scholarly journals Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

1985 ◽  
Vol 5 (5) ◽  
pp. 1058-1066 ◽  
Author(s):  
H Iba ◽  
F R Cross ◽  
E A Garber ◽  
H Hanafusa
Keyword(s):  
Rous Sarcoma Virus ◽  
Cell Transformation ◽  
Spontaneous Mutation ◽  
Cellular Protein ◽  
Cell Protein ◽  
Wild Type ◽  
Low Level ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src

1985 ◽  
Vol 5 (5) ◽  
pp. 1058-1066
Author(s):  
H Iba ◽  
F R Cross ◽  
E A Garber ◽  
H Hanafusa
Keyword(s):  
Rous Sarcoma Virus ◽  
Cell Transformation ◽  
Spontaneous Mutation ◽  
Cellular Protein ◽  
Cell Protein ◽  
Wild Type ◽  
Low Level ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

scholarly journals The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes.

1981 ◽  
Vol 1 (1) ◽  
pp. 43-50 ◽  
Author(s):  
E Erikson ◽  
R Cook ◽  
G J Miller ◽  
R L Erikson
Keyword(s):  
Rous Sarcoma Virus ◽  
Direct Consequence ◽  
Cellular Protein ◽  
Cell Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Transforming Proteins ◽  
Avian Sarcoma ◽  
Transforming Genes ◽  
Chicken Embryo Fibroblasts

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.

The same normal cell protein is phosphorylated after transformation by avian sarcoma viruses with unrelated transforming genes

1981 ◽  
Vol 1 (1) ◽  
pp. 43-50
Author(s):  
E Erikson ◽  
R Cook ◽  
G J Miller ◽  
R L Erikson
Keyword(s):  
Rous Sarcoma Virus ◽  
Direct Consequence ◽  
Cellular Protein ◽  
Cell Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Transforming Proteins ◽  
Avian Sarcoma ◽  
Transforming Genes ◽  
Chicken Embryo Fibroblasts

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.

scholarly journals Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector.

1986 ◽  
Vol 6 (11) ◽  
pp. 3900-3909 ◽  
Author(s):  
A Tanaka ◽  
D J Fujita
Keyword(s):  
Rous Sarcoma Virus ◽  
Cell Transformation ◽  
Retroviral Vector ◽  
Protein Kinase Activity ◽  
Coding Region ◽  
Specific Sequence ◽  
Coding Sequence ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus

We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that all introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.

scholarly journals A Functional Link between Dynamin and the Actin Cytoskeleton at Podosomes

2000 ◽  
Vol 150 (2) ◽  
pp. 377-390 ◽  
Author(s):  
Gian-Carlo Ochoa ◽  
Vladimir I. Slepnev ◽  
Lynn Neff ◽  
Niels Ringstad ◽  
Kohji Takei ◽  
...  
Keyword(s):  
Rous Sarcoma Virus ◽  
Fluorescence Recovery After Photobleaching ◽  
Cell Transformation ◽  
Wild Type ◽  
Functional Link ◽  
Rous Sarcoma ◽  
Attachment Sites ◽  
Actin Turnover ◽  
Sarcoma Virus ◽  
Dynamin 2

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aaG273D abolished podosomes while GFP-dynaminK44A was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.

Expression of a molecularly cloned human c-src oncogene by using a replication-competent retroviral vector

1986 ◽  
Vol 6 (11) ◽  
pp. 3900-3909
Author(s):  
A Tanaka ◽  
D J Fujita
Keyword(s):  
Rous Sarcoma Virus ◽  
Cell Transformation ◽  
Retroviral Vector ◽  
Protein Kinase Activity ◽  
Coding Region ◽  
Specific Sequence ◽  
Coding Sequence ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus

We studied the expression of a molecularly cloned human c-src gene, c-src-1, localized on chromosome 20, whose coding region consists of 11 exons and spans a 19.5-kilobase (kb) distance. Using a replication-competent retroviral vector derived from molecularly cloned Rous sarcoma virus DNA (pSRA-2), we obtained two constructs: one (pSR-CS) carrying the unmodified human c-src coding sequence and another (pSR-CVS) with a chimeric gene formed between the human c-src gene and the carboxy-terminal 12-amino acid v-src-specific coding sequence. From chicken embryo fibroblasts transfected with these DNA constructs, infectious viruses designated as WO CS and WO CVS, respectively, were recovered. WO CS virus did not cause cell transformation, whereas WO CVS induced cell transformation. Analyses of the proviral DNAs indicated that all introns were spliced out such that the 19-kb inserts were converted to 1.7-kb cDNA forms. Analyses of src proteins in infected cells, using monoclonal antibody MAb327 against v-src protein, showed the following results. The CVS and CS src proteins were about 60 and 61 kilodaltons in size, respectively; the specific protein kinase activity assayed in vitro of the CVS src protein was about 20-fold higher than that of the CS src protein and comparable to that of the v-src protein; the transforming CVS src protein reacted to an antibody against a v-src-specific peptide, whereas the CS src protein did not. These results indicate that the human c-src gene has a potential transforming ability and suggest that the v-src-specific sequence played an important role in the generation of Rous sarcoma virus.

scholarly journals Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rebecca J. Kaddis Maldonado ◽  
Breanna Rice ◽  
Eunice C. Chen ◽  
Kevin M. Tuffy ◽  
Estelle F. Chiari ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Rous Sarcoma Virus ◽  
Nuclear Export ◽  
Live Cell ◽  
Viral Rna ◽  
Wild Type ◽  
Gag Protein ◽  
Rous Sarcoma ◽  
Sarcoma Virus

ABSTRACT Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

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