Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) shares no general sequence homology with other PTPs, although it has an active site sequence motif CXXXXXR and a reaction mechanism identical to those of all PTPs. The main function of this enzyme is the down-regulation of platelet-derived growth factor and insulin receptors. Both human LMW-PTP isoenzymes are inactivated by H 2 O 2 . The enzymes are protected from inactivation by P i , a competitive inhibitor, suggesting that the H 2 O 2 reaction is directed to active site. Analysis of free thiols performed on the inactivated enzymes demonstrates that only two out of the eight LMW-PTP cysteines are modified. Time-course high performance liquid chromatography-electrospray mass spectrometry, together with specific radiolabeling and tryptic fingerprint analyses, enables us to demonstrate that H 2 O 2 causes the oxidation of Cys-12 and Cys-17 to form a disulfide bond. Because both residues are localized into the active site region, this modification inactivates the enzyme. Fluorescence spectroscopy experiments suggest that the fold of the enzyme is modified during oxidation by H 2 O 2 . Because a physiological concentration of H 2 O 2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the LMW-PTP in the cell.Protein tyrosine phosphorylation in eucaryotes is a key mechanism for cellular control, because it is involved in several processes, such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). A fine balancing of cellular protein tyrosine phosphorylation levels is determined by regulating the activities of protein-tyrosine kinases and/or protein-tyrosine phosphatases (PTPs).1 Receptor protein-tyrosine kinases are considered to be the major enzymes regulating mitogenic protein phosphorylation cascades; nevertheless, the presence of SH2 domains in particular PTPs and the receptorlike structure of some membrane PTPs clearly indicate that PTPs are also regulated in the cell. The PTP superfamily consists of four main families: the tyrosine-specific phosphatases, the VH1-like dual specificity phosphatases, the cdc25 phosphatases, and the low molecular weight phosphatases (LMW-PTPs). Despite extremely limited sequence similarity, all share an active site motif consisting of a cysteine and an arginine separated by five residues (CXXXXXR, where X is any amino acid). All PTPs have identical catalytic mechanism, which involves the formation of a cysteinyl-phosphate intermediate (2).Recent papers from our laboratory have demonstrated that LMW-PTP is involved in the regulation of cellular signaling started by the activation of PDGF and insulin receptors (3-5). In fact, the overexpression of the wild type enzyme in NIH/3T3 cells causes decrease of cellular growth rate and of phosphorylation level of the PDGF receptor (3). Furthermore, the overexpression in ...

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp. platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity. Its complete amino acid sequence was determined; this protein consists of 120 amino acid residues, contains 4 cysteines (S-S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi. One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus. Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.The European plane tree (Platanus acerifolia) is an ornamental plant species of the urban environment. A great number of plane trees in the parks and towns of southern Europe have been destroyed by Ceratocystis fimbriata (Ell. and Halst.) Davidson f. sp. platani Walter, the Ascomycete responsible for canker stain disease (1). This disease is characterized by foliar wilting and spreading lesions that involve phloem, cambium, and extensive regions of sapwood (2, 3). The pathogen spreads from tree to tree by means of root grafts of closely spaced plants and, more frequently, through wounds caused by pruning (4).The American species Platanus occidentalis has been shown to contain a source of resistance to C. fimbriata f. sp. platani that could prove of great interest in the genetic improvement of the European plane (5). Known post-infection host defense mechanisms involve physical factors such as the occlusion of the xylematic vessels and the compartmentalization of infected tissue areas as well as the production of flavans, umbelliferone, and scopoletine phytoalexins (6 -9). Unfortunately, only resistant P. occidentalis clones, not yet acclimatized to Europe, localized the disease (7,8). Recent papers (10, 11) have shown that C. fimbriata f. sp. platani displays an array of phytotoxic metabolites possibly involved in determining some of the symptoms of canker stain.In the present paper we report on the purification procedure, the amino acid sequence, and the characterization of the biological activity of a new protein (named cerato-platanin) from the cul...

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.

Low-M, phosphotyrosine protein phosphatase (PTPase), previously known as low-M, acid phosphatase, catalyzes the in-vitro hydrolysis of tyrosine phosphorylated proteins, low-M, aryl phosphates and natural and synthetic acyl phosphates. Its activity on Serm-phosphorylated proteins and on most alkyl phosphates is very poor. In this study the mechanism of benzoyl-phosphate hydrolysis was studied by means of non-mutated and mutated PTPase fusion proteins. The mechanism of benzoyl-phosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p-nitrophenyl-phosphate hydrolysis. The results demonstrated that both hydrolytic processes proceed through common enzyme-catalyzed mechanisms. Nevertheless, the performed phosphoenzyme-trapping experiments enable us to identify Cysl2 as the active-site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate. In addition, while the role of Cysl7 in the substrate binding was confirmed, its participation a second time in the step that involves the Cysl2 dephosphorylation was suggested by the results of phosphoenzyme-trapping experiments. The participation of Argl8 in the substrate-binding site was demonstrated by site-directed mutagenesis that produced the conservative Lysl8 and the non-conservative Met18 mutants. Both these mutants were almost inactive and not able to bind the substrate and a competitive inhibitor. Furthermore, phosphoenzyme-trapping experiments clearly excluded that Cys62 and Cys145 (that were indicated by another laboratory to be involved in the active site of the enzyme as powerful nucleophilic agents) are the residues directly involved in the formation of the phosphoenzyme covalent intermediate.

