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Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. I. Relationship between differentiation and tyrosine kinase of src gene product.

Quail embryonic pectoral myoblasts fuse with each other at 35.5 degrees C and 41 degrees C to essentially equal extents. When the myoblasts were transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), their fusion and biochemical processes of differentiation became temperature-sensitive: their fusion occurred at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature, suggesting that the fusion was regulated by the viral transforming gene. Fusion of the transformed cells proceeded more rapidly and synchronously than that of the parent cells at 41 degrees C, and was completely suppressed at the permissive temperature, unlike that of the parent cells. These transformed cells were used to examine the relationship between myogenic differentiation and the tyrosine kinase activity of the src gene product. In spite of the temperature sensitivity of transformation, results showed that expressions of the src gene at 35.5 degrees C and 41 degrees C were similar. However, the level of tyrosine-phosphorylated protein was decreased at 41 degrees C. Moreover, myoblast fusion could occur at 35.5 degrees C in the presence of herbimycin A, an inhibitor of the tyrosine kinase activity of the src gene product. These results indicate that the tyrosine kinase activity of the src gene product is closely associated with regulation of myogenic differentiation of the cells.

Animals↗

DNA sequence of the viral and cellular src gene of chickens. 1. Complete nucleotide sequence of an EcoRI fragment of recovered avian sarcoma virus which codes for gp37 and pp60src.

Recovered avian sarcoma virus is a class of virus obtained from chicken tumors induced by mutants of Rous sarcoma virus which have a deletion in the src gene. We have determined the entire nucleotide sequence of a 3.1-kilobase EcoRI DNA fragment of molecularly cloned recovered avian sarcoma virus DNA. This DNA fragment contains part of the env gene and the entire src gene. Amino acid sequences of both gene products were deduced from the DNA sequences; the predicted amino acid sequences were verified by protein studies. An env protein (gp37) was found to be composed of 205 amino acids with three glycosylation sites. gp37 had a long stretch of hydrophobic residues near the carboxyl terminus. The src gene product, pp60src, was composed of 526 amino acids and contained the possible sites for tyrosine and serine phosphorylation. The amino acid sequences predicted in this study differ significantly from the amino acid sequence predicted previously for the Schmidt-Ruppin strain of Rous sarcoma virus.

Amino Acid Sequence↗

The expression of c-src gene in the carcinogenesis process of human cardia adenocarcinoma.

AIM:To investigate the activation, expression of c-src gene and its role in the carcinogenetic process of human cardia adenocarcinoma (CA).METHODS:Fifty six cases of CA, 34 cases of normal, 36 cases of protiferative epithelia adjacent to carcinoma, and 20 cases of lymph node metastases of CA were studied for PP60(c-src),the expression product of c-src gene immunohistochemically by using the specific monoclonal antibody,Mab327.RESULTS: The positive rates of PP60(c-src) in the normal epithelia,protiferative epithelia, CA and lymph node metastases were 29.4% (10/34), 94.4% (34/36), 71.4% (40/56) and 60.0%(12/20), respectively, among them, the differences of the positive rates were statistically significant (P < 0.01). The expression levels of PP60(c-src) in CA and proli ferative epithelia were significantly higher than that in the normal epithelia(P< 0.01).The PP60(c-src) positive rates in the papillary, tubular, poorly different-tiated and mucous adenocarcinoma were 75.0% (6/8), 81.8% (18/22), 50.0% (10/20) and 100.0% (6/6), respectively, whereas those of tubular and mucous adenocarcinomas were significantly higher than those of papillary and poorly differentiated adenocar-cinomas (P < 0.05), and the PP60(c-src) expression levels of tubular and mucous adenocarcinomas were also significantly higher than those of papillary and poorly differentiated adenocarcinomas (P< 0.01).CONCLUSION:The activation and expression of c-src gene are associated with the initiation and development of human CA; the protein amount of PP60(c-src)increased during the process of carcinogenesis; and PP60(c-src) expression is also related to lymph node metastases.

Journal Article↗

Characterization of avian retroviruses carrying activated transforming human c-src genes and of steps involved in expression of activated src-PKases in vitro.

