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Mol Biol Cell. Mar 2000; 11(3): 929–939.

Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells

Martin Raff, Monitoring Editor


Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell–cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD416G and DSPD748G) in vitro, and point mutations substituting the Asp of DVPD416G and DSPD748G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD416G generated a 74-kDa fragment, which was in turn cleaved at DSPD748G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas–paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.


Apoptotic cell death is a fundamental biological process crucial for proper organism development and for maintenance of tissue homeostasis (Raff, 1992 blue right-pointing triangle). Apoptosis causes characteristic morphological changes that include membrane blebbing, decreased adhesion and intercellular contacts, chromatin condensation, nuclear fragmentation, and the packing of the nuclear fragments into membrane-enclosed apoptotic bodies (Wyllie et al., 1980 blue right-pointing triangle; Darzynkiewicz et al., 1994 blue right-pointing triangle). Caspases are a family of highly conserved aspartate-specific cysteine-proteases related to mammalian interleukin-1β-converting enzyme, which are involved in the final execution phase of apoptosis (Nicholson, 1996 blue right-pointing triangle; Nicholson and Thornberry, 1997 blue right-pointing triangle). The substrates for caspases are now known to include a growing number of molecules involved in cytoskeletal regulation and signaling, including α-fodrin, gelsolin, growth arrest–specific gene (Gas 2), MEKK-1, and PKC (Brancolini et al., 1995 blue right-pointing triangle; Emoto et al., 1995 blue right-pointing triangle; Martin et al., 1995 blue right-pointing triangle; Ghayur et al., 1996 blue right-pointing triangle; Cardone et al., 1997 blue right-pointing triangle; Kothakota et al., 1997 blue right-pointing triangle; Janicke et al., 1998 blue right-pointing triangle). Recent studies have shown that attachment to the extracellular matrix (ECM) actively regulates cell survival, and that cells undergoing apoptosis lose their connections to neighboring cells and to the ECM. For example, epithelial and endothelial cells undergo apoptosis (“anoikis”) when displaced from the ECM (Frisch and Francis, 1994 blue right-pointing triangle; Re et al., 1994 blue right-pointing triangle), whereas cell adhesion to ECM suppresses apoptosis in mammary epithelial cells (Frisch and Francis, 1994 blue right-pointing triangle; Boudreau et al., 1995 blue right-pointing triangle; Aharoni et al., 1996 blue right-pointing triangle).

Cell attachment to the ECM is mediated by the association of integrins with specific ECM components. The binding of integrin receptors to the ECM results in the clustering of integrins and the recruitment of a wide variety of proteins, leading to the formation of focal adhesions, polyprotein structures linking the ECM to the actin cytoskeleton and perhaps providing a structural basis for cell viability.

Focal adhesion complexes are made up of such components as focal adhesion kinase (FAK), Crk-associated substrate (Cas), paxillin, pp60src, vinculin, tensin, talin, and α-actinin. Among these, FAK, Cas, and paxillin are closely associated with changes in the cytoskeleton; thus reorganization of the actin cytoskeleton is accompanied by striking changes in the level of tyrosine phosphorylation of these proteins (Rozengurt, 1995 blue right-pointing triangle; Rozengurt and Rodriguez-Fernandez, 1997 blue right-pointing triangle; Tanaka et al., 1997 blue right-pointing triangle; Zhu et al., 1998 blue right-pointing triangle). For instance, tyrosine dephosphorylation of focal adhesion proteins is correlated with decreases in the length and number of actin stress fibers (Rankin and Rozengurt, 1994 blue right-pointing triangle; Casamassima and Rozengurt, 1997 blue right-pointing triangle).

In particular, several lines of evidence suggest that Cas is closely associated with changes in the cytoskeleton: Cas is colocalized with F-actin in peripheral regions of osteoclast-like cells where its phosphorylation is involved in actin ring formation (Nakamura et al., 1998 blue right-pointing triangle); actin stress fiber formation is severely impaired in Cas-deficient fibroblasts; and Cas-deficient embryos have poorly developed hearts in which myofibrils are disorganized and Z-disks are disrupted (Honda et al., 1998 blue right-pointing triangle). In addition, the transition from flat to round cell morphology, which is a characteristic feature in cells undergoing apoptosis, is accompanied by cytoskeletal rearrangement and changes in focal adhesion proteins. Indeed, proteolytic cleavage of FAK by caspase during apoptosis further suggests the important role played by the association of focal adhesion proteins, the cytoskeleton, and the ECM in the maintenance of cell morphology and viability.