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The low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is a cytosolic phosphotyrosineprotein phosphatase specifically interacting with the activated platelet-derived growth factor (PDGF) receptor through its active site. Overexpression of the LMW-PTP results in modulation of PDGF-dependent mitogenesis. In this study we investigated the effects of this tyrosine phosphatase on the signaling pathways relevant for PDGF-dependent DNA synthesis. NIH 3T3 cells were stably transfected with active or dominant negative LMW-PTP. The effects of LMW-PTP were essentially restricted to the G 1 phase of the cell cycle. Upon stimulation with PDGF, cells transfected with the dominant negative LMW-PTP showed an increased activation of Src, whereas the active LMW-PTP induced a reduced activation of this proto-oncogene. We observe that c-Src binding to PDGF receptor upon stimulation is prevented by overexpression of LMW-PTP. These effects were associated with parallel changes in myc expression. Moreover, wild-type and dominant negative LMW-PTP differentially regulated STAT1 and STAT3 activation and tyrosine phosphorylation, whereas they did not modify extracellular signal-regulated kinase activity. However, these modifications were associated with changes in fos expression despite the lack of any effect on extracellular signal-regulated kinase activation. Other independent pathways involved in PDGF-induced mitogenesis, such as phosphatidylinositol 3-kinase and phospholipase C-␥1, were not affected by LMW-PTP. These data indicate that this phosphatase selectively interferes with the Src and the STATs pathways in PDGF downstream signaling. The resulting changes in myc and fos proto-oncogene expression are likely to mediate the modifications observed in the G 1 phase of the cell cycle.Protein-tyrosine phosphorylation plays a key role in the regulation of many cellular processes in eukaryotes such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). Accumulating evidence indicates that the contribution of phosphotyrosine-protein phosphatases (PTPs) 1 to the control of phosphorylation state is as relevant as that of phosphotyrosine-protein kinases. PTPs activity is carefully regulated and appears to be, in most cases, highly specific. The PTPs consist of a family of over 40 enzymes often classified into three groups: 1) receptor-like PTPs; 2) intracellular PTPs; and 3) dual specificity PTPs (2). All PTPs share the signature active site motif CXXXXXR, in which the catalytic cysteine residue is involved in formation of a phosphoenzyme reaction intermediate (3). The low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is a cytosolic enzyme without extensive sequence homology with other PTPs family members, but it contains the CXXXXXR motif and shares the same catalytic mechanism of classical PTPs (4, 5). Furthermore, the LMW-PTP crystal structure (6) revealed a tridimensionally folded phosphate binding loop that is structurally identical to that contained in the human plac...

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. In fact, LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular, LMW-PTP is involved in pathways that regulate the transcription of the immediately early genes myc and fos in response to growth factor stimulation. Recently, we have found that LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its phosphorylation, LMW-PTP increases its catalytic activity about 20-fold. In this study, our interest was to investigate the role of LMW-PTP phosphorylation in cellular response to PDGF stimulation. To address this issue, we have transfected in NIH-3T3 cells a mutant form of LMW-PTP in which the c-Src phosphorylation sites (Tyr 131 and Tyr 132 ) were mutated to alanine. We have established that LMW-PTP phosphorylation by c-Src after PDGF treatment strongly influences both cell adhesion and migration. In addition, we have discovered a new LMW-PTP substrate localized in the cytoskeleton that becomes tyrosine-phosphorylated after PDGF treatment: p190Rho-GAP. Hence, LMW-PTP plays multiple roles in PDGF receptor-mediated mitogenesis, since it can bind and dephosphorylate PDGF receptor, and, at the same time, the cytoskeleton-associated LMW-PTP, through the regulation of the p190Rho-GAP phosphorylation state, controls the cytoskeleton rearrangement in response to PDGF stimulation.Many cellular processes such as cell migration, adhesion, and proliferation require the collaborative interaction between growth factors and extracellular matrix (ECM) 1 stimuli (1-3).Cell adhesion on ECM results in clustering of integrins in focal adhesions that contain both cytoskeletal and signaling proteins. Formation of focal adhesions as well as the closely associated actin stress fibers requires activation of the small GTPbinding protein Rho (4). Rho, a member of the Ras superfamily of GTP-binding proteins, cycles between a GDP-bound inactive form and a GTP-bound active state. Rho is regulated primarily by two groups of proteins: guanine nucleotide exchange factors that catalyze exchange of GDP for GTP and GTPase-activating proteins (GAPs) that stimulate the hydrolysis of GTP to GDP. Upon binding to GTP, Rho interacts with and activates proteins such as Rho kinase and phosphatidylinositol 4-phosphate 5-kinase (5). p190Rho-GAP is a GTPase-activating protein for Rho. During growth factor stimulation p190Rho-GAP becomes tyrosinephosphorylated by c-Src in Tyr 1105 (6, 7) and undergoes transient redistribution into perinuclear concentric arcs that coincide with epidermal growth factor-mediated focal adhesion assembly and reassembly (6). p190Rho-GAP tyrosine phosphorylation correlates with rapid disassembly of...

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