Caspases Cleave Focal Adhesion Kinase during Apoptosis to Generate a FRNK-like Polypeptide*

François G. GervaisDagger , Nancy A. Thornberry§, Salvatore C. RuffoloDagger , Donald W. NicholsonDagger , and Sophie RoyDagger

From the Dagger  Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Québec H9R 4P8, Canada and the § Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (e.g. fibronectin) during integrin-mediated cell adhesion. The activation of Fak leads to (i) activation of the Ras-mitogen-activated protein kinase pathway and (ii) recruitment and phosphorylation (either directly or indirectly via the recruitment of Src-like tyrosine kinases) of a number of cytoskeletal proteins, resulting in the formation of contact sites between the cell surface and the extracellular matrix called focal adhesions. By activating the Ras-mitogen-activated protein kinase pathway and promoting the assembly of focal adhesions, Fak mediates multiple cellular responses to cell adhesion including cell survival and proliferation as well as cell spreading and motility (for review, see Ref. 1). Fak function is dependent on two distinct domains: the tyrosine kinase domain within the amino-terminal half of the protein and a focal adhesion targeting (FAT1) domain within the carboxyl-terminal half of the protein. Fak is positively regulated by autophosphorylation, which allows the recruitment of signaling molecules like Src (2, 3), phosphatidylinositol 3-kinase (4, 5), and subsequently Grb2 (6). Fak is negatively regulated by the expression of FRNK (p41/p43FRNK; Fak-related non-kinase), a truncated isoform of Fak that contains a FAT domain but lacks the kinase domain (7). FRNK inhibits the cellular responses to adhesion by preventing the localization of Fak to sites of integrin clustering (8).

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 (anoikis) (9); (ii) inhibition of Fak in cultured fibroblasts results in apoptosis (10); (iii) Fak is overexpressed in some types of cancers (11-14); and (iv) antisense oligonucleotides to Fak induce apoptosis in tumor cells (15). It is therefore not surprising that Fak has been shown to be the target of proteolysis in chicken embryo fibroblasts undergoing apoptosis (16). More recently, 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). The caspases are responsible for initiating and executing apoptotic cell death by cleaving critical homeostatic, repair, and structural proteins in the dying cell (for review see Ref. 19). In this report, we identify two sites within chicken Fak that are cleaved by caspases in vitro and in apoptotic cells. Cleavage at either site results in the separation of the kinase domain from the FAT domain. Fragments containing the FAT domain, when expressed in HeLa cells, inhibit phosphorylation of endogenous Fak, suggesting that they act like FRNK and interfere with the function of uncleaved Fak. Although the molecular masses of the fragments released from the cleavage of human Fak in cells undergoing apoptosis are consistent with the sites identified in chicken Fak, cleavage of murine Fak appears to occur at a different site.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Construction and Mutagenesis-- The chicken Fak cDNA (in pBluescript II KS-) was the kind gift of Dr. M. Schaller (University of North Carolina). The D772A mutation was made by an overlap extension PCR method (20). The complementary inverse oligonucleotides 5' GAT CAA ACA GCT TCC TGG AAC 3' and 5' GTT CCA GGA AGC TGT TTG ATC 3' were used in combination with the internal oligonucleotides 5' AAGCCCTTCCAGGGAGTG 3' and 5' ATGCTGATACTTCCTGAGG 3'. From the large PCR fragment, a 417-base pair BstEII fragment containing the mutation was removed and cloned back into the Fak wild-type sequence. The region corresponding to the BstEII fragment was fully sequenced to ensure that no other mutation was introduced inadvertently. A similar strategy was used to generate the D704A mutation using the complementary inverse oligonucleotides 5' GTC ACA GTA TCC TGG GCC TCA GGA GGA TCA GAT G 3' and 5' CAT CTG ATC CTC CTG AGG CCC AGG ATA CTG TGA C 3' in combination with the internal oligonucleotides 5' AAG CCC TTC CAG GGA GTG 3' and 5' TCC TCC ATG TTT GGC TGC 3'. The mutated fragment was subcloned into the Fak wild-type sequence using the restriction sites PflMI and NdeI, and the clone was sequence-verified.

The introduction of a flag epitope (DYKDDDDK) at the carboxyl terminus of Fak was performed by PCR with the following oligonucleotides: 5' CGC TCG AGT TAC TTG TCA TCG TCG TCC TTG TAG TCG TGG GGC CTG GAC TGG CTG ATC ATT TTC AG 3' and 5' CCA GAT CAT GCC GCT CCA CC 3'.

