Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases.
Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop. Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2. Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress. Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress. Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts. In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts. Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation. Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress. Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).
Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, UCLA-CURE, Digestive Diseases Research Center and Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
J Adrian
JA
Lunn
Enrique
E
Rozengurt
D016475:3T3 Cells; D000199:Actins / Q000737:chemistry; D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000378:metabolism; D001839:Bombesin / Q000494:pharmacology; D016360:Clostridioides difficile / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020558:GTP Phosphohydrolases / Q000378:metabolism; D047468:Immunoprecipitation; D020287:In Situ Nick-End Labeling; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D009995:Osmosis; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D013312:Stress, Physiological; D013395:Sucrose / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes; D014443:Tyrosine / Q000737:chemistry*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D000199:Actins; D001427:Bacterial Toxins; D001839:Bombesin; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020558:GTP Phosphohydrolases; D008055:Lipids; D051419:Paxillin; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D010750:Phosphoproteins; D054648:Protein Phosphatase 2; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D013395:Sucrose; D014443:Tyrosine; D020764:cdc42 GTP-Binding Protein; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D019061:src-Family Kinases; C488583:Clostridium difficile lethal toxin B; C086766:Ppp2r1b protein, mouse; C496062:Pxn protein, mouse; C496051:Ptk2 protein, mouse