Sadaaki Kawai

Two cellular proteins that immunoprecipitate with the transforming protein of Rous sarcoma virus

Abstract The transforming gene (src) of Rous sarcoma virus encodes a 60,000-dalton phosphoprotein (pp60src) with the ability to phosphorylate tyrosine in certain protein substrates. The enzymatic activity of pp60src is thought to mediate neoplastic transformation by src. It would therefore be useful to identify cellular proteins that interact with pp60src on the chance that these proteins might be substrates for the kinase activity of the viral protein or be otherwise involved in neoplastic transformation of the host cell. In pursuit of this objective, we characterized the proteins that coprecipitate with pp60src in immune complexes. These proteins proved to be of two types. (i) Most immune complexes contained a series of proteins (50,000 to 58,000 daltons) that were apparently derived from pp60src by sequential degradation from the amino terminus. We do not know if this degradation has a physiological purpose in the infected cell, but it has at least two practical implications: it has proved useful in the analysis of the functional topography of pp60src; and it can give rise to experimental artifacts in the analysis of proteins obtained from cells infected with Rous sarcoma virus. (ii) Two proteins (50,000 and 89,000 daltons) coprecipitated with pp60src, probably by virtue of their ability to bind to the viral protein. Both proteins are phosphorylated, both are encoded by the cellular genome, and both can be recovered from either avian or mammalian cells transformed by Rous sarcoma virus. The 89,000-dalton protein contains phosphoserine, irrespective of its source, and its structure is otherwise highly conserved among widely diverged vertebrate species. By contrast, the forms of the 50,000-dalton protein recovered from chicken and rat cells can be readily distinguished by their peptide maps and by their phosphoamino acids (the avian form of the protein contains both phosphoserine and phosphotyrosine, whereas the mammalian form contains only phosphoserine). We used temperature-sensitive mutants in src to explore the possibility that the two cellular proteins might be substrates for the protein kinase activity of pp60src: propagation of infected cells at the nonpermissive temperature failed to affect the phosphorylation of either of the proteins. We conclude that at least two cellular proteins are associated with pp60src prior to immunoprecipitation with antisera directed against the viral protein. It is possible that neither of these proteins is a substrate for the protein kinase activity of pp60src, however, and their role in neoplastic transformation by src (if any) remains moot.

A cellular protein is immunologically crossreactive with and functionally homologous to the Fujinami sarcoma virus transforming protein

We obtained a regressing-tumor antiserum specific for the unique sequence of the transforming protein P140 of Fujinami sarcoma virus by injecting Fischer rats with syngeneic embryo cells transformed with Fujinami sarcoma virus. This serum is capable of immunoprecipitating a protein of 98,000 daltons from cell extracts of normal, uninfected chicken bone marrow cells. This normal cellular protein (NCP98) was shown to be structurally related to P140, sharing the majority of 35S-methionine-labeled tryptic peptides with the viral gene product P140. NCP98 is a phosphoprotein in vivo, with an associated in vitro protein kinase activity, capable of phosphorylating specifically at tyrosine residues of NCP98 itself and alpha-casein, an externally added substrate. This kinase activity is biochemically indistinguishable from the kinase activity associated with P140 by all criteria tested. Moreover, in vitro-phosphorylated NCP98 and P140 shared the same phosphopeptides. The expression of NCP98 is tissue-specific. It is readily detectable in bone marrow cells and detectable to a lesser extent in liver and lung cells from 6--18 day old chickens.

Isolation of the avian transforming retrovirus, AS42, carrying the v-maf oncogene and initial characterization of its gene product.

A novel avian transforming retrovirus was isolated from a chicken musculoaponeurotic fibrosarcoma. This virus (called AS42) induces tumors histopathologically indistinguishable from the original sarcoma after a long latent period when inoculated into newborn chickens. AS42 also exhibits a weak transforming activity when infected into chicken embryo fibroblasts (CEF). This virus is replication-defective and associated with a helper virus of subgroup A (called ASAV). An AS42-specific protein of about 100 kDa was immunoprecipitated from lysates of AS42-transformed CEF with antiserum directed against avian retrovirus virion proteins. Molecular analysis of the genomic structure of the AS42 virus has revealed that this 100-kDa protein represents a novel oncogene, v-maf of cellular origin, which is fused with a part of the viral gag gene (Nishizawa et al., Proc. Natl. Acad. Sci. USA 86, 7711-7715, 1989). Interestingly, some size variation was observed among the gag-maf fusion proteins found in individual clones of transformed CEF. Consistent with this observation, Southern blot analyses and nucleotide sequence determination of several independent isolates of proviral DNA indicated that this virus segregates multiple forms of deletion mutants, probably through homologous recombinations among the repetitive sequences present within the v-maf coding region.

