D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis.

Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified. Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain. D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine. Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk. D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment. The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32. In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM. N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S. These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.

Apoptosis (programmed cell death) acts to preserve peripheral T cell homeostasis, participating in the elimination of both immature thymocytes during thymic development and mature peripheral T cells following antigen stimulation under certain conditions (1)(2)(3). Fas (CD95), a member of the TNF receptor/ nerve growth factor family (4), is highly expressed in activated lymphocytes (5), and the ligand for Fas (FasL) appears to be expressed exclusively on activated T cells (6,7). Fas-mediated apoptosis is involved in down-regulation of immune reactions as well as in T cell-mediated cytotoxicity (8). Genetic mutations in murine Fas (lpr mutation) or FasL (gld mutation) lead to defective T cell receptor-induced cell death of mature T cells, resulting in autoimmune disease (10). A human dominant interfering Fas mutation has also been described that leads to autoimmune lymphoproliferative syndrome (11). More recently, Fas-induced apoptosis has been implicated in establishing immune-privileged sites such as the testes and eye, HIV elimination of T cells, and cytotoxic T lymphocyte-mediated cell killing (8,(12)(13)(14)(15)(16)(17).
ICE/CED-3 1 family cysteine proteases have been implicated directly in apoptosis as evidenced by the findings that overexpression of ICE-like proteases results in apoptosis and that co-expression of the viral proteins, CrmA and P35, which inhibit ICE family proteases can prevent the associated cell death (13, 18 -23). Deletion of ICE in mice renders thymocytes resistant to apoptosis induced by Fas, but not by dexamethasone or ␥-irradiation (24,25). A related protease, CPP32/Yama/ Apopain (18,26,27), specifically cleaves the nuclear protein poly(ADP-ribose) polymerase after induction of apoptosis, and inhibition of CPP32 activity by either a peptide inhibitor or by CrmA attenuates apoptosis in vitro (18,26). Furthermore, CPP32 is also involved in cytotoxic T lymphocyte-mediated target cell lysis following its activation through cleavage by granzyme B (28). 2 These data implicate CPP32 as an important ICE/CED-3 family protease directly involved in the initiation of apoptosis.
Relatively few apoptosis-related substrates for the ICE/ CED-3 family proteases have been reported, and the role of these substrates in apoptosis remains unclear. Due to their location and function, the ICE/CED-3 family protease substrates poly(ADP-ribose) polymerase and lamin are potentially important in the characteristic nuclear changes associated with apoptosis (18,26,30). The cellular signaling pathways involved in controlling apoptosis remain poorly defined as well. In particular, little is known about the mechanisms underlying the dramatic cytoskeletal, morphological, and membrane changes that accompany cell death and that may be important in the subsequent recognition and disposal of apoptotic cells by phagocytic leukocytes. In normally growing cells, such processes have been shown to be controlled by the action of Rho family GTPases (31,32). We establish here that D4-GDI, a hematopoietic cell-abundant regulator of the Rho family GTPases (33,34), is a substrate for CPP32 and is cleaved during apoptosis. These data suggest the likelihood of impor-tant roles for Rho GTPases in the signaling and cytoskeletal events accompanying apoptosis.

MATERIALS AND METHODS
Cell Experiments-Exponentially growing human Jurkat cells were resuspended in RPMI 1640 medium supplemented with 10% heatinactivated fetal calf serum at 4 ϫ 10 6 cells/ml and incubated with anti-Fas monoclonal antibody (CH11) at 250 ng/ml (Oncor Inc., Gaithersburg, MD) or 1 M staurosporine at 37°C. 3 ml of cells were removed at the indicated time and washed once with phosphate-buffered saline. One-third of the cells were fixed in 1% paraformaldehyde in phosphatebuffered saline and used for apoptosis assay using the ApopTag apoptosis detection kit (Oncor Inc.) according to the manufacturer's instructions. The rest of the cells were resuspended in 150 l of phosphatebuffered saline, sonicated, and boiled for 5 min in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The cell extract was analyzed by 14% SDS-PAGE (Novex, San Diego, CA). After electrophoresis, proteins were transferred to Immobilon polyvinylidene difluoride membrane (Millipore Inc., Bedford, MA) for immunodetection with D4-GDI-specific antiserum (1:1000 dilution), and visualized by enhanced chemiluminescence (ECL) (Amersham, Inc.).
