Degradation of Focal Adhesion Proteins Paxillin and p130cas by Caspases or Calpains in Apoptotic Rat-1 and L929 Cells

Biochemical and Biophysical Research Communications 286, 601– 608 (2001) doi:10.1006/bbrc.2001.5441, available online at http://www.idealibrary.com on

Degradation of Focal Adhesion Proteins Paxillin and p130cas by Caspases or Calpains in Apoptotic Rat-1 and L929 Cells Sang Ryeol Shim, Seunghyi Kook, Jae Il Kim, and Woo Keun Song 1 Department of Life Science, Kwangju Institute of Science and Technology, Kwangju 500-712, Korea

Received July 22, 2001

Immunofluorescence microscopy revealed the rearrangement and gradual dissociation of paxillin from focal adhesion sites during apoptosis. In vitro, cleavage of paxillin by caspase-3 generated a 42-kDa fragment, among other products, while cleavage by calpain generated a different set of fragments. In Rat-1 cells, cleavage of paxillin by caspase-3 was suppressed by zVAD-fmk or zDEVD-cmk, making caspase-3 a likely executioner during etoposide-induced apoptosis. In contrast, the cleavage of paxillin and p130cas in apoptotic L929 cells was blocked by calpain-specific inhibitors, which also reduced the death rate by 23 to 44%. Therefore, The disassembly and degradation of p130cas and paxillin during apoptosis may controlled by both caspases and calpains, depending upon their cellular contexts. Our findings also suggest that focal adhesion proteins paxillin and p130cas take part in integrin-mediated signaling for cell survival, and that their cleavage by caspase and/or calpain may not only disrupt focal adhesion complexes, but may also impede cell survival signaling. © 2001 Academic Press Key Words: paxillin; apoptosis; p130cas; focal adhesion; FAK.

Apoptosis is a process of programmed cell suicide that is a critical feature of organism development and maintenance of tissue homeostasis (1). Cells undergoing apoptosis exhibit characteristic morphological and biochemical changes, including chromatin condensation, nuclear fragmentation, DNA laddering, cell shrinkage and apoptotic bodies; adherent cells, moreAbbreviations used: ECM, extracellular matrix; FAK, focal adhesion kinase, TRICT, tetramethyl rhodamine isothiocyanate; zVAD-fmk, ZVal-Ala-DL-Asp-fluoromethylketone; zDEVD-cmk, Z-Asp-Glu-Val-Aspchloromethylketone; aLLnL, N-acetyl-Leu-Leu-Nle-CHO; zLnL, Z-LeuNle-CHO; PD150606, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic. 1 To whom correspondence should be addressed at Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea. Fax: 82-62-9702484. E-mail: [email protected].

over, change their shape—flattened cells become rounded and detach from the substratum. Such apoptotic processes are accompanied by the activation of caspases and other proteases, including calpain, cathepsin D, and the 20S proteosome (2). The cytoskeleton is recognized as being integral to the shape and function of cells. Once cells are anchored, integrin-mediated transmembrane linkages between the extracellular matrix (ECM) and cytoskeletal elements provide a structural basis for cell survival. Indeed, several lines of evidence suggest that disassembly of the focal adhesion proteins linking the cytoskeleton to the ECM and to other cells via integrin receptor proteins is closely associated with apoptotic morphological changes and cellular degeneration (3, 4). During apoptosis, transmembrane linkages may be degraded by apoptosis-related genes encoding “executioner” proteases, such as caspases and calpains. Because many key proteins, including fodrin, ␤-catenins, actin, gelsolin, focal adhesion kinase (FAK) and Crkassociated substrate (p130cas) are susceptible to caspase-catalyzed cleavage (5–10). Caspases appear to play a critical role in the execution phase of apoptosis, and appear to be responsible for many of the biochemical and morphological changes associated with apoptosis. Calpains, which are widely expressed in mammalian cells, are Ca 2⫹-activated cysteine proteases known to be involved in the proteolytic degradation of proteins during apoptosis (11, 12). They are localized and activated at focal adhesions (13, 14), where a growing number of calpain substrates have been identified— these include structural proteins, such as talin and integrin ␤ 3 subunit (15–17) as well as signaling molecules, such as protein kinase C, FAK, Src and protein tyrosine phosphatase-1B (18, 19). Thus, proteolytic cleavage of cytoskeletal and focal adhesion proteins by calpain or/and caspase may serve to regulate both cell adhesion and cellular signaling. In addition, disassembly of paxillin from focal adhesion sites is known to cause cells to detach from ECM and to go from flat-

