hSPRY2 decreased serum- or epidermal growth factor (EGF)-elicited activation of AKT and ERK1/2 and reduced the levels of EGF receptor. Silencing of hSPRY2 also inhibited serum-induced activation of p90RSK and decreased phosphorylation of pro-apoptotic protein BAD (BCL2-antagonist of cell death) by p90RSK. Inhibiting both the ERK1/2 and AKT pathways abolished the ability of serum to protect against apoptosis, mimicking the effects of silencing hSPRY2. Serum transactivated the EGF receptor (EGFR), and inhibition of the EGFR by a neutralizing antibody attenuated the anti-apoptotic actions of serum. Consistent with the role of EGFR and perhaps other growth factor receptors in the anti-apoptotic actions of serum, the tyrosine kinase binding domain of c-Cbl (Cbl-TKB) protected against down-regulation of the growth factor receptors such as EGFR and preserved the anti-apoptotic actions of serum when hSpry2 was silenced. Additionally, silencing of Spry2 in c-Cbl null cells did not alter the ability of serum to promote cell survival. Moreover, reintroduction of wild type hSPRY2, but not its mutants that do not bind c-Cbl or CIN85 into SW13 cells after endogenous hSPRY2 had been silenced, restored the anti-apoptotic actions of serum. Overall, we conclude that endogenous hSPRY2-mediated regulation of apoptosis requires c-Cbl and is manifested by the ability of hSPRY2 to sequester c-Cbl and thereby augment signaling via growth factor receptors.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

Essential tyrosine residues for interaction of the non-receptor protein-tyrosine phosphatase PTP1B with N-cadherin

Expression of a dominant-negative, catalytically inactive form of the nonreceptor protein-tyrosine phosphatase PTP1B in L-cells constitutively expressing N-cadherin results in loss of N-cadherin-mediated cell-cell adhesion. PTP1B interacts directly with the cytoplasmic domain of N-cadherin, and this association is regulated by phosphorylation of tyrosine residues in PTP1B. The following three tyrosine residues in PTP1B are potential substrates for tyrosine kinases: Tyr-66, Tyr-152, and Tyr-153. To determine the tyrosine residue(s) that are crucial for the cadherin-PTP1B interaction we used site-directed mutagenesis to create catalytically inactive PTP1B constructs bearing additional single, double, or triple mutations in which tyrosine was substituted by phenylalanine. Mutation Y152F eliminates binding to N-cadherin in vitro, whereas mutations Y66F and Y153F do not. Overexpression of the catalytically inactive PTP1B with the Y152F mutation in L-cells constitutively expressing N-cadherin has no effect on N-cadherin-mediated adhesion, and immunoprecipitation reveals that the mutant Y152F PTP1B does not associate with N-cadherin in situ. Furthermore, among cells overexpressing the Y152F mutant endogenous PTP1B associates with N-cadherin and is tyrosine-phosphorylated.     

Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H(2)O(2) in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H(2)O(2) as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M(r) insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases.     

Evidence for direct interaction between Sprouty and Cbl

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.     

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.     

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.     

The C terminus of sprouty is important for modulation of cellular migration and proliferation

The Drosophila Sprouty (SPRY) protein has been shown to inhibit the actions of epidermal growth factor and fibroblast growth factor. However, the role of mammalian SPRY proteins has not been clearly elucidated. We postulated that human Sprouty2 (hSPRY2) is an inhibitor of cellular migration and proliferation. Indeed, using stably transfected HeLa cells, which expressed hemagglutinin (HA)-tagged hSPRY2 or hSPRY2 tagged at the C terminus with red fluorescent protein, we demonstrated that hSPRY2 inhibits the migration of cells in response to serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. Additionally, hSPRY2 also inhibited the growth of HeLa cells in response to serum. Previously, two C-terminal domains on hSPRY2, which are necessary for its colocalization with microtubules (residues 123-177) or translocation to membrane ruffles (residues 178-194), have been identified (Lim, J., Wong, E. S., Ong, S. H., Yusoff, P., Low, B. C., and Guy, G. R. (2000) J. Biol. Chem. 275, 32837-32845). Therefore, using TAT-tagged hSPRY2 and its mutants, we determined the role of these two C-terminal domains in the inhibition of cell migration and proliferation. Our data show that the deletion of either of these two regions in hSPRY2 abrogates its ability to modulate cell migration in response to different growth factors and proliferation in response to serum. Therefore, we conclude that hSPRY2 inhibits the actions of a number of growth factors, and its C terminus, which is homologous among various SPRY isoforms, is important in mediating its biological activity.     