For stable expression of Fak in K562 cells, wild-type and mutant Fak cDNAs were subcloned from the pBluescript II KS- digested with the restriction enzymes NotI and EcoRV into the episomal eukaryotic expression vector pCep4beta (21) digested with BamHI (rendered blunt-ended with T4 DNA polymerase) and NotI. cDNAs for expression of FRNK and caspase generated FRNK-like fragments in HeLa cells were engineered by PCR using the following oligonucleotides: 5' AAT TAA CCC TCA CTA AAG GG 3' and either 5' CAG GAA TTC TAG CAA AAC CAT GGA ATC CAG GCG ACA AGT CAC AG 3' for FRNK, 5' CAG GAA TTC TAG CAA AAC CAT GTC AGG AGG ATC AGA TGA AGC TC 3' for C705-1053, or 5' CAG GAA TTC TAG CAA AAC CAT GTC CTG GAA CCA TCG ACC TCA GG 3' for C773-1053. C705-1053 and C773-1053 correspond exactly to the fragments generated by caspase cleavage at VSWD704 and DQTD772, respectively, except that an initiating methionine was introduced immediately upstream of the P1' amino acid. The fragments generated by PCR were first subcloned into pBluescript II KS- digested with the restriction enzyme EcoRI, sequence-verified, and then subcloned into pCep4beta (21) as described above.

Cell Lines-- Cell lines stably expressing the various Fak sequences were created by transfection of the pCep4beta constructs in the human K562 lymphoid cell line (ATCC GM05372E, NIGMS Human Genetic Mutant Cell Repository, NIH) with LipofectAMINETM (Life Technologies, Inc.) according to the manufacturer's instructions. Cell lines were propagated and selected in RPMI containing 0.5 mg/ml hygromycin B (Boehringer), 10% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).

Induction of Apoptosis-- Jurkat cells were serum-starved in 0.5% (v/v) fetal bovine serum for 48 h and then treated for 2 h with anti-CD95 (Fas/Apo-1) antibodies (MBL) at 1 µg/ml. HeLa and 3T3 L1 cells were rendered apoptotic by treatment with 1 µM staurosporine for 2 h. K562 cells were treated for 18 h with camptothecin at 5 µg/ml. Cell lysates were prepared for SDS/PAGE by boiling in Laemmli buffer at a concentration of 5-10 × 106 cells/ml.

Immunoblotting-- Cell extracts (approximately 2 × 105 cells per extract) were analyzed by SDS-PAGE (Novex) and transferred to nitrocellulose at 40 V for 16 h. The blots were first incubated in blocking buffer (Tris-buffered saline, pH 7.4 (TBS), 5% (w/v) milk (Blotting grade blocker non-fat dry milk; Bio-Rad), 0.05% (v/v) Tween 20) for 1 h at room temperature and then incubated for an additional hour in primary antibody diluted in blocking buffer. After washing three times in 1 × TBS with 0.1% (v/v) Tween 20 for 5 min, blots were incubated for 1 h at room temperature in goat anti-mouse IgG coupled to horseradish peroxidase diluted 1:3000 in blocking buffer. Blots were washed three times in 1 × TBS, 0.3% (v/v) Tween 20 for 5 min, and three times in 1 × TBS, 0.1% (v/v) Tween 20 for 5 min. Detection was performed using enhanced chemiluminescence (ECLTM system from Amersham).

In Vitro Cleavage of Fak-- [35S]Methionine-labeled Fak was obtained by coupled in vitro transcription/translation using the Promega TNT reticulocyte lysate system. One µg of the cDNA construct obtained by Qiaprep purification (Qiagen) was incubated with T7 polymerase, rabbit reticulocyte lysate, amino acid mixture minus methionine, and [35S]methionine (Ci/mmol) for 1 h at 30 OC. Cleavage of the in vitro transcribed/translated radiolabeled product was performed by incubation at 37 OC in the presence of either apoptotic extract or purified recombinant human (rh)-caspases in cleavage buffer (50 mM Hepes/KOH (pH 7.0), 2 mM EDTA, 0.1% (w/v) CHAPS, 10% (w/v) sucrose, 5 mM dithiothreitol). The final volume of the reaction was 25 µl. The cleavage reaction was terminated by the addition of SDS Laemmli loading buffer and analyzed by SDS-PAGE and fluorography. Seventy-five µg of cytosolic extracts prepared from apoptotic Jurkat T-cells lysed in cleavage buffer (see above) were used in the cleavage assays. Purified human caspases-1, -3, -6, and -8 and granzyme B were prepared as described previously (22). The protease inhibitor sensitivity profile was performed by preincubating the protease inhibitors with the apoptotic extracts for 20 min at room temperature prior to the addition of [35S]methionine-labeled Fak generated by in vitro transcription/translation and purified by FPLC on a Superdex 75 column (Amersham Pharmacia Biotech). The proteolytic cleavage was quantitated by laser densitometry of the resulting fluorograms.

