J Biol Chem, Vol. 275, Issue 13, 9314-9323, March 31, 2000
Identification of Caspase 3-mediated Cleavage and Functional
Alteration of Eukaryotic Initiation Factor 2
in Apoptosis*
Wilfred E.
Marissen
,
Yanwen
Guo§,
Adri A. M.
Thomas¶,
Robert L.
Matts§, and
Richard E.
Lloyd
**
From the Department of Microbiology, University of
Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, the
§ Department of Biochemistry and Molecular Biology, Oklahoma
State University, Stillwater, Oklahoma 74078, the ¶ Department of
Developmental Biology, University of Utrecht, 3584 CH Utrecht, The
Netherlands, and the Department of Molecular Virology and
Microbiology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
Induction of apoptosis in a variety of cell types
leads to inhibition of protein synthesis. Recently, the cleavage of
eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was
described as a possible event contributing to translation inhibition.
Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2
, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by
pretreatment of HeLa cells with Z-VAD-fmk. In vitro
analysis using purified eIF2 and purified caspases showed cleavage of
eIF2 by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most
efficiently cleaved eIF2 and this could be inhibited by addition of
Ac-DEVD-CHO in vitro. Comparison of cleavage of
phosphorylated versus nonphosphorylated eIF2 revealed a
modest preference of the caspases for the nonphosphorylated form. When
eIF2·2B complex was used as substrate, only caspase 3 was capable of
eIF2 cleavage, which was not affected by phosphorylation of the subunit. The eIF2·GDP binary complex was cleaved much less
efficiently by caspase 3. Sequence analysis of the cleavage fragment
suggested that the cleavage site is located in the C-terminal portion
of the protein. Analysis showed that after caspase cleavage, exchange
of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B.
Furthermore, in vitro translation experiments indicated that cleavage of eIF2 results in functional alteration of the eIF2
complex, which no longer stimulated upstream AUG selection on a
mRNA containing a viral internal ribosome entry site and was no
longer capable of stimulating overall translation. In conclusion, we
describe here the cleavage of a translation initiation factor, eIF2
that could contribute to inhibition or alteration of protein synthesis
during the late stages of apoptosis.
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INTRODUCTION |
Initiation of protein synthesis in mammalian cells is a highly
regulated process that requires multiple translation initiation factors. The eukaryotic translation initiation factor 2 (eIF2)1 plays a key role in
the initiation of translation. eIF2 is a heterotrimeric protein
consisting of three subunits , 
, and 
, which forms a so-called
ternary complex with GTP and the initiator Met-tRNA. The ternary
complex interacts with the 40 S ribosomal subunit thereby forming a 43 S preinitiation complex that is capable of recognizing the proper start
codon of the mRNA. It has been shown in yeast that eIF2 itself is
involved in this process of start codon selection (1) indicating its
importance in translation initiation. Upon joining of the 60 S
ribosomal subunit to the 43 S preinitiation complex, the GTP moiety is
hydrolyzed and an eIF2·GDP complex is released from the ribosome (2).
Participation of the eIF2·GDP complex in a new round of translation
requires the exchange of the GDP moiety for a new GTP molecule, a
process carried out by the guanine nucleotide exchange factor, eIF2B. The global rate of protein synthesis in eukaryotes is mainly regulated by the specific phosphorylation of Ser-51 of the eIF2 subunit. eIF2 is known to be phosphorylated by kinases such as PKR
(double-stranded RNA dependent eIF2 kinase) in response to viral
infection, and stress conditions (3), HRI (heme-regulated inhibitor) in
response to hemin availability and a host of environmental stress
conditions (4-6) and GCN2 from the yeast Saccharomyces
cerevisiae in response to amino acid starvation (7).
Phosphorylated eIF2 binds eIF2B tightly, forming a poorly dissociable
eIF2(P)·eIF2B complex. Since eIF2B exists in relatively low molar
quantities with respect to eIF2 in the cytoplasm in many systems,
phosphorylation of as little as 25% of eIF2 can be sufficient to
sequester virtually all the available eIF2B, thereby blocking the eIF2B
catalyzed recycling of the eIF2·GDP and subsequently inhibiting
protein synthesis completely (8).
It has been well established that protein synthesis is inhibited during
apoptosis or programmed cell death but the mechanism of translation
inhibition has remained unclear until recently. We and others have
recently shown that eukaryotic translation initiation factor 4GI
(eIF4GI), which is required for binding of capped mRNA to ribosomal
43 S subunits, was cleaved by caspase 3 during apoptosis (9, 10).
Similar cleavage of eIF4GI is one of the major causes of translation
inhibition during enterovirus infection of cells (11). Likewise, the
cleavage of eIF4GI correlates with the observed inhibition of protein
synthesis in apoptotic cells (9). Other reports indicate that
phosphorylation of eIF2 could represent another mechanism by which
translation is inhibited during apoptosis. For instance, overexpression
of PKR in transfected cells resulted in elevated levels of
phosphorylated eIF2 concomitant with reduced protein synthesis and
induction of apoptosis in these cells (12). In addition, it has been
reported that PKR is a tumor suppressor protein as it was shown that
overexpression of mutant PKR incapable of phosphorylating eIF2
resulted in tumor formation (13, 14). Similarly, others have described
that lowering the level of the eIF2 associated protein, p67, resulted in increased levels of phosphorylated eIF2 concomitant with
induction of apoptosis in these cells (15). In both cases, the decrease in the overall rate of protein synthesis was ascribed to the
phosphorylation of eIF2.
Here, we report the cleavage of eIF2 by caspase 3 during the late
stages of the execution phase of apoptosis in HeLa cells, K562 cells,
and Jurkat T cells. The cleavage of the 38-kDa subunit resulted
into a 36-kDa cleavage product as observed by both Coomassie stain and
immunoblot analysis. In vitro experiments using purified eIF2 and purified caspases showed that eIF2 could be a substrate for
caspases 3, 6, 9, and 10 but not caspase 8. Although cleavage by
caspase 3 appeared to be most efficient we do not rule out the
possibility that other caspases might be responsible for eIF2 cleavage in vivo. In vitro experiments using both
nonphosphorylated and phosphorylated eIF2 as a substrate showed that
all caspases had a preference for the nonphosphorylated form of
eIF2. Interestingly, when eIF2·2B complex was used as substrate no
preference was observed between nonphosphorylated and phosphorylated
eIF2. The cleavage site appears to be located in the C-terminal
region of the protein as determined by N-terminal amino acid sequencing
of the 36-kDa cleavage product. Close examination of this region
revealed two putative caspase 3 cleavage sites which would lead to the
release of a 14- or 11-amino acid peptide. Interestingly, results
indicate that cleavage of eIF2 does inactivate the eIF2 complex as
determined by in vitro translation assays.
