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R Ferracini

Publications and source records attributed to R Ferracini.

At least 37 records · Page 2Linked to original sources
PubMed1991

Identification of the major autophosphorylation site of the Met/hepatocyte growth factor receptor tyrosine kinase.

The MET proto-oncogene encodes a transmembrane tyrosine kinase receptor for HGF (p190MET). In this work, p190MET was immunoprecipitated, allowed to phosphorylate in the presence of [gamma-32P]ATP, and digested with trypsin. A major phosphopeptide was purified by reverse phase chromatography. The phosphorylated tyrosine was identified as residue 1235 (Tyr1235) by Edman covalent radiosequencing. A synthetic peptide derived from the corresponding MET sequence was phosphorylated by p190MET in an in vitro assay and coeluted in reverse phase chromatography. Tyr1235 lies within the tyrosine kinase domain of p190MET, within a canonical tyrosine autophosphorylation site that shares homology with the corresponding region of the insulin, CSF-1 and platelet-derived growth factor receptors, and of p60src and p130gag-fps. The p190MET kinase is constitutively phosphorylated on tryosine in a gastric carcinoma cell line (GTL16), due to the amplification and overexpression of the MET gene. Metabolic labeling of GTL-16 cells with [32P]orthophosphate followed by immunoprecipitation and tryptic phosphopeptide mapping of p190MET showed that Tyr1235 is a major site of tyrosine phosphorylation in vivo as well. Since phosphorylation activates p190MET kinase, we propose a regulatory role for Tyr1235.

Amino Acid Sequence↗
PubMed1991

Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene.

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.

Blotting, Western↗
PubMed1991

The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation.

Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.

Adenosine Triphosphate↗
PubMed1990

Cerebral ganglioglioma with anaplastic oligodendroglial component.

We report an unusual and possibly unique example of cerebral ganglioglioma with an anaplastic oligodendroglial component. The latter was documented on both morphological and immunohistochemical grounds. Immunohistochemically, the anaplastic cells were strongly positive with the monoclonal antibody anti-Leu-7, while they lacked glial fibrillary acid protein, vimentin and neurofilaments.

Antibodies, Monoclonal↗
PubMed1990

Analysis of mutant forms of the c-src gene product containing a phenylalanine substitution for tyrosine 416.

Several lines of evidence suggest that phosphorylation of tyrosine residue 416 (Tyr416) has a positive regulatory influence on the functional activity of the protein tyrosine kinase pp60c-src. To further examine the functional importance of phosphorylation at Tyr416, we have eliminated this phosphoacceptor site in four functionally unique mutant forms of the c-src gene product that are phosphorylated on Tyr416 in vivo. Substitution of phenylalanine for Tyr416 suppressed the biological activity of all of the mutant proteins as assayed by colony formation in soft agar and the induction of morphological alterations. However, the extent of this effect, and the degree to which the substitution affected the phosphorylation of substrates in vivo and in vitro, varied considerably in each of the mutants. These results support the notion that phosphorylation of Tyr416 has a positive regulatory effect on the biological activity of c-src; however, this effect does not directly correlate with a general effect on the total level of tyrosine kinase activity in vitro or the level of tyrosine phosphorylation of cellular proteins in vivo.

Animals↗
PubMed1988

p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line.

A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.

Amino Acids↗
PubMed1988

Transbronchial lung biopsy and bronchoalveolar lavage in diagnosis of diffuse infiltrative lung diseases.

We investigated 801 patients with lung infiltrates who underwent transbronchial lung biopsy (TBB) and performed cytologic studies of lavage fluid of 491 patients who also underwent bronchoalveolar lavage (BAL). A specific histopathological diagnosis was made in 232 cases (29%). Histopathological changes consistent with the clinical pattern were present in 296 cases (37%). The diagnostic yield of TBB was 66%. Routine evaluation of BAL fluid allowed us to make a specific diagnosis in 49 cases. The role of the pulmonary pathologist in the interpretation of TBB and BAL specimens is discussed.

Biopsy↗
PubMed1988

Histiocytosis-X: a rare cause of radiculopathy.

A rare case of histiocytosis-X of the spine is presented. The clinical and histological features of histiocytosis-X are reviewed. Complete neurological recovery was exhibited by the patient after surgical treatment and radiotherapy. The literature dealing with the eight cases previously reported is reviewed.

Adult↗
PubMed1988

Transbronchial lung biopsy in infiltrative lung disease. The importance of the pathologic approach.

Infiltrative diffuse lung diseases represent a heterogenous group of lesions with overlapping clinical and roentgenological features. Their diagnosis frequently needs a pathologic approach. We report our experience (671 patients with lung infiltrates) with transbronchial lung biopsy (TBB) procedure. The results of morphologic investigation were subdivided into three groups: 1) A specific morphologic diagnosis was made in 192 cases (29%) 2) Histopathologic changes well consistent with the clinical pattern were present in 258 patients (38%) 3) Non-specific lesions were found in 221 cases (33%). The diagnosis yield of the method was of 67%. UIP, DIP, Pulmonary Angiitis and Granulomatosis, Bronchiolitis Obliterans-Organizing Pneumonia and other rarer lung diseases may not be diagnosed on the basis of lesions present in TBB specimens.

Biopsy↗
PubMed1986

Immunological detection of proteins phosphorylated at tyrosine in cells stimulated by growth factors or transformed by retroviral-oncogene-coded tyrosine kinases.

