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Src and Ras are involved in separate pathways in epithelial cell scattering
B Boyer1, S Roche, M Denoyelle
1Laboratoire de Compartimentation et Dynamique cellulaires, UMR CNRS 144, Institut Curie Section de Recherche, 26 rue d'Ulm F-75248 Paris Cedex 05, France. bboyer@curie.fr
Abstract:
We have demonstrated previously that Src controls the epidermal growth factor (EGF)-induced dispersion of NBT-II carcinoma epithelial cells. Here we show that while only Src and Yes were expressed and activated by EGF, microinjected kinase-inactive mutants of Src (SrcK-) and Fyn (FynK-) were able to exert a dominant-negative effect on the scattering response. Both SH2 and SH3 domains of FynK- were required for inhibition of cell scattering. Expression of dominant-negative N17Ras also abrogated EGF-induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK- did not alter EGF-evoked Shc tyrosine phosphorylation, Shc-Grb2 complex formation and MAPK activation, three elements of the Ras pathway. Furthermore, the expression of Jun-Fos and Slug rescued the block induced by N17Ras but not by SrcK-, showing that Src kinases and Ras operate in separate pathways. In addition, actinomycin D inhibition of RNA synthesis repressed the ability of the activated mutant L61Ras but not that of F527Src to induce epithelial cell scattering. Since tyrosine phosphorylation of cytoskeleton-associated proteins pp125FAK and cortactin were abolished in EGF-stimulated SrcK- cells, we concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression and by directly regulating the organization of the cortical cytoskeleton.
Insights
Epidermal Growth Factor (EGF) triggers NBT-II carcinoma cell dispersion. Src kinases, unlike Ras, regulate this process independently of gene expression by directly organizing the cytoskeleton.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Epidermal Growth Factor (EGF) signaling pathways regulate cell behavior.
- Src family kinases play a role in epithelial cell dispersion.
- NBT-II carcinoma cells serve as a model for studying cell scattering.
Purpose of the Study:
- To elucidate the distinct roles of Src kinases and Ras in EGF-induced epithelial cell dispersion.
- To investigate the molecular mechanisms by which Src kinases control cell scattering.
Main Methods:
- Microinjection of kinase-inactive Src (SrcK-) and Fyn (FynK-) mutants.
- Expression of dominant-negative N17Ras.
- Analysis of signaling pathway components (Shc, Grb2, MAPK).
- Assessment of gene expression using actinomycin D.
- Examination of cytoskeleton-associated protein phosphorylation (pp125FAK, cortactin).
Main Results:
- EGF activates Src and Yes kinases.
- Kinase-inactive Src and Fyn mutants inhibit cell scattering, with Fyn requiring SH2 and SH3 domains.
- Dominant-negative Ras also inhibits scattering, indicating Ras as a regulator.
- SrcK- expression does not affect early Ras pathway signaling (Shc phosphorylation, MAPK activation).
- Src kinases and Ras operate in separate pathways; N17Ras-induced block is rescued by Jun-Fos/Slug, but SrcK- block is not.
- Activated Ras-induced scattering requires RNA synthesis, while activated Src does not.
- SrcK- abolishes EGF-induced tyrosine phosphorylation of pp125FAK and cortactin.
Conclusions:
- Src kinases and Ras are distinct regulators of EGF-induced epithelial cell dispersion.
- Src kinases control cell scattering independently of gene expression, likely by directly modulating the cortical cytoskeleton.
- Ras-mediated cell dispersion involves gene expression and downstream effectors like Jun-Fos and Slug.