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EXPERIMENTAL PROCEDURES |
Cells--
HeLa S3 cells were grown as described (9). K562 cells
were grown at 37 °C in RPMI (Irvine Scientific) supplemented with 10% bovine calf serum, 0.5% fetal calf serum (Summit Biotechnology), 100 units of penicillin, and 100 µg of streptomycin/ml (Sigma) in a
humidified chamber containing 5% CO2. For induction of
apoptosis, stock solutions of cisplatin, etoposide, cycloheximide
(Sigma), or TNF- (Peprotech) were diluted with medium and then
incubated with cells at 37 °C for the time indicated in each figure.
Preparation of Cell Lysates and Immunoblot Analysis of
Fractions--
Cell extracts were prepared as described (9). Briefly,
HeLa cells were washed with phosphate-buffered saline, resuspended in
CHAPS lysis buffer (20 mM Tris pH 7.2, 0.1 M
NaCl, 1 mM EDTA, 10 mM dithiothreitol, 0.5%
CHAPS, 10% sucrose) and incubated on ice for 30 min. Cell lysates were
then centrifuged for 10 min at 10,000 × g at 4 °C,
supernatants were collected and stored at 
80 °C. Cell pellets were
resuspended in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) loading sample buffer, boiled for 10 min,
and subjected to SDS-PAGE. Detection of poly(ADP-ribose) polymerase
(PARP) cleavage was accomplished by immunoblotting with a mouse
monoclonal anti-PARP antibody (Zymed Laboratories Inc.). Detection of eIF4GI cleavage was performed by immunoblot as described previously (9). Ribosomal salt wash fractions from HeLa
cells were prepared by standard methods.
Metabolic Labeling of Proteins--
Proteins were labeled with
[35S]methionine (ICN) as described (9) to determine
translation levels in the cells.
Purification of eIF2, eIF2·2B, and HRI--
The eIF2 (0.5 µg/µl) (16), eIF2·2B (5.0 µg/µl) (17), and HRI (0.5 µg/µl) (18) used in the caspase cleavage studies were purified from
reticulocyte lysate (Green Hectares, Oregon, WI) as described
previously. The eIF2 used for the guanine nucleotide exchange studies
was purified from rabbit reticulocyte post-ribosomal supernatant as
described (19).
Expression and Purification of Caspases--
The full-length
cDNAs encoding human caspase 3, 6, 9, and the cDNAs encoding
caspase 8 (Ser-217 to Asp-479) and caspase 10 (Ile-162 to Ile-479)
cloned into pET15b (caspase 8), pET21b (caspases 6, 9, and 10), and
pET23b (caspase 3) (all Novagen) were expressed in Escherichia
coli BL21(DE3)pLysS. Caspases 3 and 8 were a kind gift of Dr.
Claudius Vincenz; caspases 6, 9, and 10 were a kind gift of Dr. Emad
Alnemri. The expressed proteins were purified by affinity
chromatography on TALON metal affinity resin
(CLONTECH) according to the manufacturer's
instructions. Working concentrations of active caspases were
standardized based on in vitro colorimetric cleavage assays
using appropriate substrate peptides coupled to para-nitroanilide (pNA) (9). Caspase activity units are
defined as the amount of caspase required to hydrolyze 1 nmol of pNA
substrate/min. Various caspases were used at concentrations ranging
from 10 to 60 µg/ml as determined appropriate by these assays.
eIF2 Cleavage Assays--
Purified rabbit eIF2 (1 µl) was
incubated at 37 °C with purified recombinant caspase 3 (1 µl) in a
total volume of 10 µl for the time points indicated in each figure.
Samples were then analyzed by SDS-PAGE and immunoblotting using a mouse
monoclonal antiserum specific for eIF2 (20). Caspase 3-treated eIF2
for in vitro translation assays was prepared similarly as
described above with the following modification: after incubation at
37 °C for 8 h, caspase 3 was inactivated by the addition of the
caspase inhibitor Ac-DEVD-CHO (10 µM) (Quality Controlled
Biochemicals) for 1 h at 37 °C and subsequently stored at
80 °C.
Phosphorylation of eIF2--
Purified rabbit eIF2 (10 µl) or
eIF2·2B (1 µl) was incubated for 1 h at 37 °C with HRI (2 µl) in buffer containing 10 mM Tris (pH 7.4), 50 mM NaCl, 2 mM MgCl2, 0.1 mM ATP (final volume 30 µl). Three microliters of
phosphorylated eIF2 or eIF2·2B was then used in cleavage assays as
described above in a final reaction volume of 30 µl.
GDP Binding and Guanine Nucleotide Exchange Assay--
To
saturate eIF2 with GDP for cleavage studies, eIF2 (1 µl) was
incubated for 10 min at 37 °C in the presence of 30 µM
GDP. To "freeze" the GDP on the eIF2 and stabilize the conformation of eIF2, MgCl2 was added to a final concentration of 1 mM. For each 20-µl aliquot of guanine nucleotide exchange
assay mixture, ~1.5 pmol of eIF2 was incubated in the presence or
absence of 18 units of caspase 3 (Upstate Biotechnology) in buffer
containing 20 mM Tris·HCl (pH 7.4), 100 mM
NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 0.1%
CHAPS, and 10% polyethylene glycol for 3 h at 37 °C.
[3H]GDP (5 µM) was then added and after 10 min of additional incubation, 3 mM Mg(OAc)2 was
added to stabilize the eIF2·lsqb]3H]GDP complex. After
a further 5-min incubation, 100 µM unlabeled GDP was
added and the amount of eIF2·[3H]GDP complex remaining
was determined after various times of incubation by filtration of a
20-µl aliquot of each assay mixture through nitrocellulose filters as
described previously (17).
Isoelectric Focusing--
To determine the phosphorylation
status of eIF2 in vivo, 5 × 106 cells
from apoptotic or control cultures were taken and resuspended in 200 µl of VSIEF buffer (9.5 M urea, 5% CHAPS, 50 mM sodium fluoride, 5% -mercaptoethanol). Samples were
then clarified and subjected to isoelectric focusing on vertical
isoelectric focusing gels by a slight modification of the method of
Maurides et al. (21) as described previously (22).
Plasmid Constructs--
T7TS plasmid (gift of P. A. Krieg)
contains a multiple cloning site upstream of the Xenopus
-globin 3'-UTR and a track of 30 adenosyl residues which allows
generation of poly(A) mRNAs. The region encoding the
5'-untranslated region (5'-UTR) of foot and mouth disease virus (FMDV)
coupled to the reporter gene chloramphenicol acetyltransferase (CAT) as
described (23) was excised from FMDV-CAT with EcoRV and
BamHI and subsequently cloned into blunt-ended HindIII-BglII-digested pT7TS plasmid to generate
pT7TSFMDVCAT. pT7TSglobinCAT was generated by insertion of a
HindIII-SmaI globin-CAT fragment into a
HindIII-EcoRV-digested pT7TS plasmid. Plasmid DNAs were linearized with XbaI, purified by
phenol/chloroform extraction, and concentrated by alcohol precipitation
for use in transcription reactions (see below).
