Viral and cellular fos proteins: a comparative analysis

The FBJ murine osteosarcoma virus (FBJ-MuSV) induces osteosarcomas in mice and transforms fibroblasts in vitro. It contains an oncogene termed v-fos derived from a normal cellular gene by recombination with an associated helper virus. The product of the v-fos gene is a 55,000 dalton protein, p55v-fos. This protein was found in the nuclei of cells containing amplified levels of the v-fos gene, and also in the nuclei of virus-transformed cells. The c-fos protein was localized in the nuclei of normal mouse amnion cells and in the nuclei of cells transformed by a recombinant plasmid that expresses the c-fos gene product. However, p55c-fos undergoes more extensive post-translational modification in the nucleus than p55v-fos. Immunofluorescence data indicate that the level of p55c-fos in normal mouse amnion cells is similar to that found in fibroblasts transformed by the v-fos or c-fos proteins.     

Viral and cellular fos proteins are complexed with a 39,000-dalton cellular protein.

The structure of viral and cellular fos gene products and their association with a 39,000-dalton cellular protein (p39) were investigated by using antisera raised against synthetic peptides. The first peptide, termed M, corresponded to amino acids 127 to 152 of the v-fos sequence, a region which is identical in c-fos. The second peptide, termed V, corresponded to the nine C-terminal amino acids of v-fos; this region is not present in c-fos. Rabbit antisera were purified by affinity chromatography against their respective peptides before being used for immunoprecipitation. M peptide antisera precipitated p55v-fos and p55c-fos, whereas V peptide antisera precipitated only p55v-fos. This observation confirms the prediction from nucleotide sequence analysis that these proteins are distinct at their C termini. p39 was precipitated in association with p55v-fos and p55c-fos by M and V peptide antisera. However, V peptide antisera did not precipitate p39 from cells expressing p55c-fos, even though the presence of p39 in such cells was demonstrated with M peptide antisera. Denaturation of cell lysates completely abolished the precipitation of p39, whereas the precipitation of p55v-fos was unaffected. Taken together, the data demonstrate that p39 exists in a complex with p55.     

Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.

We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed.     

Expression of the c-fos proto-oncogene in bone, cartilage and tooth forming tissues during mouse development

The c-fos proto-oncogene is the cellular homologue of v-fos identified as the bone transforming gene of the FBJ and the FBR murine osteosarcoma viruses. We show here, using a sensitive in situ hybridization method, that the c-fos proto-oncogene is expressed in the cartilage, bone and tooth forming tissues during mouse development. This result suggests that the tumors observed after infection by the FBJ viral complex and c-fos overexpression in transgenic mice occur in those tissues in which c-fos is expressed during development.     

An avian transforming retrovirus isolated from a nephroblastoma that carries the fos gene as the oncogene.

A new avian transforming retrovirus, NK24, was isolated from a chicken with a nephroblastoma. This transforming virus induced fibrosarcomas with osteogenic cell proliferation and nephroblastomas in vivo and transformed fibroblast cells in vitro. From extracts of NK24-transformed cells, anti-gag serum immunoprecipitated a 100-kilodalton nonglycosylated protein with no detectable protein kinase activity. An NK24 provirus present in infected quail cells was molecularly cloned and subjected to nucleotide sequence analysis. The genome of NK24 was 5.3 kilobases long and had a 1,126-base-pair sequence of cellular origin in place of a viral sequence of avian leukosis virus containing the 3' half of the gag gene and the 5' half of the pol gene. Although the entire env gene was retained, it appeared to be inactive, possibly owing to the loss of function of its splice acceptor site as a result of a second deletion of 1,598 bases in the 3' half of the pol gene that extended to the acceptor site. Nucleotide sequence analysis revealed that the NK24 virus contained the fos gene, previously identified as the oncogene of FBJ and FBR murine osteosarcoma viruses. Unlike the v-fos gene products of FBJ and FBR, which suffer a structural alteration at their carboxyl termini, the NK24 v-fos gene product seemed to have the same carboxyl-terminal structure as the chicken c-fos gene product. A comparison of the structures of the products of the NK24 v-fos and mouse c-fos genes suggested that the fos gene product consists of highly conserved regions and relatively divergent regions.     

Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells

The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.     

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).     

Transformation of T lymphocytes by the v-fos oncogene

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.     

Structure-function analysis of fos protein: a single amino acid change activates the immortalizing potential of v-fos

We have undertaken a detailed structure-function analysis of v-fos protein, taking the ability to induce transformation and immortalization as criteria of biological activity. Our results demonstrate that the evolutionarily conserved center region of fos, comprising ca. 28% of the protein, is indispensable for its function. A single amino acid change (Glu 138----Val 138) in this region activates the immortalizing potential of v-fos without affecting its transforming capacity. The presence of additional either N- or C-terminal amino acids, however, is required for efficient expression of a stable protein, and significantly increases its biological activity. In contrast, sequences in the C-terminal half (between positions 228 and 267) strongly downmodulate the transforming activity of v-fos protein without decreasing its immortalizing potential.     

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.     

Generation of a murine monoclonal antibody that detects the fos oncogene product

A hybridoma producing a monoclonal antibody (MoAB) recognizing both the cellular and viral forms of fos has been generated by somatic cell hybridization techniques from spleen cells of mice immunized with a synthetic peptide corresponding to amino acids 128-152, a consensus region, of both the v-fos and c-fos oncogene products. Three proteins with molecular weights of 55,000, 44,000, and 42,000 were detected by immunoblotting. While MoAB 2G9C3 failed to immunoprecipitate fos from Finkel-Biskis-Jenkins murine osteosarcoma-virus-infected fibroblasts, both the 55,000 v-fos protein and the 39,000 cellular protein were coprecipitated using polyvalent rabbit antibodies to the same peptide. Whereas no cell surface membrane expression of fos was detected, after membrane permeabilization by a brief exposure to lysolecithin it was possible to specifically detect internal fos by immunofluorescence flow cytometry. Immunohistochemical staining of FBJ virus-infected cells revealed intense, nuclear staining.     

Activation of cellular p21ras by myristoylation

p21ras is palmitoylated on a cysteine residue near the C-terminus. Changing Cys-186 to Ser in oncogenic forms produces a non-palmitoylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. To examine whether palmitate acts in a general way to increase ras protein hydrophobicity, or is involved in more specific interactions between p21ras and membranes, we constructed genes that encode non-palmitoylated ras proteins containing myristic acid at their N-termini. Myristoylated, activated ras, without palmitate (61Leu/186Ser) exhibited both efficient membrane association and full transforming activity. Unexpectedly, we found that myristoylated forms of normal cellular ras were also potently transforming. Myristoylated c-ras retained the high GTP binding and GTPase characteristic of the cellular protein and, moreover, bound predominantly GDP in vivo. This implied that it continued to interact with GAP (GTPase-activating protein). While the membrane binding induced by myristate permitted transformation, only palmitate produced a normal (non-transforming) association of ras with membranes and must therefore regulate ras function by some unique property that myristate does not mimic. Myristoylation thus represents a novel mechanism by which the ras proto-oncogene protein can become transforming.     

A sensitive enzyme-linked immunosorbence assay for the c-fos and v-fos oncoproteins

The c-fos nuclear oncoprotein is rapidly induced when the growth of normal cells is initiated by mitogens, and it is also synthesized in several cell systems in response to stimuli that do not cause cell proliferation. When expressed inappropriately, c-fos, and its retroviral counterpart v-fos, can transform susceptible cells in vivo and in vitro. We have developed a simple and sensitive ELISA for the c-fos and v-fos proteins. Fos proteins are captured from cell lysates by an antibody specific for an amino-terminal peptide substantially conserved between v-fos and c-fos; the captured proteins are recognised by a second antibody against a different peptide sequence also conserved in the two proteins. The second antibody has been conjugated to alkaline phosphatase to provide an enzyme label; bound alkaline phosphatase is measured with a sensitive cycling enzyme system that generates a coloured end-product. We show that the fos ELISA is immunologically specific and use it to monitor increased c-fos expression in serum-stimulated HeLa cells and human fibroblasts, and in mitogen-stimulated murine thymocytes.