EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCγ signaling pathway

Abstract

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

“…This was also seen in mouse keratinocytes treated with EGFR and FAK inhibitors (electronic supplementary material, figure S1 b ). In live cells, assembly and disassembly rates of expressed talin-GFP were also reduced by inhibiting each kinase ( figure 3 b ) in agreement with previous studies [ 31 – 33 ]. Combining both inhibitors resulted in a similar increase in focal adhesion number and a comparable reduction in dynamics to similar levels seen in single inhibitor-treated cells (figure 3 a , b ).…”

ST6Gal-I–mediated sialylation of the epidermal growth factor receptor modulates cell mechanics and enhances invasion

“…Epidermal growth factor receptor can coordinate with integrins to regulate cell adhesion and growth ( 44 , 45 ). A cell’s ability to respond to extracellular signals is intimately connected to its ability to pull against ligands in the extracellular matrix, which is conferred by the tensional and architectural organization of the cytoskeleton ( 46 , 47 , 48 ).…”

Interference of Silver, Gold, and Iron Oxide Nanoparticles on Epidermal Growth Factor Signal Transduction in Epithelial Cells

“…The mitogenic protein epidermal growth factor (EGF) and its receptor family is arguably the best studied and characterized ligand–receptor system to date . Upon cellular introduction, EGF will bind to its specific receptor (EGFR) after which the receptor can dimerize and initiate intrinsic kinase activation, receptor phosphorylation, and signal transduction. − EGF has been shown to control the activation of numerous signaling pathways, including the Phosphoinositide 3-kinase (PI3K)/Akt pathway and the Ras/Extracellular signal-regulated kinase (Erk) cascade, which results in an up-regulation of the gene transcription necessary for cell proliferation, survival, and migration. , …”

“…11À13 EGF has been shown to control the activation of numerous signaling pathways, including the Phosphoinositide 3-kinase (PI3K)/Akt pathway and the Ras/Extracellular signal-regulated kinase (Erk) cascade, 14 which results in an up-regulation of the gene transcription necessary for cell proliferation, survival, and migration. 15,16 The aim of this study was to ascertain to what extent metallic nanoparticles of similar size and morphology, but of different composition, altered EGF-dependent signal transduction in the human epithelial cell line A-431. The A-431 cell line was selected for this study because of its well characterized overexpression of EGFRs 17 and its frequent use as a cell model for EGF signaling studies.…”