Chromosomal location of murine protein tyrosine phosphatase (Ptprj and Ptpre) genes

It is now widely accepted that protein tyrosine phosphatases (PTPases) play important or even critical roles in cell growth, differentiation, and development. Our recent experiments suggested that specific PTPases, PTP beta 2 and PTP epsilon, are involved in the early molecular events for in vitro differentiation of mouse erythroleukemia (MEL) as well as embryonic carcinoma (F9) cells. Using mouse cDNA for PTP beta 2 and PTP epsilon, which we have cloned recently, we attempted to locate the genes to mouse chromosomes. Interspecific backcross analysis indicated that the gene for PTP beta 2, Ptprj, is located in the middle region of chromosome 2, and the gene for PTP epsilon, Ptpre, was mapped in the vicinity of the imprinted regions in the distal part of chromosome 7. Possible biological roles of these PTPases are discussed.     

Successful pregnancy in a patient with heart failure secondary to aortic and mitral regurgitation

The transmembrane nature of the receptor-like protein tyrosine phosphatases (PTPases) suggests that they transduce as yet unidentified extracellular signals to intracellular events via a phosphotyrosyl-protein dephosphorylation step, although little is known of their regulation and cellular activities. Structure/function studies of PTP alpha demonstrate that both catalytic domains are required for full enzymatic efficiency and that interdomain interactions may modulate PTP alpha activity and specificity. Overexpression of PTP alpha results in cell transformation and tumorigenesis, likely as a consequence of the ability of PTP alpha to dephosphorylate and activate the c-src tyrosine kinase. This suggests a role for PTP alpha in normal cell proliferation. PTP alpha is so far unique among the PTPases in terms of its oncogenic potential, and overexpression or deregulation of PTP alpha may be involved in the genesis, progression or maintenance of certain tumor states.     

Ganglioside GQ1b enhances Ig production by human PBMCs

We have reported that high glucose conditions (27 mM for 4 days) induces activation of protein tyrosine phosphatases (PTPases) which are associated with impaired insulin signaling in Rat 1 fibroblasts expressing human insulin receptors [Maegawa, H. et al. (1995) J. Biol. Chem. 270, 7724-7730]. In this study, we found increased mRNA-levels of a non-receptor type PTPase, protein tyrosine phosphatase 1B (PTP1B), and receptor type PTPases, leukocyte common antigen-related phosphatase (LAR), and LAR-related phosphatase (LRP), under high glucose conditions. In accordance with these results, LAR content was significantly increased, whereas LRP content was not increased. Cytosolic PTP1B content was increased, but membrane-associated PTP1B content showed no detectable change. Pioglitazone, a thiazolidinedione, normalized increased cytosolic PTPase activity through reduction of cytosolic PTP1B content, but it had no effect on mRNA levels of these PTPases. Under the high glucose condition, we also found that epidermal growth factor (EGF)-stimulated signaling, including tyrosine-phosphorylation of EGF receptor and phosphatidylinositol 3'-kinase activities, was attenuated. Nevertheless, pioglitazone failed to restore the attenuated EGF-signaling. These results indicate that the high glucose conditions cause dysfunction of EGF receptor. However, the increased cytosolic PTP1B content is not involved in the abnormal regulation of EGF-signaling, in contrast to insulin-signaling.     

A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.     

Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.     

Protein tyrosine phosphatases in cell transformation

The role of tyrosine phosphorylation in cell transformation has been well established. It has been proposed that protein tyrosine phosphatases (PTPases) may be capable of dephosphorylating critical substrates involved in the transformation process, suggesting that they represent a tumor suppressor family of enzymes. Indeed, recent work showed that overexpression of some PTPases in malignant cells counteracted the action of oncogenic tyrosine kinases although overexpression of other forms of these enzymes increased tumorigenicity. The work described herein has provided some insight into the action, both antagonistic and synergistic, of the kinases and phosphatases on cell growth and transformation.     

The tumor-selective somatostatin analog, TT2-32 induces a biphasic activation of phosphotyrosine phosphatase activity in human colon tumor cell line, SW620

Somatostatin has been demonstrated to activate phosphotyrosine phosphatases (PTPases) in pancreatic cells. In this work we studied the effect of a tumor-selective somatostatin structural derivative, TT2-32, on the PTPase activity in the SW620 human colon tumor cell line. TT2-32 caused a strong inhibition of cell proliferation. In response to TT2-32 we found a rapid and sustained increase (5-30 min) in PTPase activity showing two maxima at 0.1 and 30 microM concentrations, respectively. During short-term incubation tyrosine kinase activity was much less affected by TT2-32. TT2-32-induced activation of PTPases may be an important early step in the signaling cascade in the inhibition of cell proliferation in colon carcinomas.     

ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.     

Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-gamma signaling complex in human natural killer (NK) cells

The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP). Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-gamma in NK cells. In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.     

