Dephosphorylation of Focal Adhesion Kinase (FAK) and Loss of Focal Contacts Precede Caspase-mediated Cleavage of FAK during Apoptosis in Renal Epithelial Cells

Bob Van De Water,J Fred Nagelkerke,James L Stevens

doi:10.1074/jbc.274.19.13328

Abstract

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and cleavage in renal epithelial cells during apoptosis caused by the nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a loss of cell-matrix contact which was preceded by a dissociation of FAK from the focal adhesions and tyrosine dephosphorylation of FAK. Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However, FAK cleavage occurred after cells had already lost focal adhesions indicating that cleavage of FAK by caspases is not responsible for loss of FAK from focal adhesions. Accordingly, although inhibition of caspase activity with zVAD-fmk blocked activation of caspase-3, FAK cleavage, and apoptosis, it neither affected dephosphorylation nor translocation of FAK or paxillin. However, zVAD-fmk completely blocked the cell detachment caused by DCVC treatment. Orthovanadate prevented DCVC-induced tyrosine dephosphorylation of both FAK and paxillin; however, it did not inhibit DCVC-induced apoptosis and actually potentiated focal adhesion disorganization and cell detachment. Thus, FAK dephosphorylation and loss of focal adhesions are not due to caspase activation; however, caspases are required for FAK proteolysis and cell detachment.

Highlights

The relationship between focal adhesion protein (FAK) activity and loss of cell-matrix contact during apoptosis is not entirely clear nor has the role of focal adhesion kinase (FAK) in chemically induced apoptosis been studied
DCVC Causes Activation of Caspases and Cleavage of FAK in renal proximal tubule epithelial cells (RPTE)—Apoptosis is associated with the activation of caspases
Since DCVC-induced apoptosis of RPTE is associated with impaired cell adhesion [57, 58, 65], and because FAK is cleaved by caspases in cell-free systems as well as in intact cells in vitro [12,13,14,15], we determined whether DCVC-induced caspase activation is associated with FAK cleavage in RPTE

Summary

EXPERIMENTAL PROCEDURES

Materials—Fetal bovine serum was from Life Technologies (Grand Island, NY). Dulbecco’s modified Eagle’s medium/Ham’s F-12, bovine serum albumin fraction V, cholera toxin, insulin, antibiotic were from Sigma. Cells were cultured on rat tail collagen (Collaborative Research, Bedford, MA) coated dishes in Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing 1% (v/v) fetal bovine serum, 0.5 mg/ml bovine serum albumin, 10 ␮g/ml insulin, 10 ng/ml epidermal growth factor, 10 ng/ml cholera toxin, and antibiotics as described [57, 67]. Following treatment with DCVC for 4 h, cells were allowed to recover in complete medium containing DPPD (20 ␮M) with or without the above inhibitors. To determine the percentage of cell detachment, floating cells (supernatant) and adherent cells (obtained after trypsinization; see below) were collected separately. Apoptosis was determined by cell cycle analysis Both floating and adherent cells that were trypsinized were pooled and fixed in 90% ethanol (Ϫ20 °C). Fluorescence derived from release of the AMC moiety was followed using a fluores-

Focal Adhesion Kinase in Renal Epithelial Cell Apoptosis

RESULTS

DISCUSSION