guanosine-triphosphate and Cell-Transformation--Viral

To investigate the possible involvement of GTP-binding proteins in transformation by the DNA tumor virus polyomavirus, the GTP-binding activities of Ras-like proteins and G protein alpha subunit proteins were examined in polyomavirus-transformed cells. No differences in the degrees or patterns of expression of Ras-like proteins were observed. However, a 39-kDa protein specifically bound GTP in membranes from polyomavirus-transformed cells. This protein was not seen in nontransformed or lytically infected cells or in phenotypically normal revertants of polyomavirus-transformed cells. It reappeared, however, in spontaneous retransformants derived from the revertants. The 39-kDa protein was not found stably associated with polyomavirus T antigens, nor was it phosphorylated on tyrosine. The 39-kDa protein was not recognized by an antiserum specific for members of the Gi alpha subfamily of G proteins or by antisera against all other known GTP-binding proteins of similar molecular mass. These results suggest that this novel 39-kDa GTP-binding membrane protein is observed as part of a long-term response that accompanies stable transformation by the virus.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Transformation, Viral; GTP-Binding Proteins; Guanosine Triphosphate; Membrane Proteins; Mice; Molecular Weight; Polyomavirus; Rats

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

An immortalized cell line, called P9, was derived from hepatocytes by transfection with SV40 DNA. These cells expressed enzyme activities characteristic of hepatocytes, namely glucose-6-phosphatase, glycogen phosphorylase, bilirubin glucuronyltransferase and both glucagon- and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities, albeit at decreased levels compared with native hepatocytes. Levels of the G-protein subunits alpha-Gi-2, alpha-Gi-3, G beta and the 'long' form of alpha-G2 (45 kDa) were approximately 4-fold higher relative to native hepatocytes, whereas those of the 'short' form of alpha-G2 (42 kDa) were lower by approximately 40%. Associated with this were marked alterations in the guanine nucleotide regulation of adenylate cyclase. Receptor-mediated stimulation, achieved by either PGE1 or glucagon, was apparent in P9 cells, although the latter was only evident upon amplification with forskolin. Glucagon-stimulated cyclic AMP accumulation in P9 cells did not exhibit desensitization, as in hepatocytes, nor was the phosphorylation of alpha-Gi-2 evident. Culture of P9 cells with insulin led to a dose-dependent decrease (EC50 0.2 +/- 0.1 nM) in the ability of PGE1 to stimulate adenylate cyclase activity, with the maximum effect attained after approximately 6 h. A comparable attenuation of stimulation was seen for glucagon- and guanine-nucleotide-stimulated adenylate cyclase activities. In cells cultured with insulin, lower levels of GTP were required to stimulate adenylate cyclase, ADP-ribosylation of the 45 kDa form of alpha-Gs with cholera toxin was attenuated, and the expression of both alpha Gi-2 and alpha-Gi-3 was increased. It is suggested that the expression of alpha-Gi-2 and alpha-Gi-3 may be directly regulated by the action of insulin in hepatocytes and P9 cells.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Alprostadil; Animals; Cell Line; Cell Transformation, Viral; Colforsin; Glucagon; GTP-Binding Proteins; Guanosine Triphosphate; Insulin; Liver; Male; Rats; Rats, Sprague-Dawley; Receptor, Insulin; Signal Transduction

The large subunit of Herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a chimera consisting of a Ser/Thr protein kinase (PK) with features of a transmembrane (TM) helical segment localized at the amino terminus, and the RR1 domain localized at the carboxy terminus. To elucidate the role of the TM segment in ICP10-mediated transformation we established cell lines that constitutively express ICP10 (JHLa1) or its TM deleted mutant p139TM (JHL15). ICP10 was associated with purified JHLa1 plasma membranes. Membrane immunofluorescence and FACS analysis with antibodies to synthetic peptides located upstream and downstream of the TM indicated that ICP10 is a membrane-spanning protein. p139TM was not associated with JHL15 plasma membranes. ICP10 kinase activity was detected in JHLa1 but not JHL15 cells as determined by immunocomplex kinase assays and metabolic labeling. JHLa1 cells displayed anchorage-independent growth whereas JHL15 cells and JHL9 cells that express a mutant deleted in the PK catalytic domain were negative. ras-GTPase activating protein (ras-GAP) was phosphorylated in JHLa1 but not JHL15 cells and GTPase activity was lower in JHLa1 than JHL15 cells. Furthermore, ICP10 but not p139TM bound the guanine nucleotide releasing factor son of sevenless 1 (Sos1) and ras-GTP (activated ras) was higher in JHLa1 than JHL15 cells. The data suggest that ICP10 constitutively increases ras activity, and its TM segment plays a critical role in transformation-related signaling pathways.

