Metabolic cell communication within tumour microenvironment: models, methods and perspectives.

Environmental cues are essential in defining tumour malignancy, by promoting tumour initiation, progression and metastatic spreading. Stromal cells may metabolically cooperate or compete with cancer cells, playing a mandatory role in defining cancer metabolic plasticity, potentially dictating the final tumour outcome. Assessing shared nutrients between different tumoural or stromal compartments is essential to understand the impact of environmental nutrients on the metabolic plasticity of tumours. Here, we review analytical and computational approaches for studying the tumour metabolic microenvironment, the destiny of nutrients shared among tumour and stromal populations, as well as the molecular modules of these metabolic relationships.

Honey extracts inhibit PTP1B, upregulate insulin receptor expression, and enhance glucose uptake in human HepG2 cells.

Honey is a food known for its medical properties. In this work, we have studied the impact of different types of honey on insulin signalling pathway. We found that honey extracts inhibit the enzyme PTP1B, one of the main negative regulators of insulin receptor signalling. HPLC-MS analysis allowed us to confirm the presence of several natural PTP1B inhibitors in the honey extracts analysed. Statistical analysis methods show a correlation between specific 1H-NMR resonance frequencies/HPLC peaks and the inhibitory power of the samples. This finding will allow the prediction of the biological properties of honey samples applying relative simple analytical methods. Finally, we demonstrated that the treatment of HepG2 cells with honey extracts enhances the expression of insulin receptor, and stimulates glucose uptake. For the first time, our results demonstrate that bioactive components of honey could improve glycaemic control by both inhibiting PTP1B and stimulating the expression of insulin receptor in liver cells.

Insulin inhibits platelet-derived growth factor-induced cell proliferation.

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

Acylphosphatase overexpression triggers SH-SY5Y differentiation towards neuronal phenotype.

An acylphosphatase (AcPase) overexpression study was carried out on SH-SY5Y neuroblastoma cells, using a green fluorescent fusion protein (AcP-GFP), with GFP acting as a reporter protein. The cellular proliferation rate was significantly reduced by overexpression of AcPase by a factor of ten. In contrast, clones transfected with two inactive AcPase mutants showed a growth rate comparable to control cells. This suggests that AcPase catalyzes the proliferative down-regulation. AcPase-overexpressing clones showed a physiological mortality rate as assessed by an MTT reduction test and by evaluation of necrotic markers. DNA fragmentation analysis and assays of caspase-3 and poly (ADP-ribose) polymerase (PARP)-active fragments showed no evidence of any apoptotic pattern. AcPase overexpression led to a marked increase in PARP activity as well as Bcl-2 content; these are commonly up-regulated during differentiative processes in neuronal cells. In fact, the typical differentiation marker, growth-associated-protein 43, was significantly up-regulated. Microscopic observations also showed a clear increase in the differentiative phenotype in AcPase-overexpressing cells. Our results clearly show that AcPase plays a primary causative role in neuronal differentiation.

Low molecular weight protein-tyrosine phosphatase controls the rate and the strength of NIH-3T3 cells adhesion through its phosphorylation on tyrosine 131 or 132.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement. Our previous results demonstrated that LMW-PTP is able to bind and dephosphorylate activated PDGF receptor, thus inhibiting cell proliferation. Recently we have shown that LMW-PTP is specifically phosphorylated by c-Src in a cytoskeleton-associated fraction in response to PDGF, and this phosphorylation increases LMW-PTP activity about 20-fold. LMW-PTP strongly influences cell adhesion, spreading, and chemotaxis induced by PDGF stimulation, by regulating the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and hence cytoskeleton rearrangement. In the present study we investigate the physiological role of the two LMW-PTP tyrosine phosphorylation sites, using LMW-PTP mutants on tyrosine 131 or 132. We demonstrate that each tyrosine residue is involved in specific LMW-PTP functions. Both of them are phosphorylated during PDGF signaling. Phosphorylation on tyrosine 131 influences mitogenesis, dephosphorylating activated PDGF-R and cytoskeleton rearrangement, acting on p190RhoGAP. Phosphorylation on tyrosine 132 leads to an increase in the strength of cell substrate adhesion, down-regulating matrix metalloproteases expression, through the inhibition of Grb2/MAPK pathway. In conclusion, LMW-PTP tyrosine phosphorylation on both Tyr(131) or Tyr(132) cooperate to determine a faster and stronger adhesion to extracellular matrix, although these two events may diverge in timing and relative amount.