Kinetic Evaluation of Fak Cleavage-- FPLC-purified radiolabeled Fak was incubated for 60 min at 37 OC with various concentrations of purified caspases in the cleavage buffer as described above. Reaction products were separated by SDS-PAGE, visualized by fluorography, and quantitated by phosphorimaging. All reactions were carried out using levels of substrate well below Km, where the appearance of product is a first-order process. Values for kcat/Km were calculated from the relationship St/So = e-kobs*t where St is the concentration of substrate remaining at time t, So is the initial substrate concentration, and kobs = kcat*[enzyme]/Km.

Tyrosine Phosphorylation of Endogenous Fak Protein-- HeLa cells were transiently transfected with FRNK, C705-1053, or C773-1053 cDNAs in the pCep4beta expression vector. Cells were collected by scraping 24 h after transfection, washed once in phosphate-buffered saline, and lysed in TNE buffer (50 mM Tris, pH 8.0, 1% Nonidet P-40, 2 mM EDTA) containing 10 µg of each of the following protease inhibitors: leupeptin, aprotinin, N-tosyl-L-phenylalanine chloromethyl ketone, Nalpha -p-tosyl-L-lysine chloromethyl ketone, and phenylmethylsulfonyl fluoride, as well as the phosphatase inhibitors sodium fluoride (50 mM) and sodium orthovanadate (1 mM). Protein concentrations were determined using the Bio-Rad protein assay. One mg of each lysate was used to dilute 5-fold a 5 × RIPA buffer stock (750 mM NaCl, 5% Nonidet P-40, 2.5% sodium deoxycholate, 0.5% SDS, 500 mM Tris, pH 8.0, 25 mM EDTA). Immunoprecipitation of endogenous Fak protein was carried out with 4 µg of an antibody directed against residues 748-1053 of human p125Fak (Upstate Biotechnology). Immune complexes were collected on protein A-Sepharose beads, washed three times in 1 × RIPA buffer containing 1 mM sodium orthovanadate, and eluted in Laemmli buffer by boiling. Tyrosine phosphorylation of endogenous Fak was visualized by SDS-4-20% PAGE, transfer to nitrocellulose, and immunoblotting with the RC20 anti-phosphotyrosine antibody (Transduction Laboratories) using enhanced chemiluminescence (ECLTM system from Amersham Pharmacia Biotech).

    RESULTS
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References

Serum deprivation of chicken embryo fibroblasts was shown recently to result in the proteolysis of Fak into fragments of 70-90 kDa (16). To identify the enzyme responsible for the cleavage of Fak during apoptosis, we set up an in vitro assay whereby [35S]methionine-labeled chicken Fak generated by in vitro transcription/translation was incubated with an apoptotic extract derived from Jurkat T lymphocytic cells treated with an antibody to the CD95 (Fas/Apo-1) death receptor (these cells were chosen because their ability to undergo apoptotic cell death has been extensively characterized). Two fragments of 90 and 35 kDa were observed after incubation with apoptotic but not with non-apoptotic cell extracts (Fig. 1, lanes 1 and 2). The protease inhibitor sensitivity profile of Fak cleavage was characteristic of that of the caspase family of cysteine proteases in that proteolysis was abolished by the cysteine-alkylating reagent iodoacetamide (lane 21), but not by the cysteine protease inhibitor E-64 (lane 14) nor by serine, aspartate, or metalloprotease inhibitors (lanes 3-13 and 15-20).


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Fig. 1.   Iodoacetamide-sensitive but E-64-insensitive cleavage of Fak in apoptotic extracts. Apoptotic Jurkat cell extracts were incubated with [35S]methionine-labeled Fak in the presence of various protease inhibitors, as indicated. The cleavage reactions were carried out for 1 h at 37 °C and visualized by 10% SDS-PAGE and fluorography. Arrows indicate full-length Fak (p125Fak) and cleaved fragments (p90, p35).

To confirm that a caspase was responsible for the cleavage of Fak and to define more precisely the group of caspases involved, we performed the cleavage assays in the presence of three different tetrapeptide aldehydes, each of which preferentially inhibits a caspase subgroup (22). The interpretation of the results was based on the known selectivity of these inhibitors for all 10 human recombinant caspases. Up to 10 µM Ac-YVAD-CHO did not prevent the generation of the 90- and 35-kDa proteolytic fragments (Fig. 2A, lanes 15-21), excluding the possibility that one of the Group I caspases (caspase-1, -4, -5; Ref. 22) was responsible for cleavage. In contrast, as little as 100 nM Ac-DEVD-CHO completely abolished Fak cleavage (lanes 1-7; IC50 10 nM) whereas Ac-IETD-CHO inhibited Fak cleavage only when present at moderately high concentrations (lanes 8-14; IC50 200 nM). The only caspase that has an inhibitor specificity consistent with these results is caspase-3, strongly suggesting that this enzyme is responsible for cleaving Fak in apoptotic extracts.


