Mechanism of Transformation by Rous Sarcoma Virus: Events within Adhesion Plaques

Rohrschneider, Larry R.; Rosok, M J; Shriver, K

doi:10.1101/sqb.1982.046.01.089

Cited by 33 publications

(22 citation statements)

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“…All of these molecules remained associated with the insoluble FACs after isolation and were found to be enriched in this fraction when compared with either whole cells, intact CSK, or a basal cell surface preparation that retains plasma membrane. pp125F and pp6Ov-src have been previously shown to localize within FACs by immunostaining (Rohrschneider et al, 1982;Schaller et al, 1992) and the Na+/H+ antiporter was found to codistribute with vinculin, talin, and F-actin in a recent report that used a different source of antibodies (Grinstein et al, 1993). Most importantly, we found the high affinity FGF receptor fig was not only present within the FAC, it was greatly enriched compared with either the whole cell or intact CSK.…”

Section: Discussionsupporting

confidence: 52%

“…It is well known that many viral tyrosine kinases (e.g., pp6Ov-src) localize to FACs in transformed cells (Cooper and Hunter, 1981;Kellie et al, 1986a,b;Tapley et al, 1989;Turner et al, 1989;Guan and Shalloway, 1992;Lipfert et al, 1992) and that phosphotyrosine (PTyr)-containing proteins localize to FACs within normal cells (Marchisio et al, 1984;Maher et al, 1985). Furthermore, one integrin-associated focal adhesion kinase, pp125FAK, exhibits both growth factor and ECM-dependent phosphorylation on tyrosine (Guan et al, 1991;Kornberg et al, 1992;Zachary and Rozengurt, 1992) and physically associates with pp6Oc-src via its SH2 domain when activated Xing et al, 1994;Cobb et al, 1994). pp125FAK is a substrate for pp6Ov-src, along with many other FAC proteins (Rohrschneider et al, 1982;Kellie et al, 1986b;Tapley et al, 1989;Guan and Shalloway, 1992) and a recent report suggests that integrin-dependent phosphorylation of pp125FAK may link integrin engagement to the Ras/MAP kinase signal transduction pathway via creation of an SH2-binding site for GRB2 (Schlaepfer et al, 1994). The distribution of ppl25FAK within FACs as well as its ability to be activated by growth factors appears, however, to be more dependent on its associations with the actin CSK (Lipfert et al, 1992;Rankin and Rozengurt, 1994).…”

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confidence: 99%

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Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Plopper

McNamee

Dike

et al. 1995

MBoC

478

315

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Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60csrc, ppl25FAKphosphatidylinositol-3-kinase, phospholipase C-'y, and Na+/H+ antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp6Ocsrc, pp125FAK, phospholipase G-y, and the Na+ /H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60csrc and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.

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Section: Discussionsupporting

confidence: 52%

mentioning

confidence: 99%

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Plopper

McNamee

Dike

et al. 1995

MBoC

478

315

View full text Add to dashboard Cite

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“…The localization of p36 in the transformed cells was distinctly different from that of pp60 v-src, which was found in focal areas on the undersurface of cells that presumably correspond to the adhesion plaques or focal contacts reported by others (Fig. 7E) (34,35) and in the plasma membranes at cell-cell contacts in dense cultures (not shown).…”

Section: Downloaded Frommentioning

confidence: 83%

“…We do not regard these observations as contradictory, since it is unlikely that more than a small proportion of pp6(V-SrC (ca. 10%) is actually congregated in adhesion plaques (35). ocalization of p36 by immunofluorescence microscopy.…”

Section: Discussionmentioning

confidence: 99%

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

Courtneidge¹,

Ralston²,

Alitalo³

et al. 1983

Mol. Cell. Biol.

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A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S. A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.

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“…Available evidence suggests that it is the tyrosine phosphokinase activity of pp6OSrc that is responsible for the transforming effect of RSV (41). Although the mechanism by which pp60sr, exerts its transformation is unclear, studies utilizing mutants of RSV have indicated a correlation between tyrosine phosphorylation of vinculin, loss of actin-containing stress fibers, and the ability to grow in soft agar (38,42). This suggests that the altered growth characteristics of transformed cells result partly from alterations in the cytoskeleton brought about via a mechanism involving the phosphorylation of vinculin at tyrosine residues.…”

Section: Resultsmentioning

confidence: 99%

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Lin¹,

Garber²,

E³

et al. 1983

Mol. Cell. Biol.

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Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-ot (specific activity, 106 U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60Wrc-associated protein kinase activity. Staphylococcuts aurelus V8 protease digestion of pp6Osr(, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-a also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp6O,rc. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp6Osrc

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Mechanism of Transformation by Rous Sarcoma Virus: Events within Adhesion Plaques

Cited by 33 publications

References 0 publications

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

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