Topics: Base Sequence; Cell Line; Cell Transformation, Viral; DNA Mutational Analysis; Fungal Proteins; Gene Expression Regulation; Genes, ras; GTPase-Activating Proteins; Guanosine Triphosphate; Herpes Simplex Virus Protein Vmw65; Herpesvirus 2, Human; Humans; Molecular Sequence Data; Protein Binding; Protein Serine-Threonine Kinases; Proteins; ras GTPase-Activating Proteins; Recombinant Fusion Proteins; Repressor Proteins; Ribonucleotide Reductases; Sequence Deletion; Signal Transduction; SOS1 Protein; Structure-Activity Relationship; Substrate Specificity

Polyomavirus middle T antigen mediates transformation of cells, at least in part, by its association with and activation of the intrinsic protein tyrosine kinase activity of pp60c-src. pp60c-src, by analogy with pp60v-src, elicits cell proliferation through a signal transduction pathway that includes p21c-ras. Therefore, we tested the possibility that middle T antigen acts upstream of and in the same proliferative signaling pathway as p21c-ras. Co-transfection of Rat-2 cells with plasmids expressing human Krev-1, a dominant suppressor of Ki-ras transformation, and mT antigen resulted in a dose-dependent reduction of mT antigen-induced foci. Krev-1 did not affect the transforming activity of SV40 large T antigen, demonstrating that the transformation-suppressing activity of Krev-1 is specific. To determine the effect of Krev-1 on stably transformed cell lines, Krev-1 DNA was introduced into middle T antigen-transformed Rat-2 cells along with a G418 resistance marker. Of the G418-resistant colonies examined, 1% were morphologically untransformed. Characterization of several morphological revertants revealed that, with the exception of one cell line, all of the cell lines expressed middle T antigen, which was associated with pp60c-src, whose tyrosine kinase activity was similar to that found in the parental transformed cell lines. To determine whether other phenotypic traits associated with transformation were altered in these cell lines, their growth rates and ability to form colonies in agar suspension were examined. The majority of the revertants had longer doubling times, and grew less efficiently in agar suspension compared with their transformed parents. A direct correlation was observed between Krev-1 RNA and protein expression and the efficiency with which the revertants formed colonies in suspension. These results suggest that p21c-ras lies downstream of middle T antigen and pp60c-src in the same proliferative signal transduction pathway.

Topics: Animals; Antigens, Polyomavirus Transforming; Cell Line; Cell Transformation, Viral; GTP-Binding Proteins; Guanosine Triphosphate; Polyomavirus; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); rap GTP-Binding Proteins; Rats; RNA; Transfection

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

Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N-ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms.

Topics: Animals; Antigens, CD; Cell Line; Cell Membrane; Cell Transformation, Viral; Cell-Free System; Cytosol; Ethylmaleimide; G(M3) Ganglioside; Glycolipids; Glycoproteins; Glycosphingolipids; Golgi Apparatus; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Lactosylceramides; Models, Biological; N-Acetylneuraminic Acid; Protein Processing, Post-Translational; Sialic Acids; Thionucleotides; Vesicular stomatitis Indiana virus

We sought to determine whether decreased in vitro GTPase activity is uniformly associated with ras p21 mutants possessing efficient transforming properties. Normal H-ras p21-[Gly12-Ala59] as well as an H-ras p21-[Gly12-Thr59] mutant exhibited in vitro GTPase activities at least fivefold higher than either H-ras p21-[Lys12-Ala59] or H-ras p21-[Arg12-Thr59] mutants. Microinjection of as much as 6 X 10(6) molecules/cell of bacterially expressed normal H-ras p21 induced no detectable alterations of NIH/3T3 cells. In contrast, inoculation of 4-5 X 10(5) molecules/cell of each p21 mutant induced morphologic alterations and stimulated DNA synthesis. Moreover, the transforming activity of each mutant expressed in a eukaryotic vector was similar and at least 100-fold greater than that of the normal H-ras gene. These findings establish that activation of efficient transforming properties by ras p21 proteins can occur by mechanisms not involving reduced in vitro GTPase activity.

Topics: Animals; Cell Line; Cell Transformation, Viral; Escherichia coli; GTP Phosphohydrolases; Guanosine Triphosphate; Mice; Mutation; Oncogene Proteins, Viral; Oncogenes; Phosphoric Monoester Hydrolases; Structure-Activity Relationship

Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Avian Sarcoma Viruses; Cell Transformation, Viral; Chick Embryo; Cholera Toxin; Colforsin; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Pertussis Toxin; Thionucleotides; Virulence Factors, Bordetella

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cell Transformation, Viral; Chromosome Mapping; Cloning, Molecular; GTP-Binding Proteins; Guanosine Triphosphate; Mice; Mutation; Oncogene Proteins, Viral; Phosphoproteins; Structure-Activity Relationship; Transfection

Topics: Animals; Annexins; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA; Epidermal Growth Factor; ErbB Receptors; Guanosine Triphosphate; Humans; Membrane Proteins; Neoplasm Proteins; Nucleoproteins; Oncogene Protein pp60(v-src); Oncogenes; Phosphotyrosine; Platelet-Derived Growth Factor; Protein Binding; Protein Biosynthesis; Protein Kinases; Receptors, Cell Surface; Retroviridae; Tyrosine; Viral Proteins

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.