In addition to its structural functions, Cas is activated and phosphorylated in response to a number of mitogens and growth factors and influences a variety of cellular functions, including growth, migration, and differentiation. Cas contains a Src homology 3 (SH3) domain, proline-rich regions, and a cluster of 15 putative SH2-binding motifs (Sakai et al., 1994 blue right-pointing triangle), characteristics that identify Cas as an adaptor molecule capable of transmitting cellular signals in association with other intracellular proteins containing SH2 and SH3 domains. Such proteins include Src, Crk, FAK, paxillin, and tensin (Sakai et al., 1994 blue right-pointing triangle; Turner and Miller, 1994 blue right-pointing triangle; Miyamoto et al., 1995 blue right-pointing triangle; Schaller and Parsons, 1995 blue right-pointing triangle; Harte et al., 1996 blue right-pointing triangle; Nakamoto et al., 1996 blue right-pointing triangle; Salgia et al., 1996 blue right-pointing triangle). Thus, Cas may play essential roles in the integrin-mediated signaling pathway.

In this study, we examined changes in focal adhesion proteins during apoptosis and observed that Cas is cleaved by caspase-3 in apoptotic cells. The degradation of Cas likely results in its loss from focal adhesions, disrupting cell adhesion to the ECM and/or integrin-mediated cell survival signaling.



Culture media and supplements were purchased from Life Technologies (Grand Island, NY). Etoposide was from Sigma (St. Louis, MO), and the ECL reagent was from Amersham (Buckinghamshire, England). Monoclonal antibodies (mAbs) against p130Cas (Cas mAb), FAK, and paxillin were from Transduction Laboratories (Lexington, KY). Anti-α-tubulin mAb, anti-Talin mAb, and anti-FLAG M2 mAb were from Sigma. Polyclonal paxillin antibody was kindly provided by Dr. Man Sik Kang (Seoul National University. Seoul, Korea). Anti-Cas polyclonal antibody (Cas-2 Ab; Sakai et al., 1994 blue right-pointing triangle) and rat Cas cDNA were generously provided by Dr. Hismaru Hirai (University of Tokyo, Tokyo, Japan), and Cas mAb (4F4) was provided by Dr. J.T. Parsons (University of Virginia, Charlottesville, VA). HRP-labeled anti-mouse immunoglobulin (Ig) and FITC- and TRITC-conjugated goat anti-mouse and anti-rabbit IgG or IgM were from Jackson ImmunoResearch (West Grove, PA). The Quik Change site-directed mutagenesis system was from Stratagene (La Jolla, CA). The TNT- coupled reticulocyte lysate system was from Promega (Madison, WI). The tetrapeptide caspase inhibitors ZVAD-fluoromethylketone (fmk) and DEVD-chloromethylketone (cmk) were from Bachem (Torrance, CA).

Cell Culture and Cell Viability Assays

Rat-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. To induce apoptosis, cells were exposed to 40 μM etoposide for selected periods. Apoptotic cells, which appeared as “floaters” in the culture medium, were harvested by centrifugation at 1000 × g for 5 min. Adherent, viable cells remaining on the culture dish and control cells (cultured in normal growth medium) were scraped from the dishes, collected by centrifugation, and subsequently lysed. When used, ZVAD-fmk or DEVE-cmk was added to the cells for 3 h before the induction of apoptosis. Cell viability was assessed by trypan blue exclusion.

Electrophoresis and Immunoblot Analysis

For immunoblot analysis, cells treated as described above were lysed in a lysis buffer (1% SDS, 1 mM sodium ortho-vanadate, 10 mM Tris, pH 7.4, 1 mM PMSF, 10 μM leupeptin, 1.5 μM pepstatin, and 10 μg/ml aprotinin) and then collected, boiled, and centrifuged for 5 min to remove insoluble material. Protein concentrations in the resultant lysate were measured using the BCA method (Pierce, Rockford, IL). The proteins present in aliquots of lysate were separated by 10–12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked for 1 h at room temperature in buffer containing 5% nonfat dry milk in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20 (TBST). The membranes were incubated first with primary antibody and then incubated in TBST containing an HRP-conjugated anti-mouse or anti-rabbit immunoglobulin (Jackson ImmunoResearch). Bands were detected using ECL according to the manufacturer's protocol. In some cases, blots were stripped by heating them to 65°C for 30 min in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and reprobed.