We have isolated four activated transforming human c-src mutants derived spontaneously from viruses carrying the normal human c-src (SRC) genes in the form of Rous sarcoma virus. These mutants induced transformed cell morphology distinguishable from each other in vitro as well as tumors in chicks, whereas normal SRC-carrying viruses did not. Analyses of the transforming SRC proteins together with the normal SRC protein showed that levels of the carboxy-terminal Tyr-phosphorylation were negatively correlated with both transforming ability and protein kinase (PKase) activity as determined by in vitro autophosphorylation. It was observed that two cell lysis methods, that is, NP-40 and RIPA, yielded two different phosphorylated forms of transforming SRC proteins: one possessed low levels of phosphorylation at the autophosphorylation site and the other possessed high levels of phosphorylation at this site. Using the two types of transforming SRC preparations, we have studied in vitro SRC-PKase reactions in relation to in vivo and in vitro autophosphorylation and in vitro phosphorylation of an exogenous substrate. A possible functional relationship between autophosphorylation and SRC-PKase expression is discussed.

Animals↗

Stimulation of growth rate of chondrocytes by Rous sarcoma virus is not coordinated with other expressions of the src gene phenotype.

Infection and transformation of chondrocytes by Rous sarcoma viruses (RSVs) (Schmidt-Ruppin, Prague) stimulated the rate of cell growth. In contrast, several transformation-defective (td) mutants (tdPRA, tdNY105, tdNY106, tdNY107, and tdNY108) retaining various sizes of the src gene did not stimulate cell growth, indicating that the stimulation of growth of chondrocytes is due to the function of the src gene. With the use of various T (transformation)-class temperature-sensitive (ts) mutants of RSV, growth stimulation of chondrocytes by the src gene was examined. It was found that there are two types of T-class ts mutants with regard to the stimulatory effect on the growth of chondrocytes. One type (tsNY68) stimulates cell growth at both permissive (36 degrees C) and nonpermissive (41.5 degrees C) temperature, as does the wild type of RSV. Another type (ts GI201 [clone 9]) stimulates cell growth only at the permissive temperature. Chondrocytes infected with either of these two types of T-class ts mutants showed ts properties in other transformation markers, such as uptake of 2-deoxy-D-glucose, change of cell morphology, and focus formation. These data indicate that the effect of the src gene on cell growth does not occur coordinately with other transformation markers.

Animals↗

Activation of the cellular src gene by transducing retrovirus.

Newly isolated strains of avian sarcoma virus, S1 and S2, were shown to have the transduced cellular src gene as their viral transforming gene (Yamagishi et al., Virology 137:266-275, 1984). In this work, the S1 and S2 genomes were molecularly cloned, and the junction sequences between the viral genomes and the c-src genes and the complete nucleotide sequences of the v-src genes transduced in these viruses were determined. Data on the junction sequences suggested that 5' recombination had occurred between the 5'-noncoding region of c-src and the 5' region of the gag sequence encoding p19 in both viruses and that 3' recombination had occurred in the last coding exon of c-src with either the middle portion of the env sequence encoding gp85 for S1 or the 3' portion of pol coding for reverse transcriptase for S2. Comparison of the amino acid sequences of the S1 and S2 src products deduced from the nucleotide sequences (pp62S1-src and pp62S2-src with that of c-src protein (pp60c-src) indicated that in pp62S1-src the 8 carboxy-terminal amino acid residues of the total of 533 in pp60c-src are replaced by 43 residues translated from the env sequence at the wrong frame. In pp62S2-src, on the other hand, the 14 carboxy-terminal amino acids of pp60c-src are replaced by the 38 carboxy-terminal residues of reverse transcriptase. The mechanism of c-src transduction and the structural changes necessary for pp60c-src activation are discussed.

Animals↗

Transformation-defective mutants of Rous sarcoma virus with src gene deletions of varying length.