Studies on the oxidation of p-aminobenzoate to p-nitrobenzoate by Streptomyces thioluteus☆

Abstract It was revealed that p-aminobenzoate was oxidized to p-nitrobenzoate by Streptomyces thioluteus. Several other aromatic nitro compounds such as p-aminobenzaldehyde and p-aminophenylacetate were oxidized in the same way although with a lower rate; however, others such as o-aminobenzoate, m-aminobenzoate, and p-aminophenol were not oxidized by the organism. p-(N-Dimethyl) aminobenzoate was oxidized to the corresponding N-oxide. It was found by mass spectrometric analysis with O18-labeled oxygen, that both oxygen atoms in the nitro group of p-nitrobenzoate were derived from molecular oxygen. It may be regarded as a new type of oxygenase reaction. Effects of pH and various enzyme inhibitors on the reaction were investigated, and the possible pathway for the formation of nitro group was discussed.

Disappearance of diacylglycerol kinase translocation in ras-transformed cells

Oncogenic transformation has been considered to be in part a consequence of the elevated levels of 1,2-diacylglycerol(DG), resulting in the permanent activation of protein kinase C. DG content in transformed cells with v-H-ras, c-K-ras and N-ras oncogene increased 1.5-fold compared to that in non-transformed NIH/3T3 cells. DG kinase activity of membrane fractions, which plays an important role in DG attenuation, was significantly lower in all ras-transformed cells. On the contrary, DG kinase activity in cytosol fractions in ras-transformed cells was found to be increased. DG kinase translocated very markedly from cytosol to membranes in non-transformed NIH/3T3 cells by the treatment of phospholipase C. On the other hand, translocation of DG kinase in ras-transformed cells was slight, though the formation of DG by the treatment of phospholipase C was almost same between ras-transformed and NIH/3T3 cells. These results strongly support the idea that the increased DG content in ras-transformed cells is, at least partly due to the defect of DG kinase translocation, which may lead to the sustained activation of protein kinase C.

Viral Oncogenes, v-yes and v-erbB, and Their Cellular Counterparts

Publisher Summary Within the past 10 years, about 20 retroviral genes have been reported to have the capacity of transforming cells and have been proposed to have causal relationships with some malignant tumors in experimental animals. These genes are called “viral oncogenes,” and each of them stems from a closely related gene in the genomes of normal cells. The normal cellular sequences of the genes, which are counterparts of viral oncogenes, are tentatively called “cellular oncogenes,” and they have been shown to be widely conserved in the genomes of various species of animals. Several transforming genes have also been identified in cellular DNA of human cancer cells, as well as in tumors of experimental animals, by the gene transfer technique (transfection), using NIH3T3 cells as a recipient. Some of these genes are closely related to two viral oncogenes, K-ras and H-ras, but others are not related to any known viral oncogenes. In addition, several cellular oncogenes are found to be amplified, or overexpressed, in human cancers. The various lines of progress in the studies of oncogenes provide ideas with which one may disentangle the complicated mechanisms of oncogenesis step by step.

Transformation of chicken embryo fibroblast cells by avian retroviruses containing the human Fyn gene and its mutated genes.

