Acylphosphatase is involved in differentiation of K562 cells.

The level of both isoforms of acylphosphatase was evaluated in the human erythroleukemia K562 cell line during differentiation. K562 cells were treated with PMA, which induces megakaryocytic differentiation, and with aphidicolin or hemin, which stimulate erythrocytic differentiation. While the MT isoform showed an average 10-fold increase independently of the differentiating agent used, only hemin treatment caused a similar increase of the CT isoform, suggesting a different role of the two isoforms in the cell. Treatment with either hemin or aphidicolin of K562 cells overexpressing the two acylphosphatase isoforms suggested the possibility that acylphosphatases play a role in the onset of differentiation.

Low molecular weight phosphotyrosine protein phosphatase translocation during cell stimulation with platelet-derived growth factor.

Low Mr phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low Mr PTP overexpression.

Inhibition of cellular response to platelet-derived growth factor by low M(r) phosphotyrosine protein phosphatase overexpression.

The role of low M(r) phosphotyrosine protein phosphatase (PTPase) in the control of cell proliferation was studied. A synthetic gene coding for PTPase was transfected and expressed in NIH/3T3 fibroblasts. The effects of the enzyme were particularly evident when cells were stimulated by platelet-derived growth factor (PDGF). The mitogenic response to PDGF was decreased and the inhibition reached 90%. This effect was more pronounced with respect to fetal calf serum stimulation. Hormone-dependent autophosphorylation of the PDGF receptor was significantly reduced. These results demonstrate that low M(r) PTPase, a cytosolic enzyme, not only affects cellular response to PDGF but also reduces the membrane receptor autophosphorylation.

Low M(r) phosphotyrosine protein phosphatase activity on fibroblast growth factor receptor is not associated with enzyme translocation.

Fibroblast growth factor receptor (class IV) shares a certain degree of similarity with class III members like platelet-derived growth factor and macrophage-colony-stimulating factor receptors, which, once activated, are substrates of low M(r) phosphotyrosine protein phosphatase. Up until now no phosphotyrosine phosphatase has been shown to act on this receptor in vivo. Here we demonstrate that low M(r) phosphotyrosine protein phosphatase is able to reduce receptor tyrosine phosphorylation and cell proliferation in response to basic fibroblast growth factor. Contrary to what was previously observed for platelet-derived growth factor, during cell stimulation with basic fibroblast growth factor, no enzyme redistribution among cellular compartments is observed.

Effect of phosphotyrosine phosphatase over-expression on glutathione metabolism in normal and oncogene-transformed cells.

We measured the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in normal and oncogene-transformed NIH/3T3 fibroblasts and 32D hematopoietic cells. NIH/3T3 cells transformed by the activated oncogenes erbB, src, and raf, showed increased levels of GSH with concomitant alterations in the levels of GSH-related enzymes. Transfection and over-expression of a synthetic gene coding for a phosphotyrosine protein phosphatase (PTPase), which inhibited the proliferation of normal and transformed NIH/3T3 cells, was accompanied by a decrease in GSH levels in normal and erbB-transformed fibroblasts, and by an increase in src and raf transformants. Among GSH-related enzymes, only gamma-glutamylcysteine synthetase was altered in normal and erbB-transformed NIH/3T3 fibroblasts following PTPase transfection. Therefore, tyrosine phosphorylation could be selectively involved in the regulation of GSH metabolism in normal and oncogene-transformed NIH/3T3 fibroblasts, possibly by a dual-type effect on receptor/oncoprotein-mediated mitogenic signal transduction. However, no relationship was observed between the GSH and PTPase effect on cell growth, either after oncogene transfection or PTPase transfection. Moreover, the changes in GSH metabolism were specifically related to cell lineage. In fact GSH and related enzymes did not change in 32D hematopoietic cells transformed by the same activated erbB oncogene and in those--normal or transformed--over-expressing the PTPase: in these cells also, over-expression of the PTPase gene was not accompanied by growth inhibition.

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps.

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.

Sequence-specific recognition of peptide substrates by the low Mr phosphotyrosine protein phosphatase isoforms.

A number of phosphotyrosine-containing peptides derived from the PDGF receptor phosphorylation sites have been synthesised. The peptides were assayed as substrates of the two isoforms (IF1 and IF2) of the low Mr PTPase. The calculated k(cat), Km, and k(cat)/Km values indicate that only one peptide is best hydrolysed by IF2 (but not IF1), whose catalytic efficiency averages those previously reported for most PTPases (except the Yersinia enzyme). This peptide is the only one containing a couple of no bulky hydrophobic residues at the phosphotyrosine N-side. The determination of the same catalytic parameters in the presence of analogues of the best hydrolysed peptide in which one or both hydrophobic residues were replaced by Asp or Lys residues confirmed the importance of the hydrophobic cluster at the phosphotyrosine N-side for optimal enzymatic hydrolysis. These findings are discussed in the light of the known IF2 X-ray structure.

