guanosine-diphosphate and Cell-Transformation--Viral

In mammalian cells, the guanine nucleotide exchange factor (GEF, eIF-2B) plays a major role in the regulation of initiation of protein synthesis. It catalyzes the exchange of eukaryotic chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the recycling of eIF-2 during polypeptide chain initiation. We used the Friend virus-transformed murine erythroleukemia (MEL) cell system to elucidate the translational regulatory processes that occur during growth and hexamethylene bisacetamide (HMBA)-induced cell differentiation. GEF activity is increased during growth and decreased during MEL cell differentiation, and this parallels the overall changes in protein synthesis during this period. Inhibition of GEF activity in induced cells may occur indirectly by phosphorylation of the alpha-subunit of eIF-2. However, the decrease in GEF activity in induced cells cannot be reversed by increasing the concentration of eIF-2-GDP added as a substrate in the GEF assay. This is diagnostic for the presence of eIF-2 alpha(P)-GDP in cell lysates and suggests that regulation of GEF activity may occur by one or more mechanisms other than eIF-2(alpha) phosphorylation. We have previously shown that the activity of GEF may be influenced directly by phosphorylation with casein kinase II (CK-II) of the 82-kD subunit of the factor. CK-II activity parallels the changes in GEF activity and the rate of protein synthesis during growth and differentiation of MEL cells. Addition of 1mM spermidine, a stimulator of CK-II but not of purified GEF, in induced MEL cell extracts enhances both CK-II and GEF activities approximately 48 and 32%, respectively. The results presented suggest that the inhibition of protein synthesis during MEL cell differentiation may be linked to the decreased CK-II and GEF activities.

Topics: Acetamides; Animals; Antineoplastic Agents; Casein Kinase II; Cell Differentiation; Cell Division; Cell Line; Cell Transformation, Viral; Clone Cells; Eukaryotic Initiation Factor-2; Friend murine leukemia virus; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Leukemia, Erythroblastic, Acute; Methionine; Mice; Models, Biological; Neoplasm Proteins; Protein Biosynthesis; Protein Serine-Threonine Kinases; Sulfur Radioisotopes; Tumor Cells, Cultured

Non-transformed revertant clones were isolated from the ras-transformed MTSV1-7 (ras) cell line, after treatment with the antibiotic azatyrosine. Azatyrosine significantly inhibited the growth of the ras-transformed cells but not of the normal MTSV1-7. After 7 days of azatyrosine treatment, approximately 30% of MTSV1-7 (ras) cells survived, and revertant cell lines were selected by random cloning. The azatyrosine-induced revertants (six clones) were considered non-transformed on the basis of (a) their substantially reduced ability to form colonies in soft agar, and (b) their inability to produce tumours in nude mice. Molecular analysis of the revertants revealed that each contains multiple copies of the v-H-ras gene and expresses high levels of v-H-ras mRNA, and all revertants sustain elevated levels of p21ras protein. Thus, the revertant phenotype induced by azatyrosine does not result from inactivation of v-H-ras oncogene or inhibition of its expression. In vivo guanine nucleotide binding to p21ras in the revertant cell lines demonstrated binding of both GTP and GDP, indicating that reversion to the non-transformed phenotype was not due to inability of p21ras to bind GTP. The expression of the human K-rev-1 gene, a known tumour-suppressor gene in ras-transformed NIH3T3 cells, was studied in the isolated azatyrosine revertants. All six revertants showed a significant increase in the K-rev-1 transcript levels compared with the ras-transformed MTSV1-7 cells. These results suggest that tumorigenic transformation of human mammary epithelial cells by v-H-ras may be influenced by the level of expression of the tumour-suppressor gene, K-rev-1.

Topics: Alanine; Animals; Blotting, Northern; Breast; Cell Division; Cell Transformation, Viral; Cells, Cultured; Genes, ras; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Mice, Nude; Oncogene Protein p21(ras); RNA, Messenger

We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target.

Topics: Arginine; Cell Transformation, Viral; Gene Expression Regulation; Genes, Viral; GTP Phosphohydrolases; Guanosine Diphosphate; Harvey murine sarcoma virus; Mutation; Oncogenes; Phosphorylation; Sarcoma Viruses, Murine; Subcellular Fractions; Threonine; Viral Proteins