Antibodies-Anti-peptide antiserum was raised against a synthetic peptide representing the internal D4-GDI sequence GPVVTDPKAPN-VVVTRC-amide (amino acids 55-70). The final Cys residue was added to allow the coupling to maleimide-activated carrier protein (Pierce). The peptide, conjugated to keyhole lymphet hemocyanin, was injected into New Zealand white rabbits, and titers of anti-D4-GDI antibody were determined by enzyme-linked immunosorbent assay using the same peptide conjugated to bovine serum albumin and by Western analysis against full-length and truncated D4-GDI. This antibody did not react against RhoGDI.
Preparation of ICE-related Proteases-Complementary cDNAs encoding CPP32, Mch2, and ICErel-II were separately subcloned in-frame into the BamHI/EcoRI site on the bacterial expression vector pGEX-2T (Pharmacia Biotech Inc.). Exponentially growing bacteria carrying the respective expression plasmid was induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside for 3 h at 37°C and then lysed by sonication in a lysis buffer containing 25 mM HEPES (pH 7.5), 5 mM EDTA, 2 mM dithiothreitol, and 0.1% CHAPS. The lysate was centrifuged at 16,000 ϫ g for 10 min, and the clear bacterial extracts were collected. The expressed CPP32 was autoprocessed as shown by Western blot analysis of bacterial extract using monoclonal anti-GST antibody (PharMingen, San Diego, CA). The bacterially expressed CPP32 was active, as demonstrated by specific cleavage of D4-GDI ( Fig. 3b) and Ac-DEVD-pNA (data not shown).
In some experiments, cDNA encoding D4-GDI was subcloned into the BamHI/EcoRI site of pcDNA3 (Invitrogen, San Diego, CA). Using the resulting plasmid, we constructed single or double mutations by the trans-polymerase chain reaction procedure (37). The TNT T7-coupled reticulocyte system (Promega, Madison, WI) was used to generate protein labeled with [ 35 S]methionine (15 mCi/ml, Amersham). 5 l of translated reticulocyte lysate was incubated with 5 l CPP32-expressing bacterial extract in 20 l of reaction buffer at 37°C for 40 min as described above. The reactions were analyzed by 14% SDS-PAGE, followed by autoradiography.

RESULTS AND DISCUSSION
D4-GDI is a highly abundant regulator of Rho GTPases in lymphoid and myeloid cells and is highly homologous to Rho-GDI, differing primarily at the N-terminal 25 amino acids (33,34). During the purification of ICE activity from THP-1 cells, we had observed two truncated forms of D4-GDI co-purifying with ICE activity. Briefly, ICE was partially purified from THP-1 cell lysates by three steps of ion exchange chromatog-raphy. 3 At this stage, ICE activity was purified about 3500-fold relative to cell lysate, and two protein bands could be seen co-migrating with ICE activity. N-terminal sequencing revealed that a band at 22 kDa was the p20 subunit of ICE, while a 19-kDa band was a truncated form of D4-GDI beginning at residue Gly 56 . Fig. 4, lane 2, shows a Western blot analysis of the partially purified ICE fraction using antipeptide antibody raised against the N terminus of the truncated D4-GDI. In addition to the 19-kDa fragment, the antibody detected a 23-kDa fragment of GDI. Inspection of the protein sequence revealed that the N-terminal region of D4-GDI contains two potential ICE/CED-3 protease cleavage sites with the sequences DELD 19 S and LLGD 55 G (Fig. 1). Neither site is present in Rho-GDI. These led us to test whether D4-GDI was a specific substrate for ICE-like proteases during the process of Fas-induced T cell apoptosis.