Acylphosphatase is a strong apoptosis inducer in HeLa cell line.

Acylphosphatase (AcP) is a low-molecular-weight protein widely distributed in many vertebrate tissues with a yet unknown physiologic function. To study the in vivo behavior of AcP, HeLa cells were transiently transfected with a vector expressing the AcP/EGFP fusion protein. Analysis of the transfected cells showed a high level of cellular death in cells expressing the AcP/EGFP fusion protein with respect to control cells expressing EGFP alone. Flow cytometry and time lapse analysis of AcP/EGFP transfected cells evidenced a typical pattern of apoptosis. Surprisingly, cells transfected with a mutated form of AcP, with negligible in vitro acylphosphatase activity, undergo apoptosis as well as cells transfected with wild-type protein, suggesting that the physiologic role of AcP could be not related to this catalytic activity.

The low M(r) protein-tyrosine phosphatase is involved in Rho-mediated cytoskeleton rearrangement after integrin and platelet-derived growth factor stimulation.

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.

LMW-PTP exerts a differential regulation on PDGF- and insulin-mediated signaling.

Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is able to specifically bind and dephosphorylate activated PDGF and insulin receptors, modulating the onset of mitogenic process. LMW-PTP is present in two distinct intracellular locations. While the cytosolic LMW-PTP pool interacts directly with activated insulin or PDGF receptors, the cytoskeleton-associated LMW-PTP is tyrosine phosphorylated upon PDGF stimulation and is involved in cytoskeleton rearrangement acting on p190Rho-GAP. We investigated the differential role of LMW-PTP in PDGF- or insulin-induced mitogenesis and cytoskeleton rearrangement. Dominant negative LMW-PTP influences both PDGF- and insulin-induced mitogenesis with a different extent and it induces a decrease in cellular adhesion and chemotaxis after PDGF but not insulin treatment. PDGF but not insulin stimulation leads to tyrosine phosphorylation of LMW-PTP. We propose that the differential effect of LMW-PTP on PDGF and insulin signaling is mainly due to the fact that during insulin signaling LMW-PTP does not become phosphorylated and thus does not act on its cytoskeleton-associated substrate/s.

Inhibitory effect of full-length human endostatin on in vitro angiogenesis.

Endostatin, a C-terminal product of collagen XVIII, is a very powerful angiogenesis inhibitor. In vivo experiments in mice indicate that endostatin dramatically reduces tumor mass without causing the onset of any resistance to the treatment. Recently, a 12-aa shorter human endostatin has been purified from plasma, but is ineffective in in vitro angiogenesis assays. Here we report that the full-length human recombinant endostatin has a potent inhibitory activity in in vitro angiogenesis assays. Two powerful angiogenic factors were used to stimulate endothelial cells: FGF-2 and VEGF-165. Endostatin prevented cell growth both in the basal condition and after stimulation with FGF-2 or VEGF-165. Migration of microvascular endothelial cells toward FGF-2 or VEGF-165 was impaired, both when cells were pretreated with the inhibitor and when endostatin was added together with the growth factors. Furthermore, experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostatin was ineffective. This study indicates that human endostatin is a potent angiogenesis inhibitor and suggests its use in human anticancer therapy.

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 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 pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, 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 Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

Low molecular weight protein-tyrosine phosphatase tyrosine phosphorylation by c-Src during platelet-derived growth factor-induced mitogenesis correlates with its subcellular targeting.

The low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. Our previous results have shown that 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. In this study we have established that, in nontransformed NIH3T3 cells, LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and to phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its tyrosine phosphorylation, LMW-PTP significantly increases its catalytic activity. After PDGF stimulation these two LMW-PTP pools act on distinct substrates, contributing in different manners to the PDGF receptor signaling. The cytoplasmic LMW-PTP fraction exerts its well known action on activated PDGF receptor. On the other hand we have now demonstrated that the cytoskeleton-associated LMW-PTP acts specifically on a few not yet identified proteins that become tyrosine-phosphorylated in response to the PDGF receptor activation. Finally, these two LMW-PTP pools markedly differ in the timing of the processes in which they are involved. The cytoplasmic LMW-PTP pool exerts its action within a few minutes from PDGF receptor activation (short term action), while tyrosine phosphorylation of cytoskeleton-associated LMW-PTP lasts for more than 40 min (long term action). In conclusion LMW-PTP is a striking example of an enzyme that exerts different functions and undergoes different regulation in consequence of its subcellular localization.

Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme, involved in the negative regulation of cell proliferation. In different mammalian species LMW-PTPs are expressed in two molecular forms produced from a single primary transcript through an alternative splicing mechanism. In this paper we report the cloning, expression and characterization of mouse isoforms of LMW-PTPs (called m-IF1 and m-IF2), very similar to the corresponding rat and human isoenzymes. Moreover we have identified a third cDNA encoding a protein (m-IF2P) that presents three substitutions compared to m-IF2. This new isoform is still active on pNPP, although to a lower extent: this reduction is mainly due to the leucine to proline substitution in position 13, within the catalytic loop. The mRNA expression level of this isoform is comparable to those of m-IF1 and m-IF2. It is likely that a gene duplication process followed by mutations has generated this new gene.

The Src and signal transducers and activators of transcription pathways as specific targets for low molecular weight phosphotyrosine-protein phosphatase in platelet-derived growth factor signaling.

The low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is a cytosolic phosphotyrosine-protein 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 G1 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-gamma1, 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 G1 phase of the cell cycle.

Src homology-2 domains protect phosphotyrosyl residues against enzymatic dephosphorylation.

The SH2 domain of c-Fgr (class 1A) has been expressed in E. coli as GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase). Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1 (388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used. The IC50 values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain. Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains. Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.

Common-type acylphosphatase: steady-state kinetics and leaving-group dependence.

A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 degrees C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme-substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O-inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. all these findings, as well as the strong dependence of kcat upon the leaving group pK1, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.

c-Src activates both STAT1 and STAT3 in PDGF-stimulated NIH3T3 cells.

Treatment of cells with PDGF and EGF specifically induces STAT1 and STAT3, which became phosphorylated on tyrosine residues to form homo and heterodimers: in these configurations they translocate into the nucleus where they act as transcription activators. However little is known about the activation of STATs in growth factor receptor signal transduction. Recently it has been shown that v-Src modulates the tyrosine phosphorylation of STAT3 but not of STAT1. Here we report that the cellular Src tyrosine kinase is involved in the activation of both STAT1 and STAT3 in PDGF stimulated NIH3T3 cells. Both tyrosine phosphorylation and DNA binding activity of STAT1 and STAT3 are up-regulated in c-Src overexpressing cells, while we observe the opposite phenomenon in cells overexpressing the dominant negative Src. Furthermore, our results show that STAT1 co-immunoprecipitates with c-Src, suggesting that the activation of STATs by Src occurs via a direct interaction. Taken together, these data suggest that c-Src is involved in activation of both STAT1 and 3 in PDGF signal transduction.

LMW-PTP is a negative regulator of insulin-mediated mitotic and metabolic signalling.

To understand the physiological role of low Mr weight phosphotyrosine protein phosphatase (LMW-PTP) in insulin mediated signaling, we established clonal cell lines overexpressing the dominant negative (C12S mutant) LMW-PTP (dnLMW-PTP) from NIH3T3 murine fibroblasts expressing insulin receptor. Upon insulin stimulation we observe an association between the dnLMW-PTP and the beta-subunit of the insulin receptor. This association is dependent on the tyrosine phosphorylation of the insulin receptor since it is not observed in unstimulated cells. Furthermore, in vitro binding experiments between dnLMW-PTP and the insulin receptor reveal that the interaction is mediated by the LMW-PTP catalytic site, as indicated by competition with orthovanadate. DnLMW-PTP overexpression influences both the mitogenic and the metabolic bioeffects of insulin. In particular, in cells overexpressing dnLMW-PTP we observe an increase in the glycogenosynthesis rate and in mitosis as indicated by glucose incorporation into glycogen and thymidine incorporation into DNA, respectively. Moreover, we studied the insulin mediated signal transduction pathways starting from insulin receptor, such as the Src kinase, the p21Ras/ERK, and the PI3K routes. Our findings are consistent with a specific regulation of mitogenesis by LMW-PTP through a pathway involving c-Src kinase but independent by both PI3K and ERK. These data strongly suggest that LMW-PTP acts as a negative regulator of both mitogenetic and metabolic insulin signalling.