Cells were lysed for 1 h at 4°C in 1 ml of Triton X-100 buffer (10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 1 mM sodium ortho-vanadate, 10 μM leupeptin, 1.5 μM pepstatin, and 10 μg/ml aprotinin). The lysates were clarified by centrifugation at 20,000 × g for 10 min at 4°C, after which aliquots (1 mg) were incubated overnight at 4°C with anti-Cas-2 Ab raised against amino acid residues 456–690 of rat Cas and then incubated for an additional 4 h at 4°C with protein A-agarose beads (Pharmacia Biotech, Uppsala, Sweden). The beads were then washed three times with Triton X-100 buffer to remove nonspecific binding proteins. Immune complexes were treated with SDS-sample buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol, and 0.006% bromophenol blue) and subjected to SDS-PAGE.

In Vitro Caspase Cleavage Assays

Wild-type and mutant p130cas cDNAs were constructed in pcDNA3.0 and used as templates for coupled transcription–translation using a T7 kit (Promega) with [35S]methionine (1175Ci/mmol; New England Nuclear, Boston, MA). Recombinant caspases were prepared from bacterial cells transformed with caspase-expressing plasmids. The p30 domains of mouse caspase-1, human caspase-8, and the full-length human caspase-3 were amplified by PCR using the following oligonucleotide primers: 5′-CGCGG ATCCT GGCAC ATTTC CAGGA C-3′ and 5′-CGCGG ATCCT AAGGA AGTAT TGGC-3′ (p30 caspase-1); 5′-CGCGG ATCCG GAGAA CACTG AAAAC TC-3′ and 5′-CGCGG ATCCT ACCAT CTTCT CACTT GG-3′ (caspase-3); and 5′-CGGGA TCCTA CCATC TTCTC ACTTG G-3′ and 5′-CCGCA AGCTT ATCAG AAGGG AGACA AG-3′ (p30 caspase-8). The PCR products were subcloned into the BamHI site (caspase-1 and -3) or the BamHI–HindIII sites (caspase-8) of pET-15b (Novagen, Madison, WI). Exponential growth of bacterial cells was induced for 2 h with 0.2 mM isopropyl-1-thio-α-d-galactopyranoside, after which the cells were harvested and lysed by sonication in buffer containing 0.05% NP-40, 20 mM HEPES, pH 7.4, and 100 mM NaCl. The lysates were cleared by centrifugation, and the protein concentrations were determined using the BCA method (Pierce). The bacterial cell lysates were then used as sources of recombinant caspases.

To assay caspase-catalyzed cleavage, in vitro–translated Cas was incubated for selected times at 30°C with bacterial cell lysates (20 μg) containing the respective recombinant caspases. The reactions were terminated by the addition of SDS-PAGE loading buffer and heating to 100°C for 5 min. Samples were then subjected to 10–12% SDS-PAGE, fixed with destaining solution for 30 min at room temperature, dried, and exposed to x-ray film. When endogenous Cas in cell lysates was the substrate, cells were first lysed on ice in 10 mM HEPES/KOH, pH 7.4, 2 mM EDTA, 5 mM dithiothreitol, 1% NP-40, 10 μM leupeptin, and 10 μg/ml aprotinin. The lysates were then cleared by centrifugation at 27,000 × g for 5 min, and 40-μg aliquots were incubated for selected times at 30°C with recombinant caspase-3 (20 μg of bacterial cell lysates).

Site-directed Mutagenesis

Cas mutants were generated from pcDNA3.0-Cas using the Quik Change site-directed mutagenesis kit (Stratagene). Asp residues at positions 416 (DVPD416) and 748 (DSPD748) of rat p130cas were replaced with Glu. Two sets of primers, 5′-GCAAG GATGT GCCTG AAGGC CCACT GCTGC G-3′ and 5′-CGCAG CAGTG GGCCT TCAGG CACAT CCTTG C-3′ for the D416 mutation and 5′-CCCAG GACTC TCCGG AAGGC CAGTA TGAGA ACAG-3′and 5′-CTGTT CTCAT ACTGG CCTTC CGGAG AGTCC TGGG-3′ for the D748 mutation, were used for construction of the mutants. The sequences of the mutants were then confirmed by DNA sequence analysis.