The RNAs of transformation-defective (td) deletion mutants of the Schmidt-Ruppin strain of Rous sarcoma virus were found to vary in size when compared by polyacrylamide gel electrophoresis. Three of seven td mutants appeared to recombine with a mutant of Rous sarcoma virus (Schmidt-Ruppin), which has a temperature-sensitive sarcoma (src) gene and is termed ts68, to give rise to recombinants with a reduced temperature sensitivity. The results suggested that different clones of td mutants exist: some in which the src gene appears to be deleted, and others in which the src gene is only partially deleted. A direct correlation between RNA size and the extent of src gene deletion measured by recombination was not obtained, possibly because the recombination assay could only detect src sequences homologous to the lesion(s) of ts68, whereas the electrophoretic analysis of the RNA measured src deletions as well as other possible alterations of the RNA.

Avian Sarcoma Viruses↗

Retroviral src gene expression in continuous marrow culture increases the self-renewal capacity of multilineage hematopoietic stem cells.

To define the action of the retroviral src gene on hematopoietic stem cells, C57BL/6 x DBA/2 (B6D2F1) mouse long-term marrow cultures were infected at initiation with Moloney murine leukemia virus (MuLV) pseudotypes of src-recombinant retroviruses with the src gene inserted in the env region of an amphotropic MuLV (src-Ampho), or in the gag region of Moloney MuLV (src-Mo). Other cultures were infected with Friend spleen focus-forming virus polycythemia-inducing strain (SFFVp), Moloney MuLV, or amphotropic MuLV, or were uninfected controls. Harvested nonadherent cells were tested weekly for multilineage, granulocyte-erythroid-megakaryocyte macrophage (CFU-GEMM) colony formation in vitro in recombinant murine IL-3 and erythropoietin, and individual colonies were removed, split 1:2, with half of each replated for in vitro self-renewal and the other half examined morphologically for number of hematopoietic cellular lineages, or tested for release of MuLV and src virus. Cultures infected with src-Ampho, src-Mo, or SFFVp demonstrated a significant increase in cumulative nonadherent cell and CFU-GEMM production. There was prolonged self-renewal over seven serial transfers of individual CFU-GEMM from src virus-infected cultures over seven serial transfers, and five of 61 individual colonies from the second or third generations contained detectable v-src gene sequences, but none released detectable src virus. Self-renewal of CFU-GEMM was similar to that with permanent IL-3-dependent cell line B6SUtA. In contrast, MuLV-infected or control uninfected cultures produced fewer cells, and self-renewal of CFU-GEMM did not exceed three generations. IL-3-dependent clonal hematopoietic progenitor cell lines, derived from each culture group, formed no detectable tumors in vivo; however, each released the original helper and/or transforming virus. Adherent cell lines, derived from src-Ampho-infected cultures released src virus and formed fibro-sarcomas in vivo. The data support the conclusion that src-recombinant virus expression in long-term marrow cultures increases the self-renewal capacity of multilineage hematopoietic stem cells.

Animals↗

Viral and cellular src genes contribute to the structure of recovered avian sarcoma virus transforming protein.

Recovered avian sarcoma viruses (rASVs) were obtained from tumors induced by certain transformation-defective (td) mutants of Schmidt-Ruppin strain Rous sarcoma virus of subgroup A (SR-A). The genomes of these td SR-A mutants lack most but not all of the src gene. rASV genomes, however, possess intact src genes, which are largely derived from cellular genetic information, presumably an endogenous cellular gene called c-src, which shares considerable homology with the viral src. To further define the genetic origin of rASV src, we examined by tryptic peptide analysis the product of this gene, pp60src, from rASV and SR-A, as well as the normal cellular homolog pp60c-src. We found peptides unique to each putative "parental" protein present together in maps of rASV p60src, demonstrating that the endogenous cellular c-src gene itself contributes to the structure of rASV pp60src. Certain isolates of rASV encode pp60srcS of altered apparent molecular weight. In these cases, the variation in structure was located in the amino-terminal portion of the protein. That such polymorphism can be tolerated suggests that this region of the protein is less critical to the ability of these agents to transform cells.

Amino Acids↗

Widespread distribution of the c-src gene product in nerve cells and axon terminals in the adult rat brain.

