scholarly journals Detection of vinculin-binding proteins with an 125I-vinculin gel overlay technique.

1983 ◽  
Vol 97 (4) ◽  
pp. 1283-1287 ◽  
Author(s):  
J J Otto
Keyword(s):  
Binding Proteins ◽  
Plaque Component ◽  
Rous Sarcoma ◽  
Sds Page ◽  
Sarcoma Virus ◽  
Overlay Technique ◽  
Triton X ◽  
Adhesion Plaque ◽  
Cytoplasmic Side ◽  
Cellular Fibronectin

Vinculin is an adhesion plaque component localized on the cytoplasmic side of the cell membrane where stress fibers end. To detect vinculin-binding proteins, we have developed an 125I-vinculin gel overlay method. SDS PAGE was used to separate different protein preparations. After fixing the proteins in the gel with methanol-acetic acid, the SDS was removed with ethanol and the proteins renatured in buffer. The gel was then incubated with 125I-vinculin. After extensive washing to remove nonspecifically associated label, the gel was dried and autoradiographed. Chick embryo fibroblasts, their Rous sarcoma virus transformants, and HeLa cells were found to contain two proteins (Mr 220,000 and 130,000) that bound 125I-vinculin strongly and another (Mr 42,000) that bound it moderately. The 130,000-mol-wt protein was identified as vinculin itself, which suggests that it may self-associate. The 42,000-mol-wt protein was identified as actin with which vinculin is known to interact. The identity of the 220,000-mol-wt protein is not known. It is not cellular fibronectin, myosin, or filamin. When fibroblast proteins were separated into Triton X-100 soluble and insoluble fractions, most of the vinculin and the 220,000-mol-wt protein was found to be in the soluble fraction. Chicken gizzard also contained these vinculin-binding proteins along with three others of Mr 190,000, 170,000, and 100,000.

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.

scholarly journals Is there a role for actin in virus budding?

1977 ◽  
Vol 75 (2) ◽  
pp. 593-605 ◽  
Author(s):  
C H Damsky ◽  
J B Sheffield ◽  
G P Tuszynski ◽  
L Warren
Keyword(s):  
Mammary Epithelial Cells ◽  
Sodium Dodecyl ◽  
Structural Features ◽  
Mammary Epithelial ◽  
Cellular Processes ◽  
Rous Sarcoma ◽  
Sds Page ◽  
Electrophoretic Data ◽  
Tumor Virus ◽  
Sarcoma Virus

Electrophoretic data from both sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and acid-urea gels reveal a protein in purified murine mammary tumor virus (MuMTV) which co-migrates with purified chick skeletal muscle actin. 125I-labeling of intact and disrupted virus preparations shows that the actin-like protein is not artifactually adsorbed to the outside of virions during isolation. Quantitative SDS-PAGE and examination of negatively stained preparations show that the actin cannot be accounted for by a contaminating population of virus-free vesicles. The ultrastructure of mammary epithelial cells and of Rous sarcoma virus-transformed chick embryo fibroblasts shows that virus extrusion is associated with filament-containing cellular processes. In particular, MuMTV is released from the ends of long microvilli which contain a bundle of 6-8-nm microfilaments and share other structural features with intestinal microvilli. We suggest that virus nucleoids require an interaction with host cell contractile proteins for their extrusion from the cell.

scholarly journals Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus.

1983 ◽  
Vol 3 (4) ◽  
pp. 731-746 ◽  
Author(s):  
L Rohrschneider ◽  
M J Rosok
Keyword(s):  
Cell Surface ◽  
Rous Sarcoma Virus ◽  
Soft Agar ◽  
Partial Transformation ◽  
Rous Sarcoma ◽  
Independent Functions ◽  
Sarcoma Virus ◽  
Transformation Parameters ◽  
Adhesion Plaques ◽  
Adhesion Plaque

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

scholarly journals Immunoreactive folate-binding proteins from human saliva. Isolation and comparison of two distinct species

1992 ◽  
Vol 286 (3) ◽  
pp. 707-715 ◽  
Author(s):  
R S Verma ◽  
A C Antony
Keyword(s):  
Binding Proteins ◽  
Folate Receptor ◽  
Gel Filtration ◽  
Distinct Species ◽  
Human Saliva ◽  
Western Blots ◽  
Sds Page ◽  
Folate Binding Proteins ◽  
Triton X

