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Query: UNIPROT:P06889 (Mol)
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Quail myogenic cells infected with temperature sensitive (ts) mutants of Rous sarcoma virus (RSV) exhibit a temperature-dependent transformation and block of differentiation. When the cells are allowed to differentiate at the restrictive temperature (41 degrees C) and then shifted back to the permissive temperature (35 degrees C), a sharp reduction in the accumulation of muscle-specific mRNAs is observed, following reactivation of the transforming protein pp60v-src. A kinetic analysis of this down-regulation reveals that the reduction in the accumulation of muscle-specific transcripts occurs fairly rapidly within 6 to 20 h after the shift back, depending on the mRNA analyzed. Studies on transcription of endogenous muscle-specific genes and a transfected chloramphenicol acetyltransferase reporter gene under the control of muscle-specific promoters, at the different temperatures, suggest that the oncogene exerts its control mainly at the transcriptional level. On the contrary, transcription of the CMD1 gene, the avian homolog of the mouse muscle regulatory MyoD gene, is not significantly affected by the oncogene both in proliferating myoblasts and in myotubes shifted back to 35 degrees C. These findings are consistent with the conclusion that v-src blocks myogenesis by controlling transcription of muscle-specific genes independently of cell proliferation. Furthermore, they suggest the existence of an alternative pathway, not requiring the silencing of CMD1 transcription, through which the oncogene exerts its effect.
Mol Cell Biol 1991 Jun
PMID:Transcription of muscle-specific genes is repressed by reactivation of pp60v-src in postmitotic quail myotubes. 164 48

Rous sarcoma virus-based retroviral vectors were constructed to compare three different approaches for coexpressing two genes in individual infected cells. All vectors expressed the upstream gene (lacZ) from the Rous sarcoma virus long terminal repeat, while the downstream gene (the chloramphenicol acetyltransferase gene [cat] or v-src) was expressed in one of three ways: from a subgenomic mRNA generated by regulated splicing, from a strong internal promoter, or from the encephalomyocarditis virus internal ribosome entry site (IRES). Both biochemical and immunohistochemical assays of cultured cells showed that the encephalomyocarditis virus IRES provided the most efficient means for coexpressing two genes from a single provirus. Most importantly, most cells infected by a LacZ-IRES-CAT virus expressed both LacZ and CAT, whereas most cells infected by internal promoter or regulated splicing vectors expressed either LacZ or CAT but not both. In addition, viral titers were highest with IRES vectors. Presumably, use of the IRES avoids transcriptional controls and RNA processing steps that differentially affect expression of multiple genes from internal promoter and regulated splicing vectors. Finally, we injected a LacZ-IRES-v-Src virus into chicken embryos and then identified the progeny of infected cells with a histochemical stain for LacZ. LacZ-positive cells in both skin and mesenchyme displayed morphological abnormalities attributable to expression of v-src. Thus, IRES vectors can be used to coexpress a reporter gene and a bioactive gene in vivo.
Mol Cell Biol 1991 Dec
PMID:The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. 165 18

Insulin-like growth factor-I (IGF-I) attenuates GH gene expression by a receptor-mediated mechanism in pituitary cells. We, therefore, isolated neomycin-resistant stable GC cell transfectants over-expressing human IGF-I receptor cDNA (IGFIR-cDNA) cloned in an Rous sarcoma virus-directed expression vector. A transfection control contained the IGFIR-cDNA cloned in the reverse orientation. Southern analysis confirmed incorporation of human IGFIR-cDNA sequences into rat genomic DNA. Immunoprecipitation of metabolically labeled [35S]methionine stably transfected cells revealed a 200-kDa human IGF-I receptor precursor protein. Growth rate and basal GH secretion were not altered in transfected cells. Although transfected and control cells had a similar Kd for IGF-I binding (0.43 and 0.40 nM, respectively), IGF-I-binding sites were induced 17-fold (384,000 vs. 22,000 sites/cell). Treatment of cells with IGF-I (6.5 nM) maximally attenuated GH secretion by 80% compared to 40% attenuation in control cells (P less than 0.0001). Maximal suppression of GH in transfectants occurred within 15 h of treatment, and GH secretion by control cells was only maximally suppressed after 42 h. The ED50 of IGF-I suppression of GH secretion in transfectants after 15 h was 0.5 nM. These results demonstrate that transfectants overexpressing human IGF-I receptor are hyperresponsive to exogenous IGF-I. These data indicate that IGF-I receptor number plays an important role in mediating the signal transduction of IGF-I to the GH gene.
Mol Endocrinol 1991 Jul
PMID:Insulin-like growth factor-I (IGF-I) attenuation of growth hormone is enhanced by overexpression of pituitary IGF-I receptors. 165 32

