INTRODUCTION

Apoptosis or programmed cell death is a physiological process that is important for the elimination of transformed cells, the elimination of self-reactive lymphocytes, and for the organization of developing tissues (1). Apoptosis causes characteristic morphological changes that include membrane blebbing, cellular shrinkage, and chromatin condensation that lead to cellular detachment from the substratum in adherent cells or a loss of cell-cell contact in suspension cells (2). Cellular attachment to the extracellular matrix (ECM)¹ is mediated by the association of integrins with extracellular matrix components such as fibronectin, collagen, and vitronectin, whereas cell-cell interactions are mediated by the association of integrins with members of the immunoglobulin gene superfamily such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (3). Recent studies suggest that integrins and ECM also promote cell survival. Inhibition of integrin function, for example, leads to loss of cell-cell adhesion and apoptosis in colon epithelial cells, loss of integrin interaction with ECM causes apoptosis of epithelial and endothelial cells ("anoikis"), and ECM suppresses apoptosis in mammary epithelial cells (4-7). Cells attach to the ECM through focal adhesions which are sites of tight structural attachment of the integrins on the cell membrane to the ECM. The focal adhesion can mediate signal transduction events that affect cellular migration, proliferation, differentiation, and survival (8). The cytoplasmic domain of the  subunit of integrins targets the integrin to the focal adhesion (for review see Ref. 9). Binding of integrins to ECM or clustering of integrins results in activation of specific kinases such as the tyrosine kinase focal adhesion kinase (FAK)/pp125^FAK (10).

FAK is a tyrosine kinase that localizes to focal adhesions and associates temporally and spatially with integrins (for reviews see Refs. 11 and 12). FAK also associates with other components of focal adhesions such as paxillin, p130^Cas, GRB2, pp60^src, pp59^fyn, talin, and phosphatidylinositol 3-kinase (13-23). FAK is expressed at low levels in normal cells but is overexpressed in some cancers such as breast and colon cancer (24). The precise function of FAK is not known, but it may regulate the assembly of focal adhesions in spreading or migrating cells or it may participate in a signal transduction pathway to inform the nucleus that a cell is bound to the ECM, which may suppress apoptosis. The first hypothesis is supported by a study that demonstrates that a truncated isoform of FAK (pp41/43^FRNK), which is identical to the COOH-terminal domain of full-length FAK, inhibits cell spreading and migration (25). Richardson and Parsons (25) observed that overexpression of FRNK inhibits tyrosine phosphorylation of FAK, suggesting that FRNK may act as a competitive inhibitor of FAK. The latter hypothesis is supported by recent studies which report the following: 1) FAK suppresses anoikis in epithelial cells; 2) inhibition of FAK in fibroblasts results in apoptosis; 3) attenuation of FAK induces apoptosis in tumor cells; and 4) FAK is cleaved early in myc-induced apoptosis of chick embryo fibroblasts and FAK cleavage, and apoptosis is inhibited by plating the cells on fibronectin or collagen (7, 26-28).

It is clear that mammalian cysteine proteases (now designated caspases) related to the Caenorhabditis elegans cell death gene CED-3 are the effectors of the apoptotic signaling pathway triggered by members of the tumor necrosis factor family (reviewed in Ref. 29). Members of the tumor necrosis factor family such as Fas ligand and Apo-2L, also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induce apoptosis in cells which express their cognate receptors (30-32). Apo-2L is more effective in killing adherent cell lines, but apoptosis induced by either ligand is inhibitable by CrmA, a cowpox-derived inhibitor of caspases (33, 34). Signaling through Apo-2L, unlike Fas, does not involve Fas-associated death domain; the members of the Apo-2L signaling pathway have not been characterized (33). Activation of interleukin-1 (ICE)-related proteases by Fas results in the cleavage of substrates that are thought to play a role in maintaining cytoplasmic and nuclear integrity such as lamin A, poly(A)DP-ribose polymerase (PARP), -Fodrin, sterol regulatory element-binding proteins, the 70-kDa protein component of the U1 small nuclear ribonucleoprotein, the retinoblastoma tumor suppressor gene product, protein kinase C , DNA-dependent protein kinase, and the GDP dissociation inhibitor D4-GDI (35-42). The functional significance of some of these cleavage events, however, is less clear.

