(Received for publication, January 19, 1996; and in revised form, February 16, 1996)
From the
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
µM 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
µM. N-terminal sequencing of the 23-kDa D4-GDI fragment
demonstrated that D4-GDI was cleaved between Asp and
Ser
of the poly(ADP-ribose) polymerase-like cleavage
sequence DELD
S. 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 ()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, 19, 20, 21, 22, 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) . (
)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 important roles for Rho GTPases in the signaling and cytoskeletal events accompanying apoptosis.
Figure 3: Dose-dependent inhibition of anti-Fas antibody- and staurosporine-induced apoptosis and D4-GDI cleavage in Jurkat cells by the tetrapeptide inhibitor, Ac-YVAD-cmk. Jurkat cells were preincubated for 3 h with various concentrations of Ac-YVAD-cmk (Bachem) and then stimulated with anti-Fas antibody (CH11) (a) or 1 µM staurosporine (c) for an additional 3 h at 37 °C. Apoptotic cells were measured as described under ``Materials and Methods.'' For the determination of D4-GDI cleavage, cell lysates prepared from anti-Fas antibody- (b) or staurosporine-treated (d) cells were analyzed by Western blotting with D4-GDI antibody as described. The D4 antibody-reactive bands observed were as described in the Fig. 2legend. The data shown are the average of two independent experiments.
Figure 2: Apoptosis and D4-GDI cleavage induced in both anti-Fas antibody- and staurosporine-treated Jurkat cells. Jurkat cells were treated with agonist anti-Fas monoclonal antibody (250 ng/ml) (CH11) (a) or 1 µM staurosporine (c) for the indicated time periods, and apoptotic cells were measured using the TUNEL assay kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. For the determination of D4-GDI cleavage, cell lysates were prepared from the anti-Fas monoclonal antibody-treated cells (b) or the 1 µM staurosporine-treated cells (d) and analyzed by Western blotting with a D4-GDI-specific polyclonal antibody, as described under ``Materials and Methods.'' Molecular masses are indicated in kilodaltons. The arrow on the right designates the 23-kDa cleavage product of D4-GDI after induction of apoptosis. The data shown are the average of two independent experiments.
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
[S]methionine (15 mCi/ml, Amersham). 5 µl of
translated reticulocyte lysate was incubated with 5 µl
CPP32-expressing bacterial extract in 20 µl of reaction buffer at
37 °C for 40 min as described above. The reactions were analyzed by
14% SDS-PAGE, followed by autoradiography.
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
chromatography. ()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
. 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
S and LLGD
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.
Figure 4: Cleavage of recombinant D4-GDI into a 23-kDa fragment identical to that seen in apoptotic Jurkat cells by recombinant CPP32 in vitro. 120 ng of recombinant D4-GDI was incubated with E. coli cell extract expressing CPP32 either in the presence or absence of various concentrations of the tetrapeptide inhibitors, Ac-DEVD-CHO, Ac-YVAD-CHO, and Ac-YVAD-cmk, at 37 °C for 1 h. The reaction mixture was loaded onto a 14% SDS-PAGE gel and analyzed as described with D4-GDI-specific antibody. Results shown are representative of two separate experiments. Lane 2 shows the THP-1 fraction that contained the truncated D4 fragments described under ``Results.''
Figure 1:
Schematic
structure and fragments resulting from proteolytic cleavage by CPP32
and ICE. The consensus cleavage site sequence of both proteases are
indicated, with cleavage occurring after the Asp and
Asp
residues of human D4-GDI,
respectively.
Jurkat T cells,
which highly express D4-GDI(33, 34) , ()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
5`-triphosphate 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
S (Fig. 1). Similarly,
Jurkat cells treated with 1 µM 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 µM or greater (Fig. 3, a and b), providing evidence that an ICE-related protease(s) was required for both Fas-induced 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 µM 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 ICE-related proteases.
Since D4-GDI appeared to be cleaved at a
CPP32-like consensus sequence (DELDS), 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 µM (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 µM 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 µM (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 µM 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. 3versusFig. 4). These results suggest that staurosporine-induced apoptosis proceeds through a distinct pathway that is independent of Ac-YVAD-cmk inhibitable protease(s).
In order
to confirm the exact CPP32 cleavage site of D4-GDI, the essential Asp
residues at the respective P positions (26, 39) of both putative ICE-protease cleavage sites
were mutated (see Fig. 1). Both wild type and mutant proteins
were translated as
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
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
and Ser
within the
DELD
S consensus cleavage site (data not shown). Thus,
CPP32 specifically cleaves D4-GDI at the DELD
S sequence to
a 23-kDa fragment consistent with the 23-kDa cleavage product observed
during apoptosis in Jurkat cells.
Figure 5:
Mutation of D4-GDI at Asp
Asn blocks cleavage by recombinant CPP32. Single or double
mutations of Asp to Asn at amino acids 19 and 55 were introduced into
D4-GDI. [
S]methionine-labeled wild type or
mutant D4-GDI was incubated with recombinant CPP32 for 40 min at 37
°C, as described under ``Materials and Methods.'' The
reactions were analyzed by 14% SDS-PAGE and subjected to
autoradiography. The bands shown on the gels were identical to those
seen in the preceding figures.
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. 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 mitogen-activated
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.