Human saliva contains a single 72,000-M(r) species which specifically reacted with rabbit anti-[human placental folate receptor (PFR)] serum on SDS/PAGE and Western blots. Although a specific radioimmunoassay for human PFR and related folate-binding proteins (FBPs) identified 55 ng of cross-reacting material (CRM) per mg of crude salivary proteins, only a minor fraction (1.6 ng) specifically bound radiolabelled folate. The major fraction of CRM did not contain bound endogenous folate and did not bind radiolabelled folates. On the basis of folate binding, salivary CRM species to PFR were designated as either functional (f-FBP) or non-functional (nf-FBP) species respectively. nf-FBPs and f-FBPs were isolated by different purification schemes. Both purified f-FBPs and nf-FBPs migrated as a single apparent 72,000-M(r) species on SDS/PAGE, but on Sephacryl S-200 gel filtration and sucrose-density-gradient ultracentrifugation they were eluted/sedimented with 40,000-M(r) markers. Each microgram of purified f-FBP and nf-FBP was measured in the radioimmunoassay for PFR as being equivalent to 18 ng and 24 ng of CRM respectively, indicating low epitope-relatedness to PFR. The Kd of f-FBPs was 50 pM and 0.94 mol of folate was bound/mol of protein. f-FBPs exhibited an unusual dependence on Triton X-100 for optimal ligand binding, despite the fact that Triton X-100 micelle binding was not demonstrated. The relative order of affinity of f-FBPs for pteroylglutamate greater than methotrexate greater than 5-formyltetrahydrofolate greater than 5-methyltetrahydrofolate was also distinct from that of purified PFR. Whereas amino acid and carbohydrate analysis revealed that nf-FBP (M(r) 51,400) and f-FBP (M(r) 39,200) were distinct glycoproteins with 8 and 13% carbohydrate respectively, isoelectric focusing and immunological studies suggested some structural identity. The presence of f-FBP and nf-FBP in normal saliva raises new questions about their possible role in vivo.

Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus

1983 ◽  
Vol 3 (4) ◽  
pp. 731-746
Author(s):  
L Rohrschneider ◽  
M J Rosok
Keyword(s):  
Cell Surface ◽  
Rous Sarcoma Virus ◽  
Soft Agar ◽  
Partial Transformation ◽  
Rous Sarcoma ◽  
Independent Functions ◽  
Sarcoma Virus ◽  
Transformation Parameters ◽  
Adhesion Plaques ◽  
Adhesion Plaque

Rous sarcoma virus (RSV)-induced transformation is mediated by the action of the viral src gene product pp60src. This transforming protein is found at several cytoplasmic locations, including the adhesion plaques of RSV-transformed cells. In these studies, we have focused on the adhesion plaque location of pp60src and determined whether any of the induced transformation parameters correlate with the presence of pp60src in the adhesion plaques. A series of partial transformation mutants of RSV that induce distinct transformation phenotypes were used, and infected chicken embryo cells were examined for (i) intracellular pp60src location, (ii) vinculin localization, (iii) abundance of phosphotyrosine on vinculin, (iv) integrity of stress fibers, and (v) expression of cell surface fibronectin. The results indicate that, among the limited number of mutants studied here, the presence of pp60src in adhesion plaques is independent of growth in soft agar and the increased phosphorylation of vinculin on tyrosine, but it does correlate with the loss of cell surface fibronectin. An elevated abundance of phosphotyrosine on vinculin is insufficient to cause stress fiber dissolution and is independent of the loss of fibronectin from the extracellular matrix. However, the increased relative amount of phosphotyrosine on vinculin is related to the ability of the cells to grow in soft agar. The adhesion plaque binding and tyrosine-specific kinase activities seem to represent two independent functions of pp60src.

scholarly journals Paxillin: a new vinculin-binding protein present in focal adhesions.

1990 ◽  
Vol 111 (3) ◽  
pp. 1059-1068 ◽  
Author(s):  
C E Turner ◽  
J R Glenney ◽  
K Burridge
Keyword(s):  
Focal Adhesion ◽  
Specific Interaction ◽  
Focal Adhesions ◽  
Adhesion Protein ◽  
Rous Sarcoma ◽  
Sds Page ◽  
Cytoskeletal Component ◽  
Sarcoma Virus ◽  
Focal Adhesion Protein

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

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.

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