Jurkat T cells, which highly express D4-GDI (33, 34), 4 were induced to undergo apoptosis using anti-human Fas antibody for varying times (Fig. 2). Apoptosis and cleavage of D4-GDI were analyzed using the TdT-mediated deoxyuridine 5Јtriphosphate nick end labeling assay and Western analysis employing antibody specific for D4-GDI, respectively. Treatment of Jurkat T cells with anti-Fas resulted in the rapid onset of apoptosis, with greater than 60% cell death occurring within 4 -5 h (Fig. 2a). Full-length D4-GDI, which runs on SDS-PAGE as a 28 -30-kDa polypeptide, was specifically cleaved to a 23-kDa fragment with kinetics that paralleled and preceded the induction of apoptosis (Fig. 2b). This 23-kDa fragment corresponded to the size expected if cleavage occurs at the N-terminal CPP32 consensus cleavage site DELD 19 S (Fig. 1). Similarly, Jurkat cells treated with 1 M staurosporine also underwent rapid apoptosis and cleavage of D4-GDI to the 23-kDa fragment, as seen in the Fas-induced Jurkat cells (Fig. 2,  c and d). In contrast, we could detect no breakdown of Rho-GDI with any apoptotic stimulus (not shown), consistent with the absence of the consensus cleavage sequence in this protein.
We next examined the ability of a peptide inhibitor of ICE to block both T cell apoptosis and cleavage of D4-GDI induced using either anti-Fas or staurosporine (Fig. 3). Jurkat T cells were preincubated for 3 h with increasing concentrations of the tetrapeptide ICE inhibitor Ac-YVAD-cmk (16,39) prior to the addition of either anti-Fas (Fig. 3, a and b) or staurosporine (Fig. 3, c and d). After induction, the cells were analyzed for apoptosis and cleavage of D4-GDI. Ac-YVAD-cmk inhibited both Fas-induced apoptosis and D4-GDI cleavage at concentrations of 5 M or greater (Fig. 3, a and b), providing evidence that an ICE-related protease(s) was required for both Fasinduced apoptosis and D4-GDI cleavage. Staurosporine-induced apoptosis was significantly less susceptible to inhibition by this inhibitor (Fig. 3c), as was inhibition of staurosporine- induced cleavage of D4-GDI to the 23-kDa fragment (Fig. 3d). The same treatment of Jurkat cells with up to 200 M of a nonspecific control inhibitor, Ac-AAPV-cmk, did not result in significant inhibition of Fas-or staurosporine-induced apoptosis or D4-GDI cleavage (data not shown). These results suggest that the inhibition of both apoptosis and D4-GDI cleavage by Ac-YVAD-cmk is due to the specific inhibition of ICE or ICErelated proteases.
Since D4-GDI appeared to be cleaved at a CPP32-like consensus sequence (DELD 19 S), we tested whether recombinant CPP32 would give appropriate cleavage of purified recombinant D4-GDI as seen in the Jurkat T cells during apoptosis. CPP32, expressed in E. coli as a GST-fusion protein, was autoprocessed to its mature form as monitored by the cleavage of the GST tag from the fusion protein with detection by Western analysis using anti-GST antibody (data not shown). Cleavage of D4-GDI by recombinant CPP32 was assessed by Western analysis using anti-D4-GDI (Fig. 4). Incubation of purified recombinant D4-GDI with the E. coli extract containing CPP32 resulted in the cleavage of mature 28-kDa D4-GDI to a 23-kDa fragment, identical in size to the fragment observed during Fas-induced apoptosis of Jurkat T cells (Fig. 4, compare lanes 1  and 3). Recombinant Mch2 and ICErel-II, two additional ICE/ CED-3 family members, did not cleave D4-GDI under the same conditions, nor did control E. coli extracts expressing only the GST gene (data not shown). To confirm further the specific cleavage of D4-GDI by CPP32, we examined three peptide inhibitors for their ability to inhibit this cleavage. The tetrapeptide aldehyde Ac-DEVD-CHO, a potent inhibitor for CPP32 (26), showed substantial inhibition of D4-GDI cleavage at concentrations as low as 0.1 M (Fig. 4, lanes 4 -6). In contrast, the ICE-specific tetrapeptide inhibitors, Ac-YVAD-CHO and Ac-YVAD-cmk (16,39), were less effective at inhibiting CPP32 activity for D4-GDI cleavage (Fig. 4, lanes 7-14). Together, these results demonstrated that CPP32 is capable of specifically cleaving D4-GDI to produce a 23-kDa fragment that is identical in size to the fragment generated during Fas or staurosporine-induced apoptosis.