Topics: Animals; Binding, Competitive; Cell Transformation, Viral; DNA-Directed DNA Polymerase; Guanosine Triphosphate; Kinetics; Simplexvirus; Species Specificity

Topics: Adenosine Triphosphate; Animals; Cell Division; Cell Transformation, Viral; Cells, Cultured; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Mice; Mice, Inbred Strains; Phosphorylation; Protein Kinases; Simian virus 40

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

We report the purification to homogeneity of a 20,000-dalton, transformation-related, rat cell membrane protein. This protein, p20, was originally identified in preparations of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus. The chromatographically purified p20 was an acidic hydrophobic protein, capable of specifically binding GTP (dissociation constant = 15 microM). This nucleotide binding property and other previously reported characteristics were similar to properties ascribed to the Harvey sarcoma virus src gene product. p20 also appeared similar to this src gene product when immunoprecipitates of both proteins were directly compared by one- and two-dimensional NaDodSO4 gel electrophoreses. However, the proteins were not identical, because their tryptic maps differed. Using a competition radioimmunoassay, we have measured the concentration of p20 in cells, viruses, and rat tissues: p20 was not encoded by rat sarcoma viruses because it was increased only slightly after Kirsten sarcoma virus transformation of rat cells and was not increased in nonrat cells transformed by the Kirsten or Harvey sarcoma virus. Remarkably, of 10 rat tissues examined, p20 was found predominantly in brain, specifically in the membranes.

Topics: Animals; Brain Chemistry; Cell Transformation, Viral; Cells, Cultured; Chromatography; Guanosine Triphosphate; Humans; Kidney; Kirsten murine sarcoma virus; Membrane Proteins; Mice; Mink; Phosphoproteins; Rats; Sarcoma Viruses, Murine

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.

Topics: Adenosine Triphosphate; Animals; Avian Sarcoma Viruses; Cell Adhesion; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Viral; Cricetinae; Fibroblasts; Guanosine Triphosphate; HeLa Cells; Kinetics; Mice; Molecular Weight; Phosphoproteins; Phosphorylation; Protein Kinases; Simian virus 40

Topics: Animals; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cloning, Molecular; Genes, Viral; Guanosine Triphosphate; Kirsten murine sarcoma virus; Phosphorylation; Rats; Recombination, Genetic; Retroviridae; RNA, Viral; Viral Proteins

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [gamma-32P]ATP and [gamma-32P]GTP as precursors. With ATP maximum overall incorporation of 32P into both membrane fractions occurred at pH 7.8 in the presence of 10 mM MgCl2 after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of 32P at pH 7.8 and 10 mM MgCl2 after incubation for 3 min. The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phosphorylation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular weight of about 100 000 and the ATP-specific main component a molecular weight of 110 000. The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl2 concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (Mr = 110 000) was no more phosphorylated whereas with GTP the main component Mr = 100 000 was essentially the sole phosphorylated protein.

Topics: Adenosine Triphosphate; Animals; Cell Fractionation; Cell Line; Cell Membrane; Cell Transformation, Viral; Endoplasmic Reticulum; Fibroblasts; Guanosine Triphosphate; Hydrogen-Ion Concentration; Kinetics; Magnesium; Membrane Proteins; Mice; Molecular Weight; Phosphorylation; Simian virus 40

Topics: Animals; Cell Line; Cell Membrane; Cell Transformation, Viral; Enzyme Activation; Fibroblasts; Guanosine Triphosphate; Mice; Protein Kinases; Ribonucleotides; Simian virus 40

Sugar deprivation of hamster fibroblasts (NIL) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the folloing fashion: the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma-transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar-supplemented medium. Refeeding glucose to sugar-depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.

Topics: Animals; Cell Line; Cell Transformation, Viral; Cricetinae; Cycloheximide; Cytidine Triphosphate; Cytosine Nucleotides; Fibroblasts; Glucose; Guanosine Triphosphate; Polyomavirus; Protein Biosynthesis; Tumor Virus Infections; Uracil Nucleotides; Uridine Triphosphate

Viral development induces changes in the permeability properties of the plasma membrane of the host cell. Here it is shown that, because of this leakiness, inhibitors of protein synthesis normally impermeable to uninfected cells are able to enter infected cells and thereby specifically inhibit viral protein synthesis.

Topics: Adenosine Triphosphate; Animals; Antiviral Agents; Cell Line; Cell Membrane; Cell Membrane Permeability; Cell Transformation, Viral; Encephalomyocarditis virus; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Membrane Proteins; Molecular Weight; Protein Biosynthesis; Time Factors; Viral Proteins; Virus Diseases