Low Mr Phosphotyrosine Protein Phosphatase Research Articles

Low Mr Phosphotyrosine Protein Phosphatase Associates and Dephosphorylates p125 Focal Adhesion Kinase, Interfering with Cell Motility and Spreading

Low M(r) phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M(r) phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M(r) phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60(src) with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M(r) phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M(r) phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.

Thiolation of Low-Mr Phosphotyrosine Protein Phosphatase by Thiol-Disulfides

Thiolation of Low-Mr Phosphotyrosine Protein Phosphatase by Thiol-Disulfides

Low Mr 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.

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr 131 or Tyr 132 by Src kinase

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60 Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr 131 and Tyr 132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr 131 and Tyr 132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr 131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr 132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

Differentin Vitroandin VivoActivity of LowMrPhosphotyrosine Protein Phosphatase on Epidermal Growth Factor Receptor

LowMrphosphotyrosine protein phosphatase is a cytosolic enzyme which dephosphorylates platelet-derived growth factor and insulin receptorin 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 lowMrphosphotyrosine protein phosphatase dephosphorylates the epidermal growth factor receptorin vitro.Here we demonstrate thatin vivothe 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 lowMrphosphotyrosine protein phosphatase substrate specificity is hypothesized.

Cloning, expression and characterisation of a new human low Mr phosphotyrosine protein phosphatase originating by alternative splicing

RT-PCR experiments on RNA from K562 and HepG2 cells and from human placenta led to the isolation of a novel cDNA, a further alternative splicing product of the primary transcript of low M r phosphotyrosine phosphatase (LMW-PTP), already known to produce isoforms 1 and 2. This new transcript represents 15–20% of the total LMW-PTP mRNA in the cell. This novel cDNA codifies for a protein that we have named SV3 (splicing variant 3): the deduced protein sequence presents the first 49 residues identical to those of isoform 1, followed by 24 unrelated amino acids, due to a frameshift introduced at the novel exon-exon boundary. The SV3 protein, expressed in E. coli is enzymatically inactive, most probably because unfolded, as suggested by far-UV circular dichroism (CD) experiments. SV3 protein appears to possess the characteristics of an unstructured polypeptide chain lacking the packing of side chain residues and the secondary structure level that are typical of globular proteins. This protein could represent an inactive variant of the human LMW-PTP.

Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1

The genes of the human low M r phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E. coli as fusion proteins with glutathione S-transferase. The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity. This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies. The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver. These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose. IF1 and IF2 crystals were also grown. IF1 crystals were X-ray-grade, diffracted to better than 2.4 Å and were suitable for high resolution X-ray structure determination.

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 M r PTPase. The calculated k cat, K m, and k cat/ K m 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.

LowMrPhosphotyrosine Protein Phosphatase Interacts with the PDGF Receptor Directly via Its Catalytic Site

Many proteins bind to the activated platelet derived growth factor receptor (PDGF-R) either directly or by means of adapter molecules. Up to now all these proteins were shown to transmit and amplify the signal started with PDGF-R stimulation. In a recent study our group has demonstrated that lowMrphosphotyrosine protein phosphatase (LMW-PTP) specifically interacts with PDGF-R in NIH3T3 cells. In the present study we have attempted to clarify the modality of interaction, bothin vivoandin vitro,of these two proteins, using a catalytically inactive LMW-PTP mutant. Our results indicate that LMW-PTP and PDGF-R interact directly, without the necessity of any adapter protein. This interaction leads to PDGF-R dephosphorylation and, presumably, interrupts one or more of the mitogenic pathways that originate from receptor activation.

PDGF receptor as a specific in vivo target for low Mr phosphotyrosine protein phosphatase

Low M r phosphotyrosine protein phosphatase (LMWPTP) is a 18 kDa cytosolic enzyme widely distributed in eukaryotic cells. LMW-PTP catalyses the hydrolysis of phosphotyrosine residues and overexpression of the enzyme in normal and transformed cells inhibits cell proliferation. Site directed mutagenesis, together with crystallographic studies, have contributed to clarify the catalytic mechanism, which involves the active site signature sequence C 12XXXXXR 18, a main feature of all PTPase family members. In order to identify the LMW-PTP substratels we have expressed in NIH-3T3 cells a catalytically inert Cys 12 to Ser phosphatase mutant which has preserved its capacity for substrate binding. Overexpression of the mutant phosphatase leads to enhanced cell proliferation and serum induced mitogenesis, indicating that the mutation results in the production of a dominant negative protein. Analysis of mutant LMW-PTP expressing cells has enabled us to demonstrate an association between LMW-PTP and platelet derived growth factor receptor that appears to be highly specific. Our data suggest a catalytic action of LMW-PTP on the phosphorylated platelet derived growth factor receptor.

Aspartic-129 is an essential residue in the catalytic mechanism of the low Mr phosphotyrosine protein phosphatase

The crystal structure of the bovine liver low M r phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by 1H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation ( k 2) and dephosphorylation ( k 3) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.

Inhibition of cellular response to platelet-derived growth factor by low Mr 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.

Negative growth control by a novel low Mr 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.

Differential role of four cysteines on the activity of a low Mr phosphotyrosine protein phosphatase

In this paper we describe the construction or five mutants of a bovine liver low M r phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein or E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme.

Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.

Two low M(r) phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as low M(r) acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40-73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.

TNF receptors and cytotoxicity on rat brain oligodendrocytes: [formula omitted] Dept. of Neurobiol. and Dept. of Molecular Genetics and Virol. Weizmann Inst. of Science, Rehovot, Israel

Hematopoietic cells, particularly megakaryoblastic ones, display a high level of low Mr phosphotyrosine protein phosphatase (LMW-PTP) expression; nevertheless, the role of this PTP in such cellular lineages has been scarcely investigated. Here, we demonstrate that LMW-PTP is able to associate and dephosphorylate signal transducer and activator of transcription-5 (STAT5) in DAMI megakaryocytic cells. Numerous researchers repeatedly hypothesized the association of a regulatory phosphotyrosine protein phosphatase with STAT5 C-terminus, but such phosphotyrosine protein phosphatase remained unknown. We show evidence indicating that the association of STAT5 and LMW-PTP does not exclusively involve the phosphatase active site and phosphotyrosine residue of STAT5, and we individuate an essential region of interaction at STAT5 C-terminus, coinciding with the previously hypothesized PTP-associating domain.