The receptors for polypeptide growth factors and proteins coded by oncogenes of the src family are endowed with protein kinase activity and share the uncommon property of autophosphorylating at tyrosine residues. It is unclear whether the tyrosine kinase activity is also directed towards other targets of physiological significance. In this work, phosphotyrosine antibodies were used to detect, by Western blots and immunoprecipitation, proteins phosphorylated at tyrosine in fibroblasts either stimulated by growth factors (PDGF and EGF) or transformed by oncogene-coded tyrosine kinases. In stimulated cells the antibodies detected the autophosphorylated receptors, but only trace amounts of other proteins phosphorylated at tyrosine. In fibroblasts transformed by retroviral oncogenes (v-src, v-abl, v-fps or v-fes) proteins other than the corresponding oncogene-coded kinase, were found. A p70 was found to be heavily phosphorylated in fibroblasts transformed by v-src, v-fes and v-fps. A p130 and a p36 were found in cells transformed by v-src and v-abl. A unique p70 was phosphorylated in v-abl-transformed fibroblasts. These proteins were also phosphorylated in vitro in an immunocomplex kinase reaction. This reaction was blocked by the specific kinase inhibitors. These data strongly suggest that tyrosine kinases phosphorylate protein targets other than themselves. These targets are barely detectable in normal cells stimulated by growth factors, where the kinase activity is triggered rapidly and transiently. By contrast, a number of intracellular proteins phosphorylated at tyrosine accumulate in cells transformed by v-onc-coded kinases, endowed with constitutive and non-regulated enzymatic activity.

Animals↗
PubMed1986

Protein phosphorylation at tyrosine residues in v-abl transformed mouse lymphocytes and fibroblasts.

Phosphotyrosine antibodies were employed to immunodecorate and immunoprecipitate proteins phosphorylated at tyrosine residues in cells transformed by Abelson murine leukemia virus (A-MuLV). In pre-B and pre-T lymphoma cells transformed by A-MuLV, the major phosphotyrosine-containing protein has an MW of 160 kDa and shares immunologically detectable sequences with the v-abl oncogene product. Moreover, two different proteins of approximately 100 and 68 kDa, heavily phosphorylated at tyrosine, were identified. Lack of immunological cross-reactivity with viral products and phosphopeptide mapping showed that the 100 and 68 kDa proteins are coded by cellular genes. Phosphoproteins were undetectable in control resting lymphocytes. The 68 and the 100 kDa proteins were phosphorylated to different extents in proliferating lymphocytes, either stimulated by the growth factor IL-2, or transformed by M-MuLV (lacking the oncogene coded kinase). In fibroblasts transformed by A-MuLV, phosphotyrosine antibodies identified 2 proteins of 120 and 70 kDa. By immunological cross-reaction and by phosphopeptide mapping, the first was identified as a 120 kDa form of the v-abl coded kinase. The 70 kDa protein is coded by a cellular gene, is not structurally related to the 120 kDa v-abl kinase, and is different from any phosphotyrosine-containing protein detected in A-MuLV-transformed lymphocytes. These data show that, upon v-abl-induced transformation, phosphorylation at tyrosine takes place also on proteins other than the 160 or 120-kDa oncogene products. In lymphocytes and fibroblasts these proteins are different, suggesting that the cascade of events triggered by the v-abl gene in different cell types involves tyrosine phosphorylation of different specific proteins.

Abelson murine leukemia virus↗
PubMed1986

Transbronchial lung biopsy in pulmonary sarcoidosis. Is it an evaluable method in detection of disease activity?

To assess the value of transbronchial lung biopsy in the evaluation of disease activity in pulmonary sarcoidosis, lung biopsy specimens obtained from 37 patients with this disease and their cellular patterns of bronchoalveolar lavage were studied. Morphologic analysis has showed peculiar lesions: predominant interstitial alveolitis consisting of mononuclear cells and scattered neutrophils, and eosinophils, diffuse in 13 cases and focal in 11 cases, interstitial nodular clusterings of mononuclear cells (five cases), diffuse intraalveolar infiltration of macrophages (one case), granulomas (27 cases), hyaline membranes (one case), intraalveolar (two cases) and interstitial (six cases) fibrosis, and finally cuboidal metaplasia of alveolar lining cells (eight cases). Hyaline membranes were always combined to a diffuse alveolitis consisting of some neutrophils. Only diffuse alveolitis was significantly associated with a high lymphocytosis in BAL (p less than 0.05).

Adult↗
PubMed1985

Primary intracranial lymphomas.

We reviewed the cases of 16 patients with primary intracranial lymphomas. The clinical and radiological data proved to be helpful in suggesting the diagnosis, but only tumoral biopsy provided unequivocal diagnosis. In our cases radiotherapy proved to be the most effective treatment.

Adult↗
PubMed1984

A cerebral ependymoma with extracranial metastases.

A case is presented of a 30-year-old woman with a malignant ependymoma with extracranial metastases involving the cervical lymph nodes. The positive reaction to antibody against glial fibrillary acidic protein confirmed the ependymoma origin of the lymph node metastases.

Adult↗
PubMed1984

Squamous cell carcinoma of the brain with sarcoma-like stroma.

A cerebral neoplasm containing a well differentiated epidermoid cyst surrounded by islands of infiltrating squamous cell carcinoma and a pleomorphic spindle cell stroma was described. By immunoperoxidase staining all these components contained keratin. The lesion was therefore similar to malignant squamous cell lesions with sarcoma-like stroma occurring in other body sites.

Brain Neoplasms↗