Transcription and Translation--
Transcription reactions for
FMDV-CAT using T7 RNA polymerase (Promega) were carried out according
to manufacturer's recommendations. For capped globin-CAT,
transcription was performed using the mMessage mMachine T7 kit from
Ambion. In both cases, the resulting RNAs were purified using spin
columns (Eppendorf-5 Prime). Translation reactions were performed in
rabbit reticulocyte lysate (Promega) according to the manufacturer's
recommendations in a 20-µl reaction volume. Addition of buffer
control, caspase inhibitor Ac-DEVD-CHO, eIF2, caspase 3-treated eIF2
plus Ac-DEVD-CHO were added (2 µl) to the translation reaction prior
to the addition of [35S]methionine (ICN), and incubated
for 10 min at 30 °C. After addition of the label, reactions were
incubated for another 90 min at 30 °C. Samples (2 µl) were
subjected to SDS-PAGE and analyzed by autoradiography. In addition,
translation products were quantified by densitometry (Storm 860 PhosphorImager) or scanning autoradiographs and densitometry using NIH
Image. Statistical analyses were performed using Excel.
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RESULTS |
Cleavage of eIF2 during Apoptosis--
In order to identify
possible target proteins that could contribute to translation
inhibition during cell death, apoptosis was induced in multiple cell
lines using a variety of apoptosis-inducing agents. We started with a
system in which we have previously extensively characterized cleavage
of eIF4GI (9) to determine if other factors are also modified. Thus,
HeLa cells were treated with 100 µM cisplatin and
aliquots were harvested over a 24-h time period as indicated (Fig.
1A). Fig. 1A shows
the eIF2 protein at 0 h with a molecular mass of approximately
38 kDa, at which time cisplatin (100 µM) was added to the
HeLa cell culture. Subsequent lanes show that cisplatin treatment of
HeLa cells led to cleavage of eIF2 starting between 6 and 7 h
post-induction, neared completion at 24 h and generated a cleavage
fragment of about 36 kDa in size. The cleavage of eIF2 appears to
initiate later than cleavage of the well defined caspase 3 substrate
PARP as shown in Fig. 1B and eIF4GI as shown in Fig.
1C. Generation of the characteristic 85-kDa caspase
3-derived PARP cleavage product and eIF4GI cleavage products can be
detected as early as 4 h post-induction. We noted that the eIF2
cleavage fragment is barely detectable at 24 h post-induction. Two
factors are likely to contribute to the poor detection of the eIF2
cleavage product: (i) we have noted that the anti-eIF2 monoclonal
antibody has a lower affinity for the cleavage fragment of eIF2 as
compared with intact eIF2; and (ii) the stability of the cleaved
eIF2 subunit appears to be lower than the intact protein in
vivo. In addition, treatment of HeLa cells with etoposide,
cycloheximide, or tumor necrosis factor- (TNF-), K562 cells with
cisplatin, etoposide, cycloheximide, or MG132, or Jurkat T cells with
cisplatin, etoposide, cycloheximide, MG123, or TNF- all resulted in
the generation of a similar 36-kDa cleavage product (data not shown),
indicating that this type of response can be generated in a wide
variety of cell types using different inducers of apoptosis.
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Fig. 1.
Cleavage of eIF2 in
cisplatin-treated HeLa cells. A, HeLa cells were
treated with 100 µM cisplatin and incubated for the
indicated time periods. Cells were lysed and proteins were separated by
SDS-PAGE and immunoblotted with a monoclonal antiserum specific for
eIF2. Migration of molecular weight markers, and eIF2 and eIF2
cleavage products are indicated on the left and
right, respectively. B, cell pellets from
cisplatin-treated HeLa cells derived from the experiment described in
panel A were resuspended in SDS-PAGE sample buffer, and
analyzed by immunoblot using a monoclonal anti-PARP antibody. PARP and
cleaved PARP (arrow) are indicated on the right.
Migration of molecular weight markers are indicated on the
left. C, proteins in lysates were separated by
SDS-PAGE and immunoblotted with antiserum specific for the N-terminal
domain of eIF4GI. Migration of eIF4GI and its N-terminal cleavage
products (eIF4GIcpN) are indicated on the right.
D, after treatment of the cells with 100 µM
cisplatin for the indicated time periods, cells were labeled with
[35S]methionine for 30 min at 37 °C. The cells were
then lysed, and analyzed by SDS-PAGE and autoradiography.
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The loss of approximately 2 kDa in molecular mass implies that only a
small portion of either the N or C terminus of eIF2 is removed,
which raises the question whether this cleavage event results in
inactivation of the eIF2 protein complex and could contribute to
inhibition of protein synthesis or may modulate eIF2 activity in
another way. Therefore, we further examined whether the cleavage of
eIF2 correlated with translation inhibition in apoptotic cells.
Cisplatin-treated HeLa cells were pulse labeled at the time points
indicated for 30 min, subsequently lysed and analyzed by SDS-PAGE. Fig.
1D shows that between 3 and 5 h post-induction the
incorporation of [35S]methionine into protein decreased
significantly, followed by an abrupt further decline to near zero
between 6 and 7 h. Much of this translation decrease is likely due
to rapid cleavage of eIF4GI or other factors, however, the latter
abrupt decline may correlate with the appearance of eIF2 cleavage products.
Cisplatin treatment of HeLa cells produces apoptosis, eIF4GI cleavage,
and translation shutoff faster than most other inducer/cell combinations we have tested. Thus, in order to reveal possible correlations between eIF2 cleavage and translation shutoff, we explored other apoptotic systems in which eIF4GI cleavage was not so
rapid. Fig. 2 summarizes data taken from
HeLa cells or K562 erythroblastoid cells treated with etoposide and
compares those to data taken from cisplatin-treated HeLa cells.
Etoposide treatment of HeLa or K562 cells resulted in slower induction
of apoptosis and eIF4GI cleavage, yet the kinetics of cleavage of eIF2 were nearly equivalent. In both cases, translation rates declined as cleavage of eIF2 proceeded. In particular, 80% of eIF4GI was still intact in K562 cells after 16 h treatment, yet translation rates had declined to 50%. Since studies with
poliovirus-infected cells have shown that translation rates can still
proceed at near 100% efficiency when up to 80-90% of eIF4GI has been
cleaved (24), this implies that additional events are occurring in
apoptosis to inhibit translation. At this same 16-h time point, eIF2
was 32% cleaved, suggesting a possible contribution to the translation inhibition phenotype. Interestingly, by 24 h, translation was still occurring in etoposide-treated cells even though 70-80% of
eIF2 had been processed. This may reflect the observation (see Fig.
