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Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide
F G Gervais1, N A Thornberry, S C Ruffolo
1Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Québec H9R 4P8, Canada.
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.
Insights
Focal adhesion kinase (Fak) is cleaved by caspases during apoptosis, generating fragments that inhibit its survival signals. This cleavage disables Fak
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase crucial for cell adhesion, motility, and survival.
- Fak transduces anti-apoptotic signals from focal adhesions, mediated by integrin clustering.
- Apoptosis involves programmed cell death, often regulated by specific enzymatic pathways.
Purpose of the Study:
- To investigate the cleavage of Fak by caspases during apoptosis.
- To map the specific cleavage sites and identify the caspases involved.
- To understand the functional consequences of Fak cleavage on its anti-apoptotic activity.
Main Methods:
- Identification and mapping of Fak cleavage sites using biochemical assays.
- Enzymatic assays using purified caspases (caspase-3, caspase-6) and granzyme B.
- Analysis of Fak fragments generated during apoptosis in cellular models.
Main Results:
- Fak is cleaved by caspases at two distinct sites: DQTD772 (preferentially by caspase-3) and VSWD704 (by caspase-6 and granzyme B).
- Cleavage separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain.
- Generated carboxyl-terminal fragments suppress endogenous Fak phosphorylation, mimicking the inhibitory function of FRNK.
Conclusions:
- Fak cleavage by caspases plays a role in apoptosis execution by disabling its anti-apoptotic function.
- This mechanism contributes to the cellular suicide program.
- Cleavage sites are not conserved across species (e.g., rodent Fak), representing a novel aspect of caspase substrate regulation.