In view of these data that FAK is important for suppressing apoptosis, we examined if Fas- and Apo-2L-induced apoptosis leads to disruption of FAK. We report that FAK is cleaved in suspension and adherent cells during Fas and Apo-2L-induced cell death. FAK cleavage occurred coincident with PARP cleavage or perhaps slightly before and it was mediated by ICE/Ced-3 caspases. Our data suggest that cleavage of FAK by Apo-2L or Fas may cause some of the morphological changes in apoptotic cells.

EXPERIMENTAL PROCEDURES

The Jurkat T cell line and H460 non-small cell lung cancer cell line were purchased from ATCC (Rockville, MD). Jurkat cells were cultured in RPMI with 10% fetal calf serum, and H460 cells were cultured in -minimum Eagle's media with 10% fetal calf serum supplemented with L-glutamine, penicillin, and streptomycin.

Soluble Apo-2L was provided by Dr. Avi Ashkenazi, Genentech (South San Francisco, CA), and used at a concentration of 50 ng/ml in Jurkat and H460 cells. Anti-Fas (IgM) mAb, clone CH-11, was purchased from Medical and Biological Laboratories (Nagoya, Japan) and used at a concentration of 100 ng/ml. Doxorubicin was provided by Dr. Branimir Sikic (Stanford University). Cell death was determined with the ApoAlert Annexin V Apoptosis Kit from CLONTECH (Palo Alto, CA) according to the manufacturer's protocol. After the cells were incubated with annexin V fluorescein isothiocyanate (1 µg/ml), they were analyzed by fluorescence-activated cell sorter analysis. The tetrapeptide caspase inhibitors DEVD-CHO and YVAD-CHO were purchased from BACHEM (Torrance, CA), and ZVAD-FMK was purchased from Enzyme Systems Products (Dublin, CA). For in vivo studies with the caspase inhibitors, DEVD-CHO, YVAD-CHO, or ZVAD-FMK were added to cells 1 h prior to the addition of Apo-2L or the anti-Fas mAb.

An anti-FAK mAb, clone 77, that recognizes the kinase domain (amino acids 354-533) was purchased from Transduction Laboratories (Lexington, KY), and a rabbit polyclonal antibody against the carboxyl terminus of FAK (C-903) was purchases from Santa Cruz Biotechnology (Santa Cruz, CA). The poly(ADP-ribose) polymerase (PARP) mAb, clone C2-10, was purchased from Dr. Guy Poirier (Laval University, Sainte-Foy, Quebec, Canada). The ERK2 rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For immunoblot analysis, Jurkat or H460 cells were treated under the conditions described above and then were lysed in HNET buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Samples were centrifuged for 5 min to remove insoluble material followed by measurement of protein concentration by the Bradford method, Bio-Rad. Samples containing equal protein concentrations were denatured by boiling and analyzed by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose. The blot was then placed in blocking buffer containing 4% milk, 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature or overnight at 4 °C. The blot was then incubated in blocking buffer with individual antibodies, as described above, followed by incubation in 10 mM Tris-HCl pH 7.5, 100 mM NaCl, and 0.1% Tween 20 (TBS-T) containing a horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG Caltag Laboratories (San Francisco, CA) at a dilution of 1:1,500 and detected by enhanced chemiluminescence (Amersham Corp.) followed by autoradiography. Where indicated the blot was stripped by heating the membrane at 90 °C for 5 min in TBS-T containing 0.5% SDS and reprobed with the polyclonal C-903 FAK antibody, PARP, or ERK2 antibody.