9 below) that eIF2 complex containing cleaved eIF2 rapidly exchanges
GDP, unlike its unprocessed precursor. Despite testing several
combinations of cell types and apoptosis inducers, we have not
discovered conditions where eIF2 cleavage precedes or occurs
independently of eIF4GI cleavage. In summary, these data reveal that
cleavage of eIF4GI and eIF2 often occur simultaneously, cleavage
rates are variable depending upon the apoptosis inducer and cell type
used, and that the mechanism of translation inhibition may be
multifactorial, involving simultaneous modification of more than one
translation factor.
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Fig. 2.
Cleavage of eIF2 in
etoposide-treated HeLa and K562 cells. HeLa or K562 cells were
treated with 50 µM etoposide and incubated for the
indicated time periods followed by a 30-min pulse label using
[35S]methionine at 37 °C. A, to determine
protein synthesis levels, cell lysates were analyzed by autoradiography
and actin protein bands were quantitated for each time point by
densitometry. The amount of protein synthesis was plotted as percentage
of control levels. B, the amount of eIF4GI cleavage was
determined by quantification of intact eIF4GI protein from immunoblots
for each time point, with time 0 set at 100%. C, the amount
of eIF2 cleavage was assessed by quantification of intact eIF2
protein on immunoblots for each time point, with time point 0 set as
100%. Quantification of protein bands was performed using NIH image
software. Triangle, cisplatin-treated HeLa cells (as
described in Fig. 1); square, etoposide-treated HeLa cells;
circle, etoposide-treated K562 cells.
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Inhibition of eIF2 Cleavage by Z-VAD-fmk--
To explore
whether caspase activation is required for the cleavage of eIF2, we
performed in vivo inhibition experiments using the broad
spectrum cell-permeable caspase inhibitor Z-VAD-fmk. Cell cultures were
preincubated with 75 µM Z-VAD-fmk, followed by treatment
with apoptosis inducers for 16 h. Analysis of cell lysates by
immunoblot (Fig. 3A) show that
eIF2 cleavage was completely inhibited in HeLa cells pretreated with
Z-VAD-fmk regardless of the inducing agent used. The observed
inhibition of cleavage was concomitant with a reduction in overall
caspase activation as measured by the decrease in release of pNA from
the caspase substrate Ac-DEVD-pNA (Fig. 3B). The DEVD-pNA
cleavage activity in cell lysates pretreated with Z-VAD-fmk was reduced
by more than 70% for each inducer tested. These results suggest that
the cleavage of eIF2 is caspase-dependent and indicate
that caspases could be directly involved in the induction or catalysis
of eIF2 cleavage.
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Fig. 3.
In vivo inhibition of
eIF2 cleavage by Z-VAD-fmk. A,
inhibition of eIF2 cleavage in HeLa cells. HeLa cells were
preincubated with 75 µM Z-VAD-fmk (indicated by +) for
1 h at 37 °C before addition of cisplatin (100 µM), etoposide (50 µM), TNF- (40 ng/ml)
plus cycloheximide (10 µg/ml). Cells were treated for 15 h,
lysed, and analyzed by immunoblot as described for Fig. 1. Migration of
molecular weight marker protein, and eIF2 and eIF2 cleavage
products are indicated on the left and right,
respectively. B, in vivo inhibition of caspase
activity. Cell lysates derived from the experiment described in
panel A were incubated in the presence of 0.2 mM
Ac-DEVD-pNA for 2 h at 37 °C. Release of pNA was analyzed by
optical density at 405 nm, and caspase activity is displayed as
nanomoles of pNA released per hour per total milligram of protein.
C, control; CP, cisplatin; Etop,
etoposide.
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In Vitro Cleavage of eIF2 by Caspases--
To identify caspases
that could be responsible for the observed eIF2 cleavage in
vivo, we screened a panel of purified recombinant human caspases
for their ability to cleave eIF2 in vitro. All purified
caspases were pre-standardized for equivalent levels of enzymatic
activity before incubation with protein substrates. Fig.
4A shows cleavage of eIF2
occurred when HeLa cell ribosomal salt wash (crude initiation factor
preparation containing eIF2) was incubated with caspase 3 and 6, and to
a lesser extent with caspase 8 and 10, but not with caspase 9. Comparison of these results with in vivo studies shown in
Figs. 1 and 3 reveals that the caspases generate an identical eIF2
cleavage fragment, further supporting our hypothesis that caspases are
indeed responsible for eIF2 cleavage. In this experiment caspases 3, 6, or 10 could indirectly induce eIF2 cleavage via activation of
other proteases present in the HeLa ribosomal salt wash, which
subsequently could use eIF2 as a substrate. To address this
question, we incubated highly purified rabbit eIF2 with purified
caspases to see if the observed cleavage in Fig. 4A could be
generated directly by caspases. Purified eIF2 was cleaved by caspase
3 (Fig. 4B) whereas incubation with caspase 6, 8, and 10 resulted in only minor or undetectable cleavage of eIF2. These
results might indicate that cleavage of eIF2 by caspases 6 and 10 in
panel A could have been enhanced by activation of other
caspases (e.g. caspase 3) in the lysate. Levels of cleavage
obtained with caspase 3 over several experiments were significantly
greater than the low, somewhat variable cleavage obtained with other
caspases, implying that eIF2 is a better substrate for caspase 3 than caspases 6 and 10. However, it is possible that purified eIF2
might not be an appropriate substrate for caspases 6 and 10, which
instead may function as a more efficient substrate when it is bound
within a higher order translation complex or require other unknown
cofactors for efficient cleavage of eIF2. Furthermore, in this
initial experiment, low amounts (2 units) of caspase units were used to
cleave eIF2, which also may explain the low level of eIF2
cleavage. However, further experiments, in which higher levels (17 units) of caspases were used in cleavage assays, indicated that the
amount of cleavage increased greatly only with caspase 3 but not with
the other caspases tested (see Fig. 7 below). Overall, these results
indicate that eIF2 can serve as a substrate for caspases and is
cleaved by the effector caspase 3 more efficiently than initiator
caspases (caspases 8, 9, and 10).
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Fig. 4.
In vitro cleavage of
eIF2 by purified caspases. Affinity
purified His-tagged caspases (2 units) were incubated with either HeLa
cell ribosomal salt wash fraction (A) or purified eIF2
(B) for 6 h at 37 °C. Samples were analyzed on a
10% acrylamide gel followed by immunoblot for eIF2. Lane
C indicates control; numbers on top indicate
the different caspases used. Molecular weight markers, and eIF2 and
eIF2 cleavage products are indicated on the left and
right, respectively.
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Direct Cleavage of eIF2 by Caspase 3--
Based on the results
from Fig. 4 we further explored the caspase 3-induced cleavage of
eIF2 in vitro. First, we performed a time course to
analyze the kinetics of the eIF2 cleavage reaction. Therefore,
purified caspase 3 was incubated with purified eIF2 at 37 °C,
aliquots were taken at the indicated time points, and analyzed by
immunoblot (Fig. 5A).
Significant cleavage of eIF2 occurred within the first hour followed
by complete cleavage of eIF2 between 5 and 18 h of incubation.