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tened to rounded in shape (10). However, the degradation of paxillin by caspases or calpains during apoptosis has not yet been fully characterized. We, therefore, investigated the disassembly and degradation of paxillin and p130cas during apoptosis, and found them to be degraded by both caspases and calpains, depending upon their cellular contexts. MATERIALS AND METHODS Cell culture. L929 murine fibrosarcoma cells (a kind gift from Dr. Su Jae Lee, Korean Cancer Center Hospital, Korea) were maintained in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (GibcoBRL), while Rat-1 fibroblasts were maintained in Dulbecco’s modified Eagle medium. The cells were plated to a density of 3 ⫻ 10 5/well in 6-well plates, or to 1 ⫻ 10 6/well in 100 mm culture and incubated at 37°C under a 5% CO 2 atmosphere. After 24 h, apoptosis was induced by exposing the cells to 40 ␮M etoposide for the indicated times. In some cases, a tetrapeptide caspase inhibitor—Z-ValAla-DL-Asp-fluoromethylketone (zVAD-fmk) or Z-Asp-Glu-Val-Aspchloromethyl-ketone (zDEVD-cmk) (Bachem)— or a calpain inhibitor—N-acetyl-Leu-Leu-Nle-CHO (aLLnL); Z-Leu-Nle-CHO (zLnL); or 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606)— was added to the medium 2 h prior to the induction of apoptosis. Another calpain inhibitor, Z-Leu-Leu-Tyr-CH 2F (zLLY-fmk), was added 30 min prior to induction of apoptosis. Cell viability was assessed by trypan blue exclusion. Immunofluorescence. Rat-1 and L929 cells grown on 0.1% (w/v) gelatin-coated coverslips were fixed for 10 min in 3.5% (w/v) paraformaldehyde, washed in PBS, and permeabilized for 5 min with 0.5% (v/v) Triton X-100 in PBS. The cells were then incubated for 1 h at 37°C with anti-paxillin monoclonal antibody (mAb) (Transduction Laboratories) and incubated for 1 h at room temperature with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat antimouse IgG. The coverslips were then washed in PBS, mounted with mounting solution (Mcllvaine’s buffer:glycerol, 1:1), and sealed with transparent fingernail polish. Immunofluorescence was analyzed under a Leica DMRBE microscope equipped with a 100⫻ objective lens and filters for epifluorescence. Fluorescence micrographs were taken on T-max P3200 film (Eastman Kodak Co.). Immunoblot analysis. Cells were harvested with lysis buffer [1% (w/v) SDS, 1 mM sodium orthovanadate, 10 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 10 ␮M leupeptin, 1.5 ␮M pepstatin and 10 ␮g/ml aprotinin] and centrifuged for 5 min at 4°C and 10,000g to remove insoluble material. The supernatant was then mixed with 5⫻ SDS-sample lysis buffer (60 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 25% (v/v) glycerol, 14.4 mM 2-mercaptoethanol, and 0.1% (v/v) bromophenol blue) and boiled for 10 min. Proteins in the lysates were separated by 10 –12% (w/v) SDS– PAGE and transferred to PVDF membranes. The membranes were then incubated first with primary antibody, and then with either Horseradish peroxidase (HRP)-labeled anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch Lab). Bands were detected using enhanced chemiluminescence (ECL) according to the manufacturer’s protocol (Amersham Corp.). In some cases, blots were stripped by heating them to 55°C for 30 min in stripping solution (100 mM 2-mercaptoethanol, 2% (w/v) SDS, and 62.5 mM Tris–HCl, pH 6.7) and reprobed. In vitro cleavage assay by caspases and calpain. Rat p130cas (from Dr. Hismaru Hirai, University of Tokyo, Japan) and avian paxillin cDNAs were used as templates for in vitro labeling with [ 35S]methionine (1175 Ci/mmol; NEN) using a coupled transcription and translation reticulolysate system. Recombinant caspases were prepared from bacterial cells (Escherichia coli, BL21) according to Kook et al. (9).

