Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397.
One of the major tyrosine phosphorylation activities linked to integrin signalling is that of focal adhesion kinase (FAK). High amounts of FAK are located at specialised subcellular compartments known as focal adhesions. FAK tyrosine phosphorylation at focal adhesions is increased by various stimuli including integrin engagement during migration processes, growth factors and oncogene transformation. Phosphorylation of FAK at various tyrosine residues regulates focal adhesion turnover by mechanisms that are not well understood. We made a fluorescent FAK mutant (Y397F-FAK/YCam) to analyse, in living cells, how phosphorylation of FAK regulates the turnover of focal adhesions. We found that expression of Y397F-FAK/YCam in human astrocytoma cells decreases the level of phosphorylation of FAK at endogenous Tyr-397 residues and at both endogenous and exogenous Tyr-576 residues, in the putative activation loop of the kinase. This corresponds to a decrease in phosphorylation of FAK at focal adhesions in Y397F-FAK/YCam cells, since the cellular localisation of FAK phosphoTyr-576 in cells expressing Y397F-FAK/YCam or FAK/YCam was not different. Furthermore, FRAP analysis showed that phosphorylation of FAK at Tyr-397 increases specifically the time-residency of FAK at focal adhesions but not in cytosol. This in turn induces disassembly of focal adhesions at the cell tail and promotes cell motility as shown by the decrease in microtubule-mediated turnover of Y397F-FAK/YCam-containing focal adhesions. Our data show that phosphorylation of FAK at Tyr-397 is a key determinant of how FAK controls focal adhesion turnover.
Hamadi
Abdelkader
A
UMR CNRS 7034, Pharmacologie et Physicochimie des Interactions Cellulaires et Moléculaires, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, BP 60024, 67401 Illkirch, France.
Bouali
Maya
M
Dontenwill
Monique
M
Stoeckel
Herrade
H
Takeda
Kenneth
K
Rondé
Philippe
P
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D045744:Cell Line, Tumor; D036681:Fluorescence Recovery After Photobleaching; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D015739:Nocodazole / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000378:metabolism*
D000970:Antineoplastic Agents; D016023:Integrins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015739:Nocodazole