To rule out the possibility that other contaminant proteases in the
purified preparations could be responsible for the eIF2 cleavage,
purified eIF2 and purified caspase 3 were incubated in the presence
of specific caspase 3 inhibitor Ac-DEVD-CHO (Fig. 5B).
Increasing concentrations of the inhibitor coincided with a decrease in
the amount of eIF2 cleavage product and as little as 1 µM of the inhibitor was sufficient to completely block
the cleavage of eIF2. Overall, these results indicate that caspase 3 is responsible for the observed cleavage of eIF2.
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Fig. 5.
Specific cleavage of eIF2
by caspase 3. A, kinetics of eIF2 cleavage by
caspase 3. Purified eIF2 and purified caspase 3 (17 units) were
incubated for the indicated time periods at 37 °C. Samples were
analyzed as described in the legend to Fig. 4. Molecular weight
markers, and eIF2 and eIF2 cleavage products are indicated on the
left and right, respectively. B,
in vitro inhibition of eIF2 cleavage. Purified eIF2 and
purified caspase 3 (17 units) were incubated in the presence of
increasing amounts of inhibitor Ac-DEVD-CHO, as indicated on the
top, for 6 h at 37 °C. Samples were analyzed as
described in the legend to Fig. 4. Molecular weight markers, and
eIF2 and eIF2 cleavage product are indicated on the
left and right, respectively.
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Characterization of the Caspase 3 Cleavage Site on
eIF2--
Next we wished to determine the cleavage site on eIF2
utilized by caspase 3. Therefore, we performed an in vitro
cleavage assay as described for Fig. 5A, transferred the
proteins to polyvinylidene difluoride, and subjected the 36-kDa
fragment to amino acid sequencing. The sequencing results showed that
the N-terminal sequence of the 36-kDa fragment matched 9 of 10 amino
acids in the authentic N-terminal amino acid sequence of intact eIF2
(Fig. 6A), strongly suggesting
that the caspase 3 cleavage site is located in the C-terminal portion
of the eIF2 protein. Close examination of the amino acid sequence in
the C-terminal portion of the protein revealed the presence of two
potential caspase 3 cleavage motifs similar to the preferred sequences
DEVD or DXXD that could be targeted for proteolysis (Fig.
6B). Although cleavage at either site would lead to a loss
in size of approximately 2 kDa consistent with our findings using
in vivo (e.g. Fig. 1) or in vitro
(e.g. Fig. 4) experiments, we have not yet been able to
confirm which site is actually recognized by caspase 3.
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Fig. 6.
Amino acid sequencing of the
eIF2 cleavage product. A,
comparison of eIF2 amino acid sequences (residues 1-10) and
sequences derived from microsequencing of the eIF2cp.
Vertical bars indicate identical residues. Microsequencing
was performed at the Protein Chemistry Core Facility, Baylor College of
Medicine. B, proposed caspase 3 cleavage sites on eIF2.
The C-terminal sequence of the eIF2 protein is shown. Classic
caspase 3 cleavage site (DXXD) and an alternate site are
indicated in bold; arrows indicate putative
cleavage sites.
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Cleavage of Phosphorylated versus Non-phosphorylated
eIF2--
The biological function of eIF2 is tightly regulated by
the phosphorylation of the eIF2 subunit. Therefore, we determined whether the phosphorylation status of eIF2 influences the ability of
caspases to cleave eIF2. Purified caspases were incubated with
either nonphosphorylated or phosphorylated eIF2 (eIF2(P)) for
18 h and samples were analyzed by immunoblotting (Fig.
7A). Phosphorylation of the
subunit modestly affected its cleavage, most easily observed with
caspase 3 but also faintly visible with caspases 6, 8, and 10 which
demonstrated weak cleavage activity in this assay. The ability of
caspase 3 to cleave phosphorylated eIF2 was confirmed by
immunoblotting of samples separated on slab gels by vertical
isoelectric focusing (see Fig. 8
below).
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Fig. 7.
Comparison of cleavage of nonphosphorylated
versus phosphorylated eIF2 by
caspases. Purified caspases (17 units) were incubated with either
purified eIF2 (A) or purified eIF2/2B (B) for
18 h at 37 °C. Both eIF2 and eIF2/2B were phosphorylated by
preincubation with HRI as described under "Experimental Procedures"
and are indicated by P on the top of the figure.
Samples were analyzed as described in the legend to Fig. 4. Molecular
weight markers, and eIF2 and eIF2 cleavage products are indicated
on the left and right, respectively.
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Fig. 8.
Effect of phosphorylation and GDP on the
cleavage of eIF2 in the eIF2 versus
the eIF2·2B complex. Unphosphorylated purified eIF2
(A), partially phosphorylated purified eIF2 (B),
or eIF2·2B (C) complex were incubated in the presence (+)
or absence () of 50 µM GDP for 10 min at 37 °C,
followed by a further incubation for 5 min after the addition of 3 mM Mg(OAc)2. Samples, treated (+) or untreated
() with caspase 3 (18 units) were then incubated for 3 h at
37 °C under the conditions described for cleavage of eIF2 for
guanine nucleotide exchange assays under "Experimental Procedures."
Samples were then separated by SDS-PAGE (A) or on slab gels
by vertical isoelectric focusing (B and C) and
eIF2 was detected by immunoblotting. Migration of unphosphorylated
eIF2 (eIF2), phosphorylated eIF2 (eIF2(P)),
unphosphorylated eIF2 cleavage product (eIF2cp), and
phosphorylated eIF2 cleavage product (eIF2(P)cp) are
indicated on the right. A second phosphorylated form of
eIF2 that has previously been reported to exist by Maurides et
al. (21) is also visible in part B of the figure.
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A portion of the available eIF2 present in the cell is complexed with
eIF2B, the guanine nucleotide exchange factor, which is required to
recycle the eIF2·GDP complex generated during translation initiation.
We were interested in whether caspases could cleave the subunit in
the context of an eIF2·2B complex, and if so, would this cleavage be
affected by phosphorylation of the eIF2. As shown in Fig.
7B, only caspase 3 was able to cleave eIF2 in the context
of an eIF2·2B complex whereas none of the other caspases displayed
measurable cleavage activity using this substrate. In addition, the
cleavage of eIF2 present in the eIF2·2B complex was not affected
by its phosphorylation (also see Fig. 8C, below).
Cleavage of the eIF2·GDP Complex by Caspase 3--
eIF2 exists
in vivo in at least two alternative conformational states
(17, 25). When the 60 S ribosomal subunit joins the 48 S initiation
complex during the last step of translation initiation, the GTP bound
to the eIF2·GTP·Met-tRNAi ternary complex is hydrolyzed
and eIF2 is released as a complex with GDP. In the presence of
physiological Mg2+ concentrations, the conformation of the
eIF2·GDP complex is "locked" in an inactive state from which the
bound GDP cannot dissociate at a physiologically relevant rate. The
subsequent exchange of GTP for the bound GDP obligatorily requires the
interaction of the eIF2·GDP complex with eIF2B (17, 25). In its
GTP-bound state, eIF2 is in its "active" conformation, which can
subsequently form a ternary complex with Met-tRNAi. In the
absence of Mg2+ in vitro, eIF2 can assume the
conformation from which bound GDP freely disassociates in the absence
of eIF2B, indicating that Mg2+ is required to freeze the
conformation of the eIF2·GDP complex.
To determine whether the conformational state of eIF2 affects the
ability of eIF2 to serve as a substrate for caspase 3, we incubated
purified eIF2 with GDP for 10 min at 30 °C followed by addition of
magnesium to "lock" the eIF2·GDP in its inactive conformation.
Surprisingly, when this substrate was tested, cleavage of eIF2 by
caspase 3 was found to be severely inhibited compared with cleavage of
eIF2 (Fig. 8A). This observation could not be ascribed to a
possible inhibitory effect of magnesium on caspase 3 activity since we
found no such effect on Ac-DEVD-pNA cleavage activity by caspase 3 under similar conditions with the same concentration of magnesium (data
not shown). Also, addition of magnesium to eIF2 and eIF2/2B had no
significant effect on caspase activity (Fig. 8, panels B and
C). Thus, these data support the existence of the previously
noted conformational shift in eIF2 caused by the GDP moiety and
suggests that the cleavage site for caspase 3 on eIF2 is either
masked or blocked in this conformation. In light of this result, the
modest inhibition of eIF2 cleavage observed upon eIF2
phosphorylation in Fig. 7A is most likely due to the fact
that GDP remains bound to a small fraction of purified eIF2 (26). As
such, the Mg2+ added to the kinase reaction would stabilize
the binding of this GDP to eIF2 and inhibit its cleavage by caspases.
To determine whether the phosphorylation state of the -subunit of
eIF2 affects the ability of caspase 3 to cleave eIF2 containing bound
GDP, purified fractions of eIF2 or eIF2·2B complex whose -subunit
was 40-50% phosphorylated were preincubated in the presence or
absence of GDP (Fig. 8, B and C). The ability of
caspase 3 to cleave eIF2 was then examined after addition of
Mg2+. Samples were separated on slab gels by vertical
isoelectric focusing to separate full-length and cleaved fragments of
unphosphorylated and phosphorylated eIF2. Immunoblot analysis
indicated that the presence of eIF2-bound GDP markedly inhibited the
cleavage of both phosphorylated and unphosphorylated eIF2 by caspase
3. In contrast, the presence of GDP only marginally inhibited the
cleavage of phosphorylated or unphosphorylated eIF2 present in the
eIF2·2B complex.
Effect of Caspase 3-induced Cleavage of eIF2 on eIF2
Activity--
To initially address the effect of eIF2 cleavage on
the function of eIF2, we determined the effect of caspase 3 on the
ability of eIF2 to bind and exchange GDP. eIF2 was incubated in the
presence or absence of caspase 3 for 3 h at 37 °C. Samples were
then incubated with [3H]GDP for 10 min followed by the
addition of 1 mM Mg2+. Binding of an aliquot of
each sample to nitrocellulose filters indicated that cleavage of
eIF2 by caspase 3 had no effect on the ability of eIF2 to bind GDP
(data not shown). Excess unlabeled GDP was then added to the samples
and the extent of guanine nucleotide exchange was measured at the times
indicated in Fig. 9. SDS-PAGE and
immunoblot analysis of aliquots of each sample taken at the end of the
incubation verified that the eIF2 had been quantitatively cleaved by
caspase 3 (data not shown). As previously reported, bound
[3H]GDP was chased from eIF2 at slow rate. In contrast,
after cleavage of eIF2 by caspase 3 over 90% of the eIF2-bound
[3H]GDP was chased in 15 min. Thus, exchange of eIF2
bound GDP was no longer dependent upon eIF2B after cleavage of eIF2
by caspase 3.
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Fig. 9.
Effect of cleavage on the exchange of
eIF2-bound [3H]GDP. Assay mixtures containing
purified eIF2 were incubated in the presence (open squares)
or absence (open circles) of caspase 3 for 3 h at
37 °C under the conditions described for cleavage of eIF2 for
guanine nucleotide exchange assays under "Experimental Procedures."
Cleaved and uncleaved eIF2 were bound to [3H]GDP, and
eIF2·[3H]GDP complexes were stabilized by the addition
of Mg(OAc)2. The amount of [3H]GDP bound to
cleaved and uncleaved eIF2 was determined by binding to nitrocellulose
filters (0 min), followed by the addition of excess unlabeled GDP. The
% [3H]GDP chased from eIF2·[3H]GDP
complexes was determined at 7.5 and 15 min by binding of the complex to
nitrocellulose filters as described under "Experimental
Procedures."
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To further address the effect of eIF2 cleavage on the activity of
the eIF2 complex we took advantage of a well defined characteristic of
the 5'-UTR of FMDV RNA. FMDV 5'-UTR initiates translation via a
cap-independent mechanism mediated by a large RNA structure called the
internal ribosome entry site (IRES). The 3' margin of this structure
possesses two initiation codons in tandem which lead to initiation of
two forms of the leader protease of the virus (27). The selection of
the initiation codon in mRNAs containing the FMDV 5'-UTR has been
shown to be influenced by the activity of eIF2 and the amount of
ternary complex eIF2·Met-tRNA·GTP (23, 28) in a mechanism that may
involve direct binding of eIF2 to the viral IRES RNA element (29). In
previous studies, it was shown that the addition of eIF2 to in
vitro translation lysates programmed with reporter mRNAs
containing the FMDV 5'-UTR resulted in an increase in initiation from
the upstream AUG (23).
Here, we used the same system to test whether caspase-cleaved eIF2
could still function in modulating initiation site selection. We used
mRNA transcripts containing the CAT gene downstream of either the
Xenopus globin 5'-UTR (globin-CAT) or the FMDV 5'-UTR (FMDV-CAT), which was fused at the second, downstream AUG to CAT. Untreated, intact eIF2, and caspase 3-treated eIF2 were added to
in vitro translation assays programmed with these
transcripts and their effects on translation were assessed by SDS-PAGE
autoradiography (Fig. 10A).
As previously reported (23), translation of FMDV-CAT RNA yielded the
normal size CAT protein (which comigrated with CAT produced using the
globin 5'-UTR, Fig. 10, lane h) which initiated from the
downstream AUG, and a larger product (preCAT), which initiated from the
upstream AUG (lane a). Addition of intact eIF2 resulted in a
slight stimulation in overall translation as expected (Fig.
