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The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the
Abstract:
Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.
Insights
Tyrosine phosphorylation of vinculin by Rous sarcoma virus (RSV) pp60v-src kinase does not solely cause cell rounding. Even with phosphorylated vinculin, cells can maintain normal morphology, challenging previous hypotheses.
Area of Science:
- Cell Biology
- Virology
- Cancer Research
Background:
- Rous sarcoma virus (RSV) transformation involves neoplastic cell changes.
- The src gene product, pp60v-src, is a tyrosine kinase implicated in transformation.
- Vinculin, a cytoskeletal protein, is a potential substrate for pp60v-src phosphorylation.
Purpose of the Study:
- To investigate the hypothesis that tyrosine phosphorylation of vinculin disrupts adhesion plaques, causing cell rounding in RSV-transformed cells.
- To analyze the relationship between pp60v-src localization, vinculin phosphorylation, and cell morphology in RSV-transformed fibroblasts.
Main Methods:
- Analysis of fibroblasts transformed by conditional and non-conditional RSV mutants.
- Assessment of microfilament bundles, adhesion plaques, extracellular matrix deposition, pp60v-src localization, and vinculin tyrosine phosphorylation.
- Utilized temperature-sensitive (ts) RSV mutant LA32 and recovered avian sarcoma virus rASV 2234.3.
Main Results:
- Cells transformed by ts-RSV LA32 at 41°C were morphologically normal but had active pp60v-src, concentrated in adhesion plaques, and tyrosine-phosphorylated vinculin.
- Cells transformed by rASV 2234.3 showed fusiform morphology with pp60v-src in adhesion plaques and elevated vinculin phosphotyrosine.
- RSV LA32 transformed cells at restrictive temperature were morphologically normal yet contained tyrosine-phosphorylated vinculin.
Conclusions:
- Tyrosine-specific phosphorylation of vinculin alone is insufficient to induce the rounded morphology characteristic of RSV-transformed cells.
- pp60v-src concentration in adhesion plaques is also not sufficient for the rounded cell phenotype.
- The study challenges the direct causal link between vinculin tyrosine phosphorylation and RSV-induced cell rounding.