2-Deoxy-2-fluoro-D-mannose (2FMan), an antiviral mannose analogue, inhibited the dolichol cycle of protein glycosylation. To specifically inhibit oligosaccharide-lipid synthesis, and not (viral) protein synthesis in influenza virus infected cells, the addition of guanosine to the 2FMan-treated cells was required. Under these conditions an early step in the assembly of the oligosaccharide-lipid was inhibited, and as a consequence, the glycosylation of proteins was strongly inhibited. Low-molecular-weight, lipid-linked oligosaccharides accumulated in cells treated with 2FMan plus guanosine, although dolichol phosphate (Dol-P) and GDP-Man were still present in the treated cells, and membranes from these cells were not defective in assembly of lipid-linked oligosaccharides. Thus, the presence of a soluble inhibitor of oligosaccharide-lipid assembly in these cells was postulated, and GDP-2FMan and UDP-2FMan, two metabolites found in 2FMan-treated cells, were synthesized and used to study in cell-free systems the inhibition of oligosaccharide-lipid assembly. GDP-2FMan inhibited the synthesis of Man(GlcNAc)2-PP-Dol from (GlcNAc)2-PP-Dol and GDP-Man, and in addition, it caused a trapping of Dol-P as 2FMan-P-Dol, whereas UDP-2FMan only inhibited Glc-P-Dol synthesis. However, it is probable that neither trapping of Dol-P nor inhibition of Glc-P-Dol synthesis by UDP-2FMan contributed to inhibition of protein glycosylation in cells treated with 2FMan. Incorporation of 2FMan from GDP-2FMan or UDP-2FMan into dolichol diphosphate linked oligosaccharides and interference of GDP-2FMan with the latter steps of assembly of the dolichol diphosphate linked oligosaccharide could not be shown.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Transformation, Viral; Cell-Free System; Chick Embryo; Glycoproteins; Guanine Nucleotides; Guanosine Diphosphate; Influenza A virus; Intracellular Membranes; Kinetics; Microsomes; Oligosaccharides; Polyisoprenyl Phosphate Sugars; Protein Processing, Post-Translational; Rhamnose

In the presence of ADP and GDP the tyrosine-phosphorylating activities of the viral as well as the cellular pp60src show a similar concentration-dependent inhibition in vitro. Addition of diadenosine 5',5"'-p1p4 tetraphosphate (Ap4A) to the kinase assay leads to an inhibition of the viral kinase activity, whereas the cellular kinase is not influenced.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Animals; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Dinucleoside Phosphates; Guanosine Diphosphate; Mice; Nucleotides; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinase Inhibitors; Viral Proteins

We have isolated eight rat lymphocyte-myeloma hybrid cell lines producing monoclonal antibodies that react with the 21,000-dalton transforming protein (p21) encoded by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV). These antibodies specifically immunoprecipitate both phosphorylated and non-phosphorylated forms of p21 from lysates of cells transformed by Ha-MuSV. All eight react with the products of closely related ras genes expressed in cells transformed by two additional sarcoma viruses (rat sarcoma virus and BALB sarcoma virus) or by a cellular Harvey-ras gene placed under the control of a viral promoter. Three of the antibodies also react strongly with the p21 encoded by the v-ras gene of Kirsten MuSV. These same three antibodies immunoprecipitate the predominant p21 species synthesized normally in a variety of rodent cell lines, including the p21 produced at high levels in 416B murine hemopoietic cells. This suggests that an endogenous gene closely related to Kirsten-ras is expressed in these cells. The monoclonal antibodies have been used to confirm two properties associated with p21; localization at the inner surface of the membrane of Ha-MuSV-transformed cells, assayed by immunofluorescence microscopy, and binding of guanine nucleotides.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; Guanosine Diphosphate; Mice; Oncogenic Viruses; Rats; Sarcoma Viruses, Murine; Viral Proteins

In earlier studies, we molecularly cloned a normal cellular gene, c-rasH-1, homologous to the v-ras oncogene of Harvey murine sarcoma virus (v-rasH). By ligating a type c retroviral promotor to c-rasH-1, we could transform NIH 3T3 cells with the c-rasH-1 gene. The transformed cells contained high levels of a p21 protein coded for by the c-rasH-1 gene. In the current studies, we have purified extensively both v-rasH p21 and c-rasH p21 and compared the in vivo and in vitro biochemical properties of both these p21 molecules. The p21 proteins coded for by v-rasH and c-rasH-1 shared certain properties: each protein was synthesized as a precursor protein which subsequently became bound to the inner surface of the plasma membrane; each protein was associated with guanine nucleotide-binding activity, a property which copurified with p21 molecules on a high-pressure liquid chromatography molecular sizing column. In some other properties, the v-rasH and c-rasH p21 proteins differed. In vivo, approximately 20 to 30% of v-rasH p21 molecules were in the form of phosphothreonine-containing pp21 molecules, whereas in vivo only a minute fraction of c-rasH-1 p21 contained phosphate, and this phosphate was found on a serine residue. v-rasH pp21 molecules with an authentic phosphothreonine peptide could be synthesized in vitro in an autophosphorylation reaction in which the gamma phosphate of GTP was transferred to v-rasH p21. No autophosphorylating activity was associated with purified c-rasH-1 p21 in vitro. The results indicate a major qualitative difference between the p21 proteins coded for by v-rasH and c-rasH-1. The p21 coded for by a mouse-derived oncogenic virus, BALB murine sarcoma virus, resembled the p21 coded for by c-rasH-1 in that it bound guanine nucleotides but did not label appreciably with 32Pi. The forms of p21 coded for by other members of the ras gene family were compared, and the results indicate that the guanine nucleotide-binding activity is common to p21 molecules coded for by all known members of the ras gene family.

Topics: Animals; Blood Proteins; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Mice; Oncogenes; Phosphoserine; Phosphothreonine; Receptors, Cell Surface; Sarcoma Viruses, Murine; Viral Proteins

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Genes, Viral; Guanine Nucleotides; Guanosine Diphosphate; Kirsten murine sarcoma virus; Mice; Rats; Sarcoma Viruses, Murine; Viral Proteins