We recently reported the 5'-flanking nucleotide sequence of a putative glutamine synthetase (GS) gene from 3T3-L1 cells (Bhandari, B., Beckwith, K. D. & Miller, R. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5789-5793). We now find that this gene (GSr) has many, but not all, of the characteristics of a typical retroposon. It lacks introns, it contains a short poly(A) tract at its 3' end; it is flanked by 10-base pair (bp) direct repeats; and it corresponds closely at its 5' end to the transcription start site of the intron-containing GS gene (GSi) (Kuo, C. F. & Darnell, J. E., Jr. (1989) J. Mol. Biol. 208, 45-56). GSr includes a full-length, uninterrupted coding sequence that differs little (less than 5%) from that of the intron-containing gene. By contrast, the 5'-flanking sequence of GSr has no similarity with that of GSi. The first 1,029 bp of the GSr 5'-flanking sequence drives expression of a promoterless bacterial chloramphenical acetyltransferase (CAT) gene in transfected HeLa cells at a level comparable to that of the Rous sarcoma virus promoter. Analysis of variably deleted GSrCAT fusions genes in both HeLa and 3T3-L1 cells indicates that full promoter activity of the 1,029-bp sequence requires greater than 348 bp. Moreover, nuclear extract from 3T3-L1 adipocytes as well as murine liver protects four segments in the GSr 5'-flanking sequence from DNase I digestion. Nevertheless, reverse transcription of RNA from 3T3-L1 adipocytes, mouse adipocytes, or mouse liver followed by primer-directed enzymatic amplification of the reverse transcripts reveals the presence of GSi transcripts but the absence of GSr transcripts. Thus, the 5'-flanking sequence of GSr is an active promoter that drives transcription of GSrCAT fusion genes and includes binding domains for proteins that have the potential to regulate transcription. We conclude that the intronless murine GS gene isolated from 3T3-L1 cells arose as a retroposon that was inserted into the genome downstream of a potentially active promoter.
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PMID:A functional promoter flanks an intronless glutamine synthetase gene. 167 62

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.
Mol Cell Biol 1991 Oct
PMID:Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase. 168 25

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.
Mol Cell Biol 1990 Apr
PMID:Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. 169 Aug 50

A set of genes is rapidly inducible when quiescent fibroblasts are stimulated by growth factors or by the activation of temperature-sensitive retroviral protein-tyrosine kinases. Most of these so-called immediate-early genes were cloned by differential cDNA hybridization. DNA sequence analysis identified many of them as putative members of the growth factor or of the transcription factor gene family, suggesting a role in signal transmission during the G0-to-G1 transition. In this study, we identified one of the genes that are rapidly inducible by the retroviral protein-tyrosine kinases v-Src and v-Fps of Rous sarcoma virus and Fujinami sarcoma virus, respectively, as the rhoB gene, a member of the ras gene superfamily whose products are GTP-binding proteins, rhoB is transiently activated at the transcriptional level by v-Fps and by epidermal growth factor. Its labile RNA is inducible in the presence of cycloheximide but not of actinomycin D. rhoB is strongly induced by epidermal growth factor and by platelet-derived growth factor both in subconfluent, serum-starved and in density-arrested Rat-2 fibroblasts. Fetal calf serum is a poor inducer, particularly in density-arrested cells, and phorbol esters do not increase rhoB expression at all. These data suggest that rhoB is inducible by protein-tyrosine kinases through a pathway not involving the activation of protein kinase C. Neither the closely related rhoC and rhoA genes nor the distantly related c-H-ras gene is rapidly inducible by mitogens. Thus, rhoB is the first known member of the small GTP-binding proteins among the immediate-early genes.
Mol Cell Biol 1991 Jul
PMID:The ras-related gene rhoB is an immediate-early gene inducible by v-Fps, epidermal growth factor, and platelet-derived growth factor in rat fibroblasts. 171 Jul 70

The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.
Mol Cell Biol 1991 Aug
PMID:The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1. 171 1

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.
Mol Cell Biol 1991 Aug
PMID:Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5' exons and possible mechanism for the genesis of the 3' end of v-src. 171 5

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.
Mol Cell Biol 1991 Oct
PMID:Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein. 171 25


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