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.

Abstract.The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60 ~src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60 ..... activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60Cs% On the other hand, overexpression of a mutated, constitutively activated form of pp60 csrc does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60 csr~ in epithelial cell scattering and indicate that pp60 ¢src might contribute unequally to the two separate biological activities engendered by a single signal.CATTERING of epithelial cells has a central role in the control of embryogenesis, and in the process of carcinoma cell dispersion. The search for inducer molecules of epithelial cell scattering has led to the discovery of scatter factor (SF) that has been recognized to also behave as a mitogenic factor (54). Other proteins with scatter activity were first described as mitogens: for example, EGF, one of the first characterized growth factors is able to induce keratinocyte migration (2) and membrane ruffling of mammary carcinoma cells (4, 11). We previously demonstrated that several growth factors (FGF-I, EGF, TGF-a), all of which bind to tyrosine kinase receptors, are endowed with two distinct activities toward NBT-II cells. On subconfluent cultures, these growth factors generate a scattering response, characterized by several properties: cells loose their epithelial features, they become fibroblastic, and they dissociate and start moving individually (7,18,49). The entire spectrum of changes has been termed epi-