Cell Protein Tyrosine Phosphatase Research Articles

The T Cell Protein Tyrosine Phosphatase Is a Negative Regulator of Janus Family Kinases 1 and 3

Background: The immune response is regulated through a tightly controlled cytokine network. The counteracting balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activity regulates intracellular signaling in the immune system initiated by these extracellular polypeptides. Mice deficient for the T cell protein tyrosine phosphatase (TCPTP) display gross defects in the hematopoietic compartment, indicating a critical role for TCPTP in the regulation of immune homeostasis. To date, the molecular basis underlying this phenotype has not been reported.Results: We have identified two members of the Janus family of tyrosine kinases (JAKs), JAK1 and JAK3, as bona fide substrates of TCPTP. Inherent substrate specificity in the TCPTP-JAK interaction is demonstrated by the inability of other closely related PTP family members to form an in vivo interaction with the JAKs in hematopoietic cells. In keeping with a negative regulatory role for TCPTP in cytokine signaling, expression of TCPTP in T cells abrogated phosphorylation of STAT5 following interleukin (IL)-2 stimulation. TCPTP-deficient lymphocytes treated with IL-2 had increased levels of tyrosine-phosphorylated STAT5, and thymocytes treated with interferon (IFN)-α or IFN-γ had increased tyrosine-phosphorylated STAT1. Hyperphosphorylation of JAK1 and elevated expression of iNOS was observed in IFN-γ-treated, TCPTP-deficient, bone marrow-derived macrophages.Conclusions: We have identified JAK1 and JAK3 as physiological substrates of TCPTP. These results indicate a negative regulatory role for TCPTP in cytokine signaling and provide insight into the molecular defect underlying the phenotype of TCPTP-deficient animals.

17β-Estradiol inhibits soluble guanylate cyclase activity through a protein tyrosine phosphatase in PC12 cells

Besides its involvement in reproductive functions, estrogen protects against the development of cardiovascular diseases. The guanylate cyclase/cGMP system is known to exert potent effects on the regulation of blood pressure and electrolyte balance. We examined whether 17β-estradiol can affect soluble guanylate cyclase in PC12 cells. The results indicate that 17β-estradiol decreases cGMP levels in PC12 cells. 17β-Estradiol decreases sodium nitroprusside (SNP)-stimulated, but not atrial natriuretic factor-stimulated cGMP formation in PC12 cells, indicating that 17β-estradiol decreases cGMP levels by inhibiting the activity of soluble guanylate cyclase. 17β-Estradiol also stimulates protein tyrosine phosphatase activities in PC12 cells and dephosphorylates at least three proteins. Addition of sodium vanadate, a protein tyrosine phosphatase inhibitor, blocks the inhibitory effects of 17β-estradiol on soluble guanylate cyclase activity in PC12 cells. Furthermore, transfection of SHP-1, a protein tyrosine phosphatase, into PC12 cells inhibits both basal and SNP-stimulated guanylate cyclase activity. Amino acid analysis also reveals that the 70-kDa subunit of soluble guanylate cyclase contains the SHP-1 substrate consensus sequence. These results suggest that 17β-estradiol inhibits soluble guanylate cyclase activity through SHP-1.

Receptor protein tyrosine phosphatases in hematopoietic cells.

PTPs and PTKs control the level of tyrosine phosphorylation of cellular proteins. Although many substrates for PTKs have been identified, the specific targets of individual PTP family members, along with the consequences of protein dephosphorylation for cellular physiology, remain largely unknown. Fine regulation of tyrosine phosphorylation events is required for the proper progression of hematopoiesis. In this review, we have summarized the characterization of tyrosine phosphatases in hematopoietic cells and delineated their potential role in the process of hematopoiesis and the development of hematopoietic disorders.

Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells: modulation by redox state.

Vanadate is a potent reversible inhibitor of protein tyrosine phosphatases (PTP) in vitro. Vanadate has been shown to increase the phosphotyrosine levels in some cell types whereas in others, like the Jurkat T-lymphoma, vanadate has no effect. The reason for the apparent lack of effect of vanadate in Jurkat cells was investigated in this study. Alteration of the redox state of these cells by reducing the glutathione level with 1-chloro-2,4-dinitrobenzene (DnpCl) had no effect on phosphotyrosine levels. However, the cells became sensitive to vanadate, as measured by an increase in phosphotyrosine levels on a wide range of proteins including the MAP kinases. The increase in phosphotyrosine levels most likely results from inhibition of cellular PTP and suggests that protein tyrosine kinases are constitutively active in cells, resulting in a dynamic phosphorylation-dephosphorylation cycle. The mode of inhibition of PTP by vanadate was investigated by measuring the PTP activity of Jurkat membranes isolated after treatment of cells with vanadate and DnpCl. In contrast to the reversible inhibition of PTP in vitro, the effect of vanadate in the presence of DnpCl was irreversible, raising the possibility that it is peroxovanadate formed in situ that is responsible for the inhibition of PTP in intact cells.