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Fig. 2.   Fak cleavage is sensitive to Ac-DEVD-CHO and Ac-IETD-CHO but not to Ac-YVAD-CHO. A, apoptotic Jurkat cell extracts were incubated with [35S]methionine-labeled Fak in the presence of the indicated concentrations of tetrapeptide aldehydes. The cleavage reactions were carried out for 1 h at 37 °C and visualized by 10% SDS-PAGE and fluorography. Arrows indicate full-length Fak (p125Fak) and proteolytic fragments generated (p90, p35). B, quantitation of Fak cleavage, assessed by laser densitometric scanning of the 35-kDa proteolytic fragment, is expressed as a percentage of the control to which no drug was added. From this graph, the IC50 values for the various tetrapeptide aldehydes were determined.

Caspase-3 is one of the most abundant Group II caspases in apoptotic Jurkat cell extracts.2 It has been implicated directly in the cleavage of most of the proteins targeted for proteolysis during apoptosis. To assess whether caspase-3 cleaves Fak and whether this cleavage event is of physiological relevance, [35S]methionine-labeled Fak was incubated with various amounts of purified recombinant human (rh) caspase-3. Fragments with molecular masses identical with those generated by the apoptotic Jurkat cell extract were produced when Fak was incubated with rh caspase-3 (Fig. 3), consistent with the inhibitor studies implicating a Group II caspase such as caspase-3. The kcat/Km for this substrate was 3.4 × 105 M-1 s-1, only slightly less than that for poly(ADP)-ribose polymerase (kcat/Km = 15.6 × 105 M-1 s-1; data not shown), the first identified and most extensively characterized substrate of caspase-3 (23, 24).


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Fig. 3.   Cleavage of Fak by rh-caspase-3 in vitro. [35S]Methionine-labeled Fak was incubated with various amounts of rh-caspase-3 for 1 h at 37 °C and visualized by 10% SDS-PAGE and fluorography. Arrows indicate full-length Fak (p125Fak) and proteolytic fragments generated (p90, p35). Fak cleavage was measured as described in Fig. 2 to determine the kcat/Km value (see "Materials and Methods").

Analysis of the fragments generated by the cleavage of [35S]cysteine-labeled Fak by rh caspase-3 revealed the presence of the 90-kDa fragment but the absence of the 35-kDa fragment (data not shown). Because cysteine residues are absent from the carboxyl-terminal portion of Fak, we predicted that the 35-kDa fragment was derived from the carboxyl terminus. Analysis of the chicken Fak cDNA sequence revealed an excellent consensus cleavage site for caspase-3, DQTD772, present 282 amino acids from the carboxyl terminus of the protein. To confirm that DQTD772 was the site being recognized by caspase-3, we substituted the P1 aspartic acid at position 772 for an alanine by site-directed mutagenesis (the presence of an aspartic acid in P1 is absolutely required for cleavage by all caspases). [35S]Methionine-labeled Fak carrying the D772A mutation was not cleaved by caspase-3 (Fig. 4A, lane 4) under conditions where the conversion of wild-type Fak to 90- and 35-kDa fragments occurred (lane 2).


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Fig. 4.   Identification of caspase cleavage sites in Fak. A, DQTD772 is cleaved by rh-caspase-3. [35S]Methionine-labeled wild-type (lanes 1 and 2) and mutant (D772A) (lanes 3 and 4) Fak were incubated in the presence (+) or absence (-) of 3.2 nM rh-caspase-3 for 1 h at 37 °C. Fak cleavage was visualized by 10% SDS-PAGE and fluorography. Arrows indicate full-length Fak (p125Fak) and cleaved fragments (p90, p35). B, DQTD772 and VSWD704 are cleaved upon induction of apoptosis in K562 cells. Polyclonal populations of lymphoid K562 cells expressing carboxyl-terminal Flag-tagged wild-type (lanes 1 and 2) and mutant (D772A, lanes 3 and 4; D704A/D772A, lanes 5 and 6) Fak were incubated in the presence (+) or absence (-) of camptothecin for 16 h. Lysates prepared by boiling in SDS-Laemmli buffer were analyzed by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted using an anti-flag M2 antibody (Kodak). Arrows indicate full-length (p125Fak) and cleaved (p40 and p35) Flag-tagged Fak.