The transforming activity of the human fyn protein, p59fyn, which is a kinase of the src family, was investigated by testing the effect of recombinant avian retrovirus (Fyn virus) expressing p59fyn on chickens or cultured chicken embryo fibroblast (CEF) cells. The Fyn virus did not induce transformed foci. After several passages of the virus stock on CEF cells, however, a few foci were detected in the presence of dimethyl sulfoxide. Chickens inoculated with Fyn virus at the stage of 12-day-old embryos developed fibrosarcomas 3 to 6 weeks after hatching. The viruses obtained from these foci and from one of the tumor tissues showed high transforming activity in the presence of dimethyl sulfoxide, suggesting that these viruses carry spontaneous mutations of the fyn gene. Four fyn genes from CEF DNAs infected with transforming viruses were molecularly cloned, and their products were confirmed to possess transforming activity. DNA sequence analysis of the fyn genes showed that two of the four mutants have Thr instead of Ile at position 338 in the kinase domain. The other two mutants carry deletions of 78 and 108 base pairs, respectively, which result in complete loss of region C of SH2. The overall level of proteins containing phosphotyrosine was significantly higher in transformed cells than in normal CEF cells. Our data indicate that when expressed at high levels in a retrovirus, normal p59fyn cannot cause cellular transformation, but that mutant p59fyn with either a single amino acid substitution in the kinase domain or a deletion including region C produces a transforming protein, perhaps due to enhanced tyrosine kinase activity. This is the first observation that deletion of region C can unmask the potential transforming activity of a src family kinase.

Defect in phorbol acetate-induced translocation of diacylglycerol kinase in erbB-transformed fibroblast cells

We here show that tetradecanoyl phorbol acetate (TPA) and 1‐oleoyl 2‐acetyl glycerol (OAG) cause the translocation of diacylglycerol (DG) kinase from the cytosol to the membrane fractions in chick embryo fibroblast (CEF) cells. However, this translocation is not marked in erbB‐transformed chick embryo fibroblast (GEV) cells. The activities of phosphatidylinositol (PI) and phosphatidylinositol 4‐phosphate (PIP) kinases in membrane fractions are not altered by TPA treatment in either CEF or GEV cells. Such reduced translocation of DG kinase by TPA is also observed in src‐transformed cells, but not in myc‐transformed cells. These results suggest that the defect in DG kinase translocation may result in failure to suppress the overactivation of protein kinase C in erbB‐2 and src‐transformed cells, which may lead to cell growth and transformation.

Characterization of two strains of avian sarcoma virus isolated from avian lymphatic leukosis virus-induced sarcomas.

Two replication-defective avian sarcoma viruses, S1 and S2, which were independently isolated from tumors of chickens inoculated with avian lymphatic leukosis virus (LLV) were characterized. The genomes of S1 and S2 contain src-related sequences and are, respectively, about 3.9 and 4.5 kilobases long. pp60src-related proteins with molecular weights of 62,000 (p62) were detected in cells infected with these viruses, and protein kinase activity was found to be associated with these proteins. No other viral proteins, such as gag, pol, and env gene products, were detected. These results suggested that the c-src sequence in normal chicken cells was incorporated into LLV genomes by recombination at the expense of most of the viral genes to generate highly defective new sarcoma viruses.

A variant Schmidt-Ruppin strain of Rous sarcoma virus with increased affinity for mammalian cells.

SR‐RSV‐D(H), a variant virus with extremely high tropism for mammalian cells, was isolated by passage of the Schmidt‐Ruppin strain of Rous sarcoma virus of subgroup D (SR‐RSV‐D) through hamster cells. This variant virus has acquired an altered envelope glycoprotein, encoded by the env gene, that has high affinity for receptors on the surface of mammalian cells. The variant virus transforms rat cells at about 100 times the efficiency of the parental virus, SR‐RSV‐D(S), as assayed by focus formation. Addition of amphotericin B (Fungizone) to the medium at a concentration of 0.2 μg/ml completely inhibited rat cell transformation by SR‐RSV‐D(H), possibly by blocking virus penetration into the cells, whereas the drug showed no inhibitory effect on transformation of chick embryo flbroblast (CEF) cells by the variant virus or on transformation of rat cells by the parental virus. The efficiency of transformation of rat cells by the variant virus was much less than its efficiency of transformation of CEF cells. Analysis of infection of rat cells suggested that the virus can infect rat cells as efficiently as CEF cells but that rat cells were not transformed by the virus as fully as CEF cells because of inefficiency of some post‐penetrational step involved in viral gene expression. The finding that El AY cells, rat cells expressing adenovirus El A gene, were transformed by SR‐RSV‐D(H) as efficiently as CEF cells supports this conclusion and suggests that expression of the E1A gene in rat cells may overcome the defect in the transforming step(s) in rat cells.