Lucia Gandino

C-terminal truncated forms of Met, the hepatocyte growth factor receptor.

The MET proto-oncogene encodes a transmembrane tyrosine kinase of 190 kDa (p190MET), which has recently been identified as the receptor for hepatocyte growth factor/scatter factor. p190MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p50 alpha) and 145 kDa (p145 beta). We have produced four different monoclonal antibodies that are specific for the extracellular domain of the Met receptor. These antibodies immunoprecipitate with p190MET two additional Met proteins of 140 and 130 kDa. The first protein (p140MET) is membrane bound and is composed of an alpha chain (p50 alpha) and an 85-kDa C-terminal truncated beta chain (p85 beta). The second protein (p130MET) is released in the culture supernatant and consists of an alpha chain (p50 alpha) and a 75-kDa C-terminal truncated beta chain (p75 beta). Both truncated forms lack the tyrosine kinase domain. p140MET and p130MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. p140MET is preferentially localized at the cell surface, where it is present in roughly half the amount of p190MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.

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.

Generation of a murine monoclonal antibody that detects the fos oncogene product

A hybridoma producing a monoclonal antibody (MoAB) recognizing both the cellular and viral forms of fos has been generated by somatic cell hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 128-152, a consensus region, of both the v-fos and c-fos oncogene products. Three proteins with molecular weights of 55,000, 44,000, and 42,000 were detected by immunoblotting. While MoAB 2G9C3 failed to immunoprecipitate fos from Finkel-Biskis-Jenkins murine osteosarcoma-virus-infected fibroblasts, both the 55,000 v-fos protein and the 39,000 cellular protein were coprecipitated using polyvalent rabbit antibodies to the same peptide. Whereas no cell surface membrane expression of fos was detected, after membrane permeabilization by a brief exposure to lysolecithin it was possible to specifically detect internal fos by immunofluorescence flow cytometry. Immunohistochemical staining of FBJ virus-infected cells revealed intense, nuclear staining.

Immortalization of macrophages from mouse bone marrow and fetal liver

Fresh bone marrow (BM)-derived cells infected with the J2 recombinant retrovirus (carrying v-myc and v-raf/mil oncogenes) grow as immortal cell lines belonging to the monocytic lineage. BM cells cultured for 24 h in conventional medium are no longer able to grow following infection with the J2 virus. We investigated whether specific growth factors affected the proliferative response of BM cells to the J2 virus. If the BM cells were cultured for 24 h in the presence of concanavalin A or CSF-1 and then infected with the J2 virus, immortalization of BM cells was observed. Under these conditions, the cell lines that we obtained were shown to belong to the monocytic lineage. We investigated whether target cells for the J2 virus existed in other hematopoietic organs. We observed J2-induced proliferation in fetal liver (FL) but not in spleen or thymus. The cells proliferating in the FL had macrophage characteristics during the early passages. However, some macrophage markers were lost upon extensive in vitro culture. We conclude that we have identified conditions in which J2 virus consistently and selectively stimulates the growth of macrophages from murine bone marrow and a wider range of hematopoietic cells from fetal liver.

Monoclonal Antibodies against Mouse γ-Interferon Inhibit Tumoricidal Macrophage Activation by T Lymphocytes

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.

The Receptor for the Hepatocyte Growth Factor-Scatter Factor: Ligand-Dependent and Phosphorylation-Dependent Regulation of Kinase Activity

Hepatocyte Growth Factor (HGF) and Scatter Factor (SF) are identical molecules produced by the stromal fibroblats and non-parenchimal cells of many organs and also present in serum. HGF exerts an array of activities on epitelial cells, i.e. mitogenesis, dissociation of epithelial sheets, stimulation of cell motility, and promotion of matrix invasion. The receptor for HGF is the tyrosine kinase encoded by the MET proto-oncogene. It is widely expressed in normal epithelial tissues as a 190 kDa heterodimer of two disulfide-linked protein subunits. HGF binding triggers tyrosine autophosphorylation of the receptor β subunit in intact cultured cells. Autophosphorylation upregulates the kinase activity of the receptor, increasing the Vmax of the phosphotransfer reaction. The major phosphorylation site has been mapped to Tyr 1235. Negative regulation of the receptor kinase activity occurs through distinguishable pathways involving protein kinase C activation or increase in the intracellular Ca2+ concentration. Both lead to the phosphorylation of serine residue(s) in a unique tryptic phosphopeptide of the receptor and to a decrease in its tyrosine phosphorylation and kinase activity. Receptor autophosphorylation also triggers the signal transduction pathways inside the target cells. The phosphorylated receptor associates phosphatidylinositol 3-kinase, indicating that the generation of the D-3 phosphorylated inositol lipids is involved in effecting the motility and/or growth response to HGF.