Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins

1991 ◽  
Vol 11 (3) ◽  
pp. 1607-1613
Author(s):  
M Matsuda ◽  
B J Mayer ◽  
H Hanafusa
Keyword(s):  
Escherichia Coli ◽  
Kinase Activity ◽  
Binding Activity ◽  
Transformed Cells ◽  
Dependent Manner ◽  
Tyrosine Kinase Activity ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Sarcoma Virus ◽  
Avian Sarcoma

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.

scholarly journals Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins.

1991 ◽  
Vol 11 (3) ◽  
pp. 1607-1613 ◽  
Author(s):  
M Matsuda ◽  
B J Mayer ◽  
H Hanafusa
Keyword(s):  
Escherichia Coli ◽  
Kinase Activity ◽  
Binding Activity ◽  
Transformed Cells ◽  
Dependent Manner ◽  
Tyrosine Kinase Activity ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Sarcoma Virus ◽  
Avian Sarcoma

The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.

scholarly journals Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells.

1985 ◽  
Vol 5 (9) ◽  
pp. 2399-2404 ◽  
Author(s):  
S Sugano ◽  
H Hanafusa
Keyword(s):  
Kinase Activity ◽  
Specific Activity ◽  
Transformed Cells ◽  
Rous Sarcoma ◽  
Phosphatidylinositol Kinase ◽  
Sarcoma Virus ◽  
Oncogene Products ◽  
Avian Sarcoma ◽  
Chicken Embryo Fibroblasts ◽  
Pi Kinase

We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140gag-fps was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from P140gag-fps, indicating that P140gag-fps has no detectable PI kinase activity.

Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells

1985 ◽  
Vol 5 (9) ◽  
pp. 2399-2404
Author(s):  
S Sugano ◽  
H Hanafusa
Keyword(s):  
Kinase Activity ◽  
Specific Activity ◽  
Transformed Cells ◽  
Rous Sarcoma ◽  
Phosphatidylinositol Kinase ◽  
Sarcoma Virus ◽  
Oncogene Products ◽  
Avian Sarcoma ◽  
Chicken Embryo Fibroblasts ◽  
Pi Kinase

We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140gag-fps was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from P140gag-fps, indicating that P140gag-fps has no detectable PI kinase activity.

Towards a molecular description of cell transformation by avian sarcoma virus

1980 ◽  
Vol 210 (1180) ◽  
pp. 387-396 ◽  
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Molecular Mass ◽  
Cellular Protein ◽  
Transformed Cells ◽  
Relative Molecular Mass ◽  
Protein Kinase Activity ◽  
Avian Sarcoma Virus ◽  
Sarcoma Virus ◽  
Avian Sarcoma

The avian sarcoma virus transforming gene product has been identified and partially purified from extracts of transformed cells. It is a phosphoprotein with a relative molecular mass of 60 000 (pp60 src ) with two major sites of phosphorylation. pp60 src appears to be a cyclic-AMP-independent protein kinase as judged by protein phosphorylation with partly purified fractions. The specificity of the phosphorylation observed was judged by inhibition with anti-pp60 src IgG but not by normal IgG and by the fact that the protein kinase activity isolated from ts transformation-mutant infected cells was more thermolabile than that from wild-type transformed cells, thus showing more directly the origin of the enzymic activity. A cellular protein substrate of pp60 src has been identified as a 34000 molecular mass protein. These data together suggest that protein phosphorylation by pp60 src may be a function of the molecule that plays a major role in transformation.

scholarly journals Involvement of a phosphotyrosine protein phosphatase in the suppression of platelet-derived growth factor receptor autophosphorylation in ras-transformed cells

1993 ◽  
Vol 293 (1) ◽  
pp. 215-221 ◽  
Author(s):  
L Tomáska ◽  
R J Resnick
Keyword(s):  
Growth Factor ◽  
Phosphatase Activity ◽  
Receptor Tyrosine Kinase ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Platelet Derived Growth Factor ◽  
Transformed Cells ◽  
Pdgf Receptor ◽  
Tyrosine Kinase Activity ◽  
Phosphotyrosine Protein Phosphatase

