scholarly journals Cellular partitioning of beta-1 integrins and their phosphorylated forms is altered after transformation by Rous sarcoma virus or treatment with cytochalasin D.

1991 ◽  
Vol 2 (4) ◽  
pp. 271-283 ◽  
Author(s):  
B Haimovich ◽  
B J Aneskievich ◽  
D Boettiger
Keyword(s):  
Rous Sarcoma Virus ◽  
Extraction Procedure ◽  
Insoluble Fraction ◽  
Cellular Adhesion ◽  
Cytochalasin D ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Laemmli Sample Buffer ◽  
Adhesion Plaque ◽  
Chicken Embryo Fibroblasts

A sequential extraction procedure of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) buffer followed by RIPA or Laemmli sample buffer was developed to define two distinct subpopulations of beta-1 integrins in primary chicken embryo fibroblasts. Extraction of cells in culture revealed an association of adhesion plaque-localized integrin with the CHAPS-insoluble fraction. Phosphorylated integrins were found in both fractions, but the specific phosphorylation was 12-fold higher in the CHAPS insoluble fraction. The phosphorylation was evenly distributed between phosphoserine and phosphotyrosine. Transformation by Rous sarcoma virus caused a redistribution of integrin to rosettes and an increase in total integrin phosphorylation. Treatment with cytochalasin D caused a redistribution of the adhesion plaque-associated integrin into lacelike structures and reduced the level of integrin phosphorylation. These treatments also caused an altered distribution of phosphorylated integrin between the CHAPS soluble and insoluble fractions. These results suggest a role for integrin phosphorylation in the assembly and disassembly of cellular adhesion structures.

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.

Analysis of Adhesion Plaque Structure and Function in the Mechanism of Transformation by Rous Sarcoma Virus

1982 ◽  
Vol 40 ◽  
pp. 110-113
Author(s):  
L.R. Rohrschneider ◽  
M.J. Rosok ◽  
L.E. Gentry
Keyword(s):  
Rous Sarcoma Virus ◽  
Mammalian Cells ◽  
Neoplastic Transformation ◽  
Structure And Function ◽  
Excellent Opportunity ◽  
Cells In Culture ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
And Function ◽  
Adhesion Plaque

Rous sarcoma virus (RSV) was originally isolated from a fibrosarcoma of a chicken. This virus also will efficiently infect and transform all avian cells in culture as well as most mammalian cells. The mechanism of transformation by RSV is therefore universal and this system offers an excellent opportunity to investigate the mechanism of neoplastic transformation.

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.

Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells

1987 ◽  
Vol 7 (1) ◽  
pp. 371-378
Author(s):  
J E DeClue ◽  
G S Martin
Keyword(s):  
Rous Sarcoma Virus ◽  
Peptide Mapping ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Tyrosine Residues ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Chicken Embryo Fibroblasts ◽  
In Cells

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.

scholarly journals The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes.

1993 ◽  
Vol 13 (12) ◽  
pp. 7408-7417 ◽  
Author(s):  
L B Vogel ◽  
D J Fujita
Keyword(s):  
Tyrosine Kinases ◽  
Rous Sarcoma Virus ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Rous Sarcoma ◽  
Cell Extracts ◽  
Pi3k Activity ◽  
Sarcoma Virus ◽  
Chicken Embryo Fibroblasts

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.

Sign in / Sign up

Export Citation Format

Share Document