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.

scholarly journals Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

1985 ◽  
Vol 100 (3) ◽  
pp. 692-703 ◽  
Author(s):  
J J Lin ◽  
D M Helfman ◽  
S H Hughes ◽  
C S Chou
Keyword(s):  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Actin Binding ◽  
Transformed Cells ◽  
Two Dimensional ◽  
Tropomyosin Isoforms ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed 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.

scholarly journals Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

1982 ◽  
Vol 2 (6) ◽  
pp. 653-665 ◽  
Author(s):  
Ricardo Martinez ◽  
Kenji D. Nakamura ◽  
Michael J. Weber
Keyword(s):  
Protein Kinases ◽  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Cellular Protein ◽  
Transformed Cells ◽  
Phosphoamino Acid ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Phosphorylation on tyrosine residues mediated by pp60srcappears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid aroundMr's of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defectivesrcdeletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded toMr's of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60srcor the combined action of pp60srcand pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.

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 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.

scholarly journals Recovery of avian sarcoma virus from tumors induced by transformation-defective mutants.

1977 ◽  
Vol 146 (6) ◽  
pp. 1735-1747 ◽  
Author(s):  
H Hanafusa ◽  
C C Halpern ◽  
D L Buchhagen ◽  
S Kawai
Keyword(s):  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Genetic Interaction ◽  
Avian Sarcoma Virus ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus ◽  
Avian Sarcoma ◽  
Chicken Embryo Fibroblasts

Transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), which contains deletions in the gene responsible for transformation (src gene), are unable to transform chicken embryo fibroblasts in vitro. Injection of some of these td mutants into newborn chickens resulted in the formation of sarcomas from which sarcoma virus was unfailingly recovered. The possibility that transforming RSV was present in the td virus preparations was excluded by further purification of the td viruses. Morphology of the foci induced by the newly recovered sarcoma virus was distinct from that of foci induced by the parental Schmidt Ruppin strain of RSV. It is suggested that the new sarcoma virus was generated as a result of the genetic interaction between the genomes of td virus and chicken cells.

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.

Sign in / Sign up

Export Citation Format

Share Document