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.

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 Biallelic neutrophil Na-antigen system is associated with a polymorphism on the phospho-inositol-linked Fc gamma receptor III (CD16)

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 213-217 ◽  
Author(s):  
TW Huizinga ◽  
M Kleijer ◽  
PA Tetteroo ◽  
D Roos ◽  
AE von dem Borne
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Antigen ◽  
Phosphatidyl Inositol ◽  
Structural Features ◽  
Fc Gamma Receptor ◽  
Gamma Receptor ◽  
Sds Page ◽  
Antigen System ◽  
Genetically Determined

Neutrophils express two distinct types of receptor for the Fc region of IgG, FcRII and FcRIII, in amounts of 10,000 to 20,000 FcRII (40 Kd) and 100,000 to 200,000 FcRIII (50 to 80 Kd) per neutrophil. We showed that the FcRIII exhibits genetically determined heterogeneity, detectable by differences in electrophoretic mobility with sodium dodecyl sulfate (SDS) as well as by reaction with antibodies against the biallelic neutrophil-specific antigen system NA. FcRIII was precipitated with an FcRIII-specific monoclonal antibody (MoAb) from the neutrophils of 35 donors. NA1NA1 donors expressed an FcRIII with a molecular weight (mol wt) of 50 to 65 Kd, NA1NA2 donors expressed an FcRIII with a mol wt of 50 to 80 Kd, and NA2NA2 donors expressed an FcRIII with a mol wt of 65 to 80 Kd. Statistical analysis showed that the electrophoretic heterogeneity corresponds with the NA polymorphism (k = 1). Sequential immunoprecipitation with a MoAb against NA1 and a MoAb against anti- FcRIII, followed by SDS-polyacrylamide gel electrophoresis (PAGE), showed that NA1-FcRIII is distinct from NA2-FcRIII. Moreover, immunoprecipitation with a MoAb against NA1 yielded a protein of 50 to 65 Kd, and immunoprecipitation with human anti-NA2 sera or an MoAb against NA2 yielded a protein of 65 to 80 Kd. Preincubation of NA1NA2 neutrophils with F(ab')2 fragments of an MoAb against anti-NA1 reduced binding of IgG dimers to these cells with about 50%, whereas it completely prevented binding of the dimers to NA1NA1 neutrophils. Inhibition experiments with the MoAb against NA2 yielded the same results for NA1NA2 cells, whereas binding of IgG dimers to NA2NA2 cells was completely prevented. Thus, the products of both NA alleles bind IgG. Immunoprecipitation from the medium of neutrophils either stimulated with formyl- methionyl-leucyl-phenylalanine (FMLP) or treated with glycosyl-phosphatidyl-inositol-specific phospholipase C (GPI- PLC) showed that both the NA1-FcRIII and the NA2-FcRIII are released from the cell surface, indicating that both forms of FcRIII have some structural features in common. Deglycosylation of FcRIII from homozygous donors yielded material that showed several bands on SDS- PAGE. GPI-PLC treatment of neutrophils indicated that all of this material is phosphatidyl-inositol linked.

scholarly journals MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture

2005 ◽  
Vol 201 (3) ◽  
pp. 431-439 ◽  
Author(s):  
Elad Katz ◽  
Mohamed H. Lareef ◽  
John C. Rassa ◽  
Shannon M. Grande ◽  
Leslie B. King ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinases ◽  
Mammary Epithelial Cells ◽  
Cell Transformation ◽  
Three Dimensional ◽  
Soft Agar ◽  
Mammary Epithelial ◽  
Matrigel Invasion ◽  
Tumor Virus

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

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.

Purification of PRL receptors from toad kidney: comparisons with rabbit mammary PRL receptors

1988 ◽  
Vol 254 (3) ◽  
pp. C372-C382 ◽  
Author(s):  
M. Dunand ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier ◽  
M. L. Aubert
Keyword(s):  
Mammalian Species ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Structural Features ◽  
Polyclonal Antiserum ◽  
Partial Purification ◽  
Molecular Characteristics ◽  
Binding Characteristics ◽  
Sds Page

The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with 125I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis on SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

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.

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

2005 ◽  
Vol 386 (9) ◽  
pp. 845-855 ◽  
Author(s):  
Stefan Hart ◽  
Oliver M. Fischer ◽  
Norbert Prenzel ◽  
Esther Zwick-Wallasch ◽  
Matthias Schneider ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Mammary Epithelial Cells ◽  
Growth Factor Receptor ◽  
Mammary Epithelial ◽  
Cellular Processes ◽  
Mitogenic Signalling ◽  
Epidermal Growth ◽  
G Protein Coupled

Abstract The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.

scholarly journals Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

1992 ◽  
Vol 3 (12) ◽  
pp. 1329-1337 ◽  
Author(s):  
H C Wang ◽  
R L Erikson
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Sodium Dodecyl ◽  
Cellular Transformation ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Rous Sarcoma ◽  
Phosphatase 2A ◽  
Sarcoma Virus

We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.

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