Protein tyrosine phosphatase

The protein tyrosine phosphatase (PTP) Shp2 (PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2 E76K mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2 E76K -dependent cell survival, which is associated with inhibition of Shp2 E76K PTP activity, Shp2 E76K -induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-γ (IFN-γ)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

A simple procedure was devrscd for Isolatmg from homogenates of mltotlc cells the human homolog to the fss~on yeast c&2 gene product The ldentlty of the purified protem was established with antI-p34""' antlbodles and pl3""', both specific hgands for p34c"c* Active-site labelmg with oxldlzed [cz"P]ATP showed the purified moIecule to be an ATP-bmdmg protein Its ablhty to phosphorylate casem but not hlstone, and Its phosphorylatlon on tyrosme, detected by ant;-phosphotyrosme antIbodIes, mchcates the form of p3slcdcz Purified quantmes of human ~34"" purified IS the mactlve or apocnzymc form should be of consldeeable value m estabhshmg the mechamsm of Its actlvatlon at nutosls by phosphatases Mltosls, Phosphotyrosmc, p34""(', Human

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor. The mechanism of action of the lead inhibitor compound 4e was studied by a combination of hydrogen/deuterium-exchange mass spectrometry and molecular modeling. The results suggest that the inhibitor interacts critically with a hydrophobic patch located outside the active site of the phosphatase. Targeting of secondary allosteric sites is viewed as a promising yet unexplored approach to develop pharmacological inhibitors of protein tyrosine phosphatases. Our † Accession Codes PDB codes: 3H2X, 2P6X.

We investigated the expression of protein tyrosine phosphatase-interacting protein 51 (PTPIP51) and its interaction with protein tyrosine phosphatase 1B (PTP1B) and 14-3-3β in mice exhibiting insulin resistance and obesity. A total of 20 mice were included in the study. Eight control animals were fed a normal standard diet, six animals were fed a high-fat diet and six animals were submitted to a treadmill training parallel to the feeding of a high-fat diet. After 10 weeks, a glucose tolerance test was performed and abdominal adipose tissue samples of the animals were collected. PTPIP51 protein was identified in the adipocytes of all samples. PTPIP51 interacted with PTP1B and with 14-3-3β protein. Compared with untrained mice fed a standard diet, the interaction of PTPIP51 with PTP1B was reduced in high-fat diet-fed animals. The highest interaction of PTPIP51 with 14-3-3β was seen in trained animals on high-fat diet, whereas untrained animals on high-fat diet displayed lowest values....

Vascular endothelial–cadherin (VE-cadherin) plays a key role in angiogenesis and in vascular permeability. The regulation of its biological activity may be a central mechanism in normal or pathological angiogenesis. VE-cadherin has been shown to be phosphorylated on tyrosine in vitro under various conditions, including stimulation by VEGF. In the present study, we addressed the question of the existence of a tyrosine phosphorylated form of VE-cadherin in vivo, in correlation with the quiescent versus angiogenic state of adult tissues. Phosphorylated VE-cadherin was detected in mouse lung, uterus, and ovary but not in other tissues unless mice were injected with peroxovanadate to block protein phosphatases. Remarkably, VE-cadherin tyrosine phosphorylation was dramatically increased in uterus and ovary, and not in other organs, during PMSG/hCG-induced angiogenesis. In parallel, we observed an increased association of VE-cadherin with Flk1 (VEGF receptor 2) during hormonal angiogenesis...

Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cblmediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

Caveolin-1 (CAV1) enhanced migration, invasion, and metastasis of cancer cells is inhibited by co-expression of the glycoprotein E-cadherin. Although the two proteins form a multiprotein complex that includes β-catenin, it remained unclear how this would contribute to blocking the metastasis promoting function of CAV1. Here, we characterized by mass spectrometry the protein composition of CAV1 immunoprecipitates from B16F10 murine melanoma cells expressing or not E-cadherin. The novel protein tyrosine phosphatase PTPN14 was identified by mass spectrometry analysis exclusively in co-immunoprecipitates of CAV1 with E-cadherin. Interestingly, PTPN14 is implicated in controlling metastasis, but only few known PTPN14 substrates exist. We corroborated by western blotting experiments that PTPN14 and CAV1 co-inmunoprecipitated in the presence of E-cadherin in B16F10 melanoma and other cancer cells. Moreover, the CAV1(Y14F) mutant protein was shown to co-immunoprecipitate with PTPN14 even in...

