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First published online 13 September 2005
doi: 10.1242/jcs.02565

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Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397

UMR CNRS 7034, Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, BP 60024, 67401 Illkirch, France

View larger version (54K): [in a new window]   Fig. 1. Expression of FAK/YCam and Y397F-FAK/YCam in U87 astrocytoma cells. (A) Schematic representation of FAK with the phospho-acceptor residues and the epitopes recognized by the different antibodies used in this study. (B) Control U87, FAK/YCam (FAK) and Y397F-FAK/YCam (Y397) cells were lysed and immunoblotted for FAK with antibodies that recognize the kinase domain (top panel), the C-terminal domain (Ct; middle panel) or the FAT domain (lower panel). The lower bands correspond to endogenous FAK (125 kDa), while the upper bands correspond to the fusion proteins FAK/YCam or Y397F-FAK/YCam (199 kDa).

View larger version (48K): [in a new window]   Fig. 2. Expression of Y397F-FAK/YCam decreases endogenous FAK-Tyr-397 phosphorylation at FAs. (A) Control U87, FAK/YCam (FAK) and Y397F-FAK/YCam (Y397) cells were lysed and site-specific FAK phosphorylation was analysed on western blots using anti-FAK[P-Tyr-397] antibody. The amounts of proteins were monitored by stripping and reblotting the membranes with anti-FAK antibody. In Y397F cells, note the absence of phosphorylation of exogenous FAK-Tyr-397 (199 kDa) and the reduced phosphorylation of endogenous FAK-Tyr-397 (125 kDa). (B) Quantification of the percentage of endogenous FAK-P-Tyr-397/total FAK in control and transfected cells. Data are mean±s.e.m. (n=5); *P<0.05, paired t-test. (C) FAK/YCam- and Y397F-FAK/YCam-transfected cells plated for 2 days on Matrigel were fixed, permeabilized and FAK-Tyr-397 phosphorylation was analysed by immunocytochemistry. Y397F-FAK/YCam transfected cells (lower panel) display very low levels of FAK-Tyr-397 phosphorylation at FAs compared to adjacent non-transfected cells and to FAK/YCam-transfected cells (upper panel). Scale bar: 10 µm. (D) Analysis of the localisation pattern of YCam (green) and P-Tyr-397 (red) pixels in FAK/YCam- and Y397F-FAK/YCam-transfected cells. The scatter plots (lower left corners) show colour and intensity distributions of pixels from a pair of images (YCam and P-Tyr-397). By selecting a ROI (outlined in white), a group of pixels with high intensities was extracted to form a new image of the cellular localisation of these pixels (white images in upper right corners). (E) Quantification using Pearson's coefficient (which describes the extent of overlap between image pairs) reveals a significant reduction (*P<0.05, paired t-test) of correlation in YCam and P-Tyr-397 images for Y397F-FAK/YCam-transfected cells compared to FAK/YCam-transfected cells.

View larger version (45K): [in a new window]   Fig. 3. Expression of Y397F-FAK/YCam decreases phosphorylation of endogenous and exogenous FAK-Tyr-576 at FAs. (A) Cells were lysed and site-specific FAK phosphorylation was analysed in western blots with anti-FAK[P-Tyr-576] antibody. The amounts of proteins were monitored by stripping and reblotting the membranes with anti-FAK antibody. Note the reduction in phosphorylation of endogenous and exogenous FAK at Tyr-576 in Y397F cells. (B) Quantification of the percentage of endogenous (125 kDa; upper panel) and exogenous (199 kDa; lower panel) phosphorylated FAK-Tyr-576/total FAK in control and transfected cells. Data represent mean±s.e.m. (n=5); * P<0.05, paired t-test. (C) FAK/YCam- and Y397F-FAK/YCam-transfected cells plated for 2 days on Matrigel were fixed, permeabilized and FAK-Tyr-576 phosphorylation was analysed by immunocytochemistry using anti-FAK[P-Tyr-576] antibody. Transfected Y397F-FAK/YCam cells (lower panel) have lower FAK Tyr-576 phosphorylation at FAs compared to adjacent non-transfected cells and to transfected FAK/YCam cells (upper panel). Scale bar: 10 µm. (D) Scatter plots show colour and intensity distribution of pixels in a pair of images (YCam, green and P-Tyr-576, red), with lower P-Tyr-576 intensities in Y397F cells. Extracted high intensity pixels (outlined white ROIs) were used to form a new image of the cellular localisation of these pixels (white images in upper right corners). (E) Quantification using Pearson's coefficient reveals no difference in colocalisation of YCam and P-Tyr-576 signals in Y397F-FAK/YCam cells compared to FAK/YCam cells.