To assess caspase-catalyzed cleavage, in vitro-translated paxillin was incubated for 90 min at 30°C with 20 ␮g of bacterial cell lysate containing the respective recombinant caspase. The reactions were terminated by the addition of SDS–PAGE sample buffer and boiling for 5 min. Samples were then subjected to 10 –12% (w/v) SDS–PAGE, fixed for 30 min at room temperature with destaining solution, dried and exposed to X-ray film (Konica Corp., Japan). To inhibit the caspase-3 activity, zDEVD-cmk was applied 1 h prior to the addition of labeled paxillin. To assess calpain-catalyzed cleavage, [ 35S]methinonine-labeled paxillin was incubated for the indicated time periods at 30°C with purified porcine m-calpain (calpain II). The reaction was initiated by addition of CaCl 2 to a final concentration of 1 mM and stopped by addition of SDS–PAGE sample buffer and boiling for 5 min. aLLnL was added 1 h prior to the reaction to inhibit the calpain activity. Samples were then subjected to SDS–PAGE and autoradiography. Assessment of caspase-3 activity. Caspase-3 activity was determined by using the CaspACE Assay System, Fluorometric (Promega), following the manufacturer’s instructions. In short, cells were grown and treated with etoposide (40 ␮M) for each time periods. Cells were collected by centrifugation at 450g for 10 min at 4°C and resuspended in hypotonic lysis buffer (25 mM Hepes pH 7.5, 5 mM MgCl 2, 5 mM EDTA, 5 mM DTT, 2 mM PMSF, 10 ␮g/ml pepstatin A, 10 ␮g/ml leupeptin). The cell suspension was lysed by three freezethaw cycles, and the cytosolic fraction was obtained by centrifugation at 16,000g for 20 min at 4°C. A total of 20 ␮g of protein extracts were incubated in 96-well plates in 100 ␮l assay buffer (100 mM Hepes, pH 7.5, 10% sucrose, 0.1% Chaps, 10 mM DTT), containing 50 ␮M Ac-DEVD-AMC at 30°C for 60 min. For the inhibition studies, 50 ␮⌴ caspase-3 inhibitor (Ac-DEVD-CHO) was pretreated for 30 min before the substrate was added. The release of AMC was detected on a Molecular Devices instrument (excitation 360nm; emission 460 nm) with SOFTmax Pro software. Standard dilutions of AMC were included in each assay to determine absolute concentrations of released AMC in samples and convert measured fluorescent units into a sate of substrate cleavage per ␮g protein (pmol cleaved DEVDAMC ⫻ min ⫺1 ⫻ ␮g ⫺1). Values were corrected for spontaneous release of AMC in the absence of cell extract. Protein was determined by Bradford assay. Assessment of calpain activity. The calpain activity assay used in based on those described by Edelstein et al. (20). The cell pellets were resuspended in imidazole-Ca 2⫹-free buffer (63.2 mM imidazole–HCl, pH 7.3, 10 mM 2-mercaptoethanol, 10 mM EDTA, 10 mM EGTA) and then incubated with 10 mM digitonin at 37°C for 5 min and centrifuged at 1000g for 10 min at 4°C to collect the cytosol. Suc-LLVYAMC was used as the calpain substrate. A total of 20 ␮g of protein extracts were incubated in 100 ␮l imidazole buffer (63.2 mM imidazole–HCl, pH 7.3, 10 mM 2-mercaptoethanol) containing 50 ␮⌴ Suc-LLVY-AMC at 37°C for 30 min. Three sets of assays were conducted for each sample: (i) reconstituting Ca 2⫹ at 10 mM; (ii) without reconstituting Ca 2⫹; (iii) in presence of Ca 2⫹ and 400 nM of aLLnL, an inhibitor of calpain protease.