PTP1B regulates cortactin tyrosine phosphorylation by targeting Tyr446

The emergence of protein-tyrosine phosphatase 1B (PTP1B) as a potential drug target for treatment of diabetes, obesity, and cancer underlies the importance of understanding its full range of cellular functions. Here, we have identified cortactin, a central regulator of actin cytoskeletal dynamics, as a substrate of PTP1B. A trapping mutant of PTP1B binds cortactin at the phosphorylation site Tyr(446), the regulation and function of which have not previously been characterized. We show that phosphorylation of cortactin Tyr(446) is induced by hyperosmolarity and potentiates apoptotic signaling during prolonged hyperosmotic stress. This study advances the importance of Tyr(446) in the regulation of cortactin and provides a potential mechanism to explain the effects of PTP1B on processes including cell adhesion, migration, and tumorigenesis.     

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.     

Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the Grb2 adaptor protein

Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction. However, little is known regarding the molecular interactions that influence the balance of these processes, especially the phosphorylation state of postinsulin receptor substrates, such as insulin receptor substrate-1 (IRS-1). The specific activity of four candidate protein-tyrosine phosphatases (protein-tyrosine phosphatase 1B (PTP1B), SH2 domain-containing PTPase-2 (SHP-2), leukocyte common antigen-related (LAR), and leukocyte antigen-related phosphatase) (LRP) toward IRS-1 dephosphorylation was studied using recombinant proteins in vitro. PTP1B exhibited the highest specific activity (percentage dephosphorylated per microg per min), and the enzyme activities varied over a range of 5.5 x 10(3). When evaluated as a ratio of activity versus IRS-1 to that versus p-nitrophenyl phosphate, PTP1B remained significantly more active by 3.1-293-fold, respectively. Overlay blots with recombinant Src homology 2 domains of IRS-1 adaptor proteins showed that the loss of IRS-1 binding of Crk, GRB2, SHP-2, and the p85 subunit of phosphatidylinositol 3'-kinase paralleled the rate of overall IRS-1 dephosphorylation. Further studies revealed that the adaptor protein GRB2 strongly promoted the formation of a stable protein complex between tyrosine-phosphorylated IRS-1 and catalytically inactive PTP1B, increasing their co-immunoprecipitation from an equimolar solution by 13.5 +/- 3.3-fold (n = 7; p < 0.01). Inclusion of GRB2 in a reaction mixture of IRS-1 and active PTP1B also increased the overall rate of IRS-1 tyrosine dephosphorylation by 2.7-3.9-fold (p < 0.01). These results provide new insight into novel molecular interactions involving PTP1B and GRB2 that may influence the steady-state capacity of IRS-1 to function as a phosphotyrosine scaffold and possibly affect the balance of postreceptor insulin signaling.     

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.     

Tyrosine phosphatase PTP1B interacts with TRPV6 in vivo and plays a role in TRPV6-mediated calcium influx in HEK293 cells

This study investigates the role of tyrosine phosphorylation and dephosphorylation in the regulation of the Ca(2+) permeant TRPV6 channel. HEK293 cells co-transfected with TRPV6 and the tyrosine phosphatase PTP1B show a constitutive Ca(2+) entry which was independent of tyrosine phosphorylation under resting conditions. Following depletion of intracellular Ca(2+) stores, TRPV6-mediated Ca(2+) entry could be increased in the presence of a tyrosine phosphatase inhibitor (bis-(N,N-dimethyl-hydroxamido) hydroxo-vanadate; DMHV). Inhibition of Src-kinases completely abolished DMHV-induced increase in TRPV6-mediated Ca(2+) influx. Co-transfection with Src led to tyrosine phosphorylation of TRPV6 which could be dephosphorylated by PTP1B. In vivo interaction of TRPV6 with PTP1B was visualized using the bimolecular fluorescence complementation (BiFC) method and proved by co-immunoprecipitation of both proteins. These data indicate that tyrosine phosphorylation is involved in the regulation of the TRPV6 channel protein.     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.     

Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation

Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 A resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H(2)O(2)-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.