Protein oxidation is increasingly recognized as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signaling, there is increasing evidence that other protein residues are oxidized in vivo with impact on cellular homeostasis and redox signaling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidized derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signaling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

Protein oxidation is increasingly recognized as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signaling, there is increasing evidence that other protein residues are oxidized in vivo with impact on cellular homeostasis and redox signaling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidized derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signaling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

TCR stimulation by Ag or anti-receptor antibodies in murine T cells results in the activation of two independent protein kinases, protein kinase C (PKC) and a protein tyrosine kinase. Similarly, stimulation of murine Thy-1 or Ly-6 with mAb also results in activation of both of these kinase pathways. Tyrosine phosphorylation in all cases occurs on the TCR zeta-chain. It is known that Ag and anti-receptor antibodies activate PKC in human T cells. In this study we demonstrate that mitogen or anti-CD3 antibodies activate tyrosine phosphorylation of the human TCR-zeta-chain. PMA, which activates PKC, does not result in zeta-chain tyrosine phosphorylation. Stimulation of human T cells by antibodies that bind the CD2 molecule is an alternate mode of inducing T cell proliferation. These antibodies surprisingly do not induce tyrosine phosphorylation of the zeta-chain. Thus, different methods of cellular activation can result in distinguishable patterns of receptor-mediated biochemical signal...

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) plays a pivotal role in immune homeostasis and has been associated with human autoimmune and chronic inflammatory diseases. Though PTPN2 is well-characterized in lymphocytes, little is known about its function in innate immune cells. Our findings demonstrate that dendritic cell (DC)-intrinsic PTPN2 might be the key to explain the central role for PTPN2 in the immune system to maintain immune tolerance. Partial genetic PTPN2 ablation in DCs resulted in spontaneous inflammation, particularly in skin, liver, lung and kidney 22 weeks post-birth. DC-specific PTPN2 controls steady-state immune cell composition and even incomplete PTPN2 deficiency in DCs resulted in enhanced organ infiltration of conventional type 2 DCs, accompanied by expansion of IFNγ-producing effector T-cells. Consequently, the phenotypic effects of DC-specific PTPN2 deficiency were abolished in T-cell deficient Rag knock-out mice. Our data add substantial knowledge about the molecular mechanisms to prevent inflammation and maintain tissue tolerance.

Molecular expression profiling of tumors initiated by transgenic overexpression of c-myc , c-neu , c-ha-ras , polyoma middle T antigen ( PyMT ) or simian virus 40 T/t antigen (T-ag) targeted to the mouse mammary gland have identified both common and oncogene-specific events associated with tumor formation and progression. The tumors shared great similarities in their gene-expression profiles as compared with the normal mammary gland with an induction of cell-cycle regulators, metabolic regulators, zinc finger proteins, and protein tyrosine phosphatases, along with the suppression of some protein tyrosine kinases. Selection and hierarchical clustering of the most variant genes, however, resulted in separating the mouse models into three groups with distinct oncogene-specific patterns of gene expression. Such an identification of targets specified by particular oncogenes may facilitate development of lesion-specific therapeutics and preclinical testing. Moreover, similarities in gene ...

T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endogenous mechanism present in T-lymphocyte membranes. This activation was sensitive to sodium orthovanadate and O-phosphotyrosine, consistent with the involvement of a phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphatase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) in pp56lck activation. Based on a recent report demonstrating that CD45, the leukocyte common antigen, is a membrane-bound PTPase, we analyzed its role in pp56lck activation. CD45 was found to be the major (gr...

Protein kinase C θ (PKC θ) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC θ-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC θ at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC θ −/− mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC θ and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.

