The expression of pp60src and its associated protein kinase activity in cells infected with different transformation-defective temperature-sensitive mutants of rous sarcoma virus

Rübsamen, Helga; Ziemiecki, Andrew; Friis, Robert R.; Bauer, Heinz

doi:10.1016/0042-6822(80)90113-0

Cited by 21 publications

(11 citation statements)

References 9 publications

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“…The half-lives of the mutant proteins differed little from the wild type and differed little at 35 and 42°C. These results are consistent with our previous observations on the amounts of pp605rc present at 35 and 42°C in cells infected with these temperature-sensitive mutants (22). Sefton et al (25), using metabolic labeling methods, have documented that the pp6Osrc of the Schmidt-Ruppin D strain is metabolically stable, with a half-life in excess of 2 h. The discrepancy between our results, primarily with the Prague A strain, and those of Sefton et al (25) may reflect either differences in the stability of the pp6"rc of different strains or the state of health of the cell or a combination of both.…”

Section: Discussionsupporting

confidence: 83%

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Ziemiecki¹,

Friis²,

Bauer³

1982

Mol. Cell. Biol.

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The half-life of metabolically labeled pp6(Yrc of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp6(Yrc with tumor-bearing rabbit serum. These experiments showed that pp6(Yrc has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp6(Src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42°C) or permissive (35°C) temperature. The half-life of the ppWsr-associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp6O!rc. The apparent contradiction between the half-lives of the kinase activity and the 135S]methionine-labeled pp6fysc protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp6(Yrc, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.The continual expression of the src gene of Rous sarcoma virus (RSV) is required for maintenance of the transformed phenotype in infected cells (19). A 60,000-dalton phosphoprotein, pp61)Src, has been shown to be a product of the src gene (4,5,18), and a protein kinase activity capable of phosphorylating in vitro several substrates at tyrosine has been demonstrated to be intimately associated with pp6&Wrc (6,7,10,13). Despite extensive biochemical investigation of pp60srC, little is known about the in vivo h'alf-life of the molecule itself and its associated kinase activity (25). In this paper we describe experiments aimed at determining the half-lives of metabolically labeled pp6(sYc and the associated protein kinase activity. We show that pp60Yrc metabolically labeled with [35S]methionine has a short half-life of approximately 60 min. In experiments in which the decay of the pp6&frc-associated kinase activity was followed in cells treated with cycloheximide and other inhibitors of protein synthesis, a long half-life of many hours was observed. These results could be reconciled by our observation that the protein synthesis inhibitors used to demonstrate the decay of pp64src-associated kinase activity greatly prolonged the half-life of metabolically labeled pp60sc.

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Section: Discussionsupporting

confidence: 83%

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Ziemiecki¹,

Friis²,

Bauer³

1982

Mol. Cell. Biol.

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“…Temperature-sensitive mutants of RSV have been shown to be defective in this kinase activity at the nonpermissive temperature (e.g. Rubsamen et al, 1980;Sefton et al, 1980;, and a similar defect appears to be present in the LA25 mutant (A. Goldberg, personal communication).…”

Section: Discussionmentioning

confidence: 94%

“…Several studies indicate that the transforming activity of pp60 srC involves its protein kinase activity (e.g. Sefton, Hunter, Beemon & Eckhart, 1980;Rubsamen, Ziemiecki, Friis & Bauer, 1980). Temperature-sensitive mutants of RSV have been shown to be defective in this kinase activity at the nonpermissive temperature (e.g.…”

Section: Discussionmentioning

confidence: 99%

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

1986

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Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.

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“…Thereby, the introduction of a separate reporter gene such as the bacterial chloramphenicol acetyltransferase (CAT) gene is redundant. The src gene expression can easily be followed by measuring its kinase activity [19]. The constructs used in this study were prepared by subcloning from EcoRIfragment B of SR-A-RSV [4][5][6] into the SV40 late region replacement vector pA 11 SVL2 (Fig.…”

mentioning

confidence: 99%

Inhibition of SV40 DNA replication by Rous sarcoma virus LTR enhancer

Binninger

Ferdinand

Rübsamen‐Waigmann

1989

Archives of Virology

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Simian virus 40 (SV40) late region recombinant constructs containing the Rous sarcoma virus (RSV) src gene along with RSV enhancer stimulated expression but completely abolished SV40 DNA replication. Constructs, in which the heterologous enhancer sequences were omitted, did replicate normally in African green monkey kidney cells and, in the presence of helper virus, gave rise to infectious progeny. Inhibition of SV40 DNA replication follows a cis-acting mechanism and is most likely due to a conformational change of the SV40 chromatin structure.

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The expression of pp60src and its associated protein kinase activity in cells infected with different transformation-defective temperature-sensitive mutants of rous sarcoma virus

Cited by 21 publications

References 9 publications

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Inhibition of SV40 DNA replication by Rous sarcoma virus LTR enhancer

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