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 Vm. 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.Tyrosine kinases are crucially involved in the transduction of growth-promoting stimuli to the cell interior. Transmembrane growth factor receptors such as the epidermal and platelet-derived growth factor, insulin, and colony-stimulating factor 1 receptors, are endowed with ligand-induced tyrosine kinase activity. Upon ligand binding, they display a short-lived pulse of activity leading to their autophosphorylation and to the phosphorylation of cellular substrates on tyrosine residues (for a review, see references 52 and 56). Membrane-associated tyrosine kinases of the src family are thought to be similarly involved in signal transduction operated by a distinct set of receptors. The most abundant class of oncogenes comprises the genes coding for both types of tyrosine kinases. Whatever the mechanism, activation of their transforming potential leads to the expression of a protein with nonregulated, often enhanced enzymatic activity (reviewed in references 28, 32, and 33).The enzymatic activity of tyrosine kinases can be modulated in several ways (reviewed in references 33, 52, and 56). Ligands are well-known activators of the receptors. Structural alterations such as N-and C-terminal truncation and point mutations are critical for the transforming proteins. In addition, phosphorylation is a ubiquitous way of transiently modulating tyrosine kinase activity. Tyrosine autophosphorylation has often been associated with activation. Protein kinase C-mediated serine or threonine phosphorylation has been shown to be inhibitory for some gr...
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. pl90MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p5Oa) and 145 kDa (p14513). 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 (pl40MET) is membrane bound and is composed of an a chain (p5Oa) and an 85-kDa C-terminal truncated P chain (p8503). The second protein (p1309ET) is released in the culture supernatant and consists of an a chain (p5Oa) and a 75-kDa C-terminal truncated , chain (p750'). Both truncated forms lack the tyrosine kinase domain. pl40fET and pl30MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. pl40MET is preferentially localized at the cell surface, where it is present in roughly half the amount of pl90MET. 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 J2 recombinant retrovirus expressing v-myc/v-raf (also known as MYC/RAF1) immortalized macrophages from the bone marrow of lipopolysaccharide-responsive mouse strains, producing the ANA-1 cell line from C57BL/6 mice and the INF-3A cell line from C3H/HeN mice. In contrast, J2 recombinant retrovirus infection of the fetal liver from C57BL/6-Ly-5a mice immortalized a cell line (GGD) that did not exhibit the characteristics of mature macrophages. The GGD cell line was classified as leukocytic on the basis of its expression of the Ly-6B.2, Fc gamma R, and Ly-5.2 antigens. Our results indicate that the J2 recombinant retrovirus selectively immortalizes macrophages from the bone marrow of C57BL/6 and C3H/HeN mice but immortalizes cells without definitive macrophage characteristics from murine fetal liver under the same culture conditions.
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