10B) and a significant increase in translation (34%,
p < 0.006) from the upstream AUG which was evidenced
by the increase in the ratio of preCAT:CAT (lane f); also in
good agreement with previous studies (23). In contrast, addition of
caspase 3-treated eIF2 did not stimulate translation in general or
specifically increase translation from the upstream AUG (lane
e). Immunoblot analysis of caspase 3-treated eIF2 showed that
eIF2 was completely cleaved before addition to translation lysates
(data not shown). This loss of eIF2 function in IRES-AUG selection was
completely attributed to the addition of cleaved eIF2 rather than
caspase 3 itself since caspase 3 activity (lane d) was
completely abolished by the caspase inhibitor Ac-DEVD-CHO (lane
c) under the conditions used in this assay. Last, addition of
2-aminopurine, an inhibitor of eIF2 kinases (30), which prevents the
phosphorylation of eIF2, caused an expected enhancement of the activity
of endogenous eIF2 and translation of pre-CAT (lane g) in a
similar fashion to addition of intact eIF2 (lane f). Fig.
10B shows averaged translation levels of pre-CAT and CAT
generated by the FMDV IRES as well as CAT translated using a globin
5'-UTR. These data reveal that addition of eIF2 modestly and
reproducibly stimulated translation of CAT from both the FMDV IRES
(10.8%, p < 0.02) and the globin 5'-UTR (10.3%,
p < 0.01) when compared with buffer controls. A
similar, slightly larger stimulation in translation was observed when
2-aminopurine was added as a positive control. Larger levels of
translation stimulation from addition of eIF2 or 2-aminopurine were not
anticipated since the translation lysate contained active endogenous
eIF2. In contrast, eIF2 which had been cleaved with caspase
reproducibly failed to stimulate translation of either mRNA used in
this assay. Furthermore, the cleaved eIF2 did not inhibit translation
levels, suggesting that this form of eIF2 does not function in a
dominant negative manner in this assay. Importantly, however, both
cap-dependent (globin-CAT) and cap-independent (FMDV-CAT)
translation was strongly inhibited in the presence of active caspase 3 (Fig. 10, A and B), presumably via combined
cleavage of eIF4GI, eIF2, and potentially other factors in the
lysate. This demonstrates that active caspase 3 is sufficient to block
translation in reticulocyte lysates. In conclusion, caspase 3 treatment
renders the eIF2 complex inactive in stimulation of upstream codon
selection on the FMDV IRES and blocks its normal ability to stimulate
overall translation initiation in vitro.
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Fig. 10.
Caspase 3 alters eIF2 function in
translation initiation in vitro. A,
caspase-treated eIF2 does not stimulate upstream AUG selection.
In vitro translation reactions were carried out as described
under "Experimental Procedures" using FMDV-CAT (lanes
a-g) or globin-CAT (lane h). Components added to the
reactions are as follows: lanes a and h, buffer
control; lane b, Ac-DEVD-CHO (10 µM);
lane c, caspase 3 plus Ac-DEVD-CHO (10 µM);
lane d, caspase 3; lane e, 200 ng of eIF2;
lane f, caspase 3-treated eIF2 (200 ng) plus Ac-DEVD-CHO (10 µM); lane g, 2-aminopurine (5 mM).
Migration of CAT and pre-CAT proteins are indicated on the left
side and the ratio of pre-CAT to CAT as determined by
PhosphorImager analysis is shown below lanes a-g.
B, caspase-treated eIF2 does not stimulate
cap-dependent or cap-independent translation. Results from
four independent experiments similar to panel A were
quantified, then results were averaged and standard deviations
(error bars) determined. Relative translation levels are
represented as arbitrary units (AU). Treatment groups
correspond to those in panel A, lanes a-g. Abbreviations:
I, caspase inhibitor (AcDEVD-CHO); C, caspase 3;
2AP, 2-aminopurine.
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DISCUSSION |
In this report, we demonstrate that the -subunit of eIF2 is a
target for cleavage by caspases upon induction of apoptosis in cultured
cells. The cleavage of eIF2 was induced in HeLa and K562 cells, but
also Jurkat T cells (data not shown) by cisplatin, etoposide, TNF-,
or MG132 (data not shown). Thus, the cleavage of eIF2 does occur
generally in apoptotic cells, however, the possible contribution of
eIF2 cleavage to translation inhibition during apoptosis may vary
dependent upon the cell type and apoptotic stimulus utilized. For
instance, eIF2 cleavage may play a more important role in
translation inhibition or modulation in etoposide-treated cells than
cisplatin-treated HeLa cells. In fact, we expect that the drastic
inhibition of translation in apoptosis results from a multifactorial
process involving modification of several initiation factors. To this
end, we have also documented cleavage of eIF4GII and poly(A)-binding
protein in apoptotic cells, also with variable kinetics.2 Further
experiments with cleavage-resistant mutants of eIF4GI and eIF2 and
other factors will need to be performed to clarify the role and impact
of each cleavage event in the process of translation inhibition.
Although eIF2 could be cleaved in vitro by several of the
caspases tested, caspase 3 appears to be the prime candidate for the
observed cleavage in vivo. Compared with caspase 3, cleavage of eIF2 by caspase 6, 8, and 10 was inefficient. In addition, only
caspase 3 cleaved eIF2 that was present in the eIF2·2B complex. Since eIF2 is a heterotrimeric complex we tested cleavage of other subunits of eIF2. Although preliminary experiments show possible cleavage of eIF2 (but not eIF2) in vitro, we do not
have evidence that this occurs in vivo.
The data suggest that the eIF2 present in the eIF2·2B complex is the
preferred target for caspase 3 cleavage in vivo. Caspase 3 efficiently cleaved the -subunit of eIF2 in vitro only
when eIF2 was in a conformation from which GDP could readily dissociate (i.e. eIF2 in the absence of Mg2+; or eIF2
present in the eIF2·2B complex). In the absence of Mg2+,
phosphorylation of the eIF2 present in eIF2 or the eIF2·2B complex
had little or no effect on its ability to be cleaved by caspase 3. However, in the presence of physiological Mg2+
concentrations, which locks the conformation of eIF2 complexes containing GDP, cleavage of eIF2 present in eIF2·GDP and
eIF2(P)·GDP complexes by caspase 3 is severely, if not totally
inhibited in vitro. Therefore, the eIF2·GDP complex
generated at the last step of initiation of translation is not likely
to be a physiologically relevant substrate for caspase 3 in
vivo. On the other hand, the presence of Mg2+ and GDP
had relatively little affect on the ability of caspase 3 to cleave
either unphosphorylated or phosphorylated eIF2 present in the
eIF2·2B complex. Thus, while we cannot currently rule out eIF2
present in the eIF2·GTP·Met-tRNAi complex as a
potential target for cleavage by caspase 3, our data firmly establishes the eIF2 present in the eIF2·2B complex as a target for caspase cleavage in vivo.