Jurkat cells were either untreated or treated with 50 ng/ml Apo-2L for 2 h and pelleted by centrifugation. The cells were washed with ice-cold phosphate-buffered saline and then resuspended in hypotonic buffer (10 mM Tris-HCl, pH 7.5, 20 mM KCl, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) and left on ice for 30 min. Cells were then lysed with 20 strokes of a Dounce homogenizer (type B pestle) and centrifuged at maximum speed for 20 min at 4 °C. Supernatants (naive and apoptotic extracts) were divided into small aliquots and stored at 80 °C until use.

1 µg of baculovirus-expressed FAK protein preparation (containing about 5% FAK (for details see Ref. 17)) was incubated with 20 µg of naive or apoptotic extract in cleavage buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM MgCl₂, and 1 mM dithiothreitol) at 37 °C for 1 h, electrophoresed on a 10% SDS gel, and immunoblotted with the clone 77 FAK mAb as described above. Tetrapeptide caspase inhibitors were added to the cell extracts and incubated at room temperature for 10 min prior to the addition of FAK protein.

1 µg of baculovirus-expressed FAK protein preparation was incubated with the indicated amount of purified caspases in the cleavage buffer at 37 °C for 1 h, separated on a 10% SDS gel, and immunoblotted with the clone 77 anti-FAK mAb. The same blot was stripped by heating the membrane at 90 °C for 5 min in TBS-T containing 0.5% SDS and reprobed with the polyclonal C-903 FAK polyclonal antibody. Studies were done both with caspases provided by Guy Salvesen, Burnham Institute (La Jolla, CA), and from Vishva Dixit's laboratory, University of Michigan (Ann Arbor, MI). Caspase-3 and caspase-7 provided by Dr. Guy Salvesen were used to generate the data shown in Fig. 5. The activity of caspase-3 and caspase-7 was determined by Dr. Salvesen with an affinity labeled Ac-Asp-Glu-Val-fluoromethyl ketone (a gift of Joe Krebbs, IDUN Pharmaceuticals). The caspases were titrated with a known amount of the affinity reagent, followed by analysis of residual activity using the caspase substrate Ac-DEVD-p-nitroanilide (Alexis Corp., San Diego, CA).

Untreated Jurkat cells or Jurkat cells treated with 50 ng/ml Apo-2L for 4 h were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 7.5) by passing through a 25-gauge needle 5 times. 0.5 mg of cellular protein was immunoprecipitated with 2 µl of KC rabbit polyclonal antibody raised against the kinase and COOH-terminal domain of FAK (for details see Ref. 17). Immune complexes were collected on protein A-Sepharose (Sigma), washed three times with RIPA buffer electrophoresed on a 10% SDS gel, and immunoblotted with the C-903 polyclonal anti-FAK antibody.

RESULTS AND DISCUSSION

Recent studies demonstrate that Apo-2L is more effective in inducing apoptosis of adherent cells than Fas, but both are similarly effective in inducing apoptosis of suspension cells (33, 34). FAK has recently been shown to play a role in suppressing apoptosis (7, 26-28). We, therefore, examined the effect of Apo-2L on FAK in suspension and adherent cells. Apo-2L induced sequential cleavage of FAK into two fragments in Jurkat T cells (Fig. 1A). The first cleavage product of approximately 85 kDa appeared 2 h after the addition of Apo-2L, and the second cleavage product of approximately 77 kDa appeared at 4 h (Fig. 1A). At 24 h after the addition of Apo-2L the 85 kDa fragment disappeared, but the 77-kDa fragment remained (Fig. 1A). The disappearance of the 85-kDa fragment late in Apo-2L-induced apoptosis suggests that either the 85-kDa fragment is cleaved further into the 77-kDa fragment or the 77-kDa fragment may be derived from cleavage of intact FAK and the 85-kDa fragment is unstable. FAK cleavage occurred early in apoptosis and correlated temporally with PARP cleavage (Fig. 1A). Similar results were obtained with an agonistic cross-linking Fas antibody, but FAK cleavage and apoptosis occurred 2-3 h later (data not shown).