RESULTS Alteration of paxillin in etoposide-treated Rat-1 and L929 cells. To understand the relationship between the loss of focal adhesion and apoptosis, we monitored the focal adhesion protein, paxillin, in cells exposed to etoposide, an apoptosis-inducing drug. Rat-1 and L929 cells, which adhere to the substratum and assume a flattened shape, contain well-developed patterns of focal adhesions, revealed by immunofluorescent labeling with paxillin mAb (Figs. 1A and 1B). In the presence of etoposide, a loss of paxillin from focal adhesions was detected in parallel with the morphological changes

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FIG. 1. Fluorescent images depicting the changes in the cellular localization of paxillin in etoposide-induced apoptotic Rat-1 and L929 cells. The cultured cells were stained with paxillin mAb at the indicated times after exposure to 40 ␮M etoposide. The distribution of paxillin within control cells and etoposide-treated Rat-1 and L929 cells revealed paxillin to be localized in focal adhesion sites in control cells (A and B), but redistributed to the periphery during the course of apoptosis (C–F). Apoptotic body (white arrow) was observed (E). Nuclear fragmentation (white arrowheads) was observed with Hoechst staining in both Rat-1 and L929 cells after exposure to 40 ␮M etoposide for 36 h (G and H). Bar, 10 ␮m.

to immunoblot analysis using paxillin mAb. In the presence of etoposide, the level of paxillin declined in concert with viability in both cell types (Fig. 2A), and a 42-kDa cleavage product was detected in Rat-1 cells after 36 h of etoposide exposure. To confirm that caspases catalyze the generation of the 42-kDa fragment in vitro, a cDNA encoding chick paxillin was in vitro-transcribed and translated with [ 35S]methionine, and then incubated with recombinant caspase-1 (ICE), caspase-3 (CPP32) or caspase-8 (FLICE) in bacterial lysates. As shown in Fig. 2B-a, paxillin was partially cleaved by caspase-3, generating cleavage products with molecular masses of 42, 35, 31, 29, and 25 kDa. The 42-kDa band seemed to be identical to that detected in etoposide-treated cells, while the 47-kDa band appeared to be an N-terminal truncated form, which might be translated from an internal initiation codon for methionine at position 133 (* labeled). Pretreatment with 0.5 ␮M zDEVD-cmk, a specific inhibitor for caspase-3, completely blocked the production of paxillin cleavage products, indicating caspase-3 to be at least partly responsible for paxillin cleavage during apoptosis (Fig. 2B-a). In parallel experiments, we attempted to cleave in vitro-translated paxillin with the purified calpain. We found that [ 35S]methionine-labeled paxillin was cleaved by calpain, yielding cleavage fragment of 32, 28, 26, 21 and 18-kDa; and that cleavage was completely blocked with 100 ␮M aLLnL, a synthetic antagonist that binds to the active site of calpain (Fig. 2B-b). Taken together, these results suggest that both caspase-3 and calpain are primary mediators of paxillin cleavage in apoptotic cells.

characteristic of cells undergoing apoptosis. As the cells retracted and became spherical in shape, paxillin was redistributed, gradually dissociating from focal adhesions (Figs. 1C and 1D) and after 36 h of exposure to etoposide, it was no longer detectable (Figs. 1E and 1F). Once detached, apoptotic cells floated in the culture medium, exhibiting the characteristic morphological features of apoptosis. The effects of etoposide on the morphology of Rat-1 and L929 cells were time-dependent. Nuclear condensation and fragmentation, observed with Hoechst staining, were noted as early as 6 h after exposure to etoposide (Figs. 1G and 1H), and the number of apoptotic cells increased significantly after 24 h of exposure.

