Identification of a polypeptide encoded by the avian sarcoma virus src gene.

Abstract

Two techniques were used to search for the polypeptide encoded by the avian sarcoma virus (ASV) src gene. First, antiserum from rabbits bearing ASV-induced fibrosarcomas was used to immunoprecipitate a transformation-specific antigen from ASV-transformed chick embryo fibroblasts. This antigen has an apparent molecular weight (Mr) of 60,000. Second, the 3' one-third of the ASV genome, selected by oligo(dTcellulose chromatography and sucrose gradient sedimentation, was translated in a mRNA-dependent reticulocyte cell-free lysate. This RNA species programmed the synthesis of a polypeptide that comigrated with the transformation-specific antigen of Mr 60,000 immunoprecipitated from transformed cells. The methionine-containing tryptic peptides from the polypeptides of Mr 60,000 obtained from translation in vitro and from immunoprecipitation were found to be identical upon twodimensional fractionation.Some RNA-containing tumor viruses are able to cause rapid and efficient transformation of cells in culture as well as the production of sarcomas in animals. The avian sarcoma viruses (ASV) are particularly interesting because they produce tumors in both avian and mammalian species (1, 2). The high efficiency of cell transformation by these viruses, the availability of viral mutants, and the considerable structural information about the viral genome make ASV useful for the study of the molecular events that occur during viral oncogenesis.Although analyses of viral mutants indicate that a polypeptide encoded by the src gene of ASV is likely to be responsible for the maintenance of cell transformation, such a polypeptide had not been identified biochemically or immunologically when we began our studies. Several ASV conditional, temperature-sensitive (ts) mutants, and nonconditional, deletion mutants, termed transformation-defective (td), have been isolated that have an altered capacity to transform cells (3-5). Studies with ts mutant-infected cells indicate that a virus-encoded product affects gross cytoskeletal structure, growth in soft agar, and rate of sugar uptake, characteristics that are phenotypic markers of transformation in culture. Avian or mammalian cells infected with a ts mutant can be reversibly switched to either the transformed or "normal" state within a few-hours after the appropriate temperature shift. Inhibitors of protein synthesis do not affect the conversion from the transformed to normal state upon shift to the nonpermissive temperature, but do interfere with the appearance of the transformed phenotype when cultures are shifted from the nonpermissive to permissive temperature. These virus mutants can replicate normally at both temperatures, suggesting that a virus-encoded, heat-labile protein, which is not an essential structural component of the virus, is responsible for cell transformation (6).All ts transformation mutations occur in a region of the ASVThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "...

src gene product from different strains of avain sarcoma virus: Kinetics and possible mechanism of heat inactivation of protein kinase activity from cells infected by transformation-defective, temperature-sensitive mutant and wild-type virus

“…1 Heat Inactivation Kinetics of the Kinase Associated with p60s"c. It is well established that the 3' end of the ASV genome contains a genetic element called src that is responsible for the ability of the virus to transform chicken embryo cells (3, 4). Because the p6Osrc is believed to be the product of the src gene (5,6), one would expect that some transformation-defective (T class) ts mutants should have a thermolabile kinase if the kinase activity were a true enzymatic activity of p6Os8c. We have, therefore, compared the kinetics of heat inactivation of the kinase activity after lysates were incubated at 42'C for various lengths of time prior to immune precipitation.…”

“…The in vitro cell-free translation of the 3' one-third of the RSV genome, the region known to. contain the viral transforming information [the src gene (3, 4)1, yielded a protein of the same molecular weight and similar peptide composition (5,6). Hence, this protein is thought to be responsible for RSVinduced cell transformation and has been named p6Osrc.…”

c-Src and IL-6 inhibit osteoblast differentiation and integrate IGFBP5 signalling

“…This is a non-receptor tyrosine kinase, which belongs to a family of cytoplasmic tyrosine kinases (Protein Tyrosine Kinase family, PTKs), capable of communicating with a large number of different receptors 14,15 . c-Src activity maintains osteoblasts in a less differentiated status, negatively regulating the expression of differentiation markers 16 .…”

Cellular information in the genome of recovered avian sarcoma virus directs the synthesis of transforming protein.

“…3A, lanes b, c, believed to be the message for p60src (8,26). A 26S species containing sarc-specific sequences, and presumed to be the messenger for p60sarc, has been identified in the cytoplasm of normal cells (4,5).…”

“…Erikson and coworkers have identified a protein (7), called p6Osrc, that is encoded by the src region of the ASV genome (8). It is a phosphoprotein (9) of 60 kilodaltons (kDal) that is found closely associated with, and may be identical to, a protein kinase activity (10)(11)(12).…”