Temperature-sensitive transformation by Rous sarcoma virus and temperature-sensitive protein kinase activity (original) (raw)
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Journal of Virology
The transforming protein of Rous sarcoma virus, p60"rc, has associated with it a protein kinase activity. We examined whether a correlation exists between the cellular concentration of enzymatically active p60Brc and the degree to which chick cells are transformed by mutants of Rous sarcoma virus which are temperaturesensitive for transformation. Such a correlation does exist, but cells infected with some mutants could be shown to contain, at the nonpermissive temperature, an amount of protein kinase activity equal to 30 to 40% of that in a wild-type transformed cell. We quantified the amount of virus-induced protein kinase activity by precipitation of p6Osrc with an excess of antitumor antiserum. Our initial measurements of activity were serious underestimates, due to the lability of the protein kinase activity associated with p60`rc of at least four temperaturesensitive mutants. In fact, no activity at all was associated with p60src of tsLA90 when immunoprecipitation was performed by standard means. However, when immunoprecipitation was performed with procedures which minimize inactivation, it became apparent both that cells transformed by tsLA90 contained protein kinase activity and that cells infected with either NY68 or BK5 contained at the nonpermissive temperature, one-third to one-half as much activity as wild-type transformed cells. This level of activity was much more than that arising from p60arc in uninfected cells. In uninfected cells we found an amount of protein kinase activity which varied from 3 to 5% as much as that in a virally transfonned cell. The lability of the protein kinase activity of each of these mutants is a further demonstration that this activity is essential for the transformation of cells by Rous sarcoma virus. So as to explain the high protein kinase levels in cells infected with NY68 and BK5 at the nonpermissive temperature, the idea that transformation may be a response to a small quantitative change in the total activity of p60`rc and the possibility that there may be more than one viral function which is essential for transformation are discussed.
Journal of Virology
In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p6O', the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p6O0 which was synthesized in vitro in the reticulocyte lysate, just as it is with p6O' which is obtained from transformed chick and mnmalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 410C. In vitro translation of ts NY68 virion RNA at 300C resulted in efficient synthesis of immunoprecipitable p60w, but very inefficient synthesis of an immunoprecipitable protein kinase. The p60w obtained by in vitro translation of wild-type virion RNA was more than 20-fold more active as a protein kinase than was that obtained from ts NY68 RNA. The correlation in the case of ts NY68 of a deficiency in protein kinase activity with an inability to transform celLs at high temperature suggests that the protein kinase activity associated with p60w is indeed critical to cellular transformation.
Journal of Virology, 1981
Two temperature-sensitive mutants of Fujinami sarcoma virus were isolated and characterized. Cells infected with the mutants were temperature sensitive in focus formation, colony formation, increased sugar uptake, and synthesis of plasminogen activator. The changes between transformed and nontransformed states of cultures were completely reversible by shifting the temperature. A Fujinami sarcoma virus-specific protein of 130,000 daltons, p130, was synthesized in mutant-infected cells regardless of the temperature, but the immunoprecipitates of p130 from extracts of infected cells were active in protein kinase only when cells had been incubated at the permissive temperature. These results appear to indicate that p130 is the transforming protein of Fujinami sarcoma virus, and that its protein kinase activity plays a crucial role in cell transformation by this virus.