The nature of the suppression of platelet-derived growth factor (PDGF) receptor autophosphorylation in ras-transformed NIH 3T3 fibroblasts was investigated. The PDGF receptor from ras-transformed cells that had been purified by wheatgerm-lectin affinity chromatography displayed normal PDGF-induced autophosphorylation, indicating that the receptor is not irreversibly modified. Various phosphotyrosine-protein-phosphatase inhibitors did not reverse the inhibition of PDGF-receptor kinase in crude membrane preparations from ras-transformed cells. However, treatment of intact ras-transformed cells both with 2 mM sodium orthovanadate and with 20 microM phenylarsine oxide restored PDGF-receptor tyrosine-kinase activity to a level similar to that observed in normal cells. Direct measurement of the phosphatase activities in crude cellular fractions revealed a 2.5-fold higher membrane-associated phosphotyrosine-protein-phosphatase activity in ras-transformed cells, whereas phosphoserine-protein-phosphatase activity remained unchanged between the cell lines. These data suggest that the suppression of the PDGF-receptor tyrosine-kinase activity in ras-transformed cells is mediated via an inhibitory component, distinct from the receptor, that may be positively regulated by the dephosphorylation of tyrosine residue(s).

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants

1984 ◽  
Vol 4 (8) ◽  
pp. 1508-1514
Author(s):  
A W Stoker ◽  
P J Enrietto ◽  
J A Wyke
Keyword(s):  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Tyrosine Kinase Activity ◽  
Rous Sarcoma ◽  
Heat Labile ◽  
Src Gene ◽  
Sarcoma Virus

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

v-src inhibits differentiation via an extracellular intermediate(s)

1985 ◽  
Vol 5 (10) ◽  
pp. 2847-2850
Author(s):  
P R Langer-Safer ◽  
S R Lehrman ◽  
A M Skalka
Keyword(s):  
Conditioned Medium ◽  
Transformed Cells ◽  
Parental Line ◽  
Avian Sarcoma Virus ◽  
Stable Factor ◽  
Heat Stable ◽  
Sarcoma Virus ◽  
The Relationship ◽  
Avian Sarcoma

Murine 3T3L1 preadipocytes transformed by avian sarcoma virus were unable to differentiate in response to insulin or dexamethasone plus 1-methyl-3-isobutylxanthine, both potent inducers of differentiation of the nontransformed 3T3L1 parental line. Conditioned medium from transformed cells contained a relatively heat-stable factor(s) which inhibited the differentiation of untransformed parental 3T3L1 cells but did not induce any changes in their morphology. A protease-sensitive mitogen was also detected in the medium. The relationship between the two activities remains to be elucidated.

scholarly journals Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.

1985 ◽  
Vol 5 (1) ◽  
pp. 263-267 ◽  
Author(s):  
A M Antler ◽  
M E Greenberg ◽  
G M Edelman ◽  
H Hanafusa
Keyword(s):  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Cell Transformation ◽  
Transformed Cells ◽  
Detectable Change ◽  
Tyrosine Residues ◽  
Microfilament Bundles ◽  
Sarcoma Virus ◽  
Avian Sarcoma ◽  
Chicken Embryo Fibroblasts

The level of phosphotyrosine in vinculin was determined in chicken embryo fibroblasts transformed by various strains of avian sarcoma virus. As previously reported (Sefton et al., Cell 24:165-174, 1981), vinculin was phosphorylated at tyrosine residues in most cultures examined, but the level varied greatly and no detectable change was found in cultures infected with Fujinami sarcoma virus or UR2 sarcoma virus. Regardless of the level of vinculin phosphorylation, the number of organized microfilament bundles was found to be decreased in all transformed cells. These results strongly suggest that tyrosine phosphorylation of vinculin is not an obligatory step in cell transformation by this class of oncogenes, nor is it correlated with the associated cytoskeletal disarray.

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