PhyA from Selenomonas ruminantium (PhyAsr), is a bacterial protein tyrosine phosphatase (PTP)-like inositol polyphosphate phosphatase (IPPase) that is distantly related to known PTPs. PhyAsr has a second substrate binding site referred to as a standby site and the P-loop (HCX 5 R) has been observed in both open (inactive) and closed (active) conformations. Site-directed mutagenesis and kinetic and structural studies indicate PhyAsr follows a classical PTP mechanism of hydrolysis and has a broad specificity toward polyphosphorylated myo-inositol substrates, including phosphoinositides. Kinetic and molecular docking experiments demonstrate PhyAsr preferentially cleaves the 3-phosphate position of Ins P 6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D-Ins(1,2,4,5,6)P 5 , Ins(2,4,5,6)P 4 , D-Ins(2,4,5)P 3 , and D-Ins(2,4)P 2 . The data support a distributive enzyme mechanism and suggest the PhyAsr standby site is involved in the recruitment of substrate. Structural studies at physiological pH and high salt concentrations demonstrate the ''closed'' or active P-loop conformation can be induced in the absence of substrate. These results suggest PhyAsr should be reclassified as a D-3 myo-inositol hexakisphosphate phosphohydrolase and suggest the PhyAsr reaction mechanism is more similar to that of PTPs than previously suspected.

myo-inostiol phosphate isomers are abbreviated according to IUPAC rules of nomenclature. D/L -prefix is noted to indicate that the two stereoisomers are not discriminated. In those cases where no prefix is listed the compound is a symmetric meso-compound. Where enantiomers are not known, possible isomers are given and separated by a slash.

PhyA from Selenomonas ruminantium (PhyAsr), is a bacterial protein tyrosine phosphatase (PTP)‐like inositol polyphosphate phosphatase (IPPase) that is distantly related to known PTPs. PhyAsr has a second substrate binding site referred to as a standby site and the P‐loop (HCX5R) has been observed in both open (inactive) and closed (active) conformations. Site‐directed mutagenesis and kinetic and structural studies indicate PhyAsr follows a classical PTP mechanism of hydrolysis and has a broad specificity toward polyphosphorylated myo‐inositol substrates, including phosphoinositides. Kinetic and molecular docking experiments demonstrate PhyAsr preferentially cleaves the 3‐phosphate position of Ins P6 and will produce Ins(2)P via a highly ordered series of sequential dephosphorylations: D‐Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D‐Ins(2,4,5)P3, and D‐Ins(2,4)P2. The data support a distributive enzyme mechanism and suggest the PhyAsr standby site is involved in the recruitment of substrate. S...

Inositol polyphosphatases (IPPases), particularly those that can hydrolyze myo-inositol hexakisphosphate (Ins P 6 ), are of biotechnological interest for their ability to reduce the metabolically unavailable organic phosphate content of feedstuffs and to produce lower inositol polyphosphates (IPPs) for research and pharmaceutical applications. Here, the gene coding for a new protein tyrosine phosphatase (PTP)-like IPPase was cloned from Megasphaera elsdenii (phyAme), and the biochemical properties of the recombinant protein were determined. The deduced amino acid sequence of PhyAme is similar to known PTPlike IPPases (29-44% identity), and the recombinant enzyme displayed strict specificity for IPP substrates. Optimal IPPase activity was displayed at an ionic strength of 250 mM, a pH of 5.0, and a temperature of 60°C. In order to elucidate its stereospecificity of Ins P 6 dephosphorylation, a combination of high-performance ion-pair chromatography and kinetic studies was conducted. PhyAme displayed a stereospecificity that is unique among enzymes belonging to this class in that it preferentially cleaved Ins P 6 at one of two phosphate positions, 1D-3 or 1D-4. PhyAme followed two distinct and specific routes of hydrolysis, predominantly degrading Ins P 6 to Ins(2)P via: (a) 1D-Ins(1,2,4,5,6)P 5 , 1D-Ins(1,2,5,6)P 4 , 1D-Ins(1,2,6)P 3 , and 1D-Ins(1,2)P 2 (60%) and (b) 1D-Ins(1,2,3,5,6)P 5 , 1D-Ins(1,2,3,6)P 4 , Ins(1,2,3)P 3 , and D/L-Ins(1,2)P 2 (35%).