Proceedings of the National Academy of Sciences, 1981
A single viral protein (pp60c) mediates neoplastic transformation of cells infected with Rous sarcoma virus. Immunoprecipitation of pp60W has revealed two cellular proteins (Mr 50,000 and 89,000) that appear to associate with pp60 in a specific manner. Neither of the cellular proteins has been well characterized, but it is thought that both may participate in the function of pp60'. Treatment of avian cells with unphysiological temperature or certain chemical agents amplifies the production of several proteins in the manner of the "heat shock' response earlier described for Drosophika We report here that one of these proteins, with a molecular weight of 89,000, is identical to the 89kilodalton protein found associated with pp60. The 89-kilodalton protein is a major constituent of both uninfected and infected cells, even in the absence of inducing agents, but only a small fraction of this protein appears to associate with pp60" in cells transformed by Rous sarcoma virus. The complex containing pp6jIC and the 89-kilodalton protein can be precipitated by an immune reaction involving pp601 alone. The complexed form of the 89kilodalton protein did not react directly with antibodies but regained its reactivity subsequent to release from the complex. We conclude that the 89-kilodalton protein is bound to pp60 in a relatively stable complex. We suggest that the 89-kilodalton pro
Product of in vitro translation of the Rous sarcoma virus src gene has protein kinase activity
Journal of Virology, 1979
In vitro translation of Rous sarcoma virus virion RNA resulted in the synthesis of a protein kinase which, when immunoprecipitated with antitumor serum, phosphorylated the immunoglobulin heavy chain. Even though in vitro translation of virion RNA resulted in the synthesis of a number of polypeptides which were recognized by antitumor serum, control experiments demonstrated that an immunoprecipitable protein kinase activity was found only when an immunoprecipitable p60src, the polypeptide product of the src gene, was synthesized. A protein kinase with similar properties was therefore intimately associated with p60src which was synthesized in vitro in the reticulocyte lysate, just as it is with p60src which is obtained from transformed chick and mammalian cells. It is therefore highly unlikely that this association is artifactual. ts NY68 is a mutant of Rous sarcoma virus which is able to transform cells at 36 but not at 41 degrees C. In vitro translation of ts NY68 virion RNA at 30 d...
Journal of Virology
The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild-type src sequences. Three of the mutants, which were derived from the Schmidt-Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant.
Membrane and cytoskeletal changes in cells after transformation by Rous Sarcoma virus
Biochemical Society Transactions, 1987
Rous sarcoma virus (RSV) transforms cells by the production of a single viral gene product, pp60'"". This protein is a 60 kDa phosphoprotein with tyrosine kinase activity and in several cell types is localized at the plasma membrane in particular in areas of membrane-cytoskeletal interaction (Collett et al., 1980; Rohrschneider, 1980). Although several intracellular targets for pp60'"" have been identified, in no case has tyrosine-specific phosphorylation of any of these substrates been shown to be necessary for transfor-Abbreviations used: RSV, Rous sarcoma virus; TPA, 12-tetradecanoyl phorbol-13-acetate; CEF, chick embryo fibroblasts; ts. temperature sensitive; HPFN, human plasma fibronectin.
Journal of Virology, 1981
The transformation-specific protein pp60 src coded for by avian sarcoma viruses and its associated protein kinase activity is present in virus particles of Rous sarcoma virus, Schmidt-Ruppin strain, subgroup D. Quantitative comparison of the immunoglobulin G-phosphorylating activity in Schmidt-Ruppin D virus and Schmidt-Ruppin D virus-transformed fibroblasts indicated that there was two- to fourfold less activity in the virus particles. Disruption of virus particles with nonionic detergent demonstrated that the protein kinase activity fractionated together with the viral membrane protein gp85. Therefore, viral membranes were isolated by floating detergent-disrupted virus through a discontinuous sucrose density gradient. At a characteristic density corresponding to 26% sucrose, viral membranes were identified by the radioactively labeled viral glycoprotein and furthermore by the membrane marker enzyme Na + -K + -stimulated, Mg 2+ -activated ATPase and were visualized by electron micr...
Molecular and Cellular Biology, 1984
The phosphorylation of a 34,000-molecular-weight (34K) cell protein, purported to be a substrate of the avian retrovirus pp60src-associated protein kinase activity, was compared in three types of Rous sarcoma virus-infected vole cells: fully transformed cells, partial revertants which are morphologically normal in appearance but retain their tumorigenic potential, and full revertants which are similar to normal vole cells in all parameters including a lack of tumorigenicity. Although similar amounts of 34K protein are present in all three cell types, phosphorylation of the 34K protein was significantly reduced in the full revertant cell type. The reduced phosphorylation occurred at the tyrosine residue.