We then examined the effect of Apo-2L on FAK in the H460 non-small cell lung cancer adherent cell line. Apoptosis in H460 cells after the addition of Apo-2L followed the same time course that was observed in Jurkat T cells (Fig. 1B). Apo-2L induced cleavage of FAK into a fragment of approximately 85 kDa 3 h after the addition of Apo-2L (Fig. 1B). In H460 cells, interestingly, no second 77-kDa cleavage product was observed at 5 or 24 h after the addition of Apo-2L (Fig. 1B and data not shown). We did observe, however, that FAK was cleaved into the 85- and 77-kDa fragments in the adherent MCF-7 breast cancer cell line (data not shown). An anti-Fas cross-linking mAb also induced apoptosis and FAK cleavage in H460 cells, but it was temporally later and quantitatively less than Apo-2L (data not shown). The chemotherapeutic agent doxorubicin (2 µM), in addition, induced FAK cleavage in H460 cells (data not shown). These data demonstrate that FAK cleavage occurs during apoptosis induced by different apoptotic stimuli.

A recent study reported that FAK is cleaved during c-Myc-induced apoptosis of chick embryo fibroblasts. Cell death and FAK cleavage were suppressed by plating the cells on ECM components such as collagen and fibronectin (26). FAK cleavage was also inhibited by a 1-specific integrin antibody. Crouch et al. (26) concluded that c-myc-induced cell death in chick embryo fibroblasts requires disruption of the integrin signaling pathways which are mediated, at least in part, by the interaction of FAK with its downstream effectors. We examined if plating the H460 adherent cell line on ECM inhibits Apo-2L-induced apoptosis and FAK proteolysis. H460 cells were plated on an ECM mixture of fibronectin, vitrogen, and bovine serum albumin. Focal adhesions were detected in the H460 cells plated on ECM by immunofluorescent staining with an anti-vinculin monoclonal antibody (data not shown). There was no significant increase in cell survival or inhibition of FAK proteolysis after plating the cells on ECM (data not shown). These data coupled with our observation that FAK is cleaved in suspension cells suggests that integrin signaling through ECM is not involved in suppressing apoptosis induced by Apo-2L or Fas.

We then tested the effects of tetrapeptide cysteine protease inhibitors on FAK cleavage and cell survival after Apo-2L treatment of Jurkat cells. ZVAD-FMK (40 µM) completely blocked FAK cleavage in vivo and significantly increased cell survival (Fig. 2). ZVAD-FMK is a general inhibitor of cysteine proteases and Fas-induced apoptosis (43). DEVD-CHO inhibited formation of the second 77-kDa cleavage product at lower concentrations (40 µM), but it did not affect formation of the first 85-kDa fragment at that concentration. At higher concentrations DEVD-CHO (200 µM) suppressed formation of both FAK cleavage products and increased cell survival. DEVD-CHO preferentially inhibits activation of YAMA/CPP32-like caspases (44, 45). YVAD-CHO did not suppress FAK cleavage or augment survival even at a concentration of 200 µM (Fig. 2 and data not shown). YVAD-CHO is more effective as an inhibitor of ICE and ICE-like proteases, but YVAD-CHO is less effective than YVAD-CMK as an ICE-like protease inhibitor (44, 46). We then tested the ability of these inhibitors to suppress cleavage of purified FAK by apoptotic extract in vitro. DEVD-CHO and ZVAD-FMK, but not YVAD-CHO, at 0.5 µM concentrations or more inhibited formation of both FAK cleavage products by apoptotic extract in vitro (Fig. 2B). These data suggest that DEVD-CHO is less potent as a caspase inhibitor in vivo because of reduced cellular permeability. CPP32/YAMA-like caspases appear to mediate the cleavage of FAK, and in vivo the second 77-kDa cleavage event is more sensitive to a DEVD-inhibitable caspase(s) than the first 85-kDa cleavage event. Similar results were also obtained with these inhibitors following activation of the Fas pathway in Jurkat cells (data not shown).