View larger version (29K): [in a new window]   Fig. 4. FRAP experiments at FAs uncover regulation of FAK/YCam molecular dynamics by phosphorylation. (A) U87 cells expressing FAK/YCam (left) or Y397F-FAK/YCam (right) were imaged before bleaching of FA-containing ROIs (white squares). Scale bar: 10 µm. The time lapse sequences (in seconds) below show recovery after bleaching of corresponding FAs for FAK/YCam (upper row) and Y397F-FAK/YCam (lower row). Bpb, before photobleaching; apb, immediately after photobleaching. Scale bar: 2 µm. (B) Kinetics of recovery of FAK/YCam (-µ-) and Y397F-FAK/YCam (--) in cytosolic (top left) and FA compartments (top right) after bleaching. The fluorescence intensity in the bleached region was measured and expressed as the relative recovery. At FAs (bottom right), note the significantly shorter recovery half-time for Y397F-FAK/YCam compared to FAK/YCam.

View larger version (41K): [in a new window]   Fig. 5. Effects of mutation at Tyr-397 on the dynamics of FAK/YCam after nocodazole washout observed by confocal imaging during 1 hour of cells transfected with FAK/YCam (A), Y397F-FAK/YCam (B) and non transfected cells (C). White arrows show positions of FAK/YCam-containing FAs that were highly dynamic with clear cell edge movements (A). White arrow heads indicate positions of Y397F-FAK/YCam-containing FAs that were stable (B). A non transfected cell adjacent to the cell transfected with Y397F-FAK/YCam displays normal motility, with cell edge retraction (grey arrow, C). Images are representative of cells from a minimum of for separate experiments. Scale bar: 5 µm.

View larger version (65K): [in a new window]   Fig. 6. Quantification of the effects of mutation at Tyr-397 of FAK/YCam on FA disassembly after nocodazole washout. (A) In FAK/YCam-transfected cells, cellular motility was clearly apparent (directions is indicated by the white arrow), during the 1 hour recovery period after nocodazole (see supplementary material Movie 1), unlike Y397F-FAK/YCam-transfected cells (see supplementary material Movie 2). Scale bar: 10 µm. (B) FA turnover was also inhibited in Y397F-FAK/YCam cells compared with FAK/YCam. Left images at t=0, centre images at t=60 minutes; right images are the result of subtraction of the images at t=60 from those at t=0. In the subtracted images, disassembled FAs are black, newly formed FAs are white and constant FAs are grey. (C) Disassembled, newly formed and constant (immobile) FAs plotted as a percentage of the total FAs in FAK/YCam and Y397F-FAK/YCam cells during recovery from nocodazole treatment. Values are the mean of four separate experiments. (D). Migration speed of FAK/YCam and Y397F-FAK/YCam cells was assessed using a wound-healing model (see Materials and Methods). Y397F-FAK/Ycam cells (n=120) had a significantly decreased migration speed compared with FAK/Ycam cells (n=120).

View larger version (58K): [in a new window]   Fig. 7. Analysis of the expression of FAK/YCam or Y397F-FAK/YCam and paxillin in U87 astrocytoma cells before and after FA turnover induced by nocodazole wash-out. FAK/YCam- and Y397F-FAK/YCam-transfected cells plated for 2 days on Matrigel were fixed before addition of nocodazole (0'), or 60 (60') minutes after nocodazole wash-out, permeabilized and paxillin was revealed by immunocytochemistry. (A) Before addition of nocodazole, FAK/YCam and Y397F-FAK/YCam colocalized with paxillin. (B) Quantification, using Pearson's coefficient, reveals no difference in colocalisation of YCam and paxillin before addition of nocodazole or after FA disassembly induced by nocodazole wash-out in FAK/YCam cells and Y397F-FAK/YCam cells. Scale bar: 10 µm.

View larger version (20K): [in a new window]   Fig. 8. Model for FAK-mediated FA dynamics. (A) FAK exists in equilibrium between two compartments: in assembled FAs and in cytosol. Increasing kon or koff will either stabilize or disassemble FAs. (B) In FAs, FAK is phosphorylated by cis/trans activation before leaving the FA compartment. (C) Decreasing k3, for example by expression of mutant Y397F-FAK, will lead to accumulation of non-phosphorylated FAK at FAs and thus a greater k2 (consistent with the shorter recovery half-time for Y397F-FAK/YCam compared to FAK/YCam), and in turn, to a decrease in FA disassembly.