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PubMed1985

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver.

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.

Adenylyl Imidodiphosphate↗
PubMedDate not supplied

Association of the polyomavirus middle-T antigen with c-yes protein.

Expression of the middle-T antigen of polyomavirus is sufficient to induce transformation of fibroblasts in culture and tumour formation in whole animals. Middle-T can form a complex with the cellular src gene product (p60c-src) and can be phosphorylated by p60c-src in vitro. Studies using middle-T mutants have suggested that the association of middle-T with p60c-src may be necessary but not sufficient for transformation. Therefore, we addressed the possibility that middle-T could interact with other tyrosine protein kinases structurally related to p60c-src. Using antibody raised against a fusion protein between beta-galactosidase and amino-terminal sequences of p90gag-yes from Y73 virus (anti-yes antibody), we have found that middle-T can associate with and be phosphorylated by the c-yes proto-oncogene product, a protein of relative molecular mass (Mr) 62,000 (62K). This raises the possibility that the middle-T-p62c-yes complex contributes to transformation by polyomavirus.

Antigens, Viral, Tumor↗
PubMed1977

Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

Animals↗
PubMed1985

Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.

Animals↗
PubMed1993

Increase in activity and level of pp60c-src in progressive stages of human colorectal cancer.

Activation of the tyrosine kinase of the c-src gene product, pp60c-src, has been shown to occur in nearly every primary colorectal carcinoma, and is found as early as in polyps of high malignant potential. However, no studies have addressed potential pp60c-src changes which occur during progression. To examine this question, we have studied kinase activity and protein levels in 7 colonic polyps, 19 primary lesions, and 19 liver metastases relative to normal colonic mucosa. Significant increases in tyrosine kinase activity were seen as early as in colonic polyps of high malignant potential. Further increases were observed in activity and level in primary tumors. However, the greatest increases in activity and protein levels were observed in liver metastases. Additionally, six metastatic lesions were obtained in which synchronous primary tumor was resected. In each of these liver metastases, pp60c-src activity and level were significantly increased relative to the corresponding primary tumor, as well as to normal colonic mucosa. Our results demonstrate that progression of colon primary tumors to liver metastases correlates with increased pp60c-src kinase activity and protein level.

Adenocarcinoma↗
PubMed1988

Alteration of p60c-src expression in human leukemia-lymphoma cells correlated with induced differentiation.

Expression of cellular src-gene product (p60c-src) in human leukemia-lymphoma cell lines was analysed by flow cytometry using a monoclonal antibody (McAb), H2B4 which recognizes p60c-src protein in human cells. In several human leukemia-lymphoma cell lines (K562, Namalva, HL60, U937), p60c-src expression was higher than in peripheral mononuclear cells from healthy volunteers. Some non-lymphoid leukemia cells can be induced to differentiate into monocyte-macrophages by 12-O-tetradecanoyl phorbol-13-acetate (TPA). K562 cells were also induced to differentiate not only morphologically but also functionally into monocyte-macrophages by TPA. Flow cytometric analyses using the McAb H2B4 revealed that the amount of p60c-src expression in K562 cells markedly decreased during TPA induced differentiation. The activity of protein kinase associated with p60c-src in K562 cells was determined employing IgG of immunized rabbit serum specific for p60c-src. The immunized rabbit IgG heavy chain phosphorylation by protein kinase also decreased after the induced differentiation. We detected p60c-src protein in acute lymphoid leukemia cells as well as acute non-lymphoid leukemia cells freshly isolated from patients. The amount of p60c-src protein decreased in some acute non-lymphoid leukemia cases, but it increased in others after TPA induced differentiation. No correlation was observed between FAB classification of acute leukemias and the amount of endogenous p60c-src expression.

Cell Differentiation↗
PubMed1989

Preferential expression of a pp60c-src related protein tyrosine kinase activity in nerve cells of the early metazoan Hydra (Coelenterates).