The Noncatalytic C-terminal Segment of the T Cell Protein Tyrosine Phosphatase Regulates Activity via an Intramolecular Mechanism

Human T cell protein tyrosine phosphatase (TCPTP) is a nontransmembrane enzyme, the first of the protein tyrosine phosphatase family to be cloned. Alternative mRNA splicing results in variation in the sequence at the extreme C terminus of TCPTP and generates a 45-kDa form (TC45) that is targeted to the nucleus and a 48-kDa variant (TC48) associated with membranes of the endoplasmic reticulum. In this report, we assessed the role of the C-terminal, noncatalytic segment of TCPTP in regulating activity, concentrating primarily on the TC45 variant. We have demonstrated that limited tryptic proteolysis of TC45 releases first a 42-kDa fragment, then a 33-kDa catalytic domain. Using reduced carboxyamidomethylated and maleylated lysozyme as substrate (RCML), the catalytic domain displays 20-100-fold more activity than the full-length enzyme. Analysis of the time course of limited trypsinolysis revealed that proteolytic activation occurred following cleavage of a protease-sensitive region (residues 353-387) located at the C terminus of TC45. The activity of truncation mutants illustrated that removal of 20 C-terminal residues was sufficient to activate the enzyme fully. The 33-kDa catalytic domain, but not the full-length enzyme, was inhibited in a concentration-dependent manner by addition of the noncatalytic C-terminal segment of TC45. A monoclonal antibody to TCPTP, CF4, which recognizes an epitope located between residues 350 and 363, was capable of fully activating TC45. These data indicate that the noncatalytic segment of TC45 contains an autoregulatory site that modulates activity via a reversible intramolecular interaction with the catalytic domain. These studies suggest that the C-terminal noncatalytic segment of TC45, and possibly TC48, may not only direct the enzyme to different subcellular locations but may also modulate activity in response to the binding of regulatory proteins and/or posttranslational modification.

DNA-binding antibodies from viable motheaten mutant mice: implications for B cell tolerance.

C57BL/6 me(v)/me(v) (viable motheaten) mice have defective hemopoietic cell protein-tyrosine phosphatase (HCP, SHP-1) and are a model in which to study the role of this phosphatase in immune regulation and hemopoietic development. Immune defects in these mice include hypergammaglobulinemia, production of multiple autoantibodies, and glomerular nephritis. The presence of self-reactive Abs in these mice is an enigma, since it has been demonstrated that they have increased negative selection against self-reactive transgenic Abs. It was of interest to examine autoantibodies from these mice to identify reactivity or structural features that might account for their persistence and expansion in the repertoire. Abs to DNA were selected for investigation, since they have been characterized extensively from normal and autoimmune strains of mice. Viable motheaten mice had high titers of IgM, anti-dsDNA Abs, and all 12 anti-DNA hybridomas generated were also IgM. They had high avidity for dsDNA, also bound to ssDNA, but were not polyreactive. They used V(H) and V(L) gene families found in anti-DNA Abs from other strains, which reinforces evidence that there is preferential use of a restricted number of germline elements in anti-DNA Abs. The results indicate that Hcph-mutant mice do not delete DNA-reactive B cells, nor are they able to maintain these B cells in a state of anergy. Evidence for positive selection and clonal expansion after V-D-J rearrangement was also seen, in that several Abs expressed the same heavy chain in combination with different light chains, which is also a hallmark of the autoimmune anti-DNA response.

Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice.

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex.

