FAK Acts as a Suppressor of RTK-MAP Kinase Signalling in Drosophila melanogaster Epithelia and Human Cancer Cells
Figure 8
FAK decreases EGFR at the plasma membrane via reduced recycling.
(A–B) MDA-MB-231 cells transfected with non-targeting (NT) siRNA or FAK siRNA were immunostained with anti-EGFR antibody (green, A″ and B″), Rhodamine-phalloidin (red, A′ and B′) and DAPI (blue). Note the differential localisation of EGFR; while in siNT cells the receptor is distributed in plasma membrane and internal vesicles, FAK downregulation leads to an increase of EGFR levels at the cellular membrane. Scale bar, 10 µm. (C) Quantification of relative EGFR membrane levels, values are expressed as relative levels of the receptor against the mean value of siNT cells; four confocal fields for each condition were analysed: n = 347 cells (siNT), and n = 414 cells (siFAK). p<0.0286 in a Mann-Whitney test. (D) MDA-MB-231 cells were transfected with either non-targeting (siNT) or FAK-specific siRNA (siFAK) and deprived of serum prior to addition of 80 µM Dynasore. siNT-transfected cells showed an increased pERK1/2 level in response to both Dynasore treatment (80 µM, 30 minutes) and FAK knockdown. Total levels of EGFR and ERK were not changed and actin levels were probed as an additional loading control. (E) The internalization of membrane EGFR (top panel) and recycling of internalised EGFR (bottom panel) were determined in MDA-MB-231 cells transfected with non-targeting siRNA (siCTR) or FAK siRNA (siFAK). Values are means ± Standard Deviation (SD) of two independent experiments with four to eight replicates of each time point per genotype. See materials and methods for more details. FAK knockdown did not affect receptor internalization but increased the recycling of the internalised EGFR pool. (F) A working model for the regulatory mechanism of FAK. Ectopic expression and/or hyperactivation of RTKs activate FAK and Ras among other signalling pathways. FAK mediates a negative regulation of receptor recycling; when FAK is reduced or absent, there are more RTKs molecules at the plasma membrane, thus enabling a higher flux of signalling through Ras/MAPK pathway. See the text for more details.