Biomedical subjects

G Camici

Publications and source records attributed to G Camici.

At least 19 recordsLinked to original sources

PubMed2004

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.

Animals↗

PubMed2004

Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and Saccharomyces cerevisiae growth.

Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.

Casein Kinase II↗

PubMed2002

Some protein tyrosine phosphatases target in part to lipid rafts and interact with caveolin-1.

A profile-based search of the SWISS-PROT database reveals that most protein tyrosine phosphatases (PTPs) contain at least one caveolin-1-binding motif. To ascertain if the presence of caveolin-binding motif(s) in PTPs corresponds to their actual localization in caveolin-1-enriched membrane fractions, we performed subcellular fractionating experiments. We found that all tested PTPs (PTP1B, PTP1C, SHPTP2, PTEN, and LAR) are actually localized in caveolin-enriched membrane fractions, despite their distribution in other subcellular sites, too. More than 1/2 of LAR and about 1/4 of SHPTP2 and PTP-1C are localized in caveolin-enriched membrane fractions whereas, in these fractions, PTP-1B and PTEN are poorly concentrated. Co-immunoprecipitation experiments with antibodies specific for each tested PTP demonstrated that all five phosphatases form molecular complexes with caveolin-1 in vivo. Collectively, our findings propose that particular PTPs could perform some of their cellular actions or are regulated by recruitment into caveolin-enriched membrane fractions.

Amino Acid Motifs↗

PubMed2001

Hydrogen peroxide triggers the formation of a disulfide dimer of muscle acylphosphatase and modifies some functional properties of the enzyme.

Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H(2)O(2) at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the reduced enzyme does not bind polynucleotides. Experiments performed with H(2)O(2) in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 microm, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 --> Ser mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.

Acid Anhydride Hydrolases↗

PubMed2001

Tyrosine-phosphorylated caveolin is a physiological substrate of the low M(r) protein-tyrosine phosphatase.

Low M(r) phosphotyrosine-protein phosphatase is involved in the regulation of several tyrosine kinase growth factor receptors. The best characterized action of this enzyme is on the signaling pathways activated by platelet-derived growth factor, where it plays multiple roles. In this study we identify tyrosine-phosphorylated caveolin as a new potential substrate for low M(r) phosphotyrosine-protein phosphatase. Caveolin is tyrosine-phosphorylated in vivo by Src kinases, recruits into caveolae, and hence regulates the activities of several proteins involved in cellular signaling cascades. Our results demonstrate that caveolin and low M(r) phosphotyrosine-protein phosphatase coimmunoprecipitate from cell lysates, and that a fraction of the enzyme localizes in caveolae. Furthermore, in a cell line sensitive to insulin, the overexpression of the C12S dominant negative mutant of low M(r) phosphotyrosine-protein phosphatase (a form lacking activity but able to bind substrates) causes the enhancement of tyrosine-phosphorylated caveolin. Insulin stimulation of these cells induces a strong increase of caveolin phosphorylation. The localization of low M(r) phosphotyrosine-protein phosphatase in caveolae, the in vivo interaction between this enzyme and caveolin, and the capacity of this enzyme to rapidly dephosphorylate phosphocaveolin, all indicate that tyrosine-phosphorylated caveolin is a relevant substrate for this phosphatase.

3T3 Cells↗

PubMed2001

Skeletal muscle pathology in autosomal dominant Emery-Dreifuss muscular dystrophy with lamin A/C mutations.

We present our observations on the skeletal muscle pathology of nine cases from seven families of autosomal dominant Emery-Dreifuss muscular dystrophy (ADEDMD) with identified mutations in the lamin A/C gene, aged 2-35 years at the time of biopsy. The severity of pathological change was moderate and the most common features were variation in fibre size (hypertrophy and atrophy), an increase in internal nuclei and smaller diameter fibres with high oxidative enzyme activity. Only one case showed necrosis, which was present in two separate samples taken from the quadriceps and tibialis anterior, at different ages. Immunocytochemistry detected an age-related reduction of laminin beta1 on the muscle fibres in adolescent and adult cases. Antibodies to lamins A and A/C, and emerin did not reveal any detectable differences from controls. Electron microscopy of two out of three cases showed an abnormal distribution of heterochromatin in many fibre nuclei. Our results show that dystrophic changes in skeletal muscle are not a major feature of ADEDMD, and that nuclear abnormalities may be detected with electron microscopy. Immunodetection of reduced laminin beta1 may be a useful secondary marker in adults with this disorder, as immunocytochemistry of lamins is not yet of diagnostic use.

Adolescent↗

PubMed2000

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.