To confirm that the aspartic acid at position 772 was being cleaved in cells undergoing apoptosis, we subcloned wild-type and mutant Fak (D772A) into the eukaryotic expression vector pCEP4beta and established polyclonal stable K562 lymphoid cell lines expressing these proteins. The proteins were engineered with a Flag-epitope tag at the carboxyl terminus to allow visualization of the carboxyl-terminal fragments released by proteolysis. Large amounts of the wild-type (wt) and D772A mutant proteins were expressed in these stable cell lines, as assessed by immunoblot analysis using a monoclonal antibody to the Flag epitope (Fig. 4B, lanes 1 and 3). When the cells were rendered apoptotic by treatment with the topoisomerase inhibitor camptothecin, Fak cleavage occurred, as visualized by the appearance of the Flag-tagged 35-kDa carboxyl-terminal fragment (lane 2). As expected, the 35-kDa fragment was not detected in cells expressing the D772A mutant Fak, confirming that cleavage at Asp772 was responsible for the liberation of the 35-kDa fragment. Instead, low amounts of a fragment of 40 kDa were detected in apoptotic K562 cells expressing the D772A mutant Fak (lane 4). The corresponding 85-kDa fragment was visualized by Western blot analysis of extracts from apoptotic cells expressing a D772A mutant protein with a Flag-epitope tag at the amino terminus (data not shown). We predicted that the potential cleavage site VSWD704 present upstream of the aspartic acid at position 772 was also being cleaved under these conditions. This was confirmed by the absence of Fak cleavage in camptothecin-treated K562 cells expressing a Fak mutant whereby the aspartic acids at both positions 704 and 772 were mutated to alanines (lanes 5 and 6).

Cleavage assays using recombinant human caspase-3 excluded the possibility that the VSWD704 site was recognized by caspase-3 (Fig. 3). The substrate specificity profile of the caspase family members (22) suggested that VSWD704 represented a good consensus site for the Group III activator caspases and a poor substrate for Group II enzymes such as caspase-3. Although one of the Group III enzymes, caspase-8, did not cleave at VSWD704, caspase-6 cleaved both VSWD704 and DQTD772 (Fig. 5), as determined in vitro using the recombinant human enzyme. Incubation of wild-type [35S]methionine-labeled Fak with caspase-6 generated both 40- and 35-kDa carboxyl-terminal fragments and the corresponding amino-terminal derived 85- and 90-kDa fragments, respectively (lane 2). Substitution of the aspartic acids at positions 704 and 772 for alanines confirmed that the 40- and 85-kDa fragments were generated by cleavage at VSWD704 and the 35- and 90-kDa fragments, by cleavage at DQTD772 (lanes 3-5). The kcat/Km values for cleavage at VSWD704 and DQTD772 by rh caspase-6 were 6.5 × 104 M-1 s-1 and 5.9 × 104 M-1 s-1, respectively. The serine protease granzyme B, which is present in the granules of cytotoxic T lymphocytes and has been shown to have a very similar substrate specificity to that of Group III activator caspases (22), also cleaved these sites with kcat/Km values similar to those determined for caspase-6 (kcat/Km for VSWD704 = 3.1 × 104 M-1 s-1; kcat/Km for DQTD772 = 8.9 × 104 M-1 s-1). Although both caspase-3 and caspase-6 cleave DQTD772, it is noteworthy that the catalytic efficiency of caspase-3 for this site is approximately 6 times greater than that of caspase-6, suggesting that caspase-3 (or another Group II effector caspase) is more likely to be the protease responsible for cleavage at this site in vivo.


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Fig. 5.   Cleavage of Fak by rh-caspase-6 in vitro. [35S]Methionine-labeled Fak wild-type (lanes 1 and 2) and mutant (D704A, lane 3; D772A, lane 4; D704A/D772A, lane 5) were incubated with 7.7 nM rh-caspase-6 for 1 h at 37 °C and visualized by 10% SDS-PAGE and fluorography. Arrows indicate full-length Fak (p125Fak) and cleaved fragments (p90, p85, and corresponding p35 and p40).

The physiological significance of the presence of two distinct caspase cleavage sites in Fak was underscored by examining the cleavage of endogenous protein in human cell lines undergoing apoptosis (both DQTD772 and VSWD704 are conserved in human Fak). Total cell lysates were prepared from Jurkat cells at various times after stimulation of the CD95 (Fas/Apo-1) death receptor by antibodies to CD95 (Fas/Apo-1) (Fig. 6A). As early as 2 h after the addition of anti-CD95 (Fas/Apo-1), Fak cleavage to a 90-kDa fragment was detected by Western blot analysis using two different antibodies to Fak, one directed against residues 748 to 1053 (alpha 748-1053) and one directed against residues 354-533 (alpha 354-533). The antibody raised against residues 354-533 recognized an additional fragment of 85 kDa. These results are consistent with the cleavage of Fak at both VSWD704 and DQTD772 sites in apoptotic Jurkat cells (see Fig. 6C). Curiously, only the 90-kDa fragment was generated in the human cervical carcinoma cell line HeLa in response to treatment for 2 h with staurosporine, a nonspecific kinase inhibitor that induces apoptosis (Fig. 6B). A similar result was obtained when we induced apoptosis by serum starvation or by treatment with camptothecin (data not shown). We confirmed that only DQTD772 is recognized in apoptotic HeLa cells by demonstrating that cleavage of transiently transfected chicken Fak was abolished when the P1 aspartic acid at position 772 was substituted for alanine without appearance of fragments corresponding to cleavage at VSWD704 (data not shown). The absence of cleavage at VSWD704 may be due to the absence of caspase-6 (or another caspase capable of cleaving VSWD704) or to the inaccessibility of VSWD704 in HeLa cells.