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA)

Phosphatidylcholine-phospholipase D has been proposed to play a key role in the transduction of the proliferative responses of a wide range of mitogens and growth factors. We now report that the antigen receptors on T lymphocytes derived from human tonsillar or murine splenic preparations are coupled to phosphatidylcholine (PtdCho)-phospholipase D (PLD) activation following stimulation of these T cells with anti-CD3 antibodies. However, since we also demonstrate that the antigen receptors on murine thymocytes are coupled to PtdCho-PLD activation, we propose that it is unlikely that this PLD pathway plays a central role in the transduction of T-cell proliferative responses, but rather, may be involved in either driving cells into cycle or maintaining cell cycle progression, processes required both for proliferation and activation-induced cell death. Whilst the molecular mechanisms underlying T-cell receptor (TCR)-coupling to PtdCho-PLD activation in these cells have not been fully defined, kinetics studies and experiments using pharmacological inhibitors of protein tyrosine phosphatases (PTPases) and reconstituting CD3-coupled PtdCho-PLD activity in streptolysin-O permeabilized cells, suggest that the TCR/CD3 complex, under optimal conditions of activation, may be predominantly coupled to PtdCho-PLD activation downstream of tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma), phosphatidylinositol (PtdIns)P2 hydrolysis, calcium mobilization and protein kinase C (PKC) activation.     

Differential regulation of glucose transport and glucose transporter (GLUT-1) gene expression by vanadate, phorbol ester and okadaic acid in L6 skeletal muscle cells

Vanadate, an inhibitor of protein tyrosine phosphatases (PTPases), elicited time-and-dose-dependent increases in glucose transport in rat muscle L6 cells in culture: the rate was increased by 150-175% over control in 24 h at 75-100 microM. In contrast, molybdate, another inhibitor of PTPases, failed to stimulate glucose transport. The effect of vanadate was not blocked by tyrosine kinase inhibitors, genistein or tyrphostin RG 50864, implying that tyrosine kinase activation may not mediate the action of vanadate. The ability of vanadate to stimulate glucose transport was preserved in cells whose protein kinase C (PKC) activity was down-regulated by prior exposure to phorbol esters (TPA), suggesting that the vanadate effect was unrelated to the TPA-sensitive PKC isoform(s). Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, was a potent activator of glucose transport increasing the rate 7-fold in 24 h at a concentration of 50 nM. The increases in GLUT-1 mRNA level in response to vanadate and TPA were paralleled bh much smaller increases in immunoreactive GLUT-1 protein level, whereas okadaic acid treatment markedly elevated GLUT-1 protein without a concomitant change in GLUT-1 mRNA levels.     

The selective reduction in PTPdelta expression in hepatomas

The mRNA levels for receptor-like protein tyrosine phosphatases (PTPases), PTPalpha, PTPdelta, PTPgamma and LAR, were evaluated by Northern blot analysis in two types of chemically-induced rat primary hepatomas. In the four PTPases the PTPdelta mRNA was selectively reduced in these hepatoma tissues. It was also diminished in HepG2 hepatoblastoma cell line and in all of the poorly differentiated ascites hepatoma cells examined. PTPalpha, PTPgamma and LAR did not show such a characteristic decrease. This selective reduction in PTPdelta expression strongly suggests PTPdelta plays an important role in hepatocarcinogenesis, possibly as a tumor suppressor gene.     

A new region of conservation is defined between human and mouse X chromosomes

Comparative mapping of the X chromosome in eutherian mammals has revealed distinct regions of conservation as well as evolutionary rearrangements between human and mouse. Recently, we and others mapped the murine homologue of CLCN4 (Chloride channel 4) to band F4 of the X chromosome in Mus spretus but to chromosome 7 in laboratory strains. We now report the mapping of the murine homologues of APXL (Apical protein Xenopus laevis-like) and OA1 (Ocular albinism type I), two genes that are located on the human X chromosome at band p22. 3 and in close proximity to CLCN4. Interestingly, Oa1 and Apxl map to bands F2-F3 in both M. spretus and the laboratory strain C57BL/6J, defining a new rearrangement between human and mouse X chromosomes.     

PSTPIP 2, a second tyrosine phosphorylated, cytoskeletal-associated protein that binds a PEST-type protein-tyrosine phosphatase

