Subcellular localization of glycoproteins encoded by the viral oncogene v-fms

Abstract

The McDonough strain of feline sarcoma virus encodes a polyprotein that is cotranslationally glycosylated and proteolytically cleaved to yield transforming glycoproteins specified by the viral oncogene v-fms. The major form of the glycoprotein (gpl20fms) contains endoglycosidase H-sensitive, N-linked oligosaccharide chains lacking fucose and sialic acid, characteristic of glycoproteins in the endoplasmic reticulum. Kinetic and steadystate measurements showed that most gpI20fms molecules were not converted to mature forms containing complex carbohydrate moieties. Fixed-cell immunofluorescence confirmed that the majority of v-fms-coded antigens were internally sequestered in transformed cells. Dual-antibody fluorescence performed with antibodies to intermediate filaments (IFs) showed that the IFs of transformed cells were rearranged, and their distribution coincided with that of v-fms-coded antigens. No specific disruption of actin cables was observed. The v-fms gene products cofractionated with IFs isolated from virus-transformed cells and reassociated with IFs self-assembled in vitro. A minor population of v-fms-coded molecules (gpl40fms) acquired endoglycosidase H-resistant, N-linked oligosaccharide chains containing fucose and sialic acid residues, characteristic of molecules processed in the Golgi complex. Some gpl4f`ms molecules were detected at the plasma membrane and were radiolabeled by lactoperoxidase-catalyzed iodination of live transformed cells. We suggest that v-fmscoded molecules are translated as integral transmembrane glycoproteins, most of which are inhibited in transport through the Golgi complex to the plasma membrane.

The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

“…4A, the p55gag fragment was detected after a short labeling period and rapidly turned over. Very similar results were previously obtained for other cell lines transformed by SM-FeSV (2,24,26). In lysates from cells transfected with pZIPneo-fms, gpl20-fms was rapidly labeled and turned over with similar kinetics (Fig.…”

“…Under the above labeling conditions, all three forms of the v-fms-coded glycoprotein were observed, but p55gag, which has a short half-life (2), was not readily detected. Shorter labeling intervals were necessary to demonstrate the presence of the gag-coded fragment (1,2,4,26). Thus, p55gag was observed in some experiments (see Fig.…”

“…Cells were rinsed in ice-cold phosphatebuffered saline and lysed in RIPA buffer (50 mM Tris hydrochloride [pH 7.4], 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.5% sodium dodecyl sulfate [SDS]) containing 2% aprotinin and 1% phenylmethylsulfonyl fluoride as protease inhibitors. Immune precipitation with antiserum to the v-fms gene products or to FeLV gag-coded determinants (p30) was performed as described previously (2), and the washed precipitates were denatured and analyzed by electrophoresis on polyacrylamide gels containing SDS (SDS-PAGE) (2). Under the above labeling conditions, all three forms of the v-fms-coded glycoprotein were observed, but p55gag, which has a short half-life (2), was not readily detected.…”

Novel INK4 Proteins, p19 and p18, Are Specific Inhibitors of the Cyclin D-Dependent Kinases CDK4 and CDK6

“…Following transcription and translation, rabbit reticulocyte lysates containing [ 35 S]methionine-labeled CDKs (20 to 40 l) were diluted to 0.5 ml in IP kinase buffer (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol [DTT], and 0.1% Tween 20) containing 10 mg of BSA per ml and mixed with 1 g of purified GST or GST-p19 immobilized on glutathione-Sepharose beads. After 2 h of incubation at 4ЊC, the beads were collected by centrifugation and washed four times in IP kinase buffer, and the bound proteins were denatured and analyzed by electrophoresis on 12.5% polyacrylamide gels containing SDS (1).…”

Immunogold labeling of oncogenic and tumor related proteins

“…Recent development in molecular biology provided significant information on the cellular, subcellular, and tissue-specific localization of oncogene products. However, most of these data are derived from fractionation procedures (Cirillo et al, 1984;Dominguez et al, 1991;Tanaka et al, 1986;Wilson and Wilson, 1992), and light microscopic observations (Anderson et al, 1984;Greenberg and Edelman, 1983;Klempnauer et al, Fig. 1. Prembedding EM immunogold labeling of tubulin and pp39""" in c-mosxe expressing X3 cells.…”