Negative growth control by a novel low M(r) phosphotyrosine protein phosphatase in normal and transformed cells.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as 'PTPase') in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

[Data collection: evaluation of a pilot model]. / Coleta de dados: avaliação de um modelo piloto.

The overall purpose of this study was to explore nurses' feelings about the applicability and adequacy of a pilot model of nursing assessment (PMNA) developed for coronary care units (CCU) in order to obtain data that could help in establishing a definitive model. The evaluation, performed by 11 CCU nurses, showed that they considered the development and implementation of PMNA as valuable, and that its design was adequate for interviewing cardiac patients. These results will be employed in the elaboration of a definitive model of nursing assessment.

Different in vitro and in vivo activity of low Mr phosphotyrosine protein phosphatase on epidermal growth factor receptor.

Low Mr phosphotyrosine protein phosphatase is a cytosolic enzyme which dephosphorylates platelet-derived growth factor and insulin receptor in vivo, thus reducing cellular mitogenic response to such growth factors. Following cell stimulation with platelet-derived growth factor the phosphatase undergoes a redistribution from the citosol to the Triton X-100-insoluble fraction where its activity upon the growth factor receptor is intense. Previous research uncovered evidence that low Mr phosphotyrosine protein phosphatase dephosphorylates the epidermal growth factor receptor in vitro. Here we demonstrate that in vivo the enzyme is not active on the phosphorylated epidermal growth factor receptor and it does not influence the mitogenic response of cells. Since the enzyme distribution is not affected by epidermal growth factor stimulation, involvement of a recruitment mechanism in the definition of low Mr phosphotyrosine protein phosphatase substrate specificity is hypothesized.

pp60v-src phosphorylates and activates low molecular weight phosphotyrosine-protein phosphatase.

Low M(r) phosphotyrosine-protein phosphatase belongs to the non-receptor cytosolic phosphotyrosine-protein phosphatase subfamily. It has been demonstrated that this enzyme dephosphorylates receptor tyrosine kinases, namely the epidermal growth factor receptor in vitro and the platelet-derived growth factor receptor in vivo. Low M(r) phosphotyrosine-protein phosphatase is constitutively tyrosine-phosphorylated in NIH/3T3 cells transformed by pp60v-src. The same tyrosine kinase, previously immunoprecipitated, phosphorylates this enzyme in vitro as well. Phosphorylation is enhanced using phosphatase inhibitors and phenylarsine oxide-inactivated phosphatase, consistently with the existence of an auto-dephosphorylation process. Intermolecular dephosphorylation is demonstrated adding the active enzyme in a solution containing the inactivated and previously phosphorylated one. This tyrosine phosphorylation correlates with an increase in catalytic activity. Our results provide evidence of a physiological mechanism of low M(r) phosphotyrosine-protein phosphatase activity regulation.

Evidence for glutathione involvement in platelet-derived growth-factor-mediated signal transduction.

Recent studies show that glutathione, while being involved in the well-known physiological processes of amino acid transport and detoxification, can also play a part in cell proliferation events. Cell treatment with l-buthionine sulphoximine, which causes glutathione depletion, is accompanied by a decrease in cell proliferation. At present no precise relationship between this thiol and any critical intermediate of the mitogenic cascade has been proved. In this study, conducted on NIH/3T3 murine fibroblasts, we demonstrate a strict correlation between glutathione levels and platelet-derived growth-factor-receptor activation in response to stimulation and cell proliferation. The receptor autophosphorylation is severely impaired at low glutathione cellular levels. The interaction of glutathione with this growth-factor receptor in vivo, while being rather specific, is complex and may involve both cytosolic and extracellular receptor domains.

The low-molecular-weight phosphotyrosine protein phosphatase, when overexpressed, reduces the mitogenic response to macrophage colony-stimulating factor and tyrosine phosphorylation of its receptor.

The interference of low-molecular-weight phosphotyrosine protein phosphatase with the macrophage response to macrophage colony-stimulating factor was investigated. This paper shows that this phosphatase, already known to be involved in platelet-derived growth factor receptor signaling, is physiologically expressed in murine macrophages and dephosphorylates in vitro macrophage colony-stimulating factor receptor molecules immunoprecipitated from macrophage colony-stimulating factor-stimulated macrophages. We obtained the first demonstration that a phosphotyrosine-specific protein phosphatase dephosphorylates the macrophage colony-stimulating factor receptor in vivo and reduces the mitogenic response to macrophage colony-stimulating factor. The data indicate that low-molecular-weight phosphotyrosine protein phosphatase is a negative regulator of macrophage colony-stimulating factor receptor signaling.

Properties of Cys21-mutated muscle acylphosphatases.

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.