Phytohemagglutinin-activated peripheral CD95+ T cells (day 1 T cells) are resistant to CD95-mediated apoptosis. After prolonged interleukin-2 treatment, these T cells become CD95-mediated apoptosis-sensitive (day 6 T cells). To elucidate the molecular mechanism of apoptosis resistance, day 1 and day 6 T cells were tested for formation of the CD95 death-inducing signaling complex (DISC). DISC-associated active Fas-associated DD protein (FADD)-like interleukin-1 beta-converting enzyme-like protease (FLICE) also referred to as MACH/caspase 8 was only found in apoptosis-sensitive day 6 T cells. Further-analysis of mRNA and protein expression levels of apoptosis-signaling molecules FADD, receptor interacting protein, hematopoietic cell protein tyrosine phosphatase, Fas-associated phosphatase-1, FLICE, bel-2, bcl-xL, and, bax-alpha showed that only the expression level of bcl-xL correlated with T cell resistance to CD95-mediated apoptosis (day 1 T cells: bcl-xhiL; day 6 T cells: bcl-XloL). In T cells activated in vitro, up-regulation of bcl-xL, has previously been correlated with general apoptosis resistance. However, the experiments presented suggest that resistance to CD95-mediated apoptosis in T cells can also be regulated at the level of recruitment of FLICE to the DISC.

Identification of a developmentally regulated protein tyrosine phosphatase in embryonic stem cells that is a marker of pluripotential epiblast and early mesoderm

A sensitive RT-PCR display technique was used to examine the expression of protein tyrosine phosphatases (PTPs) during the differentiation of mouse embryonic stem (ES) cells. The majority of PTPs are expressed constitutively but one is present only in undifferentiated ES cells. This PTP was cloned and named ES cell phosphatase (ESP). ESP mRNA is detectable in oocytes and throughout early mouse embryo development. At early egg cylinder stages, transcripts are localised in the pluripotential epiblast. As gastrulation commences, however, epiblast expression is lost. Transcripts are present transiently in newly formed embryonic mesoderm. These data suggest that this transmembrane signaling molecule is associated with developmental lability in early embryogenesis.

Hematopoietic cell protein-tyrosine phosphatase-deficient motheaten mice exhibit T cell apoptosis defect.

We previously demonstrated that hematopoietic cell protein-tyrosine phosphatase is one of the molecules that can transduce Fas-mediated apoptosis signals in lymphoid cells. The present study analyzed the effect of defective Fas signaling on the T cell phenotype and apoptosis function in hematopoietic cell protein-tyrosine phosphatase-deficient motheaten mice. Viable motheaten (me(v)/me(v)) mice exhibited increased T cell proliferation and defective activation-induced apoptosis of Fas+ T cells in the lymph node, which was not ascribed to defective Fas ligand function. Furthermore, the Fas-mediated apoptosis defect in activated T cells from me(v)/me(v) mice was confirmed by their resistance to anti-Fas-induced apoptosis. No protein tyrosine dephosphorylation signal was delivered after anti-Fas cross-linking in the lymph node cells of me(v)/me(v) mice as revealed by 32Pi labeling of protein phosphatase substrates. The defective activation-induced apoptosis of Fas+ T cells in me(v)/me(v) mice led to lymphadenopathy with an accumulation of CD4- CD8- B220+ CD3+ T cells. Pneumonitis in me(v)/me(v) mice was associated with infiltration of cycling T cells detected by bromodeoxyuridine uptake in vivo. Thus, T cells from me(v)/me(v) mice are resistant to Fas-mediated apoptosis which results in lymphoproliferative disease and tissue infiltration.

COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus.

The noncatalytic domain of the human T cell protein tyrosine phosphatase (TCPTP) is alternatively spliced to generate a 45-kD form, p45TC, and a 48-kD form, p48TC (Champion-Arnaud et al., 1991; Mosinger et al., 1992). This manuscript concerns structural motifs in the noncatalytic segment of the enzyme responsible for targeting the two forms to different subcellular compartments. Endogenous and transiently expressed p48TC associates with the ER, as determined by sucrose gradient fractionation and indirect immunofluorescence, respectively. By contrast, p45TC localizes in the nucleus even though upon cell lysis it is not retained and fractionates with markers for soluble enzymes. Using fusion proteins consisting of beta-galactosidase and COOH-terminal fragments of p48TC, two motifs necessary for ER retention within a 70-residue targeting segment have been identified. These include the terminal 19 hydrophobic residues which comprise a potential membrane-spanning segment and residues 346-358 which encompass a cluster of basic amino acids that may represent another type of ER retention motif. The sequence RKRKR, which immediately precedes the splice junction, functions as a nuclear localization signal for p45TC.

