Protein tyrosine phosphatase 1B participates in the down-regulation of erythropoietin receptor signalling.
ABSTRACT: Erythropoietin (EPO) is the principal hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Binding of ligand to the cell-surface EPO-R (EPO receptor) induces dimerization and JAK2 (Janus kinase 2)-mediated tyrosine phosphorylation of the receptor. Less than 1% of the EPO-Rs are displayed on the cell surface; most of the receptor molecules are retained in intracellular compartments, including the ER (endoplasmic reticulum). Using pervanadate (PV) as a potent tool to inhibit cellular PTPs (protein tyrosine phosphatases), we demonstrated previously the accumulation of mature (endoglycosidase H-resistant) tyrosine-phosphorylated EPO-R [Cohen, Altaratz, Zick, Klingmuller and Neumann (1997) Biochem. J. 327, 391-397]. In the present study, we investigated the participation of the ER-associated PTP1B in the dephosphorylation of intracellular EPO-R. We demonstrate tyrosine phosphorylation of EPO-R in BOSC-23T cells co-expressing EPO-R and the 'substrate-trapping' mutant form of PTP1B, PTP1B D181A (referred to as PTP1BD). In vivo interaction between EPO-R and PTP1B suggested that PTP1B dephosphorylates the EPO-R intracellularly. Endoglycosidase H resistance of tyrosine-phosphorylated EPO-R in cells expressing PTP1BD suggested that mature EPO-R is dephosphorylated by PTP1B. Stimulation with EPO of cells co-expressing EPO-R and either PTP1BD or PTP1B resulted in an increase or decrease respectively in phosphotyrosine EPO-R. We thus suggest that PTP1B dephosphorylates EPO-stimulated EPO-R and participates in the down-regulation cascade of EPO-mediated signal transduction.
Project description:PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.
Project description:The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a "substrate trap." Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B in vivo and that one important function of PTP1B is to prevent the inappropriate, ligand-independent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.
Project description: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.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-eIF2? phosphorylation and IRE1?-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 eIF2? 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.Collectively, the data demonstrate that PTP1B is a physiologically-relevant modulator of ER stress in brown adipocytes and that PTP1B deficiency modulates PERK-eIF2? phosphorylation and protein synthesis.
Project description:Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.
Project description:Erythropoietin (EPO) is the major hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Ligand binding to the erythropoietin receptor (EPO-R), a member of the cytokine receptor family, triggers Tyr phosphorylation of the surface form of the receptor, presumably mediated by the Janus kinase (JAK) 2. To study whether non-surface EPO-R can be phosphorylated, Ba/F3 cells stably transfected with EPO-R were treated with pervanadate (PV), which is widely used as a potent tool to inhibit cellular protein Tyr phosphatases, thus resulting in enhanced Tyr phosphorylation of cellular proteins. PV treatment caused the EPO-R to undergo Tyr phosphorylation in a time-dependent and dose-dependent manner. PV-mediated Tyr phosphorylation of EPO-R occurred at several intracellular sites including the endoplasmic reticulum (ER), because both endoglycosidase H (endo H)-resistant EPO-R and the ER-retained EPO-R mutant (DeltaWS1 EPO-R) were Tyr phosphorylated in response to PV. Moreover, in metabolic labelling experiments, endo H-sensitive EPO-R was also phosphorylated. The phosphorylated fraction accounted for only 30-50% of the newly synthesized EPO-R, the fraction that normally exits from the ER. Tyr phosphorylation could not be detected on proteolytic fragments of the EPO-R, suggesting that this is a highly regulated process. Unlike the wild-type (wt) EPO-R, which was phosphorylated both on EPO binding and after inhibition of Tyr phosphatases by PV treatment, an EPO-R mutant (W282R EPO-R) that does not activate JAK2 was phosphorylated after PV treatment but not by EPO binding. Both EPO-R and JAK2 were phosphorylated with similar kinetics by PV treatment, suggesting that JAK2, as well as protein Tyr kinases different from JAK2, might mediate PV-dependent EPO-R phosphorylation. Furthermore the Tyr-phosphorylated ER-retained EPO-R mutant DeltaWS1 co-immunoprecipitated with JAK2 kinase, indicating that the EPO-R might interact with JAK2 while in the ER.
Project description:PTP1B is an endoplasmic reticulum (ER) anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC) in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM) associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC). Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.
Project description:Vascular endothelial growth factor (VEGF) binding induces phosphorylation of VEGF receptor (VEGFR)2 in tyrosine, which is followed by disruption of VE-cadherin-mediated cell-cell contacts of endothelial cells (ECs), thereby stimulating EC proliferation and migration to promote angiogenesis. Tyrosine phosphorylation events are controlled by the balance of activation of protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Little is known about the role of endogenous PTPs in VEGF signaling in ECs. In this study, we found that PTP1B expression and activity are markedly increased in mice hindlimb ischemia model of angiogenesis. In ECs, overexpression of PTP1B, but not catalytically inactive mutant PTP1B-C/S, inhibits VEGF-induced phosphorylation of VEGFR2 and extracellular signal-regulated kinase 1/2, as well as EC proliferation, whereas knockdown of PTP1B by small interfering RNA enhances these responses, suggesting that PTP1B negatively regulates VEGFR2 signaling in ECs. VEGF-induced p38 mitogen-activated protein kinase phosphorylation and EC migration are not affected by PTP1B overexpression or knockdown. In vivo dephosphorylation and cotransfection assays reveal that PTP1B binds to VEGFR2 cytoplasmic domain in vivo and directly dephosphorylates activated VEGFR2 immunoprecipitates from human umbilical vein endothelial cells. Overexpression of PTP1B stabilizes VE-cadherin-mediated cell-cell adhesions by reducing VE-cadherin tyrosine phosphorylation, whereas PTP1B small interfering RNA causes opposite effects with increasing endothelial permeability, as measured by transendothelial electric resistance. In summary, PTP1B negatively regulates VEGFR2 receptor activation via binding to the VEGFR2, as well as stabilizes cell-cell adhesions through reducing tyrosine phosphorylation of VE-cadherin. Induction of PTP1B by hindlimb ischemia may represent an important counterregulatory mechanism that blunts overactivation of VEGFR2 during angiogenesis in vivo.
Project description:To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in beta1-integrin- mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with beta1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.
Project description:Protein tyrosine phosphatase (PTP)1B is an abundant non-transmembrane enzyme that plays a major role in regulating insulin and leptin signaling. Recently, we reported that PTP1B is inhibited by sumoylation, and that sumoylated PTP1B accumulates in a perinuclear distribution, consistent with its known localization in the endoplasmic reticulum (ER) and the contiguous outer nuclear membrane. Here, we report that, in addition to its localization at the ER, PTP1B also is found at the inner nuclear membrane, where it is heavily sumoylated. We also find that PTP1B interacts with emerin, an inner nuclear membrane protein that is known to be tyrosine phosphorylated, and that PTP1B expression levels are inversely correlated with tyrosine phosphorylation levels of emerin. PTP1B sumoylation greatly increases as cells approach mitosis, corresponding to the stage where tyrosine phosphorylation of emerin is maximal. In addition, expression of a non-sumoylatable mutant of PTP1B greatly reduced levels of emerin tyrosine phosphorylation. These results suggest that PTP1B regulates the tyrosine phosphorylation of a key inner nuclear membrane protein in a sumoylation- and cell-cycle-dependent manner.
Project description:Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: alpha-and beta- or gamma- catenin. Phosphorylation of beta-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from beta-catenin, thus maintaining the cadherin-actin connection (). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and beta-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion.