Increase In Protein Tyrosine Phosphatase Activity Research Articles

Postactivated B cells in systemic lupus erythematosus: update on translational aspects and therapeutic considerations.

This review summarizes recent insights and current understanding of the role of postactivated B cells in SLE and related pathogenic and potential therapeutic implications. B cells are considered key players in SLE and experience from various B-cell-targeted therapies underlines their clinical relevance. In the last years, new insights have been obtained on B-cell abnormalities within the complex pathophysiology of SLE. These insights involve a revised understanding of BCR signaling, that has been reported to be hyperresponsive in the past, but newer studies suggest a postactivation functiotype in terms of reduced BCR and TLR signaling. Despite comprehensive efforts to delineate B-cell abnormalities on assessing large-scale genomic, epigenomic and proteomic data, understanding functional impairments of cellular interactions and subcellular functions remains crucial. A recently identified enhanced protein tyrosine phosphatase (PTP) activity was found in relation to diminished BCR responses in SLE. This finding together with reduced cytokine production upon TLR9 activation appears to mark postactivated lupus B cells. Other studies identified increased PTP activity in line with a gain-of-function mutation of phosphatase PTPN22, one of the strongest SLE risk alleles. Improved understanding of these B cell abnormalities in SLE holds promise to gain further insights in mechanisms of autoimmunity and pave the way for selective therapies targeting key principles of chronic autoimmunity. SLE B cells (similar as previously described for lupus T cells) are characterized by a postactivation (exhausted) functiotype mandating consideration for innovative therapies.

Role of Protein-tyrosine Phosphatase SHP2 in Focal Adhesion Kinase Down-regulation during Neutrophil Cathepsin G-induced Cardiomyocytes Anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

PTPN11 (Shp2) Mutations in LEOPARD Syndrome Have Dominant Negative, Not Activating, Effects

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.

Nitric oxide-mediated alterations of protein tyrosine phosphatase activity and expression during hypoxia in the cerebral cortex of newborn piglets

The present study tested the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase (PTP) activity in the membranes and increased activity and expression of PTPs (PTP-1B, PTP-SH1 and 2) in the cytosol of the cerebral cortex of newborn piglets are mediated by nitric oxide (NO). To test this hypothesis, PTP activity in cell membranes and activity and expression were measured in the cytosol of normoxic (Nx, n=5), hypoxic (Hx, n=5), and 7-nitro-indazole sodium salt (7-NINA), a selective inhibitor of neuronal nitric oxide synthase (nNOS), pretreated hypoxic (7-NINA+Hx, n=6) newborn piglets. PTP activity in cortical cell membranes was lower in the Hx group as compared to the Nx group and this decrease was prevented in the 7-NINA+Hx group. The density of cytosolic PTP-1B, cytosolic PTP-SH1 and PTP-SH2 was increased in the Hx group and this increase was prevented in the 7-NINA+Hx group. Immunohistochemistry results show an increased immunoreactivity to PTP-1B in the Hx as compared to Nx animals. The data show that pretreatment with 7-NINA, a selective inhibitor of nNOS, prevents the hypoxia-induced decrease in PTP activity in membranes. nNOS inhibition also prevented the hypoxia-induced increase in PTP activity and expression in cytosol, and therefore we conclude that modification of PTP during hypoxia is NO-mediated.

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343–19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase α (RPTPα). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.

TIMP-2 Mediated Inhibition of Angiogenesis: An MMP-Independent Mechanism

Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require α3β1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with β1 integrin subunits as well as dissociation of the phosphatase SHP-1 from β1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.

A Novel Protein-Protein Interaction between a G Protein-coupled Receptor and the Phosphatase SHP-2 Is Involved in Bradykinin-induced Inhibition of Cell Proliferation

Mitogenic G protein-coupled receptor (GPCR) signaling has been extensively studied. In contrast, little is known about anti-mitogenic GPCR signaling. We show here that anti-mitogenic signaling of a GPCR, the bradykinin B2 receptor, involves a novel direct protein-protein interaction. The antiproliferative effect of bradykinin was accompanied by a transient increase in protein-tyrosine phosphatase activity. Using surface plasmon resonance analysis, we observed that an immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the C-terminal part of the B2 receptor interacted specifically with the protein-tyrosine phosphatase SHP-2. The interaction was confirmed in primary culture renal mesangial cells by co-immunoprecipitation of a B2 receptor.SHP-2 complex. The extent of the interaction was transiently increased by stimulation with bradykinin, which was accompanied by an increase in specific SHP-2 phosphatase activity. Mutational analysis of the key ITIM residue confirmed that the B2 receptor ITIM sequence is required for interaction with SHP-2, SHP-2 activation, and the anti-mitogenic effect of bradykinin. Finally, in mesangial cells transfected with a dominant-negative form of SHP-2, bradykinin lost the ability to inhibit cell proliferation. These observations demonstrate that bradykinin inhibits cell proliferation by a novel mechanism involving a direct protein-protein interaction between a GPCR (the B2 receptor) and SHP-2.

Two mechanisms activate PTPalpha during mitosis.

We show that, dependent on serine hyperphosphorylation, protein tyrosine phosphatase alpha (PTPalpha) is activated by two different mechanisms during mitosis: its specific activity increases and its inhibitory binding to Grb2 decreases. The latter effect probably abates Grb2 inhibition of the phosphotyrosine displacement process that is required specifically for Src dephosphorylation and causes a mitotic increase in transient PTPalpha-Src binding. Thus, part of the increased protein tyrosine phosphatase activity may be specific for Src family members. These effects cease along with Src activation when cells exit mitosis. Src is not activated in mitosis in PTPalpha-knockout cells, indicating a unique mitotic role for this phosphatase. The activation of PTPalpha, combined with the effects of mitotic Cdc2-mediated phosphorylations of Src, quantitatively accounts for the mitotic activation of Src, indicating that PTPalpha is the membrane-bound, serine phosphorylation-activated, protein tyrosine phosphatase that activates Src during mitosis.

A Redox Signaling Mechanism for Density-dependent Inhibition of Cell Growth

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.

Induction of IW32 erythroleukemia cell differentiation by p53 is dependent on protein tyrosine phosphatase.

The biological activity of p53 in IW32 erythroleukemia cells was investigated. IW32 cells had no detectable levels of p53 mRNA and protein expression. By transfecting a temperature-sensitive mutant p53 cDNA, tsp53val135, into the cells, we have established several clones stably expressing the mutant p53 allele. At permissive temperature, these p53 transfectants were arrested in G1 phase and underwent apoptosis. Moreover, differentiation along the erythroid pathway was observed as evidenced by increased benzidine staining and mRNA expression of beta-globin and the erythroid-specific delta-aminolevulinic acid synthase (ALAS-E). Treatment of cells with protein tyrosine phosphatase inhibitor vanadate blocked the p53-induced differentiation, but not that of cell death or growth arrest. Increased protein tyrosine phosphatase activity as well as mRNA levels of PTPbeta2 and PTPepsilon could be observed by wildtype p53 overexpression. These results indicate that p53 induced multiple phenotypic consequences through separate signal pathways in IW32 erythroleukemia cells, and protein tyrosine phosphatase is required for the induced differentiation.

Restoration of potent protein-tyrosine phosphatase activity into the membrane-distal domain of receptor protein-tyrosine phosphatase alpha.

Most transmembrane, receptor-like protein-tyrosine phosphatases (RPTPs) contain two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains, of which the membrane-proximal domain, D1, contains the majority of the activity, while the membrane-distal domain, D2, exhibits little or no activity. We have investigated the structural basis for reduced activity in RPTP-D2s, using RPTPalpha as a model system. Sequence alignment of PTP domains indicated that two motifs, the KNRY motif and the WpD motif, are highly conserved in all PTP domains, but not in RPTP-D2s. In RPTPalpha-D2, the Tyr in the KNRY motif is substituted by Val (position 555) and the Asp in the WpD motif by Glu (position 690). Mutation of Val555 and Glu690 had synergistic effects on RPTPalpha-D2 activity, in that the PTP activity of RPTPalpha-D2-V555Y/E690D was greatly enhanced to levels that were similar to or approaching those of RPTPalpha-D1. Therefore, Val555 and Glu690 are responsible in large part for reduced RPTPalpha-D2 activity. In addition, we established that the increased PTP activity is due to restoration of effective transition-state stabilization in RPTPalpha-D2-V555Y/E690D. Since the KNRY motif and the WpD motif are mutated in all RPTP-D2s, it is highly unlikely, due to lack of transition-state stabilization, that the residual RPTP-D2 catalytic activity plays a role in the function of RPTPs.

Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

AbstractPolycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

Identification of Increased Protein Tyrosine Phosphatase Activity in Polycythemia Vera Erythroid Progenitor Cells

Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

Modulation of major histocompatibility complex class I genes by interferon-gamma and ganglioside GT 1b in astrocytes: involvement of protein tyrosine phosphatases.

We have previously reported that the polysialoganglioside GT1b suppresses the induction of major histocompatibility complex class I molecules by interferon-gamma in astrocytes. Suppression by GT1b depended on the presence of sialic acid moieties because asialo-GM1 was not suppressive. In the present report, GT1b was found to act transcriptionally to suppress class I genes because both the interferon-gamma induction of RNA and the activity of class I promoter constructs were inhibited. Furthermore, GT1b suppressed promoter activity through interferon regulatory factor elements, indicating an effect on the transcription activation factor, interferon regulatory factor 1. Interferon-gamma induced interferon regulatory factor 1 within 8 h, and GT1b suppressed this induction. The suppression of interferon regulatory factor 1 by GT1b correlated with the suppression of gamma-activated factor binding at the promoter of the interferon regulatory factor 1 gene. The suppression of gamma-activated factor by GT1b appeared to involve increased protein tyrosine phosphatase activity because treatment of the cells with pervanadate reversed the effect of GT1b on the gamma-activated factor and, correspondingly, phosphotyrosine content. In sum, GT1b displays specific effects on interferon-gamma signaling and negative feedback regulatory molecules in astrocytes.

Inhibition of Cell Proliferation and Activation of Protein Tyrosine Phosphatase Mediated by Angiotensin II Type 2 (AT2) Receptor in R3T3 Cells

Angiotensin II (Ang II) type 2 receptor (AT2) mediated inhibition of cell proliferation and activation of protein tyrosine phosphatase (PTP) were investigated in R3T3 fibroblast cells selectively expressing the AT2subtype. Ang II did not alter the cell number of serum depleted R3T3 cells but inhibited basic fibroblast growth factor (bFGF)-stimulated cell proliferation in a dose dependent manner (IC50; 1-5 nM). This inhibitory response was abolished by an concomitant incubation with an AT2antagonist, PD123319, and was mimicked by an AT2agonist, CGP42112A. Stimulation of Ang II resulted in a rapid and transient increase in PTP activity as determined by using para-nitrophenyl phosphate (p-Npp) as a substrate in serum depleted R3T3 cells. This PTP activation was mimicked by CGP42112A. We conclude that the AT2receptor is involved in the inhibition of cell proliferation and activation of protein tyrosine phosphatase.

Preferential requirement for protein tyrosine phosphatase activity in the 12- O-tetradecanoylphorbol-13-acetate-induced differentiation of human colon cancer cells

Some lines of colon cancer cells are forced to undergo differentiation by 12- O-tetradecanoylphorbol-13-acetate (TPA). The increases in activities of both protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK) have been reported to be associated with the TPA-induced differentiation of HL-60 leukemia cells. In the present study, a 2-fold increase in PTP activity was observed in SW620 human colon cancer cells after 30 min of TPA treatment; a maximal level (4- to 5-fold) was reached at 60 min and continued for more than 6 hr. In addition, two TPA-induced differentiated characteristics, morphological alteration and release of cellular surface proteoglycan, were effectively blocked by PTP inhibitors, such as sodium orthovanadate (50 μM), zinc chloride (100 μM), and iodoacetate (250 μM), but not by the protein serine/threonine phosphatase inhibitor okadaic acid (20 nM). On the other hand, although TPA induced a transient slight increase in PTK activity (1.4-fold) at 60 min, four PTK inhibitors (genistein, herbimycin A, tyrphostin-23 and quercetin) had different effects on the TPA-induced release of cell surface proteoglycan. Genistein (60 μM) potentiated this process, but in contrast, quercetin (45 μM) could partially inhibit the TPA effect. Taken together, these observations suggest that both PTP and PTK activities were increased in SW620 cells in response to TPA; however, the activation of PTP seems to be preferentially required for the TPA-induced differentiation of SW620 human colon cancer cells.