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Fig. 6.   Different caspase cleavage sites are recognized in different cell lines. A, cleavage of Fak in Jurkat cells. Human Jurkat T-cells were treated for various periods of time with anti-CD95 (Fas/Apo-1) antibodies to induce apoptosis. Lysates prepared by boiling in SDS-Laemmli buffer were analyzed by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either a monoclonal antibody directed against the carboxyl-terminal portion of Fak (residues 748-1053; Upstate Biotechnology) or a monoclonal antibody directed against the amino-terminal portion of Fak (residues 354-533; Transduction Laboratories). B, cleavage of p125Fak in human HeLa cervical carcinoma cells and in murine 3T3 L1 fibroblast cells. HeLa and 3T3 L1 were incubated in the presence (+) or absence (-) of staurosporine for 2 h after which lysates were prepared by boiling in SDS-Laemmli buffer, analyzed by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with a monoclonal antibody directed against the amino-terminal portion of Fak (residues 354-533; Transduction Laboratories). Arrows indicate full-length Fak p125Fak and cleaved fragments (p90 and p75). C, schematic representation of Fak. The positions of the kinase domain, proline-rich region (hatched box), and focal adhesion targeting domain (FAT) are illustrated. Also shown are the regions encompassing the epitopes recognized by the two antibodies (alpha 354-533; alpha 748-1053) and the 85-kDa (p85) and 90-kDa (p90) fragments generated by cleavage at VSWD704 and DQTD772, respectively. D, comparison of the proteolytic fragments generated by caspase cleavage of Fak during apoptosis with the natural competitive inhibitor of Fak, FRNK.

Cleavage at either DQTD772 or VSWD704 results in the separation of the kinase domain from the focal adhesion targeting (FAT) domain. Noteworthy is the fact that the naturally occurring inhibitor of Fak, FRNK, corresponds to the carboxyl-terminal half of Fak, as illustrated in Fig. 6D. When overexpressed, FRNK prevents the localization of Fak to sites of integrin engagement resulting in decreased Fak phosphorylation and inhibition of Fak-mediated cellular responses to cell adhesion (25). In view of the presence of an intact FAT domain within the 35- and 40-kDa fragments generated by caspase cleavage of Fak during apoptosis, we reasoned that these fragments may also act as competitive inhibitors of Fak. To test this hypothesis, Fak deletion mutants identical with the carboxyl-terminal fragments generated during apoptosis were engineered and transiently transfected into HeLa cells. The levels of phosphorylated Fak protein were measured by immunoprecipitation with an anti-Fak antibody and immunoblotting with an anti-phosphotyrosine antibody 24 h after transfection (Fig. 7). Expression of either the 35- (lane 4) or the 40-kDa fragment (lane 3) suppressed the phosphorylation of endogenous Fak, as was observed after transfection of FRNK (lane 2). Like FRNK, these fragments acted as competitive inhibitors because their inhibitory effects were abrogated by overexpressing full-length Fak protein (data not shown).


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Fig. 7.   Inhibition of endogenous Fak tyrosine phosphorylation by the expression of FRNK or the related carboxyl-terminal fragments generated by caspase cleavage. HeLa cells were transiently transfected with the wild-type pCEP4beta vector (lane 1) or pCEP4beta containing FRNK (lane 2), C705-1053 (lane 3), or C773-1053 (lane 4). C705-1053 and C773-1053 correspond exactly to the carboxyl-terminal fragments generated by caspase cleavage at VSWD704 and DQTD772, respectively, except that an initiating methionine was introduced immediately upstream of the P1' amino acid. Twenty-four h after transfection, cells were lysed, and tyrosine-phosphorylated Fak was detected by immunoprecipitation using an antibody directed against the carboxyl terminus of Fak followed by immunoblotting using the anti-phosphotyrosine antibody RC20 (alpha PY; Transduction Laboratories). In parallel, total amounts of Fak were assessed by immunoprecipitation as described above followed by immunoblotting with an anti-Fak antibody (alpha Fak; anti-kinase domain from Transduction Laboratories).