Regulation of the levels of tyrosine phosphorylation is essential to maintain the functions of proteins in different signaling pathways and other cellular systems, but how the steady-state levels of tyrosine phosphorylation are coordinated in different cellular systems to initiate complex cellular functions remains a formidable challenge. The receptor protein tyrosine phosphatase (RPTP)β/ζ is a transmembrane tyrosine phosphatase whose substrates include proteins important in intracellular and transmembrane protein-signaling pathways, cytoskeletal structure, cell–cell adhesion, endocytosis, and chromatin remodeling. Pleiotrophin (PTN the protein and Ptn the gene) is a ligand for RPTPβ/ζ; PTN inactivates RPTPβ/ζ, leaving unchecked the continued endogenous activity of tyrosine kinases that increase phosphorylation of the substrates of RPTPβ/ζ at sites dephosphorylated by RPTPβ/ζ in cells not stimulated by PTN. Thus, through the regulation of the tyrosine phosphatase activity of RPTPβ/ζ...

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) first discovered as the constitutively active nucleophosmin-ALK oncoprotein in anaplastic large cell lymphomas (ALCL). Fulllength ALK has a critical role in normal development and differentiation. Activated full-length ALK also is found in different malignant cancers. Nevertheless, the ligand to activate ALK remained unknown until recently, when ALK was proposed to be the physiological receptor of the cytokine pleiotrophin (PTN, Ptn). However, earlier studies had demonstrated that receptor protein tyrosine phosphatase (RPTP) ␤/ is a physiological PTN receptor. We now demonstrate that phosphorylation of ALK in PTN-stimulated cells is mediated through the PTN/RPTP␤/ signaling pathway. ALK is phosphorylated independently of a direct interaction of PTN with ALK. The data thus support a unique model of ALK activation. In cells not stimulated by PTN, RPTP␤/ dephosphorylates ALK at the site(s) in ALK that is undergoing autophosphorylation through autoactivation. In contrast, when RPTP␤/ is inactivated in PTN-stimulated cells, the sites that are autophosphorylated in ALK no longer can be dephosphorylated by RPTP␤/; thus, autoactivation and tyrosine phosphorylation of ALK rapidly increase. The data indicate that the PTN/RPTP␤/ signaling pathway is a critical regulator of the steady state levels of tyrosine phosphorylation and activation of ALK; the data support the conclusion that ALK phosphorylation and activation in PTNstimulated cells are increased through a unique "alternative mechanism of RTK activation."

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.

P 27 Kip1 (p27) can prevent cell proliferation by inactivating cyclindependent kinases. This function is impaired upon phosphorylation of p27 at tyrosine residue 88. We observed that FLT3 and FLT3-ITD can directly bind and selectively phosphorylate p27 on this residue. Inhibition of FLT3-ITD in cell lines strongly reduced p27 tyrosine 88 phosphorylation and resulted in increased p27 levels and cell cycle arrest. Subsequent analysis revealed the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors.

P 27 Kip1 (p27) can prevent cell proliferation by inactivating cyclindependent kinases. This function is impaired upon phosphorylation of p27 at tyrosine residue 88. We observed that FLT3 and FLT3-ITD can directly bind and selectively phosphorylate p27 on this residue. Inhibition of FLT3-ITD in cell lines strongly reduced p27 tyrosine 88 phosphorylation and resulted in increased p27 levels and cell cycle arrest. Subsequent analysis revealed the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors.

A non-transmembrane protein-tyrosine phosphatase, PTP-S, has been shown to bind to DNA in vitro through its C-terminal noncatalytic domain. The cellular PTP-S gene product was identified as 42-44 kDa polypeptides in the rat fibroblast cell lines Rat2 and F111, by immunoblotting using polyclonal and monoclonal antibodies. Indirect immunofluorescence-staining experiments showed that PTP-S was predominantly associated with the nuclear compartment in interphase cells, although some fluorescence was present in cytoplasm. This protein appeared to be uniformly distributed throughout the cell during mitosis. Subcellular fractionation followed by immunoblotting supports the immunofluorescence data showing that PTP-S was present in the nucleus as well as cytoplasm. PTP-S could be released from the isolated nuclei by moderate salt concentration (400 mM NaCl) or mild treatment with DNAase I, suggesting that at least part of this protein present in the nucleus was associated with chromatin. PTP-...

PTP‐S2 is a widely expressed nuclear protein tyrosine phosphatase which shows increased expression upon mitogenic stimulation of a variety of cells. In order to elucidate the role of this enzyme in cell division, stable clones of HeLa cells expressing rat PTP‐S2 were isolated and their growth properties analysed. Overexpressed PTP‐S2 was located in the cell nucleus and there was no significant change in the total tyrosine phosphatase activity of PTP‐S2 overexpressing cells. PTP‐S2 overexpressing clones, D3 and B5, showed increased rate of cell division and lower serum requirement as compared with control cells. D3 and B5 cells formed larger colonies in soft agar, were not contact inhibited upon confluency, grew in multilayers, and showed altered morphology. These results are consistent with the suggestion that PTP‐S2 may be a positive regulator of cell proliferation.

PTP‐S2/TC45 is a nuclear protein tyrosine phosphatase, which induces p53‐dependent apoptosis. Here we show that the p53 protein level increased in MCF‐7 cells in response to PTP‐S2 overexpression. PTP‐S2‐induced p53 protein was transcriptionally active and it could activate caspase‐1 gene expression from endogenous as well as ectopic promoter. Coexpression of an active site mutant of procaspase‐1 strongly inhibited PTP‐S2‐induced apoptosis. Mutant procaspase‐1 also inhibited apoptosis induced by p53 overexpression or doxorubicin treatment, which induce caspase‐1 gene expression. In contrast, apoptosis induced by staurosporine or cycloheximide, which do not increase caspase‐1 gene expression, was not affected by mutant procaspase‐1. These results suggest that caspase‐1 may be one of the mediators of p53‐dependent apoptosis in human cells.

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

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Ho ¨ltta ¨, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60 v-src , known to associate also with v-Crk and designated p130 CAS . We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src-and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130 CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130 CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130 CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphor...

Alzheimer's disease (AD). Previous studies have shown that AD pathophysiology involves increased expression of a central nervous system-enriched protein tyrosine phosphatase called STEP (STriatal-Enriched protein tyrosine Phosphatase). STEP opposes the development of synaptic strengthening by dephosphorylating substrates, including GluN2B, Pyk2 and ERK1/2. Genetic reduction of STEP as well as pharmacological inhibition of STEP improves cognitive function and hippocampal memory in the 3xTg AD mouse model. Here, we show that the improved cognitive function is accompanied by an increase in synaptic connectivity in cell cultures as well as in the triple transgenic AD mouse model, further highlighting the potential of STEP inhibitors as a therapeutic agent.

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR substrate (specific activity ranging from 0.23 to 40 pmol/min/g). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ITAMs, with PTPH1 directly dephosphorylating . This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR .

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR substrate (specific activity ranging from 0.23 to 40 pmol/min/g). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR ITAMs, with PTPH1 directly dephosphorylating . This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR .

PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype. It appears that PRL-1 is important in norm...

Protein tyrosine phosphatase of liver regeneration-1 ( Prl-1) is an immediate-early gene that is significantly induced during liver regeneration. Several in vitro studies have suggested that Prl-1 is important for the regulation of cell cycle progression. To evaluate its function in liver regeneration, we ablated the Prl-1 gene specifically in mouse hepatocytes using the Cre-loxP system. Prl-1 mutant mice ( Prl-1loxP/loxP;AlfpCre) appeared normal and fertile. Liver size and metabolic function in Prl-1 mutants were comparable to controls, indicating that Prl-1 is dispensable for liver development, postnatal growth, and hepatocyte differentiation. Mutant mice demonstrated a delay in DNA synthesis after 70% partial hepatectomy, although ultimate liver mass restoration was not affected. At 40 h posthepatectomy, reduced protein levels of the cell cycle regulators cyclin E, cyclin A2, cyclin B1, and cyclin-dependent kinase 1 were observed in Prl-1 mutant liver. Investigation of the major ...

PRL-1 gene, which encodes a unique nuclear protein tyrosine phosphatase, is positively associated with cellular growth during liver development, regeneration, and oncogenesis but with differentiation in intestine and other tissues. Here, we analyzed the structure of the human PRL-1 gene and localized it to chromosome 6 within band q12. Human, rat, and mouse PRL-1 are 100% conserved at the amino acid level and 55% identical to a newly identified Caenorhabditis elegans PRL-1. The presence of two promoter activities, P1 and P2, in the human PRL-1 gene were identified by primer extension and RNase protection assays. A functional TATA box was identified in promoter P1 upstream of the non-coding first exon. A non-canonical internal promoter, P2, was found in the first intron that results in PRL-1 transcripts beginning 8 base pairs downstream of the 5-end of exon 2 and causes no alteration in the encoded protein. The first 200-base pair region of either promoter P1 or P2 conferred high basal transcriptional activity. An enhancer that bound a developmentally regulated factor, PRL-1 intron enhancer complex (PIEC), was localized to the first intron of the human PRL-1 gene. The presence of PIEC correlated with the ability of the intron enhancer to confer transcriptional activation in HepG2 and F9 cells. The intron enhancer contributed significantly to PRL-1 promoter activity in HepG2 cells which contain PIEC but not to NIH 3T3 cells which do not.

The cellular signals that initiate cell growth are incompletely understood. Insight could be provided by understanding the signals regulating the transcriptional induction of immediate-early genes which occurs within minutes of the growth stimulus. The expression of the PRL-1 gene, which encodes a unique nuclear protein-tyrosine phosphatase, is rapidly induced in regenerating liver and mitogen-treated cells. Transcription of the PRL-1 gene increased in the rat liver remnant within a few minutes after partial hepatectomy and largely explained the increase in steady-state PRL-1 mRNA in the first few hours posthepatectomy. Egr-1 (early growth response factor) specifically bound a region of the proximal PRL-1 promoter P1 (؊99). Egr-1 binding activity was more rapidly induced in regenerating liver than mitogen-treated H35 and NIH 3T3 cells, remained elevated through 4 h posthepatectomy, and appeared to be dependent not only on new Egr-1 protein synthesis but on post-translational regulation of Egr-1. Egr-1 efficiently transactivated a PRL-1 promoter reporter construct containing an intact not mutant Egr-1 site, and the Egr-1 site largely accounted for PRL-1 gene up-regulation in response to mitogen stimulation. These data predict that Egr-1 activation is an early event in liver regeneration and mitogen-activated cells that provides a regulatory stimulus for a subset of immediateearly genes.

c-Jun N-terminal kinase (JNK) regulates gene expression in response to various extracellular stimuli. JNK can be activated by the tumor promoting agent, 12-Otetradecanoylphorbol-13-acetate (TPA) in normal human oral keratinocytes but not in human keratinocytes that have been immortalized (HOK-16B and HaCaT) or transformed (HOK-16B-Bap-T) nor in a cervical carcinoma cell line (HeLa). The refractory JNK activation response to TPA is not due a defect in the JNK pathway, because JNK can be activated by other stimuli, e.g. UV irradiation and an alkylating agent N-methyl-N-nitrosoguanidine in these immortalized or transformed cells. More importantly, the refractory JNK and JNKK activation response to TPA can be restored by treatment of the cells with a combination of TPA and a proteintyrosine phosphatase inhibitor, sodium orthovanadate. Furthermore, pretreatment of cells with TPA partially inhibited UV-or N-methyl-N-nitrosoguanidine-induced JNK activity. These results suggest that a TPA-inducible, orthovanadate-sensitive protein-tyrosine phosphatase may specifically down-regulate JNK signaling pathway in these immortalized/transformed epithelial cells. In contrast, ERK and p38/Mpk2 are not regulated by this TPA-induced phosphatase. This putative protein-tyrosine phosphatase appears to be JNK pathway-specific.

The Src-homology domain 2 (SH2)-containing cytoplasmic tyrosine phosphatase, SHP-1 (SH2-containing protein tyrosine phosphatase-1), interacts with several B cell surface and intracellular signal transduction molecules through its SH2 domains. Mice with the motheaten and viable motheaten mutations are deficient in SHP-1 and lack most mature B cells. To define the role of SHP-1 in mature B cells, we expressed phosphatase-inactive SHP-1 (C453S) in a mature B cell lymphoma line. SHP-1 (C453S) retains the ability to bind to both substrates and appropriate tyrosine-phosphorylated proteins and therefore can compete with the endogenous wild-type enzyme. We found that B cells expressing SHP-1 (C453S) demonstrated enhanced and prolonged tyrosine phosphorylation of proteins with molecular masses of 110, 70, and 55–60 kDa after stimulation with anti-mouse IgG. The tyrosine kinase Syk was hyperphosphorylated and hyperactive in B cells expressing SHP-1 (C453S). SHP-1 and Syk were coimmunoprecipit...

