scholarly journals Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

1983 ◽  
Vol 3 (9) ◽  
pp. 1656-1664 ◽  
Author(s):  
S L Lin ◽  
E A Garber ◽  
E Wang ◽  
L A Caliguiri ◽  
H Schellekens ◽  
...  
Keyword(s):  
Growth Rate ◽  
Interferon Alpha ◽  
Rous Sarcoma Virus ◽  
Specific Activity ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
Rous Sarcoma ◽  
Protease Digestion ◽  
Related Phenotype ◽  
Sarcoma Virus

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells

1983 ◽  
Vol 3 (9) ◽  
pp. 1656-1664
Author(s):  
S L Lin ◽  
E A Garber ◽  
E Wang ◽  
L A Caliguiri ◽  
H Schellekens ◽  
...  
Keyword(s):  
Growth Rate ◽  
Interferon Alpha ◽  
Rous Sarcoma Virus ◽  
Specific Activity ◽  
Protein Kinase Activity ◽  
Dependent Manner ◽  
Rous Sarcoma ◽  
Protease Digestion ◽  
Related Phenotype ◽  
Sarcoma Virus

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates

1984 ◽  
Vol 4 (7) ◽  
pp. 1213-1220
Author(s):  
M S Collett ◽  
S K Belzer ◽  
A F Purchio
Keyword(s):  
Tyrosine Phosphorylation ◽  
Rous Sarcoma Virus ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Terminal Region ◽  
Transformed Cell ◽  
Cell Lysates ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Correlation between phosphorylation of a 34,000-molecular-weight protein, pp60src-associated kinase activity, and tumorigenicity in transformed and revertant vole cells

1984 ◽  
Vol 4 (1) ◽  
pp. 212-215
Author(s):  
J F Nawrocki ◽  
A F Lau ◽  
A J Faras
Keyword(s):  
Molecular Weight ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Cell Types ◽  
Cell Protein ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Tumorigenic Potential ◽  
Sarcoma Virus ◽  
Weight Protein

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.

scholarly journals Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

1982 ◽  
Vol 2 (2) ◽  
pp. 199-206 ◽  
Author(s):  
T D Gilmore ◽  
K Radke ◽  
G S Martin
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

scholarly journals Correlation between phosphorylation of a 34,000-molecular-weight protein, pp60src-associated kinase activity, and tumorigenicity in transformed and revertant vole cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 212-215 ◽  
Author(s):  
J F Nawrocki ◽  
A F Lau ◽  
A J Faras
Keyword(s):  
Molecular Weight ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Cell Types ◽  
Cell Protein ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Tumorigenic Potential ◽  
Sarcoma Virus ◽  
Weight Protein

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.

Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src

1982 ◽  
Vol 2 (2) ◽  
pp. 199-206
Author(s):  
T D Gilmore ◽  
K Radke ◽  
G S Martin
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Defective Mutant ◽  
Sarcoma Virus

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

scholarly journals Staurosporine inhibits tyrosine-specific protein kinase activity of Rous sarcoma virus transforming protein p60.

1987 ◽  
Vol 40 (5) ◽  
pp. 706-708 ◽  
Author(s):  
HIROFUMI NAKANO ◽  
EIJI KOBAYASHI ◽  
ISAMI TAKAHASHI ◽  
TATSUYA TAMAOKI ◽  
YASUKO KUZUU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Specific Protein Kinase
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