Cell surface expression of v-fms-coded glycoproteins is required for transformation.

Abstract

The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type vfms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolyticaHly processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane.Retroviral oncogenes, acquired by recombination between viruses and protooncogenes found in the DNA of normal cells, encode proteins which morphologically transform cultured cells and induce tumors in animals. Several of the known viral oncogene products appear to affect cell growth by mechanisms which mimic the response of cell surface receptors to regulatory polypeptide hormones (or growth factors). For example, the product of the v-sis oncogene is homologous and possibly identical to the A chain of plateletderived growth factor (8, 37) so that cells acquiring v-sis elaborate an autoendocrine signal. In contrast, the v-erbB gene encodes a glycoprotein closely related to a portion of the epidermal growth factor receptor (9) Cotransfection experiments were performed with a plasmid (pSV2) containing the dominant selectable marker, eco gpt; transfected colonies were -selected in hypoxanthineaminopterin-thymidine medium containing mycophenolic acid (24) before metabolic labeling or cell sorting experiments. To allow the spread of SM-FeSV genomes in transfected cultures, cotransfection was also performed with pSM-FeSV DNA and proviral DNA from a molecularly cloned, amphotropic murine leukemia virus (pA-MuLV). Transfected culture supernatants, shown to produce infectious virus with a test for virion-associated, RNA-dependent DNA polymerase, were filtered and used to infect rat NRK cells. Virus infection, polymerase assays, and titration of focus-forming SM-FeSV were performed as described previously (32) with 10-fold serial dilutions of virus-containing culture supernatants.Metabolic radiolabeling of glycoproteins, preparation of cell lysates, and immunoprecipitatation. Metabolic radiolabeling medium consist...

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation

“…There is evidence that cell surface expression of vFms is required for ®broblast transformation, possibly because v-Fms needs to activate key substrates at the cell membrane (Nichols et al, 1985;Roussel et al, 1984;Hadwiger et al, 1986). The correlation between lack of cell surface expression of the mutant 802 receptors and their inability to transform ®broblasts is particularly strong (compare Figure 1 and Figure 2b).…”

gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

“…gpl20v-fms is specified entirely by the viral oncogene v-fms and remains membrane associated. About 10 to 15% of the molecules are transported to the plasma membrane and concomitantly processed by additional glycosylation of the amino-terminal domain to yield gpl40v-fms (1,8,10,12). It has been reported previously that the extracellular domain of gpl40-fms binds the colony-stimulating factor 1 of macrophages (13).…”

Evidence that the phl gene encodes a 160,000-dalton phosphoprotein with associated kinase activity.

“…K562 cells were labeled in vivo with 32p;, and analyzed for immunoprecipitation with 5' phl-specific antisera 486 and Bi. Cells (108) were labeled with 32p, (12 mCi) for 4 h and disrupted in 10 ml of RIPA buffer (19). Cell extracts were clarified by centrifugation (13), and a 1-ml extract was incubated overnight at 4°C with 5 ,ul of antiserum.…”