Caspase-catalyzed cleavage of paxillin in apoptotic Rat-1 cells. When Rat-1 cells were incubated with etoposide for 36 h, the death rate was 48.2% (Fig. 3A). Preincubation of zVAD-fmk or zDEVD-cmk reduced the death rate in a concentration-dependent manner; as an example, the death rate was reduced to 15.2% or less by 100 ␮M zVAD-fmk and to 27.4% by 200 ␮M zDEVD-cmk (Fig. 3A). Immunoblot analysis confirmed that the appearance of the 42-kDa paxillin cleavage product was completely suppressed by zVAD-fmk and partially suppressed by zDEVD-cmk (Fig. 3B). In parallel with the inhibition of paxillin degradation, the morphological changes induced by etoposide were also significantly inhibited by both inhibitors. Calpain inhibitors, by contrast, had no effect on etoposide-induced apoptosis in Rat-1 cells (Fig. 3B). The 42-kDa cleavage product was detectable even in the presence of calpain inhibitors, implying that paxillin in Rat-1 cells is cleaved primarily by caspase during etoposide-induced apoptosis.

Cleavage of paxillin by caspases or calpains. To assess the modification of paxillin during apoptosis, etoposide-treated Rat-1 and L929 cells were subjected

Calpain-catalyzed cleavage of paxillin in apoptotic L929 cells. To assess cleavage of paxillin in caspaseindependent cell death, L929 cells were exposed to

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FIG. 2. (A) Degradation of paxillin was detected in Rat-1 (a) and L929 cells (b) during etoposide-induced apoptosis. After cells were exposed to 40 ␮M etoposide, paxillin was detected with monoclonal (upper panel) or polyclonal paxillin Ab (lower panel). Cell viability was assessed by trypan blue exclusion. (B) In vitro cleavage of paxillin with caspases and calpain. (a) [ 35S]Methionine-labeled paxillin was generated by in vitro translation and then cleaved using recombinant caspase-1 (ICE), caspase-3 (CPP32), or caspase-8 (FLICE) in bacterial lysates. Paxillin (filled arrowhead) was partially cleaved by caspase-3, but was unaffected by caspase-1 and caspase-8. zDEVD-cmk inhibited paxillin cleavage in a concentration-dependent manner. (b) [ 35S]Methionine-labeled paxillin was incubated with purified porcine calpain II, yielding small cleavage fragments. Calpain activity was blocked by aLLnL. The 47-kDa band (*) seems to be an N-terminal truncated from of paxillin. Cleavage fragments of paxillin were indicated by open arrowheads.

etoposide, which induced death in 64% of control cells within 36 h. Interestingly, caspase inhibitors appeared to promote cell death in L929 cells; i.e., the death rate increased to 80% in L929 cells pretreated with zVADfmk for 2 h (Fig. 4A). This result is consistent with an earlier reports that TNF-treated or anti-Fas-treated L929cells underwent necrotic cell death in the presence of caspase inhibitors, zVAD-fmk or zDEVD-cmk (21, 22). On the other hand, addition of calpain inhibitors significantly inhibited etoposide–induced apoptosis and morphological changes in L929 cells—50 ␮M aLLnL reduced the death rate to 20.4%; 50 ␮g/ml zLnL reduced it to 37.9%; and 50 ␮M zLLY-fmk reduced it to 35.3% (Fig. 4A). To verify the specificity of the effects of the calpain inhibitors, we assessed the effects of PD150606, a calpain antagonist that acts by blocking the Ca 2⫹-binding sites of calpain, and found that PD150606 (250 ␮M) reduced the apoptosis rate by 39.4%. Immunoblot analysis revealed that paxillin degradation was diminished by calpain inhibitors in apoptotic L929 cells (Fig. 4B, right), which is consistent with the reduction in the apoptosis rate. In contrast, caspase

