Preferential Phosphorylation of Focal Adhesion Kinase Tyrosine 861 Is Critical for Mediating an Anti-apoptotic Response to Hyperosmotic Stress

Enrique Rozengurt,Rodrigo Jacamo,J. Adrian Lunn

doi:10.1074/jbc.m607780200

Enrique Rozengurt, Rodrigo Jacamo + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m607780200

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Abstract

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.

Highlights

Are exposed to hyperosmotic stress [1, 2]
We determined whether hyperosmotic stress stimulates phosphorylation of specific focal adhesion kinase (FAK) tyrosine residues in epithelial cells derived from rat intestinal crypts (IEC-18), human colonic carcinoma (Caco2), and human gastric carcinoma (NCI-N87)
We propose that FAK signaling from phosphorylated Tyr-861 plays a critical role in the response of epithelial cells to withstand hyperosmotic stress

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—IEC-18 cells were maintained in DMEM with 5% FBS between 50 and 80% confluency. CDNA Plasmid and Retroviral Transfection Methods—To generate the MSCV-FAK-GFP construct, we first engineered an intermediate vector named pFAK-EGFP. After FACS sorting for GFP-positive cells, cells transfected with FAK WT or FAK 861F were grown to 90% confluence in DMEM. Membranes were blocked using 5% nonfat dried milk in PBST, pH 7.2, and incubated overnight at 4 °C with the anti-FAK-Tyr(P)-397 antibody (0.1 ␮g/ml), anti-FAK-Tyr(P)576 antibody (0.1 ␮g/ml), anti-FAK-Tyr(P)-861 antibody (0.1 ␮g/ml), or anti-FAK C20 antibodies They were incubated in horseradish peroxidase-conjugated secondary antibodies (donkey anti-rabbit or sheep anti-mouse) at 1:5000 for 1 h at room temperature. After washing five times in PBS, cell were blocked in PBS containing 1% bovine serum albumin (fraction V, Sigma) and 2% fetal bovine serum for 2 h at room temperature. All other reagents used were of the highest grade available

RESULTS

DMEM alone or DMEM containing

DISCUSSION