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.

scholarly journals 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.

scholarly journals Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.

1985 ◽  
Vol 5 (5) ◽  
pp. 916-922 ◽  
Author(s):  
M D Resh ◽  
R L Erikson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Membrane Vesicles ◽  
Terminal Region ◽  
Transformed Cell ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxy Terminal

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.

Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes

1985 ◽  
Vol 5 (5) ◽  
pp. 916-922
Author(s):  
M D Resh ◽  
R L Erikson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Membrane Vesicles ◽  
Terminal Region ◽  
Transformed Cell ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxy Terminal

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.

v-src mutations outside the carboxyl-coding region are not sufficient to fully activate transformation by pp60c-src in NIH 3T3 cells

1988 ◽  
Vol 8 (2) ◽  
pp. 704-712
Author(s):  
S Reddy ◽  
P Yaciuk ◽  
T E Kmiecik ◽  
P M Coussens ◽  
D Shalloway
Keyword(s):  
Rous Sarcoma Virus ◽  
Terminal Region ◽  
Cell Protein ◽  
Coding Region ◽  
3T3 Cells ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxyl Terminal ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Previous studies have shown that carboxyl-terminal mutation of pp60c-src can activate its transforming ability. Conflicting results have been reported for the transforming ability of pp60c-src mutants having only mutations outside its carboxyl-terminal region. To clarify the effects of such mutations, we tested the activities of chimeric v(amino)- and c(carboxyl)-src (v/c-src) proteins at different dosages in NIH 3T3 cells. The focus-forming activity of Rous sarcoma virus long terminal repeat (LTR)-src expression plasmids was significantly reduced when the v-src 3' coding region was replaced with the corresponding c-src region. This difference was masked when the Rous sarcoma virus LTR was replaced with the Moloney murine leukemia virus LTR, which induced approximately 20-fold more protein expression, but even focus-selected lines expressing v/c-src proteins were unable to form large colonies in soft agarose or tumors in NFS mice. This suggests that pp60c-src is not equally sensitive to mutations in its different domains and that there are at least two distinguishable levels of regulation, the dominant one being associated with its carboxyl terminus. v/c-src chimeric proteins expressed with either LTR had high in vitro specific kinase activity equal to that of pp60v-src but, in contrast, were phosphorylated at both Tyr-527 and Tyr-416. Total cell protein phosphotyrosine was enhanced in cells incompletely transformed by v/c-src proteins to the same extent as in v-src-transformed cells, suggesting that the carboxyl-terminal region may affect substrate specificity in a manner that is important for transformation.

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.

Glycoprotein tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryo fibroblasts

1990 ◽  
Vol 10 (2) ◽  
pp. 837-841
Author(s):  
L M Kozma ◽  
A B Reynolds ◽  
M J Weber
Keyword(s):  
Tyrosine Phosphorylation ◽  
Rous Sarcoma Virus ◽  
Cellular Membrane ◽  
Soft Agar ◽  
Morphological Transformation ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Molecular Masses ◽  
Phenotypic Transformation ◽  
Chicken Embryo Fibroblasts

The level of tyrosine phosphorylation of cellular glycoproteins isolated by wheat germ agglutinin chromatography in cells infected with a variety of kinase-positive/transformation-defective src mutants was examined in an effort to identify cellular membrane proteins whose phosphorylation correlates with phenotypic transformation. We have identified two glycoproteins, with molecular masses of 95 and 135 kilodaltons, whose phosphorylation correlates with morphological transformation, growth in soft agar, and an increase in the rate of 2-deoxyglucose uptake. The strong correlation obtained between transformation and phosphorylation of these proteins suggests that they may be substrates for pp60src which are important in the process of transformation.

Reconstitution of the Rous sarcoma virus transforming protein pp60v-src into phospholipid vesicles

1988 ◽  
Vol 8 (5) ◽  
pp. 1896-1905
Author(s):  
M D Resh
Keyword(s):  
Lipid Bilayers ◽  
Rous Sarcoma Virus ◽  
Membrane System ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Amino Terminal Domain

An artificial membrane system was developed to study the molecular basis for interaction of pp60v-src, the Rous sarcoma virus transforming protein, with lipid bilayers. pp60v-src was extracted from cell membranes by detergent solubilization and reincorporated into phospholipid vesicles. Reconstituted pp60v-src retained tyrosine kinase activity and was integrally associated with the liposome through a 10-kilodalton (kDa) amino-terminal domain. The same 10-kDa domain was shown to anchor pp60v-src to the plasma membrane of transformed cells. Reconstitution experiments performed with nonmyristylated pp60v-src proteins revealed that these polypeptides did not interact with phospholipid vesicles. In contrast, myristylated, soluble pp60v-src molecules (including a highly purified pp60v-src preparation) could be reconstituted into liposomes, but their interaction with the liposomal bilayer was not mediated by the 10-kDa amino-terminal domain. When membrane proteins were included during reconstitution of purified pp60v-src, binding through the 10-kDa anchor was restored. A model is presented to accommodate the different types of interactions of pp60v-src with liposomes; the model postulates the existence of an additional membrane component that anchors the pp60v-src polypeptide to the phospholipid bilayer.

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.

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