inhibitors zVAD-fmk and zDEVD-cmk did not reduce paxillin degradation, and may have even enhanced it (Fig. 4B, left); paxillin became virtually undetectable in the presence of 50 ␮M zVAD-fmk Thus, apoptotic degradation of paxillin is apparently calpain-specific in L929 cells. Caspase- or calpain-specific degradation of p130cas in Rat-1 and L929 cells. To address that caspase-3 or calpain is responsible for paxillin degradation during apoptosis of L929cells, we have observed the activity of caspase-3 and calpain in apoptotic cells. In apoptotic L929 cells, calpain activity was gradually increased, which is in accord with the time course of paxillin degradation but caspase-3 activity was remained in basal level, suggesting that calpain is a primary mediator of paxillin degradation in L929 cells (Fig. 5A). To verify that, depending on their cellular contexts, focal adhesion proteins undergo degradation by different sets of proteases during apoptosis, we monitored another focal adhesion protein, p130cas. The cleavage product of p130cas (fragment of 31-kDa) was respectively detected in apoptotic Rat-1 and L929 cells using p130cas mAb (Fig. 5B-a, b). Caspase inhibitors zVAD-

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FIG. 3. Caspase-catalyzed cleavage of paxillin in etoposide-treated Rat-1 cells. (A) The viability of Rat-1 cells was analyzed after exposure to 40 ␮M etoposide in the presence of caspase- or calpain-specific inhibitors. Cells were pretreated with caspase inhibitors [zVAD-fmk (zVAD) or zDEVD-cmk (zDEVD)] or with calpain inhibitors [aLLnL, ZLnL, or PD150606] prior to etoposide treatment. Trypan blue exclusion was then assessed after 36 h of exposure to etoposide. Results are representative of three separate experiments. (B) Cells were pretreated with caspase or calpain-specific inhibitors, harvested after 36 h of exposure to etoposide, and immunoblotted with monoclonal (upper panel) or polyclonal (lower panel) antibodies.

fmk and zDEVD-cmk significantly inhibited the degradation of p130cas in apoptotic Rat-1 cells (Fig. 5B-a). Conversely, caspase inhibitors had little effect in L929 cells, where the degradation of p130cas was inhibited in the presence of calpain inhibitors—formation of the p130cas 31-kDa fragment was dramatically blocked by zLnL, and partially blocked by aLLnL (Fig. 5B-b). However, the 31-kDa fragment produced by calpain is not identical with the 31-kDa fragment produced by

caspases, even both fragments are reactive with the same monoclonal anti-cas antibody. Caspase-3 did not produce the 31-kDa fragment in the double mutant (D416E/D748E) of p130 cas (10) whereas calpain produced the 31-kDa fragment even in the double mutant (Fig. 5B-c). This result indicates that both caspase-3 and calpain cleave and produce the similar size fragment (⬃31-kDa) of p130cas reactive with the monoclonal anti-cas antibody.

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FIG. 4. Calpain-catalyzed cleavage of paxillin in etoposide-treated L929 cells. (A) The viability of L929 cells was analyzed after exposure to 40 ␮M etoposide in the presence of specific caspase and calpain inhibitors. Cells were pretreated with the respective antagonists as in Fig. 3. Results are representative of three separate experiments. (B) Immunoblot illustrating the effects of caspase and calpain antagonists on apoptosis-induced cleavage of paxillin. Cells were pretreated with the indicated antagonist at the indicated concentration (␮M) prior to exposure to etoposide.

DISCUSSION For most eukaryotic cells, anchorage is directly related to survival; detachment from neighbors or the ECM induces a series of apoptotic processes and vice versa. We found that in Rat-1 and L929 cells exposed to etoposide, degradation of focal adhesion proteins was the primary cause of the morphological changes characteristic of apoptosis: cleavage of paxillin and p130cas by caspase-3 or calpain was accompanied by cell rounding, shrinking and detachment from the substratum. These findings are in accord with several lines of evidence that suggest detachment from ECM and the resultant changes in morphology are caused by structural disruption of focal adhesion complexes and/or changes in the molecular interactions among them (3, 4). Furthermore, because focal adhesion complexes also serve as molecular adaptors and signal transduc-

ers, their disruption may impede survival signaling, or even constitute a death signal, contributing to the apoptotic process (23–26). Paxillin and p130cas were degraded to several smaller fragments by caspase in etoposide-treated Rat-1 cells and the degradation was blocked by caspase inhibitors, zVAD-fmk and zDEVD-cmk. This is consistent our earlier study (9, 10), as well as with other studies showing that another focal adhesion protein, FAK, was cleaved by several caspases during apoptosis (27–30). We conclude, therefore, that focal adhesion proteins paxillin and p130cas serve as caspase substrates during apoptosis (6, 9, 10). L929 fibrosarcoma cells differed from Rat-1 cells in that etoposide-induced cleavage of paxillin and p130cas was diminished and cell survival was enhanced by pretreatment with calpain inhibitors (aLLnL, zLnL,