Our in vivo findings suggested that FAK cleavage is mediated by DEVD-sensitive caspase(s). To identify the caspase(s) involved in FAK cleavage, we examined the effect of different purified caspases on baculovirus expressed FAK in vitro. The addition of purified caspase-3 or caspase-7 to FAK generated an 85-kDa FAK cleavage product that migrated with the same mobility as the first cleavage product which was observed in apoptotic extracts in vivo (Fig. 3A). Neither caspase-3 nor caspase-7 generated the second cleavage product in vitro, but each generated other fragments that probably represent specific caspase-3- and caspase-7-sensitive cleavage sites in FAK (Fig. 3A). Caspase-6, interestingly, partially cleaved FAK into a 77-kDa cleavage product that migrated with the same mobility as the second cleavage product observed with the apoptotic extract in vivo, but it did not generate levels of the first cleavage product that were above control (Fig. 3A). Caspase-6 induced only partial FAK cleavage so that we examined if cleavage to the 88-kDa FAK fragment would facilitate generation of the 77-kDa fragment by caspase-6 in vitro. Incubation of purified FAK with caspase-3 or caspase-7 followed by incubation with caspase-6 did generate more of the 77-kDa product than caspase-6 alone (data not shown).

Caspase-6, but not caspase-3 or caspase-7, has recently been shown to cleave lamin A (47, 48). Caspase-8 has recently been shown to be capable of proteolytically activating caspases such as caspase-3, caspase-4, caspase-7, and caspase-9 in the absence of naive extract (49, 50). Caspase-6 and caspase-2 were efficiently cleaved by caspase-8 only in the presence of naive extract (50). We tested if caspase-8 alone or in the presence of naive extract cleaves FAK in vitro. Caspase-8 induced partial FAK cleavage to form the first 85-kDa product, but it induced more FAK cleavage in the presence of naive extract suggesting that caspase-8 activates other caspases such as caspase-3 and caspase-7 in naive extract (Fig. 3). We used twice as much caspase-8 in the assay because caspase-3, caspase-6, and caspase-7 are twice as active as caspase-8.²

These data shown above were generated with an antibody that recognizes the FAK kinase domain. To help us map the potential cleavage sites in FAK we reprobed the blots with an antibody that recognizes the FAK carboxyl terminus. The COOH-terminal antibody recognized a FAK cleavage product of approximately 33 kDa after FAK was incubated with caspase-3, caspase-7, or with caspase-8 plus naive extract in vitro (Fig. 3B). The 33-kDa fragment is the predicted size of a COOH-terminal fragment that would be detected after the first FAK cleavage in vivo. The same antibody detected a cleavage product of approximately 41 kDa after incubation with caspase-6, but the band was visible only after a longer gel exposure (data not shown). To detect the COOH-terminal FAK fragment in intact cells, we immunoprecipitated with a polyclonal FAK antibody raised against the kinase and COOH-terminal domain of FAK followed by immunoblotting with a COOH-terminal FAK antibody (Fig. 3C). We detected a band of 33 kDa in apoptotic extract that was the same size as the FAK cleavage fragment generated by caspase-3 or caspase-7 in vitro (Fig. 3C).

Based on the size of these cleavage products that we observed with the kinase domain antibody and COOH-terminal antibody, we predict that the first cleavage in FAK occurs after the P1 aspartate in DQTDS (amino acid 772) and the second cleavage occurs after the aspartate in VSWDS (amino acid 704) (see Fig. 4 for schematic of FAK cleavage). The first cleavage would, therefore, generate a 33-kDa COOH-terminal fragment leaving an intact 85-kDa fragment. The second cleavage would remove an additional 8 kDa from the 85-kDa fragment, but the 33-kDa carboxyl-terminal fragment would not change. Our data also suggest that the first FAK cleavage may be caused by caspase-3 or caspase-7 and the second cleavage by caspase-6 or by a related caspase.

