Effect of v-Src on cell motility : speed, directionality and signalling pathways

Cell motility, a key parameter in cancer invasion and metastasis, is driven by the actin cytoskeleton and can be oriented by extracellular signals. We have examined the role of oncogenic v-Src in spontaneous and oriented motility. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34°C but inactivated at 40°C. v-Src activation induced transformation of both cell lines and accelerated their spontaneous motility by 2-3-fold, as evidenced by time-lapse recording in Dunn chambers and wound-healing assay. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src, suggesting that these enzymes mediate v-Src-induced spontaneous migration. We further analysed in Dunn chambers the chemotactic response to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) gradients. Oncogenic v-Src decreased by 2-3-fold the surface pool at steady-state of PDGF-receptors on Rat-1 fibroblasts and of EGF-receptors on MDCK cells. At levels of receptors occupancy comparable to those of non-transformed cells, v-Src abrogated chemotaxis towards these growth factors (GFs). Thus, v-Src accelerates spontaneous motility but abrogates chemotaxis. In the second part of the study we addressed the question why v-Src decreases the surface pool of GF-receptors and causes the loss of the chemotactic response. Overnight v-Src activation resulted in ~3-fold decrease in PDGFRb mRNA (as measured by quantitative real-time PCR), and total protein (Western blotting). PDGFR signalling, assessed using anti-Y751 phosphopeptide antibodies corresponding to the docking site for PI3-K, was not detected at 40°C in the absence of PDGF and was strongly induced by its addition. At 34°C, Y751 phosphorylation was already detected in the absence of ligand and further increased upon PDGF-stimulation, indicating that PDGFR in v-Src-transformed cells still responds to its cognate ligand. At 40°C, in a PDGF gradient, immunolabelled PDGFRs and active Src were concentrated in vesicles, and PI3-K was translocated to lamellipodia, both oriented towards the PDGF source which also caused polarised orientation of centrosomes. By live cell imaging, PI(3,4,5)P3 locally produced by PI3-K was further visualised by recruitment of the plekstrin homology (PH) domain of Akt fused with GFP. At 40°C, Akt-PH-GFP was diffusely localised in the cytoplasm in untreated cells and was translocated to the plasma membrane after PDGF stimulation; when PDGF was added as gradient, it induced a polarised membrane spreading and generation of PI3-K second messengers toward a chemoattractant source. Upon v-Src activation at 34°C, Akt-PH-GFP was partially located to the plasma membrane in untreated cells and did not respond to addition of PDGF, either diffusely nor as gradient. v-Src also abrogated polarised orientation of centrosomes. Taken together, these observations suggest that up-regulation of PI3-K by v-Src overcomes the effect of growth factors by abolishing the polarisation of lipidic second messengers generation ('signalling requires silence').