Defective expression of hematopoietic cell protein tyrosine phosphatase (HCP) in lymphoid cells blocks Fas-mediated apoptosis

Protein tyrosine dephosphorylation after Fas cross-linking occurred In Fas apoptosis-sensitive CEM-6 cells but not in Fas apoptosis-resistant MOLT-4 cells, and apoptosis in the CEM-6 cells could be inhibited by the protein tyrosine phosphatase inhibitor, pervanadate. The time course and level of dephosphorylation were correlated with Increased hematopoletic cell protein tyrosine phosphatase (HCP) activity, but not with the activity of two other tyrosine phosphatases. The level of expression of HCP was correlated with Fas apoptosis function in eleven human and murine Fas-positive lymphoid cell lines. Expression of recombinant HCP In the MOLT-4 cell line converted this Fas apoptosis-resistant cell line to Fas apoptosis sensitive. HCP-mutant me v/ me v mice exhibited increased expression of Fas but decreased Fas-mediated apoptosis function in lymphold organs after anti-mouse Fas antibody treatment in vivo. Thus, HCP-mediated protein dephosphorylation is involved in the delivery of the Fas apoptosis signal in lymphoid cells.

Activation of B-cells by sIgM cross-linking induces accumulation of CD5 mRNA.

The surface membrane molecule CD5 is expressed on mature T cells and on the B-1a subpopulation of B cells. These CD5 positive B cells express an antibody repertoire with a relatively high frequency of self-reactivity. There is uncertainty about the origins of CD5 B cells and the reasons for this are reviewed. Recent reports which relate to the lineage/selection debate are discussed. For instance, an increase in the frequency of CD5 B cells is a feature of several genetically determined polysystem autoimmune syndromes. In the case of motheaten (me, mev) the pathogenesis of this increase in CD5 B cells is not yet understood, even though the mutation has been mapped to the Hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Another mutation which affects B cell development, X-linked immunodeficiency (xid), encodes a point mutation in a B cell cytoplasmic tyrosine kinase. Expression of xid in otherwise normal mice causes a lack of CD5 B cells and a shift in the antibody repertoire. Interestingly, expression of both xid and motheaten results in an amelioration of autoantibody production. Evidence is presented that in B cells regulation of expression of CD5 can occur at the level of mRNA and that cross-linking of sIgM can induce the accumulation of CD5 mRNA. The overall concept advanced is that cells expressing natural autoantibodies are triggered via sIgM ligation to become CD5 B cells.

Islet cell antigen 512 is a diabetes-specific islet autoantigen related to protein tyrosine phosphatases.

Islet cell Ag 512 (ICA512) is a recombinant human Ag that was isolated from an islet cDNA expression library by screening with human insulin-dependent diabetes mellitus sera. Specificity of reaction with diabetic sera was demonstrated initially by immunoprecipitation with a small number of diabetic and normal serum samples. To permit quantitative and rapid serum testing, ICA512 was purified and adapted to an ELISA format. In this way, a sensitivity of 48% with newly diagnosed diabetic sera has been measured with a panel of 80 sera. DNA sequencing of ICA512-3, a cDNA that contains a 1644 bp open reading frame, suggests that it codes for a transmembrane protein having a single membrane-spanning segment and a cytoplasmic domain that is closely related to the first intracellular (catalytic) domain of the T cell protein tyrosine phosphatase, CD45. Northern blot analysis of poly(A)+ RNAs from several human tissues indicates that ICA512 mRNA is expressed in brain and pancreas.

Chromosomal localization of the protein tyrosine phosphatase G1 gene and characterization of the aberrant transcripts in human colon cancer cells

We have recently described the isolation of the human PTPG1 gene which encodes a member of intracellular protein tyrosine phosphatases that may be candidates for tumor suppressor genes. In order to investigate the abnormality of the PTPG1 transcript in various human cancer cell lines, we have analyzed the consensus catalytic region of PTPG1 cDNA, using the reverse transcriptionpolymerase chain reaction. In a colorectal carcinoma cell line, DLD-1, we found three aberrant transcripts. Sequencing analysis revealed that one had a missense point mutation and the remainders contained 77 bp and 173 bp deletions, respectively. These alterations might directly affect their phosphatase activities. Our findings provide the first evidence for the aberrant transcripts of the protein tyrosine phosphatase in human cancer cells, and suggest that the aberration of PTPG1 gene might be involved in the tumororigenesis. Moreover, the human PTPG1 gene is localized on chromosome 7q11.23, a region with frequent abnormalities implicated in some human cancers.