If the disabling of Fak by caspase cleavage represents a critical step in the execution of the apoptotic program, the two cleavage sites identified should be conserved in other species. Whereas the caspase-6 cleavage site VSWD704 is present in all species examined (human, rat, mouse, chicken, frog; Fig. 8), the caspase-3 cleavage site DQTD772 is present in chicken and human but not rat or mouse (Fig. 8; the DHMD772 sequence present in frog Fak is predicted to be cleaved efficiently by a Group II effector caspase). Given that one of the caspase cleavage sites (DQTD772) is absent in mouse Fak, we examined whether Fak cleavage occurred in murine cells in response to an apoptotic stimulus. Lysates were prepared from staurosporine-treated 3T3 L1 fibroblasts and analyzed by immunoblotting using the alpha  354-533 antibody that recognizes both 85- and 90-kDa fragments in human Fak (Fig. 6B). Neither 85- nor 90-kDa fragments of Fak could be detected in apoptotic murine cells. Instead, murine Fak was cleaved at an upstream site, the identity of which remains to be determined, which results in the appearance of a 75-kDa fragment (we have not excluded the possibility that murine Fak is cleaved first at VSWD704 and then at an upstream site).


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Fig. 8.   Conservation of caspase cleavage sites in human but not rodent Fak. Amino acid sequences of human (GenBankTM accession number L13616), rat (S83358), mouse (M95408), chicken (M86656), and frog Fak (1362692) were aligned in areas corresponding to the chicken Fak caspase cleavage sites DQTD772 and VSWD704 (numbers refer to the amino acid positions in the chicken sequence, 1 being the initiating methionine).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The cleavage of Fak observed in both adherent and suspension cell lines in response to various apoptotic inducers suggests that this proteolytic event plays an important role in the execution of the suicide pathway. Preliminary results by Wen et al. (17) and Levkau et al. (18) demonstrated recently the cleavage of Fak by caspases during apoptosis. We have identified two caspase cleavage sites within the carboxyl-terminal half of Fak; DQTD772 which is preferentially cleaved by the group II effector caspase, caspase-3, and VSWD704, which is preferentially cleaved by the group III activator caspase, caspase-6. Although caspase-3 does not recognize VSWD704, caspase-6 cleaves both DQTD772 and VSWD704 with virtually identical kinetics (kcat/Km = 5.9 × 104 M-1 s-1 and 6.5 × 104 M-1 s-1, respectively). The relative promiscuity of caspase-6 versus caspase-3 is consistent with results obtained from a combinatorial tetrapeptide library used to define the substrate specificity of the caspase family members (22). This combinatorial approach revealed that in the critical specificity-determining P4 subsite, caspase-6 tolerates a number of amino acids whereas caspase-3 exhibits a very strict requirement for aspartic acid.

Our studies underscore the importance of determining the catalytic efficiency of cleavage for a given substrate with a given caspase. Indeed, although caspase-6 can cleave DQTD772 (kcat/Km = 5.9 × 104 M-1 s-1), this site is clearly preferred by caspase-3 (kcat/Km = 3.4 × 105 M-1 s-1). Of interest is our observation that whereas DQTD772 is cleaved in all human cell lines examined, presumably because Group II effector caspases are present in these cell lines, cleavage at VSWD704 appears to be cell type-dependent, possibly because Group III activator caspases capable of cleaving this site are not ubiquitously expressed. It is of interest that the caspase cleavage sites lie on either side of a proline-rich region shown to interact with two SH3-containing proteins: Graf (a Rho and Cdc42 GTPase-activating protein; Ref. 26) and p130Cas (an adapter molecule phosphorylated by various tyrosine kinases; Ref. 27). It remains to be determined whether protein-protein interactions modulate the sensitivity of Fak to caspase cleavage.

It is curious that the predominant caspase-3 consensus cleavage site identified in chicken and human Fak is absent in rodent Fak. Species alignments for several identified caspase substrates have revealed that in all cases examined, the P1 aspartic acid in the caspase cleavage site was conserved. It remains to be determined whether the absence of this predominant caspase cleavage site in rodent Fak modulates the cellular responses to cell adhesion. Of interest is the report that the murine cell line, NIH3T3, is relatively resistant to anoikis (9).

Cleavage at either DQTD772 or VSWD704 generates carboxyl-terminal fragments that inhibit Fak phosphorylation and thus act like FRNK, the naturally occurring variant of Fak (Fig. 6D). Results presented in this report suggest two mechanisms by which Fak-mediated cellular responses to cell adhesion are abrogated during apoptosis: (i) by decreasing the overall amount of Fak in the cell and (ii) by generating fragments that act as competitive inhibitors of the remaining full-length Fak protein. That the cell has devised two mechanisms to inactivate Fak underscores the importance of this cleavage event in the execution of apoptotic cell death. We propose that the disabling of Fak liberates the cell from anti-apoptotic signals generated by the extracellular matrix and allows removal of the apoptotic cell from the tissue.

    ACKNOWLEDGEMENTS

We thank Dr. Michael D. Schaller for helpful comments during the course of this work. We thank Isolde Seiden and Vicky Houtzager for sequencing some of the constructs used in this study.