Contruction of Expression Plasmids and Transient Tranfection

The full-length cDNA of wild-type and mutant p130cas were PCR amplified from pcDNA3.0-cas using the forward and reverse primers 5′-TGTGCTGGAATTCGGCGGCCG-3′ and 5′-ACCGGATCCGGGCGGCAGCCAGCTGG-3′. The PCR reaction was carried out using pfu DNA polymerase (Stratagene), digested with BamHI and EcoRI, and cloned into pFLAG-cytomegalovirus (CMV)-5c (Eastman Kodak, New Haven, CT) to generate pFLAG-CMV-Cas. Rat-1 cells were maintained in Dulbecco's modified Eagle's medium with 10% FBS (Life Technologies, Gaithersburg, MD). To generate cells transiently expressing either the wild-type Cas or each mutant, Rat-1 cells were transfected with the constructed DNAs using LipofectAMINE following the manufacturer's instructions (Life Technologies). The expression of the constructed DNAs was analyzed by Western blotting using Flag M2 mAb (Sigma).


Rat-1 cells grown on 0.1% gelatin-coated coverslips were fixed with 1.5% paraformaldehyde for 10 min and washed in PBS. They were then permeabilized with 0.5% Triton X-100 in PBS for 5 min. The cells were then incubated for 1 h in a humidified chamber at 37°C with appropriate combinations of the primary Ab in PBS containing 1% BSA, washed four times with 0.1% Triton X-100 in PBS, and incubated for 1 h at room temperature with FITC-conjugated donkey anti-IgG/IgM in PBS containing 1% BSA. The coverslips were then washed with 0.1% Triton X-100 in PBS and mounted with 90% glycerol and 0.1% o-phenylenediamine in PBS. Immunofluorescence was analyzed under a Leica (Nussloch, Germany) DMRBE microscope equipped with a 100× objective lens and filters for epifluorescence. Fluorescence micrographs were taken on T-max P3200 film (Kodak).


Cleavage of Cas during Etoposide-induced Apoptosis in Rat-1 Cells

Exposing cells to etoposide induces apoptosis and gross changes in cell shape. The transition from flat to round cell morphology likely results from detachment from the ECM and leads to the appearance of apoptotic cells floating in the medium. Because cell detachment from the ECM is well known to result from loss of focal adhesion sites, we examined in vivo changes in focal adhesion proteins during apoptosis.

Rat-1 cells were incubated with 40 μM etoposide for selected periods, lysed, and subjected to immunoblot analysis using Cas mAb and Cas-2 Ab as probes. When blots were probed with Cas mAb, it was found that Cas had undergone proteolytic cleavage during etoposide-induced apoptosis (Figure (Figure1A).1A). A 31-kDa proteolytic fragment was first detected at 12 h, markedly increased for up to 24 h in the continued presence of etoposide, and then gradually declined until, after 48h, it was no longer detectable. In contrast, the 31-kDa fragment was barely detected in untreated control cells (Figure (Figure11A). The appearance of the 31-kDa proteolytic fragment is in accord with the previous report by Bannerman et al. (1998) blue right-pointing triangle. In similar manner, a 47-kDa cleavage fragment was detected when blots were probed with Cas-2 Ab (Figure (Figure1B).1B).

Figure 1
Proteolytic cleavage of Cas during etoposide-induced apoptosis in Rat-1 cells. Rat-1 cells were exposed to 40 μM etoposide for the indicated periods. Cell lysates were then subjected to immunoblot analysis using Cas mAb (A) and Cas-2 (B). Intact ...

The appearance of Cas degradation products correlated well with the reduction of FAK, which time dependently declined in the presence of etoposide and was undetectable within 36 h (Figure (Figure1C).1C). A ~77-kDa cleavage product was faintly detected at 12 h and became intense within 18–24 h after treatment. This suggests proteolytic cleavage of FAK, which is consistent with several lines of evidence indicating that caspases also degrade FAK in cells undergoing apoptosis (Wen et al., 1997 blue right-pointing triangle; Levkau et al., 1998 blue right-pointing triangle).

In Vitro Cleavage of Cas by Caspase-3

Analysis of its amino acid sequence showed that Cas has several probable caspase cleavage sites: 10 sites with the DXXD consensus sequence preferentially cleaved by caspase-3-like caspases and 5 sites with the sequence (IVL)XXD preferentially cleaved by caspase-6-like caspases (Talanian et al., 1997 blue right-pointing triangle). We therefore investigated whether Cas is cleaved by caspases in vitro. The cDNA encoding rat Cas was transcribed and translated in vitro and then incubated with recombinant caspases-1, -3, and -8. The specific activities of recombinant caspases-1 and -3 were previously confirmed by cleaving GDP dissociation inhibitor type D4, and the activity of caspase-8 was confirmed by cleaving poly(ADP-ribose) polymerase (our unpublished data).