Although cytoskeletal regulation is critical to cell function during interphase and mitosis, the components of the cytoskeleton involved with its control are only beginning to be elucidated. Recently, we reported the identification of a cytoskeletal-associated protein, proline-serine-threonine phosphatase-interacting protein (PSTPIP), whose level of tyrosine phosphorylation was controlled by PEST-type protein-tyrosine phosphatases (PTPs) bound to a novel protein interaction site in the PSTPIP predicted coiled-coil domain. We also showed that the PSTPIP SH3 domain interacts with the Wiskott-Aldrich syndrome protein (WASP), a cytoskeletal regulatory protein, in a manner modulated by tyrosine phosphorylation. Here we describe the identification of PSTPIP 2, a widely expressed protein that is related to PSTPIP. PSTPIP 2 lacks an SH3 domain but contains a region predicted to bind to PEST-type PTPs, and structure-function analyses demonstrate that PSTPIP 2 interacts with the proline-rich C terminus of the PEST-type PTP hematopoietic stem cell factor in a manner similar to that previously demonstrated for PSTPIP. Confocal microscopy revealed that PSTPIP 2 colocalizes with PSTPIP in F actin-rich regions. PSTPIP 2 was found to be efficiently phosphorylated in v-Src-transfected or pervanadate-treated cells at two tyrosines conserved in PSTPIP, but in contrast to PSTPIP, tyrosine phosphorylated PSTPIP 2 was only weakly dephosphorylated in the presence of PTP HSCF. Finally, analysis of oligomer formation demonstrated that PSTPIP and PSTPIP 2 formed homo- but not heterodimers. These data suggest that a family of tyrosine phosphorylated, PEST PTP binding proteins may be implicated in cytoskeletal regulation.     

GLEPP1, a renal glomerular epithelial cell (podocyte) membrane protein-tyrosine phosphatase. Identification, molecular cloning, and characterization in rabbit

Podocytes are specialized epithelial cells with delicate interdigitating foot processes which cover the exterior basement membrane surface of the glomerular capillary. They are in part responsible for the extraordinary charge and size filtration characteristics of the glomerulus. To better understand disease processes affecting the glomerular filter, we searched for proteins with relative specificity to the podocyte using a monoclonal antibody strategy. The first such protein characterized (designated glomerular epithelial protein 1 (GLEPP1)) is a membrane protein-tyrosine phosphatase (PTPase) with a large extracellular domain containing eight fibronectin type III-like repeats, a hydrophobic transmembrane segment, and a single PTPase domain. The GLEPP1 PTPase domain shows homology with two other single domain transmembrane PTPases (PTP beta and Drosophila central nervous system PTP10D). This homology includes 2 cysteines in the PTPase domain not present in intracellular or tandem domain membrane PTPases. GLEPP1 PTPase protein is distributed to the podocyte foot processes themselves. RNase protection assay shows that GLEPP1 mRNA is also present in brain. By analogy with the CD45 PTPase of T cells, we expect that this receptor might play a role in maintaining foot process structure and/or function by regulating tyrosine phosphorylation of podocyte proteins.     

Association of PTPmu with catenins in cancer cells: a possible role for E-cadherin

Protein tyrosine phosphorylation and dephosphorylation is regulated by the action of protein tyrosine kinases (PTK) and phosphatases (PTP) respectively. The receptor type phosphatase, PTPmu, is located at the cell surface where it may function to regulate the phosphoryl status of members of the cadherin adhesion complex and thus cadherin function. We have investigated the association of PTPmu with E-cadherin and catenin molecules in human tumour cells and report that PTPmu; is associated with E-cadherin and alpha and beta-catenin in E-cadherin-positive cell lines. However, no association between PTPmu and catenin members could be detected in E-cadherin negative cells. These observations suggest that the association of PTPmu with catenin molecules may occur via E-cadherin rather than a direct interaction.     

The receptor-like protein-tyrosine phosphatase DEP-1 is constitutively associated with a 64-kDa protein serine/threonine kinase

Protein-tyrosine phosphatases (PTPs) are involved in the regulation of diverse cellular processes and may function as positive effectors as well as negative regulators of intracellular signaling. Recent data demonstrate that malignant transformation of cells is frequently associated with changes in PTP expression or activity. Our analysis of PTP expression in mammary carcinoma cell lines resulted in the molecular cloning of a receptor-like PTP, also known as DEP-1. DEP-1 was found to be expressed at varying levels in mammary carcinoma cell lines and A431 cells. In all tumor cell lines analyzed, DEP-1 was constitutively phosphorylated on tyrosine residues. Phosphorylation of DEP-1 increased significantly after treatment of cells with the PTP inhibitor pervanadate. In A431 cells, tyrosine phosphorylation of DEP-1 was also observed after stimulation with epidermal growth factor, however, only after prolonged exposure of the cells to the ligand, suggesting an indirect mechanism of phosphorylation. In addition, DEP-1 coprecipitated with several tyrosine-phosphorylated proteins from pervanadate-treated cells. In vitro binding experiments using a glutathione S-transferase fusion protein containing the catalytically inactive PTP domain of DEP-1 (Gst-DEP-1-C/S) identify these proteins as potential substrates of DEP-1. In addition, we found a 64-kDa serine/threonine kinase to be constitutively associated with DEP-1 in all tumor cell lines tested. The 64-kDa kinase forms a stable complex with DEP-1 and phosphorylates DEP-1 and DEP-1-interacting proteins in vitro. These data suggest a possible mechanism of DEP-1 regulation in tumor cell lines involving serine/threonine and/or tyrosine phosphorylation.