The regional and cellular distribution of the proto-oncogene product pp60c-src, a member of the family of membrane-associated tyrosine-specific protein kinases, was analysed in adult rat brain. High-resolution SDS-PAGE allowed analysis of both the 'fibroblast' 60-kDa form and a variant, 61-kDa neuron-specific form of the c-src gene product which is encoded by an alternately processed c-src mRNA. Studies of microdissected brain regions showed that all CNS regions contained both forms of the enzyme, the 61-kDa form predominating in most regions with high content of gray matter and high density of synapses. Lesion-induced degenerations of specific neuronal elements in the basal ganglia decreased the level of both forms of the c-src gene product both in regions where cell bodies had been lesioned and in regions where nerve terminals had degenerated. The 61-kDa form of the enzyme appeared somewhat more sensitive to the effects caused by these lesions than the 60-kDa form. These results indicate that, within the mature mammalian brain, both cell body regions and nerve terminals of many, and possibly all, nerve cells contain both forms of the c-src gene product, the 61-kDa form being most highly enriched in the nerve cells. These results suggest that the enzyme may be involved in pleiotropic functions, including signal transduction in nerve terminals.

Animals↗

Structural organization of a src gene from Xenopus laevis.

By sequence analysis of genomic clones, the exon-intron structure of one of the two src genes from Xenopus laevis has been determined. The coding region of the gene is interrupted by 10 introns whose locations are identical to the introns in the coding regions of the src genes of human and chicken. The 5' untranslated region is contained on a separate exon with no sequence conservation relative to the corresponding region of the chicken gene. The 5' untranslated region of the Xenopus gene contains a G + C-rich stem-loop sequence and two ATG triplets. A 1.4-kb fragment containing the 5' untranslated region and sequences upstream of it acts as a promoter when introduced in the correct orientation into X. laevis cell lines. The DNA sequence of this fragment lacks the typical arrangement of TATA and CCAAT sequences but contains the ATGCAAAT octamer sequence and a (TA)39 sequence.

Amino Acid Sequence↗

Calvasculin, as a factor affecting the microfilament assemblies in rat fibroblasts transfected by src gene.

Cell transformations accompany alterations in cell morphology and microfilament patterns. Calvasculin encodes mRNA termed pEL-98, 18A2, 42A, p9Ka, or mts1, found to be elevated in several metastatic cell lines. We report the elevation of calvasculin expression in SR-3Y1 cells, which show disappearance of ordered microfilaments, compared to that in 3Y1 cells and that the similar distribution of calvasculin to that of actin filaments. Interestingly, calvasculin co-sediments with F-actin and bundles actin filaments in a Ca(2+)-dependent manner. This activity, along with the elevation of calvasculin following transformation, suggests that the disorganization of filaments in SR-3Y1 cell is due to the cross-linking activity of calvasculin.

Actin Cytoskeleton↗

Is the product of the src gene a promoter?

Addition of a potent promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), to primary avian tendon or chicken embryo fibroblast cells infected with a temperature-sensitive mutant of Rous sarcoma virus produced a complete transformed phenocopy at the nonpermissive temperature by the criteria tested. While normal, uninfected cultures also shifted towards a transformed phenotype after TPA addition, they did not achieve the same degree of morphological and biochemical alteration seen in virus-infected, TPA-treated cells. It is proposed that viral carcinogenesis, despite its rapidity, may occur in two stages: an "initiation" step caused by expression of a part of viral genome other than src (or by integration) and a promotion step (itself a multistep process) caused by the activation of the src gene. The src gene product could be enhanced or replaced by other promoting agents.

Avian Sarcoma Viruses↗

Site-directed mutagenesis of the src gene of Rous sarcoma virus: construction and characterization of a deletion mutant temperature sensitive for transformation.