As shown in Figure Figure2A,2A, caspase-3 completely cleaved in vitro–translated Cas, generating six cleavage products with molecular masses of ~ 47, 46, 43, 35, 32, and 31 kDa, respectively. The 31-kDa band appeared identical to the band detected by Cas mAb in etoposide-treated cells. Most likely because of differences in substrate specificity, Cas was cleaved to a lesser extent by caspase-1 and was not cleaved at all by caspase-8.

Figure 2
In vitro cleavage of Cas by recombinant caspases. (A) 35S-Labeled rat Cas was generated by in vitro translation and incubated with bacterial cell lysates (20 μg) containing overexpressed recombinant caspases (caspase-1, -3, and -8). Cas was cleaved ...

To confirm that the Cas proteolysis was catalyzed by caspase, recombinant caspases were preincubated with membrane-permeant caspase antagonists ZVAD-fmk and DEVD-cmk before incubation with in vitro–translated Cas. DEVD-cmk (100 nM), a specific caspase-3 inhibitor, completely blocked cleavage of Cas (Figure (Figure2B),2B), whereas ZVAD-fmk (100 nM), which is a less specific inhibitor, was a less effective in the inhibition of Cas cleavage. Thus, cleavage of Cas appears to be catalyzed primarily by caspase-3.

When Rat-1 cells were preincubated with either ZVAD-fmk or DEVD-cmk for 3 h and then exposed to etoposide for an additional 12 h, ZVAD-fmk suppressed the appearance of the 31-kDa cleavage product in a concentration-dependent manner, whereas DEVD-cmk did so only partially even at 300 μM (Figure (Figure3A).3A). DEVD-cmk was less effective at inhibiting FAK cleavage as well (Figure (Figure3A).3A). However, when apoptotic cell lysates obtained from the cells exposed to etoposide for 36 h were incubated with in vitro–translated Cas in the presence of either ZVAD-fmk or DEVD-cmk, cleavage of in vitro–translated Cas was blocked by both caspase inhibitors even at 50 nM (Figure (Figure3B).3B). Thus, although other caspases may be involved in Cas cleavage during apoptosis, caspase-3 appears to be the primary mediator of Cas cleavage. In addition, the observed effects of the caspase inhibitors from in vivo and in vitro cleavage experiments reflect relative differences in cell membrane permeability.

Figure 3
Inhibition of Cas proteolysis in vivo by ZAVD-fmk and DEVD-cmk. (A) Rat-1 cells were pretreated for 3 h with the indicated concentrations of ZVAD-fmk or DEVD-cmk and then exposed to 40 μM etoposide for an additional 12 h. Cas proteolysis was analyzed ...

Determination of the Sites of Cas Cleavage by Site-directed Mutagenesis

To define the sites at which caspase-3 sequentially cleaves Cas, both in vitro–translated and endogenous Cas were digested with caspase-3, and the time courses of the proteolysis were analyzed. In vitro–translated Cas generated a total of nine different cleavage products (Figure (Figure4A).4A). Within 1 min after exposure to caspase-3, 100-, 90-, 74-, 47-, 46-, 43-, and 31-kDa fragments were detected; levels of the 47-, 46-, and 43-kDa fragments increased up to 15 min, and 35- and 32-kDa fragments first appeared after 15 min. The cleavage pattern of endogenous Cas was then probed using Cas mAb raised against the amino acid sequence (644–819) of rat Cas. Incubating recombinant caspase-3 with cell lysates generated the 74-kDa (AF1) and 31-kDa (AF2) fragments (Figure (Figure4B),4B), which appear to correspond to the 74- and 31-kDa fragments detected when in vitro–translated Cas was digested by caspase-3. Immunoblot analysis using Cas-2 Ab and Cas mAb showed that the 74-kDa fragment generates two cleavage products with apparent molecular masses of 47 and 31 kDa, the former being detected by Cas-2 Ab (Figure (Figure1B)1B) and the latter by Cas mAb (Figure (Figure4B).4B). In immunoblot analysis, the 31- and 47-kDa fragments shown in cell lysates are matched with the cleavage fragments of in vitro–translated wild type (our unpublished data).

Figure 4
Time course of the in vitro proteolysis of Cas by caspase-3. (A) In vitro–translated Cas was incubated with caspase-3 for the indicated times. Samples were subjected to SDS-PAGE, and Cas cleavage fragments were detected by autoradiography. Seven ...