Characterization of an SH2 containing protein tyrosine phosphatase in rat parotid gland acinar cells

Rat parotid glands were shown to possess protein phosphatase activity capable of catalyzing the dephosphorylation of several model phosphotase substrates, including p-nitrophenyl phosphate, tyrosine phosphorylated myelin basic protein and serine phosphorylated casein. A portion of this activity closely resembled dephosphorylation patterns of known protein tyrosine phosphatases. The reaction showed sensitivity to sodium orthovanadate, proceeded efficiently in the presence of metal chelators and favored acidic pH for optimum activity. Cell lysates from EGF-or isoproterenol-stimulated parotid glands, when immunoprecipitated with anti-Syp antibody, showed the induction of protein tyrosine phosphatase activity significantly higher than the unstimulated controls. The protein of M r = 65kDa also had elevated levels of tyrosine phosphorylation following isolation from cells treated to undergo proliferation. Thus parotid gland acinar cells possess protein tyrosine phosphatase activity of the PTPase 1D class associated with inducible cell growth, in addition to other phosphatases.

Anti-CD11b antibody prevents immunopathologic changes in viable moth-eaten bone marrow chimeric mice.

The effect of in vivo treatment with anti-CD11b (MAC-1) antibody (Ab) was examined in an inflammatory disease model, the viable moth-eaten (mev) mutant mouse. The autosomal recessive mev gene occurred spontaneously as a point mutation of the hematopoietic cell protein tyrosine phosphatase in C57BL/6 mice. Homozygotes (mev/mev) develop a chronic myelomonocytic inflammation, involving accumulation of myelomonocytic cells in lungs and skin, resulting in interstitial pneumonitis and severe edema in the paws. These mice also exhibit abnormalities in lymphoid development, thymic atrophy, with T cell and NK cell dysfunction. These inflammatory changes are transferrable by bone marrow cells of mev/mev mice, indicating that mev mutation is due to a stem cell defect in the myelomonocytic pathway. An anti-CD-11b (5C6) Ab inhibited the immunopathologic changes in the bone marrow chimeras, when the Ab treatment was initiated on day -1 or day 0 of the bone marrow transplant. The lungs, paws, and thymus all remained normal after treatment. Furthermore, the Ab also delayed the onset of the mev syndromes when the Ab was given 10 days after the bone marrow transfer. Therefore anti-CD11b Ab inhibited inflammation both prophylactically and therapeutically, and restored normal function of T and NK cells in this disease model. These results support the contention that CD11b molecules expressed in the myelomonocytic cells play a critical role in this naturally occurring inflammatory disease.

The leukocyte common antigen, CD45 and other protein tyrosine phosphatases in hematopoietic cells

Numerous protein tyrosine phosphatases have been characterized that are expressed by hematopoietic-derived cells. CD45, a transmembrane protein tyrosine phosphatase expressed by all nucleated cells of hematopoietic origin, has been a prototype for studying the function of these enzymes. In T lymphocytes, CD45 serves to dephosphorylate and activate members of the Src-tyrosine kinase family. Without CD45 function, T cells cannot respond to antigen. Therefore, CD45 functions positively to regulate lymphocyte activation. The characteristics of other protein tyrosine phosphatases found in hematopoietic-derived cells suggests that they are also essential in regulating cell differentiation and activation.

Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in response to analogues of cAMP or diacylglycerol plus okadaic acid

The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mammalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca2+/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50 kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide 32P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated 'downstream' of cAMP- and Ca2+/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283-364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity.

Differential activities of protein tyrosine phosphatases in intact cells.

We have employed transient co-overexpression of protein tyrosine phosphatases (PTPs) with a panel of receptor tyrosine kinases (RTKs) to investigate molecular parameters that regulate dephosphorylation activity and specificity in intact cells. Our results demonstrate clear differences in susceptibility of various forms of different RTKs to the action of PTP 1B, T-cell phosphatase (TC-PTP), and CD45, which suggests cellular compartmentalization as a major factor defining activity and overall function. TC-M PTP, a nonlocalized cytosolic mutant, is deregulated and is therefore able to efficiently suppress v-erbB- and v-fms-induced cell transformation, which is not observed with the intact TC-PTP or PTP 1B. The transmembrane PTP CD45 displays more selectivity but appears to be already active during transport to the cell surface. Dephosphorylation activity is also dependent on relative RTK/PTP expression levels and can be modulated by the SH2 domain-containing noncatalytic subunit of phosphatidylinositol 3'-kinase, p85. Overexpression of high affinity binding proteins could therefore contribute to RTK-induced cell transformation and cancer.