It has been suggested that the proto-oncogene c-src plays a functional role in developing neurons, and in the mature nerve cells of higher vertebrates. The coelenterate Hydra represents the most primitive known organism possessing nerve cells. With Southern blot hybridizations we have demonstrated src-related sequences in Hydra. Antisera specific for the c-src gene product (pp60c-src) of birds and mammals precipitate a protein from Hydra cell extracts with a tyrosine-specific protein kinase activity. Studies of tissues and cells fractionated from a temperature sensitive mutant of Hydra which is depleted of interstitial (including nerve) cells at the non-permissive temperature, have indicated the src-like kinase of Hydra to be preferentially expressed in nerve cells. The high conservation of structural features and of the expression pattern indicates a basic function for pp60c-src in neurons.

Animals↗
PubMed1987

[The erbB-related protooncogenes encoding growth factor receptors].

The c-erbB-2 gene was first identified by virtue of its cross-hybridization with v-erbB. Nucleotide sequence analysis of complementary DNA clones suggested that the c-erbB-2 gene encodes a growth factor receptor similar to that for EGF. Antibodies against the carboxyl terminal sequence of the c-erbB-2 protein immunoprecipitated a 185-kDa glycoprotein which showed protein-tyrosine kinase activity in vitro. Despite the extensive similarity between the c-erbB-2 protein and EGF receptor, neither EGF nor TGF-alpha bound to the c-erbB-2 protein. Phosphorylation of the c-erbB-2 protein was stimulated by TPA via protein kinase C in vivo. EGF also induced phosphorylation of the c-erbB-2 protein. This phosphorylation occurred not only on serine and threonine residues but also on tyrosine residues. Preliminary data suggested that the latter was mediated by the kinase activity of the EGF receptor. Southern blot analysis of DNAs from primary tumors revealed that the c-erbB-2 gene tends to be amplified in adenocarcinomas, mostly of the stomach and the breast. By screening both human genomic and cDNA libraries using v-yes DNA as a probe, we obtained DNA clones of the c-yes gene, the pseudogene of c-yes, c-fgr gene and c-src gene and two novel yes-related genes, fyn and lyn. Complete nucleotide sequence analysis of the cDNA clones of c-yes, fyn and lyn revealed that these genes encode proteins similar to p60src both in size and sequence.

Adenocarcinoma↗
PubMed1980

Characterization of the transforming gene of Fujinami sarcoma virus.

The src gene present in all avian sarcoma viruses is not present in the genome of Fujinami sarcoma virus, a potent sarcoma-inducing virus in chickens. Fujinami virus is defective and requires helper virus for replication. RNA from a mixture of helper and transforming viruses consists of two components, 35S and 28S. Oligonucleotide fingerprinting of each RNA component revealed that the 35S component was identical to the RNA of the helper virus. Thus, the genome of Fujinami virus must be the 28S RNA, which corresponds approximately to a molecular weight of 1.7 x 10(6) or 5300 nucleotides. Fujinami viral RNA shares several oligonucleotides with helper viral RNA at both 3' and 5' ends but contains a unique sequence of at least 3000 nucleotides in the middle of the genome. Fujinami viral RNA contains no src-specific oligonucleotides of the Rous sarcoma virus genome and did not hybridize with DNA complementary to the src sequences. The 60,000-dalton src protein of Rous sarcoma virus was undetectable in Fujinami virus-transformed cells. Instead, these transformed cells contain a protein of 140,000 daltons precipitable by antisera against virion proteins, which is likely to be the transforming protein of this virus.

Alpharetrovirus↗
PubMed1993

Increased expression of the src proto-oncogene in hairy cell leukemia and a subgroup of B-cell lymphomas.

The c-src proto-oncogene encodes a M(r) 60,000 phosphoprotein, pp60c-src, with tyrosine-specific protein kinase activity. We have used an immune complex protein kinase assay for pp60c-src to analyze a spectrum of B-cell neoplasms. pp60c-src activity was elevated in all five hairy cell leukemia specimens and in a number of the large cell and immunoblastic lymphomas; neoplasms representing later stages in B-cell development. pp60c-src activity was low in neoplastic cells which correspond to early and intermediate stages in B-cell development (acute and chronic lymphatic leukemia, lymphoblastic lymphoma, small lymphocytic lymphoma). The enhanced pp60c-src activity was associated with high levels of pp60c-src protein. However, increased expression of c-src was not associated with amplification or gross structural rearrangement of the c-src gene. This preliminary study demonstrates elevated levels of pp60c-src protein and tyrosine protein kinase activity in neoplasms corresponding to the later stages of B-cell ontogeny.