To define the cleavage sites generating the 74- and 31-kDa fragments, we diagrammed the amino acid sequence of rat Cas (Figure (Figure5)5) and determined that there were two probable cleavage sites at DVPD416G and DSPD748G. That these sites were cleaved by caspase-3 was confirmed by inserting point mutations replacing Asp at the sites with Glu and then exposing the translated products to caspase-3. As shown in Figure Figure6,6, the 74-kDa fragment completely disappeared after mutation at DVPD416G, whereas the 31-kDa fragment disappeared after mutation at DSPD748G. The 47-kDa fragment was still detected after mutation at DSPD748G, but it was not the same 47-kDa fragment reactive with Cas-2 Ab (our unpublished data). The disappearance of the 74- and 31-kDa fragments was more apparent in a double mutant (D416E and D748E) with which only a single, 90-kDa cleavage fragment appeared. To further confirm in vivo cleavage of Cas, mutant Cas DNAs were cloned into pFLAG-CMV-5c vectors and transiently transfected into Rat-1 cells. After 24 h of exposure to 40 μM etoposide, cell lysates were immunoblotted with anti-FLAG M2 mAb (Figure (Figure7A),7A), and the same membrane was reprobed with Cas mAb (Figure (Figure7B).7B). Without etoposide treatment, full-length Cas (130-kDa) was well detected in wild-type Cas-transfected cells by anti-FLAG M2 mAb and Cas mAbs. After treatment with etoposide for 24 h, a 31-kDa fragment was apparently detected in wild-type Cas- and single-mutant Cas-transfected cells (D416E) by anti-FLAG M2 mAb. However, the 31-kDa fragment was not detected in single-mutant (D748E) and double-mutant Cas-transfected cells, respectively. As shown in Figure Figure7B,7B, Cas mAb was able to detect a 31-kDa fragment in etoposide-treated cells, which is a cleavage product of endogenous Cas. These data further confirm that DVPD416G and DSPD748G are cleavage sites of Cas.

Figure 5
Schematic representation of the structure of Cas with the predicted cleavage sites and the functional domains. The following regions within Cas are indicated: the SH3 domain (SH3), the proline-rich sequence (Pro), the substrate domain (YXXP, tyrosine ...
Figure 6
Determination of the caspase-3 cleavage sites in Cas using site-directed mutagenesis. (A) Cas mutants containing point mutations replacing Asp (DVPD416 and/or DSPD748) with Glu were translated in vitro and incubated with extracts (20 μg) prepared ...
Figure 7
In vivo cleavage of mutant Cas. Rat-1 cells transiently transfected with pFLAG-CMV-5c containing each mutant Cas DNA such as vector only (lane 1), wild-type Cas (wt, lanes 2 and 4), single-mutant Cas (D416E, lane 5; and D748E, lane 6), and double-mutant ...

Cleavage of Cas Alters Its Association with Paxillin

Our findings thus far indicate that in etoposide-treated cells, the decrease in Cas is most likely due to caspase-mediated proteolysis during apoptosis. We, therefore, examined how this cleavage affects the interaction between Cas and another focal adhesion protein, paxillin. In this experiment, cells were grouped into adherent and apoptotic cells, the former being cells adhering to the bottom of culture dish and the latter being apoptotic cells floating in the medium after 36 h of exposure to etoposide. Although paxillin was unchanged in control and adherent cell lysates, Cas was dramatically decreased in adherent cells and was not even detected in apoptotic cells. Furthermore, the cleavage fragment (31-kDa) was also detected in both adherent and apoptotic cells (Figure (Figure8A).8A). In a parallel experiment, Cas was coprecipitated with paxillin in control adherent cells, whereas full-length Cas (130-kDa) associated with paxillin dramatically decreased, and a 100-kDa truncated Cas additionally appeared in adherent cells after treatment with etoposide for 12 h. In adherent cells exposed with etoposide for 36 h, a 100-kDa truncated Cas but not full-length Cas was only coprecipitated with paxillin (Figure (Figure8B).8B). This reflects the decline in the availability of Cas by apoptotic degradation.

Figure 8
Interaction between Cas and paxillin in apoptotic cells. (A) Cell lysates were immunoblotted with Cas mAb or paxillin mAb. (B) Cas and paxillin were immunoprecipitated from control (C), adherent (H), or apoptotic (A) cells by Cas-2 Ab or paxillin mAb, ...

In addition, Cas was detected as a 100-kDa truncated form in adherent cells rather than its full-length, 130-kDa form (Figure (Figure8B),8B), which suggests that initiation of Cas degradation inhibits the binding of Cas to paxillin, rendering Cas incapable of functioning as a docking molecule. It is interesting that, if already bound, the partially cleaved Cas remained bound to paxillin in adherent cells. This likely reflects the essential role played by Cas in the assembly of focal adhesion complexes.