A significant difference in the sensitivity to inhibition by Ac-YVAD-cmk on CPP32-mediated D4-GDI cleavage in vitro versus Fas-induced Jurkat cell apoptosis was observed in these experiments. 5 M Ac-YVAD-cmk blocked approximately 90% cell death and D4-GDI cleavage during Fas-induced apoptosis in Jurkat cells (Fig. 3, a and c). In contrast, CPP32-mediated cleavage of D4-GDI in vitro was only minimally inhibited by Ac-YVAD-cmk at a concentration of 10 M (Fig. 4, lane 13). This difference in sensitivity may be attributed to inhibition of an ICE-related enzyme that is required for processing and activation of CPP32 following Fas induction. This possibility is supported by the findings that transgenic deletion of ICE in mice abrogated Fas-mediated apoptosis of thymocytes (24) and that CPP32 can be processed in vitro by ICE (18). In contrast to Fas-induced apoptosis, 50 M or higher concentrations of Ac-YVAD-cmk were required to block staurosporine-induced apoptosis and associated D4-GDI cleavage in Jurkat cells. These concentrations were more in line with in vitro inhibition of CPP32-mediated D4-GDI cleavage (Fig. 3 versus Fig. 4). These results suggest that staurosporine-induced apoptosis proceeds

CPP32-mediated Cleavage of D4-GDI
through a distinct pathway that is independent of Ac-YVADcmk inhibitable protease(s).
In order to confirm the exact CPP32 cleavage site of D4-GDI, the essential Asp residues at the respective P 1 positions (26,39) of both putative ICE-protease cleavage sites were mutated (see Fig. 1). Both wild type and mutant proteins were translated as 35 S-Met-labeled protein in an in vitro transcription/translation system. Recombinant CPP32 was added to the lysates containing either wild type or mutant D4-GDI, and the cleavage of 35 S-Met-labeled D4-GDI was followed using autoradiography (Fig. 5). Both wild-type D4-GDI (Fig. 5, compare lanes 1 and 2) and the D55N mutant form of D4-GDI (Fig. 5, compare lanes 7  and 8) were cleaved by CPP32 to the 23-kDa fragment. In contrast, D19N and D19/D55N mutant proteins were not cleaved by CPP32 (Fig. 5, lanes 3-6). The cleavage site was also determined by N-terminal sequencing of the 23-kDa fragment. This revealed that CPP32 cleaves D4-GDI between Asp 19 and Ser 20 within the DELD 19 S consensus cleavage site (data not shown). Thus, CPP32 specifically cleaves D4-GDI at the DELD 19 S sequence to a 23-kDa fragment consistent with the CPP32-mediated Cleavage of D4-GDI 23-kDa cleavage product observed during apoptosis in Jurkat cells.
D4-GDI was first identified as a hematopoietic cell-specific homolog of Rho-GDI, a negative regulator of Rho family GTPases (33,34). These proteins form a complex with members of the Rho GTPase family (Rho, Rac, and Cdc42) and thereby maintain an inactive, cytosolic form of the GTPase (40). The cellular signals that cause disruption of the complex, thereby resulting in conversion to the active, GTP-bound form of the GTPase through the action of guanine nucleotide exchange factors (41), are not known, although biologically active lipids can display this activity in vitro (40). Here we demonstrate that D4-GDI is specifically cleaved by CPP32 during Fas-induced apoptosis, suggesting a novel means of regulation of such complexes. Cleavage through the action of CPP32 produces an irreversible modification specifically of D4-GDI (versus Rho-GDI), removing the most divergent portion of the protein. Indeed, the initial 20 amino acids of Rho-GDI appear to be critical for its ability to inhibit GTP hydrolysis and effector activity. 4 Deregulation of Rho GTPase function by proteolytic cleavage of D4-GDI in Jurkat cells could have profound effects on cellular activities regulated by these GTPases, including the actin cytoskeleton (31) and activation of the stress-activated mitogenactivated protein kinases, p38, and c-Jun amino-terminal kinase (38,42,43). The latter has been recently reported to be a critical determinant of whether cells undergo an apoptotic response (29). We will direct future studies toward understanding the relevance of D4-GDI cleavage by CPP32 to the dramatic membrane, cytoskeletal, and biochemical changes that accompany the apoptotic process.