Blotting, Southern↗
PubMed1997

Splicing efficiency of human immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2.

We have previously demonstrated that an exon splicing silencer (ESS) is present within human immunodeficiency virus type 1 (HIV-1)tat exon 2. This 20 nucleotide (nt) RNA element acts selectively to inhibit splicing at the upstream 3'splice site (3'ss #3) flanking this exon. In this report, we have used in vitro splicing of mutated RNA substrates to determine the sequences necessary and sufficient for the activity of the ESS. The activity of the ESS within tat exon 2 maps to a 10 nt core sequence CUAGACUAGA. This core sequence was sufficient to inhibit splicing when inserted downstream from the 3'ss of the heterologous Rous sarcoma virus src gene. Mutagenesis of the interspersed purines in the polypyrimidine tract of the tat exon 2 3'ss to pyrimidines resulted in a significant increase in splicing efficiency indicating that 3'ss#3 is suboptimal. The ESS acts to inhibit splicing at the optimized 3'splice sites of both the HIV-1 tat and RSV src constructs but with a reduced efficiency compared to its effect on suboptimal 3'splice sites. The results indicate that both the ESS and a suboptimal 3'splice site act together to control splicing at the 3'splice site flanking at exon 2.

Avian Sarcoma Viruses↗
PubMed1983

Cultured cells transformed by Rous sarcoma virus: a genetically defined model and its phenotype.

The mechanism by which Rous sarcoma virus transforms cells is better understood at the molecular level than that of any other oncogenic agent. The gene (src) responsible for transformation has been identified and its nucleotide sequence has been determined. The transforming protein (pp60src) has been identified and an enzymatic activity assigned to it. The unusual enzymatic activity of pp60src (phosphorylation of proteins on tyrosine) has allowed us to identify a large number of putative targets of this protein. And genetic evidence indicates that the phosphorylation of various targets is responsible for generating the various manifestations of the transformed phenotype. What can this model system contribute to understanding of hereditary large bowel cancer? First of all, it provides an intellectual paradigm for analyzing the mechanism by which a single autosomal dominant gene can alter the metabolism and regulatory behavior of a cell. A cellular homolog of src or of some other onc gene could be responsible for hereditary colon cancer. Second, it provides a model for understanding why some "markers" of malignancy are not invariably associated with cancer: since the oncogenic protein can interact with a variety of primary targets giving rise to the various parameters of transformation, not every sort of biological effect need be necessary for malignancy. Third, it points out that the various syndromes which constitute hereditary colon cancer may well be due to a single gene: since mutations in the src gene are capable of generating a variety of distinct phenotypic alterations in infected cells, different from that generated by the wild-type virus, it certainly is conceivable that different alleles of a single transforming gene could give rise to the different types of hereditary colon cancer. Whether this is the explanation for the various forms of hereditary colon cancer, or whether they result from the activities of several different onc genes can only be determined by identification of the gene(s) at the molecular level. Finally, this model system has provided information which may prove useful in improving the specificity of cancer chemotherapy. Since production of plasminogen activator seems to correlate well with growth in soft agar and tumorigenicity, an anti-cancer prodrug which is activated specifically by cells producing plasminogen activator might be selectively toxic to malignant cells. We have in fact synthesized such drugs and shown them to be selectively toxic in vitro to malignant cells (Carl et al 1980). In vivo tests of these agents are in progress.

Avian Sarcoma Viruses↗
PubMed2002

Third position codon composition suggests two classes of genes within the Cauliflower mosaic virus genome.