Caspase-3 and caspase-7 both cleave substrates with DXXD motifs, but there is no known published data that show differential sensitivity of a substrate, such as PARP, to caspase-3 versus caspase-7. We observed, interestingly, that purified caspase-7 is approximately 100-fold more active in generating the first FAK 85-kDa cleavage product than caspase-3 in vitro (Fig. 5A). We also observed identical results using the COOH-terminal FAK antibody to detect FAK cleavage (Fig. 5B). These purified caspases are equally active against a defined substrate (see "Experimental Procedures").² The enhanced sensitivity to caspase-7 in vitro, therefore, suggests that FAK and possibly other substrates may show differences in sensitivity to caspase-3 and caspase-7 in vivo.

Our in vitro and in vivo caspase data suggest that the first FAK cleavage is caused by caspase-7 preferentially over caspase-3 which is followed by an additional cleavage in Jurkat T cells by caspase-6 or a related caspase. Our data which show that the second cleavage event is very sensitive to inhibition by DEVD-CHO suggest that a DEVD-inhibitable caspase such as caspase-7 and/or caspase-3 activates caspase-6 or a related caspase to produce the 85- and 77-kDa FAK cleavage products. This ordering puts caspase-6 downstream of caspase-3 or caspase-7 which appears to be in contrast to a recent report that shows caspase-6 activates caspase-3 and caspase-7 in vitro (51). It is possible, however, that caspase-6 is not the caspase that generates the second FAK cleavage in vivo, a possibility supported by our data that show that caspase-6 induces only partial cleavage of FAK in vitro. It is also possible that caspase-6 or a related caspase can cleave FAK only after it has been cleaved by caspase-7 or caspase-3 which is supported by our observation that cleavage into the 88-kDa fragment by caspase-3 or caspase-7 facilitates cleavage into the 77-kDa fragment by caspase-6 in vitro. Further studies using FAK as a substrate may help decipher the ordering of the caspases that are activated during Apo-2L- and Fas-triggered apoptosis. It will also be important to determine if FAK is a preferred substrate for caspase-7 over caspase-3 in vivo.

FAK plays an important role in regulating focal adhesion formation in adherent cells, but a role in suspension cells has not been defined. Recent studies suggest that FAK suppresses apoptosis of adherent cells and that disruption of FAK can lead to apoptosis. Our data demonstrate that Apo-2L and Fas induce sequential proteolytic cleavage of FAK in Jurkat T cells. In the H460 adherent lung cancer cell line only the first cleavage product was detected. The first cleavage is predicted to generate a 33-kDa COOH-terminal fragment which would leave an intact kinase domain. The COOH-terminal region of FAK contains the targeting sequence that is required for efficient recruitment of FAK to focal adhesions. FAK cleavage would therefore lead to an uncoupling of the kinase domain from the focal adhesion targeting COOH-terminal domain. We have observed that Jurkat cells have basal FAK kinase activity, and kinase activity is markedly reduced in apoptotic extract from Apo-2L-treated Jurkat cells, which supports our hypothesis that FAK function is compromised during Apo-2L-induced apoptosis.³ Richardson and Parsons (25) identified a naturally occurring truncated isoform of FAK (pp41/43^FRNK) that is identical to the COOH-terminal domain of full-length FAK. Overexpression of FRNK inhibited tyrosine phosphorylation of FAK and caused a delay in cell spreading suggesting that FRNK may be a competitive inhibitor of FAK. It is possible that the COOH-terminal FAK fragment that is generated during Apo-2L- and Fas-induced apoptosis will also act as a competitive inhibitor of FAK. It will be interesting, therefore, to determine if overexpression of the COOH-terminal FAK fragment which is generated during Apo-2L- and Fas-induced apoptosis will also perturb FAK function and perhaps induce apoptosis. Our observation that FAK is cleaved in suspension cells and that cell-cell interactions are disrupted during apoptosis suggests that FAK may play a role in maintaining cell-cell interactions in suspension cells. In summary, our data which show early cleavage of FAK into at least two fragments during Apo-2L- and Fas-triggered apoptosis suggest that disruption of FAK contributes to the morphological changes observed in apoptotic suspension and adherent cells.