To test whether the molecular changes attenuating the interaction of Cas and paxillin correlate with changes in the cellular architecture of focal adhesions, we used a Cas mAb (4F4), which recognizes the tyrosine-phosphorylated form of Cas, to probe the cellular distribution of Cas in cells exposed to etoposide for 24 h. In control cells, Cas was well localized in focal adhesion sites (Figure (Figure9A).9A). But in etoposide-treated cells, Cas was lost from focal adhesions at the bottom of cells and was instead found in cell peripheries to occur membrane retraction during the process of cell detachment from the substratum (Figure (Figure9B).9B). Paxillin and talin underwent the same change in cellular localization as well (Figure (Figure9,9, C–F). In addition, actin stress fibers in control cells formed well-arranged straight lines in the cytoplasm and in peripheral regions (Figure (Figure9G),9G), whereas most of the cytoplasmic stress fibers had disappeared in etoposide-treated cells, and only discontinuous, truncated fibers were seen in the cell periphery (Figure (Figure9H).9H). This finding is consistent with changes in the actin cytoskeleton observed in endothelial cells undergoing apoptosis (Palladini et al., 1996 blue right-pointing triangle) and suggests that degradation of Cas during apoptosis leads to relocalization of focal adhesions into the periphery of the cell and the rearrangement of the cytoskeleton and consequently results in disassembly of focal adhesions.

Figure 9
Fluorescent images depicting the changes in the cellular localization of Cas, paxillin, talin, and actin in apoptotic cells. The distribution of Cas (4F4, A and B), paxillin (C and D), and talin (E and F) within control cells or etoposide-treated cells ...

To examine further the role of Cas in the assembly of focal adhesions, cells were transiently transfected pFLAG-CMV-5c vectors containing double-mutant Cas or wild-type DNAs and double immunostained with anti-FLAG M2 mAb and paxillin Ab (Figure (Figure10).10). Cells expressing double-mutant Cas are indicated by positive staining with anti-FLAG M2 mAb. Immunofluorescence analysis using paxillin Ab in double-mutant Cas-transfected cells demonstrated that focal adhesions still remained in the bottom of cells after treatment with etoposide for 18 h, whereas focal adhesions in untransfected cells and wild-type Cas-transfected cells were completely lost from the bottom of cells and instead relocalized into the pheripery of cells (Figure (Figure10,10, A–D). After treatment with etoposide for 24 h, both transfected and untransfected cells displayed the relocalization of focal adhesions into the pheriphery of cells (Figure Figure10,10, E and F). These data suggest that mutation in cleavage sites may attenuate Cas degradation at the beginning of apoptosis and consequently protect, at least in part, the disassembly of focal adhesions in the bottom of cells. However, the prolonged treatment with etoposide seems to induce the cleavage of mutant Cas and results in the loss of focal adhesions from the bottom of cells.

Figure 10
Changes of cellular localization of Cas in double-mutant Cas-transfected Rat-1 cells. Rat-1 cells were transiently transfected with pFLAG-CMV-5c containing double-mutant (D416E and D748E) or wild-type Cas DNAs and incubated with 40 μM etoposide ...


In the present study, we identified the focal adhesion molecule Cas as a substrate for apoptotic caspases. Previously, another focal adhesion molecule, FAK, was also identified as a substrate for cleavage by caspases, a proteolytic event that may be key to the execution of the suicide pathway (Crouch et al., 1996 blue right-pointing triangle; Wen et al., 1997 blue right-pointing triangle; Gervais et al., 1998 blue right-pointing triangle). Our data show that etoposide induces proteolytic cleavage of Cas in Rat-1 cells undergoing apoptosis. Cleavage of Cas has also been observed when apoptosis was induced by staurosporine or nocodazole, a microtubule-depolymerizing agent (Kook et al., 2000 blue right-pointing triangle), which suggests that Cas degradation is a general phenomenon associated with apoptosis.

The cleavage patterns of in vitro–translated and native Cas were similar and interesting in several respects. The time course of these cleavages suggests that there is a hierarchy in the proteolytic cascade, perhaps in part because not all potential cleavage sites are simultaneously exposed. For example, a 100-kDa band appeared at early times (Figure (Figure4),4), which was likely generated by cleavage of the C terminus, because the N terminus lacks a putative cleavage site that would yield a fragment of a corresponding size. Considering the potential sites for Cas proteolysis, it is reasonable to predict that the DAVD860A sequence in the C terminus would be preferentially cleaved. The C-terminal cleavage fragment would not be detected in our experiment, because it would lack a methionine residue. Cleavage near the C terminus may induce a conformational change that in turn causes other potential cleavage sites to be exposed. Using Cas mAb, we were able to detect 74- and 31-kDa fragments.