The translation of viral mRNAs by host ribosomes is essential for infection. Hence, codon usage of virus genes may influence efficiency of infection. In addition, composition of nucleotides in the third position within codons of genes can reflect evolutionary relationships. In this study, third position codon composition was examined for the seven genes of eight Cauliflower mosaic virus isolates. Genes IV-VII had similar codon composition values and were termed Class 1 genes. Genes I-III possessed corresponding codon composition values and were termed Class 2 genes. The codon composition values of Class 1 and genes differed significantly. Neither Class 1 nor Class 2 genes had codon composition values identical to that of the host plant, Arabidopsis thaliana. However, Class 1 genes possessed codon composition values closer to those of the host than Class 2 genes. Examination of the genomes of three Rous sarcoma virus isolates indicated that codon composition values were similar for the gag, pol, and env genes but these genes differed significantly from the src genes. Since codon composition values for Rous sarcoma virus distinguished a "foreign" gene from the rest of the viral genome, it is possible that the Cauliflower mosaic virus genome is composed of genes from two different sources. Others have suggested that Cauliflower mosaic virus evolved in this manner and our data provide support for this hypothesis.

Arabidopsis↗
PubMed1987

Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart.

Neuronal cells express a pp60c-src variant that displays an altered electrophoretic mobility and a different V8 peptide pattern relative to pp60c-src expressed in tissues of non-neuronal origin. To determine whether the neuronal form of pp60c-src is encoded by a brain-specific messenger RNA, a mouse brain complementary DNA (cDNA) library was screened with a chicken c-src probe and a 3.8-kilobase c-src cDNA clone was isolated. This clone encodes a 60-kilodalton protein that differs from chicken or human pp60c-src primarily in having six extra amino acids (Arg-Lys-Val-Asp-Val-Arg) within the NH2-terminal 16 kilodaltons of the molecule. S1 nuclease protection analysis confirmed that brain c-src RNA contains an 18-nucleotide insertion at the position of the extra six amino acids. This insertion occurs at a position that corresponds to a splice junction in the chicken and human c-src genes. The isolated c-src cDNA clone encodes a protein that displays an identical V8 peptide pattern to that observed in pp60c-src isolated from tissues of neuronal origin.

Amino Acid Sequence↗
PubMed1986

Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1.

We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses.

Adenoviruses, Human↗
PubMed1985

RSV provirus with same flanking sequences is found on different size classes of Chinese hamster chromosomes.

Rous sarcoma virus(RSV)-transformed Chinese hamster fibroblasts, containing approximately ten copies of the DNA domain comprising a single provirus and its flanking cellular sequences, were arrested in metaphase, and the chromosomes were fractionated by size in a sucrose gradient. The resolution of polymorphic ribosomal genes, the dihydrofolate reductase gene, and the c-src gene demonstrated that the gradient can distinguish between small, medium, and large chromosomes. The same DNA domain carrying the RSV provirus was found to be associated with chromosomes of all three size classes. Polymorphic copies of the domain in small and large chromosomes could be distinguished from those in medium-sized chromosomes because of the polymorphism in the XhoI and EcoRI sites on 5' and 3' adjacent cellular sequences, respectively. The presence of the same provirus domain on different chromosomes, together with the karyological data showing trisomies in the same chromosome size classes, suggest that the provirus domain, possibly the entire replicon, was duplicated and transferred to different nonhomologous chromosomes, and the transfer was followed by duplication of the target chromosomes. The possibility that proviral long terminal repeats might be involved in replicon transfer is discussed.

Animals↗
PubMed1994

Activation of human immunodeficiency virus 1 gene expression by the src oncoprotein.

Several genes are induced constitutively in cells transformed by the v-src oncoprotein. This induction is generally dependent on the activation of transcription factors binding to src-responsive elements of the promoter. In previous studies, we showed that the induction of the CEF-4/9E3 cytokine gene by pp60v-src is dependent on the PRDII/kappa B domain of the promoter (Dehbi et al., 1992). In this investigation, we describe the activation of the HIV-1 LTR by pp60v-src and show that a region of 30 bp containing the two NF-kappa B binding sites is critical for activation of the promoter. The induction was dependent on transformation since non-transforming forms of pp60v-src had little or no effect on the promoter. The expression of proviral DNA and the release of p24 antigen were also increased by v-src indicating that viral replication was stimulated in src-transformed cells. The effect of v-src on HIV-1 gene expression occurred in the presence or in the absence of the tat viral trans-activator, in fibroblasts and in Jurkat T lymphocytes. These results indicate that several promoters controlled by PRDII/kappa B may be activated constitutively in v-src transformed cells and suggest that oncogenic tyrosine kinases may play a role in the induction of viruses with a PRDII/kappa B-controlled promoter.

Animals↗