eIF2 is the second initiation factor we have identified to be cleaved
during apoptosis and that potentially contributes to alterations in
translation or inhibition of translation in apoptotic cells. eIF2 is a
G-protein, which is known to be a critical factor in translation
initiation. In its GTP-bound conformation, eIF2 binds
Met-tRNAi to the 40 S ribosomal subunit to form a 43 S
preinitiation complex and aids in the recognition of the initiation
codon (1). In its GDP-bound conformation, eIF2 is inactive. Recycling
of eIF2·GDP requires its interaction with the guanine nucleotide exchange factor, eIF2B. As discussed in the Introduction, the activity
of eIF2 and eIF2B is regulated by phosphorylation of the -subunit of
eIF2 by several kinases (e.g. PKR and HRI) in response to
certain stimuli which leads to global inhibition of translation. Our
data indicate that cleavage of eIF2 alters the activity of the eIF2
complex. Cleavage of eIF2: (i) generated an eIF2 complex from which
GDP can dissociate independent of eIF2B; (ii) eliminated the ability of
eIF2 to stimulate translation from upstream AUG codons; and (iii)
inhibited the ability of the eIF2 complex to stimulate translation
in vitro. Even though our data indicate that eIF2
cleavage followed or was coincident with eIF4GI cleavage in cultured
cells, it is possible that the alterations in eIF2 activity induced by
eIF2 cleavage contribute significantly to the inhibition of protein
synthesis at later stages in apoptotic cells. Furthermore, it is known
that phosphorylation of as little as 25% of all eIF2 is sufficient to
cause inhibition of protein synthesis through the ability of
phosphorylated eIF2 to sequester eIF2B. Since the eIF2 present in
the eIF2·2B complex has been identified as the likely target for
cleavage in vivo, it is possible that cleavage of only a
small portion of eIF2 suffices to alter translation.
Based on the results presented in this report, we postulate that
caspase 3-induced removal of the C-terminal portion of eIF2 could
contribute to the regulation of translation in apoptotic cells through
three potential mechanisms which may combine to provide a complex
phenotype. (i) The cleaved form of eIF2 which exchanges GDP may
partly counteract PKR-driven translation inhibition after partial
caspase activation occurs. (ii) Large levels of cleaved eIF2 in
apoptotic cells might mimic the effect of eIF2 phosphorylation and
contribute to a general block in translation. (iii) Cleavage of eIF2
by caspase 3 might result in an eIF2 complex similar to that described
for yeast, a complex capable of inhibiting protein synthesis, and
perhaps, causing a specific up or down-regulation of certain
apoptosis-related genes containing ORFs in their 5'-UTR. Those yeast
studies indicated that removal of the short, highly acidic C-terminal
region of eIF2 had the same regulatory effect on GCN4 mRNA
translation as phosphorylation of eIF2 (31). Phosphorylation of eIF2
in yeast stimulates the translation of GCN4 mRNA by slowing the
recycling of eIF2·GDP by eIF2B. Reduction in the rate of eIF2 recycling stimulates initiation of translation of GCN4 mRNA at the
authentic downstream AUG start codon by eliminating the inhibitory effect of upstream ORFs in the 5'-UTR of GCN4 mRNA (32).
Could cleavage of eIF2 contribute to regulation of apoptosis?
Conceivably, cleavage of eIF2 could result in translation inhibition
that potentially contributes to the induction of apoptosis by
suppressing anti-apoptotic genes that normally block apoptosis. Indeed,
several reports have shown that treatment of cells with the protein
synthesis inhibitor cycloheximide does induce apoptosis by itself,
indicating that blocking protein synthesis can lead directly to cell
death (33-35). However, several other publications showed that
cycloheximide prevented apoptosis, so the necessity of protein
synthesis appears to be dependent on cell type and trigger of apoptosis
(36-38). While extensive phosphorylation of eIF2 by an eIF2
kinase, such as PKR, could lead to a complete global arrest of
translation which induces apoptosis in many cell types, cleavage of
eIF2 by caspase 3 would yield eIF2 from which GDP can dissociate
independent of active eIF2B and potentially relieve global arrest
during early stages. Alternatively, cleaved eIF2 might function similar
to the yeast GCN4 system (32) to enhance the translation of mRNAs
coding for pro-apoptotic proteins that contain upstream ORFs in their
5'-UTRs as postulated above, thus contributing to the completion of the
apoptotic process.
PKR itself has been shown to induce apoptosis in several systems
(39-41) whereas mutant forms of PKR have been implicated in malignant
transformation (13). Srivastava et al. (12) showed that
during TNF-induced cell death levels of PKR activity were increased and
concomitantly levels of phosphorylated eIF2 were increased as well.
Expression of a nonphosphorylated form of eIF2, S51A, partially
protected against TNF-induced apoptosis, suggesting that PKR is
required for and mediates stress-induced apoptosis through eIF2
phosphorylation. Other studies showed that increased PKR activity leads
to transcriptional and translational up-regulation of Fas and Bax, two
pro-apoptotic proteins (42). The mRNAs for both Fas and Bax have
upstream ORFs in their 5'-UTRs which could account for translational
up-regulation through a eIF2 phosphorylation-mediated mechanism
similar to the regulation of yeast GCN4 mRNA (31, 32). Finally,
while this article was in review, another group (43) has also shown
that eIF2 is cleaved in apoptosis, generating results similar to
those herein. In that study it was noted that cleaved eIF2 was able
to repress PKR-mediated suppression of translation, but no mechanistic
explanation for that observation was provided (43). It is likely that
the enhanced ability of cleaved eIF2 to exchange GDP reported here
helps counteract this effect of PKR. Much more investigation is
required to unravel these complexities and to fully discern the role of
eIF2 cleavage in apoptosis.
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ACKNOWLEDGEMENT |
We thank Alike van der Velde for construction
of plasmids.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants AI27914 and GM59803 (to R. E. L.), Oklahoma
Agricultural Experiment Station project number 1975, and NIEHS,
National Institutes of Health Grant ES 042299 (to R. L. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed. Tel.: 713-798-8993;
Fax: 713-798-5075; E-mail: rlloyd@bcm.tmc.edu.
2
W. E. Marissen, A. Gradi, N. Sonenberg, and
R. E. Lloyd, manuscript in preparation.
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ABBREVIATIONS |
The abbreviations used are:
eIF, eukaryotic
translation initiation factor;
ORF, open reading frame;
5'-UTR, 5'-untranslated region;
FMDV, foot and mouth disease virus;
CAT, chloramphenicol acetyltransferase;
PKR, double-stranded
RNA-dependent eIF2 kinase;
HRI, heme-regulated eIF2 kinase;
Ac-DEVD-CHO, acetyl-DEVD-aldehyde;
Z-VAD-FMK, benzyloxycarbonyl-VAD-fluoromethylketone;
pNA, para-nitroanilide;
TNF-, tumor necrosis factor-;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
PARP, poly(ADP-ribose) polymerase;
IRES, internal ribosome entry site.
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TOP
ABSTRACT
INTRODUCTION