3T3 Cells↗

PubMed2000

Acylphosphatase possesses nucleoside triphosphatase and nucleoside diphosphatase activities.

We have demonstrated that acylphosphatase possesses ATP-diphosphohydrolase (apyrase-like) activity. In fact, acylphosphatase first catalyses the hydrolysis of the gamma-phosphate group of nucleoside triphosphates, and then attacks the beta-phosphate group of the initially produced nucleoside diphosphates, generating nucleoside monophosphates. In contrast, it binds nucleoside monophosphates but does not catalyse their hydrolyses. The calculated k(cat) values for the nucleoside triphosphatase activity of acylphosphatase are of the same order of magnitude as those displayed by certain G-proteins. An acidic environment enhances the apyrase-like activity of acylphosphatase. The true nucleotide substrates of acylphosphatase are free nucleoside di- and triphosphates, as indicated by the Mg(2+) ion inhibition of the activity. We have also demonstrated that, although nucleoside triphosphates are still hydrolysed at pH 7.2 and 37 degrees C, in the presence of millimolar Mg(2+) concentrations this occurs at a lower rate. Taken together with the previously observed strong increase of acylphosphatase levels during induced cell differentiation, our findings suggest that acylphosphatase plays an active role in the differentiation process (as well as in other processes, such as apoptosis) by modulating the ratio between the cellular levels of nucleoside diphosphates and nucleoside triphosphates.

Acid Anhydride Hydrolases↗

PubMed2000

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.

Animals↗

PubMed2000

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.

3T3 Cells↗

PubMed2000

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.

3T3 Cells↗

PubMed1999

Purification, characterization, and amino acid sequence of cerato-platanin, a new phytotoxic protein from Ceratocystis fimbriata f. sp. platani.

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(TM)) 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.

Amino Acid Sequence↗

PubMed1999

The contribution of acidic residues to the conformational stability of common-type acylphosphatase.

Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, DeltaG(H2O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.

Acid Anhydride Hydrolases↗

PubMed1999

Thiolation of low-Mr phosphotyrosine protein phosphatase by thiol-disulfides.

Thiol-disulfides cause a time- and a concentration-dependent inactivation of the low-M(r) phosphotyrosine protein phosphatase (PTP). We demonstrated that six of eight enzyme cysteines have similar reactivity against 5,5'-dithiobis(nitrobenzoic acid): Their thiolation is accompanied by enzyme inactivation. The inactivation of the enzyme by glutathione disulfide also is accompanied by the thiolation of six cysteine residues. Inorganic phosphate, a competitive enzyme inhibitor, protects the enzyme from inactivation, indicating that the inactivation results from thiolation of the essential active-site cysteine of the enzyme. The inactivation is reversed by dithiothreitol. Although all PTPs have three-dimensional active-site structures very similar to each other and also have identical reaction mechanisms, the thiol group contained in the active site of low-M(r) PTP seems to have lower reactivity than that of other PTPs in the protein thiolation reaction.

Binding Sites↗

PubMed1998

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.

3T3 Cells↗

PubMed1998

The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2.

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 H2O2. The enzymes are protected from inactivation by Pi, a competitive inhibitor, suggesting that the H2O2 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 H2O2 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 H2O2. Because a physiological concentration of H2O2 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.

Chromatography, High Pressure Liquid↗

PubMed1998

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.

3T3 Cells↗

PubMed1998

The amino acid sequences of two acylphosphatase isoforms from fish muscle (Lamna nasus).

Two acylphosphatase isoenzymes have been purified from Lamna nasus muscle, and their complete amino acid sequences have been determined. The former (E1) consists of 99 amino acid residues, while the latter (E2) consists of 102 residues. Both are acetylated at their N termini. E1 has the FFRK active site motif characteristic of all common-type acylphosphatase isoenzymes, whereas E2 contains the CFRM active site motif characteristic of all muscle-type acylphosphatase isoenzymes. They have quite similar kinetic properties. The comparison of sequences of fish E1 and E2 isoenzymes with other known mammalian and bird acylphosphatases reveals that the E2 isoenzyme has an N terminus tail, four residues long, similar to those previously found in all known bird species muscle-type isoenzymes. Among organ-common-type acylphosphatases about 50% of residues are completely conserved, whereas about 60% of muscle-type acylphosphatase residues are completely conserved, indicating that the latter type of isoenzyme has a slower evolutionary rate than the former. The sequences of E1 and E2 acylphosphatases from L. nasus represent the first primary structures of this kind of enzyme determined among fish species.

Acid Anhydride Hydrolases↗