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FIG. 5. (A) L929 cells were treated with 40 ␮M etoposide for various time periods and the calpain and caspase activities were measured (see Materials and Methods). Calpain activity was gradually increased but caspase-3 activity was remained in basal level up to 36 h after etoposide treatment. Results are representative of three separate experiments. (B) Rat-1 cells were pretreated with caspase antagonists as in Figs. 3 and 4 and then exposed to etoposide for 36 h (a). L929 cells were pretreated with calpain antagonists and then exposed to etoposide for 36 h (b). p130cas degradation was detected with p130cas mAb. The in vitro translated p130cas wild type and double mutant (D416E/D748E) were cleaved by caspase-3 or calpain and the cleavage fragments were detected with p130cas mAb (c).

zLLY or PD150606). In addition, calpain activity but not caspase-3 activity was increased during apoptosis, which is in accord with paxillin degradation. However, caspase inhibitors even enhanced cell death in the etoposide-treated L929 cells. Etoposide-induced cell death of L929 cells in the presence of caspase inhibitors, zVAD-fmk or zDEVD-cmk, seems to be a necrotic cell death. It could be supported by the earlier reports that caspase inhibitors enhance cell susceptibility to death signals by production of reactive oxygen radical, which leads to cell death by necrosis (21, 22). Calpains have been localized to the cytoskeletal fraction and to focal adhesions in several cell types, which suggests they participate in the proteolytic cleavage of focal adhesion proteins and in the disassembly of cytoskeletal signaling complexes induced by apoptotic signaling (13, 18, 31). Although activation of calpain has generally been considered a component of caspaseindependent apoptotic processes—such as occur in U937 and neuronal cells, where apoptosis is associated

with abrupt increases in [Ca 2⫹] i—a growing number of molecules, affecting a variety of cell functions, have been reported to be cleaved by calpains (32). What’s more, recent studies have shown that calpains and caspases share some of the same substrates, and that the former may act as upstream regulators or downstream effectors of caspase activation (33–35). The fact that rapid elevations in [Ca 2⫹] i are often not detected in cells undergoing apoptosis has raised a question as to whether [Ca 2⫹] i in those cells ever gets high enough to activate calpains. However, Saido et al. (14) and Strobl et al. (36) showed that calpains were activated in vivo at [Ca 2⫹] i of 100 –300 nM, levels comparable to those seen in L929 cells during the early phase of etoposide stimulation (37). In addition, the present study showed that degradation of paxillin and p130cas was inhibited in L929 cells by PD150606, a calpain antagonist that blocks the Ca 2⫹ binding site. Apparently then, [Ca 2⫹] i is indeed sufficiently high to enable calpain to serve as the primary mediator of

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paxillin and p130cas cleavage in L929 cells during etoposide-induced apoptosis. The effects of calpain antagonists that bind to the active site of the enzyme on the cell death rate, and on the degradation of paxillin and p130cas, should be considered with a certain amount of caution; most also block papain-like protease or proteosome activities to some extent. Clarification of the specific roles played by these enzymes will require further study. Calpastatin, an endogenous calpain inhibitor, and lactacystin, a potent proteosome inhibitor, should be useful in addressing this question. In summary, we present here that degradation of focal adhesion proteins, paxillin and p130cas by both calpain or caspases, depending upon their cellular contexts and the degradation may not only disrupt focal adhesion complex, but also impede cell survival signaling. ACKNOWLEDGMENTS This study was supported by in part by grants from Protein Network Research Center (KOSEF) and Life Phenomena and Function Research Group (The Ministry of Science & Technology). Dr. S. Kook (postdoctoral fellow) and S. R. Shim were supported by Brain Korea 21 Program.

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