Caveolin-1 (CAV1) enhanced migration, invasion, and metastasis of cancer cells is inhibited by co-expression of the glycoprotein E-cadherin. Although the two proteins form a multiprotein complex that includes β-catenin, it remained unclear how this would contribute to blocking the metastasis promoting function of CAV1. Here, we characterized by mass spectrometry the protein composition of CAV1 immunoprecipitates from B16F10 murine melanoma cells expressing or not E-cadherin. The novel protein tyrosine phosphatase PTPN14 was identified by mass spectrometry analysis exclusively in co-immunoprecipitates of CAV1 with E-cadherin. Interestingly, PTPN14 is implicated in controlling metastasis, but only few known PTPN14 substrates exist. We corroborated by western blotting experiments that PTPN14 and CAV1 co-inmunoprecipitated in the presence of E-cadherin in B16F10 melanoma and other cancer cells. Moreover, the CAV1(Y14F) mutant protein was shown to co-immunoprecipitate with PTPN14 even in...

and partner of EphB2 and EphA7 receptor tyrosine kinases, Howard Hughes Medical Institute and of ephrinB ligands (Bruckner et al., 1999; Lin et al., Massachusetts General Hospital and 1999; Torres et al., 1998). GRIP additionally interacts Harvard Medical School with GRASP-1, a Ras guanine-nucleotide exchange fac-Boston, Massachusetts 02114 tor that affects the synaptic distribution of AMPA recep-2 Center for Learning and Memory tors (Ye et al., 2000). The biological function of GRIP is RIKEN-MIT Neuroscience Research Center and still not clear, but it presumably acts as a scaffold for Howard Hughes Medical Institute assembly of large and heterogeneous protein com-Massachusetts Institute of Technology plexes (Sheng and Sala, 2001; Ye et al., 2000). Cambridge, Massachusetts 02139 GRIP and AMPA receptors are highly associated in 3 Department of Biological Sciences rat brain (Wyszynski et al., 1999). A subpopulation of Korea Advanced Institute of Science and GRIP is present at excitatory synapses by immunocyto-Technology chemistry, but GRIP also exists abundantly in nonsynap-Taejon 305-701 tic locations such as in dendritic shafts (where it is asso-Korea ciated with vesicles and microtubules), axons, axon 4 Department of Cell Biology and Anatomy terminals, and cell soma (associated with endoplasmic University of North Carolina at Chapel Hill reticulum) (Dong et al., 1999; Srivastava et al., 1998; Chapel Hill, North Carolina 27599 Wyszynski et al., 1999). Biochemical fractionation stud-5 Department of Cancer Immunology and AIDS ies show that GRIP is only modestly enriched in the Dana-Farber Cancer Institute and postsynaptic density (PSD) compared with PSD-95, and Department of Pathology is readily detectable in cytosol and intracellular mem-Harvard Medical School brane fractions (Wyszynski et al., 1998). This widespread Boston, Massachusetts 02115 subcellular distribution suggests that GRIP has functions distinct from, or in addition to, its putative role of anchoring AMPA receptors at postsynaptic sites. For Summary example, the association of GRIP with intracellular vesicles suggests a possible role in protein transport in Interaction with the multi-PDZ protein GRIP is required neurons. for the synaptic targeting of AMPA receptors, but the The liprin-␣ family of proteins (liprin-␣1, -␣2, -␣3, and underlying mechanism is unknown. We show that -␣4) was first identified by their interaction with the LAR GRIP binds to the liprin-␣/SYD2 family of proteins that family of receptor protein tyrosine phosphatases (LARinteract with LAR receptor protein tyrosine phospha-RPTPs), which includes LAR, PTP, and PTP␦ (Pulido tases (LAR-RPTPs) and that are implicated in presynet al., 1995; Serra-Page `s et al., 1995, 1998). Liprin-␣ aptic development. In neurons, liprin-␣ and LAR-RPTP consists of an N-terminal coiled-coil region that mediare enriched at synapses and coimmunoprecipitate ates homo-and heteromultimerization, and a C-terminal with GRIP and AMPA receptors. Dominant-negative region that binds to LAR-RPTPs (Pulido et al., 1995; constructs that interfere with the GRIP-liprin interac-Serra-Page `s et al., 1995, 1998). In cultured cell lines, tion disrupt the surface expression and dendritic clusliprin-␣ colocalizes with LAR-RPTP at focal adhesions, tering of AMPA receptors in cultured neurons. Thus, suggesting a possible role in cell-matrix interactions by mediating the targeting of liprin/GRIP-associated (Serra-Page `s et al., 1995). proteins, liprin-␣ is important for postsynaptic as well The C. elegans liprin-␣ homolog syd-2 was isolated as presynaptic maturation. in a screen for mutants affecting localization of the synaptic vesicle protein synaptobrevin (Zhen and Jin, 1999). Syd-2 mutants display an abnormally diffuse distribution Introduction of presynaptic markers, lengthening of active zones, and impaired synaptic transmission (Zhen and Jin, AMPA-type glutamate receptors mediate excitatory 1999). Zhen and Jin (1999) suggested that SYD-2 might synaptic transmission and are composed of heteromeric function in presynaptic differentiation as a structural combinations of GluR1-4 subunits. The GRIP family of component of active zones and/or as a molecular anchor recruiting other molecules such as LAR-RPTP to the

