Abstract
Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.
Highlights
Focal adhesion kinase (Fak) is a 125-kDa non-receptor protein-tyrosine kinase that is recruited to and activated by the engagement of transmembrane integrins by components of the extracellular matrix during integrin-mediated cell adhesion
The importance of Fak in transducing an anti-apoptotic signal upon integrin engagement is underscored by a number of studies: (i) constitutively activated forms of Fak prevent epithelial cell death upon cell detachment [9]; (ii) inhibition of Fak in cultured fibroblasts results in apoptosis [10]; (iii) Fak is overexpressed in some types of cancers [11,12,13,14]; and (iv) antisense oligonucleotides to Fak induce apoptosis in tumor cells [15]
These observations were extended in human cell lines undergoing apoptosis and the caspase family of cysteinyl aspartate-specific proteases were directly implicated in Fak cleavage [17] and disassembly of focal adhesions [18]
Summary
Plasmid Construction and Mutagenesis—The chicken Fak cDNA (in pBluescript II KSϪ) was the kind gift of Dr M.
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