Transformation of cells by Rous sarcoma virus results from the expression of the viral src gene product, pp60src. Site-directed mutagenesis techniques have been used to construct defined deletion mutations within the src gene of Prague A strain of Rous sarcoma virus. The deletion of DNA sequences at the Bg/II restriction site in the src gene yielded both transformation-defective mutants (tdCH4, 64, and 146) and a mutant temperature sensitive for morphological transformation (tsCH119). The genome of tsCH119 contains an in-phase deletion of approximately 160 base pairs, which mapped to the immediate 3' side of the Bg/II restriction site. Upon infection of chicken cells, tsCH119 encoded a structurally altered src protein, pp53src, containing a deletion of amino acid residues 202 to 255. Immune complexes containing pp53src isolated from tsCH119-infected cells grown at 41 degrees C exhibited only 50% less tyrosine-specific kinase activity than immune complexes isolated from cells grown at 35 degrees C. pp53src immunoprecipitated from tsCH119-infected cells grown at either 35 or 41 degrees C contained phosphoserine and phosphotyrosine. We suggest that tsCH119 represents a class of mutants containing mutations mapping within a functionally important domain of the src protein, distinct from the domain specifying the protein kinase activity.

Animals↗

Characterization of the protein kinase activity of avian sarcoma virus src gene product.

The avian sarcoma virus src gene product, p60src, has been purified 650-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on omega-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p60src is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0. In immunoprecipitates, p60src catalyzed phosphorylation of anti-p60src IgG heavy chains within the variable (VH) domain, which contains the heavy chain portion of the antigen combining site. Crude preparations of p60src contained phosphatase activity able to cleave phosphate from IgG heavy chains; this activity was removed by the purification procedure, and partially purified p60src could phosphorylate the heavy chain of specific antibody in solution. Furthermore, purified p60src catalyzed phosphorylation in solution of the general protein kinase substrate, alpha-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vivo.

Alpharetrovirus↗

Evidence for autoinhibitory regulation of the c-src gene product. A possible interaction between the src homology 2 domain and autophosphorylation site.

In the previous study (Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T., and Fukami, Y. (1990) Biochem. Biophys. Res. Commun. 171, 1152-1159), we found a synthetic peptide, termed peptide A, that inhibited the kinase activity of p60v-src. The peptide A sequence corresponds to residues 137 to 157 of p60v-src which are included in the amino-terminal portion of the src homology 2 domain. In this study, we attempted to specify the inhibitory sequence in this domain and to identify its target site. The most potent peptide A derivative was one that corresponds to residues 140 through 157. The target site of peptide A was assumed to reside in the autophosphorylation site of p60v-src, since synthetic peptides containing the sequence Phe424-Pro-Ile-Lys-Trp428 which is present downstream of the autophosphorylated Tyr416 partially counteracted the inhibitory effect of peptide A. An antibody was prepared against one of such target peptides, termed pepY. Cross-linking experiments showed that 125I-labeled peptide A could bind to p60v-src blotted on a membrane, and the binding was blocked by the anti-pepY antibody but not by other anti-p60v-src antibodies. Conversely, immunoblotting of p60v-src with anti-pepY antibody was blocked by the cross-linking of peptide A to p60v-src. To our surprise, anti-pepY antibody did not affect the p60v-src activity. Furthermore, p60c-src was activated 2- to 6-fold by this antibody. These results suggest that the pepY region in the catalytic domain of p60v-src or of p60c-src is not essential for the catalytic activity but rather is involved in the negative regulation of the kinase activity of p60c-src.

Amino Acid Sequence↗

Two divergent cellular src genes are expressed in Xenopus laevis.

Genomic and cDNA clones of the X. laevis src gene have been isolated and characterized by hybridization and DNA sequence analyses. The haploid genome of X. laevis contains two src genes, which can be distinguished from one another by virtue of sequence divergence in the 3' untranslated regions. Both of the genes are functional as indicated by the fact that oocytes contain RNAs transcribed from each of the genes. The two genes each encode an RNA which is 3.3 kb in length, or twice the length required to encode the 60,000 dalton src protein (pp60). Sequence analysis of the cDNA clones revealed that nearly all of the non-coding sequence is located at the 3' end. The availability of sequence data from cDNA clones has also made it possible for the first time to identify with certainty the carboxyl terminal sequence of a cellular pp60 molecule.

Amino Acid Sequence↗