Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress.
The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.
Lunn
J Adrian
JA
Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine, UCLA-CURE, Digestive Diseases Research Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095.
Jacamo
Rodrigo
R
Rozengurt
Enrique
E
D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D003572:Cytochalasins / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology*; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D020125:Mutation, Missense; D009997:Osmotic Pressure / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000235:genetics; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C412373:AG 1879; D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003572:Cytochalasins; D007211:Indoles; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C416927:SU 6656; D013449:Sulfonamides; D053778:Thiazolidines; C031218:cytochalasin A; D014443:Tyrosine; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases; C037067:latrunculin A