An increasing number of caspase-3 substrates sharing the DXXD consensus sequence for caspase-3 are being identified (Harvey et al., 1998 blue right-pointing triangle; Wang et al., 1998 blue right-pointing triangle). Ten potential cleavage sites found in Cas fit with the preferred consensus sequence for caspase-3, and using site-directed mutagenesis, we confirmed that two of them, DVPD416G and DSPD748G, were cleaved by caspase-3. Cleavage of DVPD416G generates a 74-kDa fragment, further cleavage of which at the DSPD748G site generates the 31- and 47-kDa fragments. Substituting D416 or D748 with Glu resulted in the complete disappearance of the 74- and 31-kDa fragments, respectively, confirming that these two sites are in fact cleaved by caspase-3. Other potential cleavage sites could not be investigated, because the specificity of our antibodies is limited.

Cas degradation correlated temporally with the onset of apoptosis. Initiation of Cas cleavage blocks the binding of paxillin to Cas and the resultant localization of paxillin within focal adhesion sites. Consistent with that finding, Cas-deficient embryos exhibit disorganized myofibrils, disrupted Z-disks, and impaired actin stress fiber formation (Honda et al., 1998 blue right-pointing triangle). We also found that Cas was concentrated in the periphery of cells where membrane retraction is ongoing during apoptosis, and that actin fibers were truncated, no longer forming continuous stress fibers. In addition, we demonstrated that the double-mutant Cas (D416E and D748E) could attenuate the Cas degradation and consequently partially blocked the relocalization of focal adhesion proteins into the periphery of cells. Because focal adhesions provide a structural link between the actin cytoskeleton and ECM, these data suggest that Cas degradation during apoptosis disrupts the architecture of focal adhesions, disrupts molecular interactions within focal adhesions, and, consequently, disrupts the cytoskeleton. Thus, Cas can be added to the list of proteins associated with regulation of the cytoskeleton and cell attachment that undergo proteolytic cleavage during apoptosis.

Cas was initially identified as a prominent tyrosine-phosphorylated substrate of the oncoproteins v-Crk and v-Src (Reynolds et al., 1989 blue right-pointing triangle; Mayer and Hanafusa, 1990 blue right-pointing triangle; Kanner et al., 1991 blue right-pointing triangle). It was also reported that Cas was phosphorylated when cells adhere to the ECM and β1-integrin is cross-linked (Nojima et al., 1995 blue right-pointing triangle; Petch et al., 1995 blue right-pointing triangle). This phosphorylation is apparently involved in recruiting signaling molecules into focal adhesions. Moreover, Cas is an adaptor protein with an SH3 domain, a proline-rich domain, a substrate domain containing nine Tyr-Asp-X-Pro motifs, and a C-terminal domain containing the consensus SH3 and SH2 binding sites for Src (Sakai et al., 1994 blue right-pointing triangle). The association of Cas with these molecules is likely to be functionally important in the formation and maintenance of focal adhesion complexes, and SH2- and SH3-containing molecules associated with Cas may transduce a common set of signals that regulate cell growth, differentiation, and survival (Chan et al., 1999 blue right-pointing triangle). Degradation of Cas during apoptosis may thus have downstream effects interrupting survival signals from the ECM and reducing the likelihood of cell survival.

The sequence RPLPSPP (733–739) and Tyr762 near the C terminus of Cas are involved in the association of Cas with Src; mutation of either the sequence or Tyr762 significantly reduced Cas binding to Src (Nakamura et al., 1998 blue right-pointing triangle). It is of interest, therefore, that one of the Cas cleavage sites identified in this study is located at DSPD748G, because cleavage at that site would be expected to separate the two Src-binding motifs causing Cas to dissociate from Src, which would in turn cause disassembly of focal adhesion complexes. Confirmation of this scenario, however, will require further study.

In summary, we have demonstrated that early caspase-catalyzed proteolysis of Cas during etoposide-induced apoptosis contributes to the morphological characteristics of apoptotic cells that reflect disassembly of focal adhesion complexes and the interruption of survival signals from the ECM.


We thank Dr. Hirai (Tokyo University) and Dr. Parsons (University of Virginia) for kindly providing the rat Cas cDNA and Cas monoclonal antibody (4F4). This study was supported in part by a grant from the Korea Science and Engineering Foundation (KOSEF-97-0401-07-01-5) and from the Ministry of Science and Technology (98-NQ-07-01-A).


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