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and und...

Protein tyrosine phosphatases (PTPases) play critical roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. The structures of several different PTPases have revealed a conserved active site architecture in which a phosphate-binding loop, together with an invariant arginine, cradle the phosphate of a phosphotyrosine substrate and poise it for nucleophilic attack by an invariant cysteine nucleophile. We previously reported that binding of tungstate to the Yop5 l PTPase from Yersiniu induced a loop conformational change that moved aspartic acid 356 into the active site, where it can function as a general acid. This is consistent with the aspartic acid donating a proton to the tyrosyl leaving group during the initial hydrolysis step. In this report, using a similar structure of the inactive Cys 403 + Ser mutant of the Yersinia PTPase complexed with sulfate, we detail the structural and functional details of this conformational change. In response to oxyanion binding, small perturbations occur in active site residues, especially Arg 409, and trigger the loop to close. Interestingly, the peptide bond following Asp 356 has flipped to ligate a buried, active site water molecule that also hydrogen bonds to the bound sulfate anion and two invariant glutamines. Loop closure also significantly decreases the solvent accessibility of the bound oxyanion and could effectively shield catalytic intermediates from phosphate acceptors other than water. We speculate that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.

SummaryYopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C‐terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N‐terminal non‐catalytic domain of YopH binds p130Cas in a phosphotyrosine‐dependent manner and functions as a novel substrate‐targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate‐targeting site (‘site 2’) within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine‐d...

Protein tyrosine phosphatases (PTPases) play critical roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. The structures of several different PTPases have revealed a conserved active site architecture in which a phosphate-binding loop, together with an invariant arginine, cradle the phosphate of a phosphotyrosine substrate and poise it for nucleophilic attack by an invariant cysteine nucleophile. We previously reported that binding of tungstate to the Yop5 l PTPase from Yersiniu induced a loop conformational change that moved aspartic acid 356 into the active site, where it can function as a general acid. This is consistent with the aspartic acid donating a proton to the tyrosyl leaving group during the initial hydrolysis step. In this report, using a similar structure of the inactive Cys 403 + Ser mutant of the Yersinia PTPase complexed with sulfate, we detail the structural and functional details of this conformational change. In response to oxyanion binding, small perturbations occur in active site residues, especially Arg 409, and trigger the loop to close. Interestingly, the peptide bond following Asp 356 has flipped to ligate a buried, active site water molecule that also hydrogen bonds to the bound sulfate anion and two invariant glutamines. Loop closure also significantly decreases the solvent accessibility of the bound oxyanion and could effectively shield catalytic intermediates from phosphate acceptors other than water. We speculate that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.