Mechanism of acylphosphatase inactivation by Woodward's reagent K.

The organ common-type (CT) isoenzyme of acylphosphatase is inactivated by Woodward's reagent K (WRK) (N-ethyl-5-phenylisoxazolium-3'-sulphonate) at pH6.0. The inactivation reaction follows apparent pseudo first-order kinetics. The dependence of the reciprocal of the pseudo first-order kinetic constant (kobs) on the reciprocal WRK concentration reveals saturation kinetics, suggesting that the WRK forms a reversible complex with the enzyme before causing inactivation. Competitive inhibitors, such as inorganic phosphate and ATP, protect the enzyme from WRK inactivation, suggesting that this reagent acts at or near to the enzyme active site. The reagent-enzyme adduct, which elicits a strong absorption band with lambdamax at 346 nm, was separated from unreacted enzyme by reverse phase HPLC and the modified protein was cleaved with endoproteinase Glu-C to produce fragments. The HPLC fractionation gave two reagent-labelled peptides (peak 1 and peak 2) that were analysed by ion-spray MS and sequenced. The former is VFFRKHTQAE (residues 20-29 of human CT acylphosphatase) and the latter IFGKVQGVFFRKHTQAE (residues 13-29). MS demonstrated that both peptides are WRK adducts. A fragment ion with m/z of 1171, which is present in the mass spectrum of peak 1, has been identified as a WRK adduct of the peptide fragment 20-26. The lambdamax at 346 nm of WRK adduct suggests that the modified residue is His-25. Five recombinant enzymes mutated in residues included in the 20-29 polypeptide stretch have been produced. Analysis of their reactivities with WRK demonstrates that His-25 is the molecular target of the reagent as its modification causes the inactivation of the enzyme. Since both His-25-->Gln and His-25-->Phe mutants maintain high catalytic activity, we suggest that the observed enzyme inactivation is caused by the reagent (covalently bound to His-25), which shields the active site.

Identity of zinc ion-dependent acid phosphatase from bovine brain and myo-inositol 1-phosphatase.

A 62 kDa Zn(2+)-dependent acid phosphatase has been purified from bovine brain. The protein was carboxymethylated and then cleaved by endoproteinase Glu-C, trypsin and CNBr. Several fragments were subjected to structural analysis either by using mass spectrometry or automated peptide sequencing. The four sequenced peptides were compared with the known protein sequences contained in the EMBL Data Bank. All four peptide sequences were identical to the corresponding amino-acid sequences present in myo-inositol 1-phosphatase from bovine brain. Furthermore we found that the amino-acid composition of Zn(2+)-dependent acid phosphatase purified in our laboratory is very similar to that of myo-inositol 1-phosphatase, and that several peptide fragments have molecular weights (measured by mass spectrometry techniques) identical to those expected for cleavage-fragments originated from the authentic myo-inositol 1-phosphatase. This is one of the key enzymes in the receptor-stimulated inositol phospholipid metabolism and it has been considered as the probable target of Li+ ion during LiCl therapy in manic-depressive patients. The comparison of the Zn(2+)-dependent acid phosphatase and the Mg(2+)-dependent myo-inositol-1-phosphatase activities, measured at different purification steps, shows that the ratio between the two activities was remarkably constant during enzyme purification. We also demonstrated that in the presence of Mg2+ this enzyme efficiently catalyses the hydrolysis of myo-inositol 1-phosphate, and that the Li+ ion inhibits this activity. Furthermore, the thermal treatment of the enzyme causes a time-dependent parallel decrease of both Zn-dependent p-nitrophenyl phosphatase (assayed at pH 5.5) and Mg(2+)-dependent myo-inositol-1-phosphatase (assayed at pH 8.0) activities, suggesting the hypothesis that the same protein possesses both these activities.

Low M(r) phosphotyrosine 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 had demonstrated that low M(r) phosphotyrosine 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, both in vivo and in 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.