The oncoprotein v-Src and its cellular homologue (c-Src) are tyrosine kinases that modulate the actin cytoskeleton and cell adhesions. Through the concerted action of their protein-interaction and kinase domains, they are targeted to cell matrix integrin adhesions or cadherin-dependent junctions between epithelial cells, where they phosphorylate substrates that induce adhesion turnover and actin re-modelling. Recent experiments have defined some of the key targets and effector pathways that mediate the pleiotropic oncogenic effects of v-Src.
Abstract. We have isolated Swiss 3T3 subclones that are resistant to the rnitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Racmediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.
To determine how an oncogenic tyrosine kinase disturbs cell cycle control we examined expression of cell cycle proteins and growth of ®broblasts reversibly transformed by a temperature sensitive mutant of v-Src (ts LA 29). ts LA 29 Rat-1 cells and normal Rat-1 cells had similar growth rates but the transformed cells traversed the G 1 phase of the cell cycle more rapidly and failed to exit cycle e ciently in response to serum starvation and cell con¯uence. Cyclin D1 and cyclin E levels were not elevated in growing ts LA 29 Rat-1 cells and the abbreviated G 1 was further accelerated by overexpression of cyclin E. A fall in cyclin E and cyclin A dependent kinase activities in Rat-1 cells in response to inhibitory growth conditions was abrogated in ts LA 29 Rat-1 cells and correlated with lack of p27 accumulation or cyclin A down regulation, the latter due to sustained cyclin A promoter activity. The expression of p27 mRNA was lower in ts LA 29 Rat-1 cells than Rat-1 cells and was elevated following v-Src inactivation concurrent with an increase in p27 promoter activity and temporary cell cycle exit. The suppression of mRNA or transcription is a novel way an oncoprotein can induce down regulation of p27 and contributes to the G 1 shortening and perturbed cell cycle regulation of the v-Src transformed cells.
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