Src and Ras are involved in separate pathways in epithelial cell scattering.
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.
Boyer
B
B
Laboratoire 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
Roche
S
S
Denoyelle
M
M
Thiery
J P
JP
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002465:Cell Movement; D003609:Dactinomycin / Q000494:pharmacology; D004285:Dogs; D004815:Epidermal Growth Factor / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D012313:RNA / Q000096:biosynthesis; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism*; D019061:src-Family Kinases
D019384:Nucleic Acid Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D003609:Dactinomycin; D004815:Epidermal Growth Factor; D012313:RNA; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D018631:ras Proteins