    FOOTNOTES

* This work was supported in part by the Medical Research Council of Canada (MRC) (to F. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe Claire-Dorval, Québec H9R 4P8, Canada. Tel.: 514-428-3430; Fax: 514-695-0693; E-mail: sophie_roy{at}merck.com.

1 The abbreviations used are: FAT, focal adhesion targeting; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; rh, recombinant human; FPLC, fast protein liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

2 D. Rasper, S. Xanthoudakis, S. Roy, and D. W. Nicholson, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Otey, C. A. (1996) Int. Rev. Cytol. 167, 161-183[Medline] [Order article via Infotrieve]
  2. Cobb, B. S., Schaller, M. D., Leu, T.-H., and Parsons, J. T. (1994) Mol. Cell. Biol. 14, 147-155[Abstract]
  3. Xing, Z., Chen, H.-C., Nowlen, J. K., Taylor, S. J., Shalloway, D., and Guan, J.-L. (1994) Mol. Cell. Biol. 5, 413-421
  4. Chen, H.-C., and Guan, J.-L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10148-10152[Abstract/Free Full Text]
  5. Guinebault, C., Payrastre, B., Racaud-Sultan, C., Mazarguil, H., Breton, M., Mauco, G., Plantavid, M., and Chap, H. (1995) J. Cell Biol. 129, 831-842[Abstract]
  6. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and Van der Geer, P. (1994) Nature 372, 786-791[Medline] [Order article via Infotrieve]
  7. Schaller, M. D., Borgman, C. A., and Parsons, J. T. (1993) Mol. Cell. Biol. 13, 786-791
  8. Richardson, A., and Parsons, T. (1996) Nature 380, 538-540[CrossRef][Medline] [Order article via Infotrieve]
  9. Frisch, S. M., Vuori, K., Ruoslahti, E., and Chan-Hui, P-Y. (1996) J. Cell. Biol. 134, 793-799[Abstract]
  10. Hungerford, J. E., Compton, M. T., Matter, M. L., Hoffstrom, B. G., and Otey, C. A. (1996) J. Cell Biol. 135, 1383-1390[Abstract]
  11. Owens, L. V., Xu, L., Craven, R. J., Dent, G. A., Weiner, T. M., Kornberg, L., Liu, E. T., and Cance, W. G. (1995) Cancer Res. 55, 2752-2755[Abstract]
  12. Owens, L. V., Xu, L., Dent, G. A., Yang, X., Sturge, G. C., Craven, R. J., and Cance, W. G. (1996) Ann. Surg. Oncol. 3, 100-105[Abstract]
  13. Weiner, T. M., Liu, E. T., Craven, R. J., and Cance, W. G. (1993) Lancet 342, 1024-1025[CrossRef][Medline] [Order article via Infotrieve]
  14. Weiner, T. M., Liu, E. T., Craven, R. J., and Cance, W. G. (1994) Ann. Surg. Oncol. 1, 18-27[Abstract]
  15. Xu, L-H., Owens, L. V., Sturge, G. C., Yang, X., Liu, E. T., Craven, R. J., and Cance, W. G. (1996) Cell Growth Differ. 7, 413-418[Abstract]
  16. Crouch, D. H., Fincham, V. J., and Frame, M. C. (1996) Oncogene 12, 2689-2696[Medline] [Order article via Infotrieve]
  17. Wen, L.-P., Fahrni, J. A., Troie, S., Guan, J.-L., Orth, K., and Rosen, G. D. (1997) J. Biol. Chem. 272, 26056-26061[Abstract/Free Full Text]
  18. Levkau, B., Herren, B., Koyama, H., Ross, R., and Raines, E. W. (1998) J. Exp. Med. 187, 579-586[Abstract/Free Full Text]
  19. Nicholson, D. W., and Thornberry, N. A. (1997) Trends Biochem. Sci. 22, 299-306[CrossRef][Medline] [Order article via Infotrieve]
  20. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve]
  21. Groger, R., Morrow, D. M., and Tyckocinski, M. L. (1989) Gene 81, 285-294[Medline] [Order article via Infotrieve]
  22. Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T., and Nicholson, D. W. (1997) J. Biol. Chem. 272, 17907-17911[Abstract/Free Full Text]
  23. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346-347[CrossRef][Medline] [Order article via Infotrieve]
  24. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, E. M., Yamin, T.-T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37-43[CrossRef][Medline] [Order article via Infotrieve]
  25. Richardson, A., Malik, R. K., Hildebrand, J. D., and Parsons, J. T. (1997) Mol. Cell. Biol. 17, 6906-6914[Abstract]
  26. Hildebrand, J. D., Taylor, J. M., and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178[Abstract]
  27. Harte, M. T., Hildebrand, J. D., Burnham, M. R., Bouton, A. H., and Parsons, J. T. (1996) J. Biol. Chem. 271, 13649-13655[Abstract/Free Full Text]


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