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Mol Cell Biol, June 1998, p. 3300-3309, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Inhibitor of Apoptosis Proteins Physically Interact with and
Block Apoptosis Induced by Drosophila Proteins HID and
GRIM
Domagoj
Vucic,1
William J.
Kaiser,1 and
Lois K.
Miller1,2,*
Department of
Genetics1 and
Department of
Entomology,2 The University of Georgia,
Athens, Georgia 30602
Received 5 December 1997/Returned for modification 12 February
1998/Accepted 13 March 1998
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ABSTRACT |
Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed
to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21
cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and
L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183-10188, 1997).
In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and
GRIM-induced apoptosis and also physically interacted with them
through the BIR motifs of the IAPs. The region of sequence similarity
shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each
protein, was required for the induction of apoptosis by HID and
its binding to IAPs. When stably overexpressed by fusion to an
unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids
of HID and GRIM were sufficient to induce apoptosis and confer
IAP binding activity. However, GRIM was more complex than HID since the
C-terminal 124 amino acids of GRIM retained apoptosis-inducing
and IAP binding activity, suggesting the presence of two
independent apoptotic motifs within GRIM. Coexpression of IAPs with HID
stabilized HID levels and resulted in the accumulation of HID in
punctate perinuclear locations which coincided with IAP localization.
The physical interaction of IAPs with RPR, HID, and GRIM provides a
common molecular mechanism for IAP inhibition of these
Drosophila proapoptotic proteins.
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INTRODUCTION |
Programmed cell death, or
apoptosis, is a genetically regulated mechanism that plays an
important role in development and homeostasis in vertebrates and
invertebrates (25). Three Drosophila melanogaster
genes, reaper (rpr), head involution
defective (hid), and grim, were identified
within the chromosomal 75C1,2 region as inducers of apoptosis
in cells destined to die during embryogenesis (5, 12,
30). In Drosophila, ectopic expression of
rpr, hid, and grim, which encode a
65-amino-acid polypeptide (RPR), 410-amino-acid
polypeptide (HID), and 138-amino-acid polypeptide (GRIM), respectively, induces cell death through activation of caspase(s) (5, 12, 15, 19, 29). rpr and
grim transcripts accumulate in a defined subset of Type II
neurons prior to the onset of apoptosis (20), and
rpr and hid seem to act synergistically to induce
programmed cell death during development of the Drosophila central nervous system midline (32). RPR, HID, and GRIM
share no extensive homology, although there is resemblance between the sequences of the N-terminal 14 amino acids of these three proteins (5). The functional significance of this short homologous
region remains ambiguous as mutations of the most conserved amino acid residues (28), or elimination of the whole region
(4), only partially diminish the proapoptotic activity of
RPR upon overexpression in insect cell cultures.
Two types of antiapoptotic proteins, caspase inhibitors and members of
the inhibitors of apoptosis (IAPs) family, are known to block
apoptosis in insect cells. Baculovirus P35, which normally blocks apoptosis during baculovirus replication in lepidopteran SF-21 cells (7), is a broad-spectrum caspase inhibitor
(3, 31) that blocks apoptosis induced by ectopic
expression of RPR, HID, or GRIM in Drosophila (5, 12,
15). The founder members of the IAP family, baculovirus Op-IAP
and Cp-IAP, were identified by their ability to substitute functionally
for the baculovirus P35 (2, 8, 9). In addition, baculovirus
IAPs also block apoptosis induced by RPR (28), Doom
(13), FADD (28), actinomycin D (8),
and UV irradiation (17) in SF-21 cells. Both Op-IAP and
Cp-IAP are able to block the activation of Sf-caspase-1 during baculovirus infection (23) and Cp-IAP can partially block
RPR-dependent apoptosis in Drosophila
developing eyes. Drosophila-encoded IAPs, D-IAP1 and
D-IAP2, block HID- and RPR-induced apoptosis in
Drosophila developing eyes (15). Members of the
IAP family are characterized by the presence of at least one and
usually two or three tandem baculovirus IAP repeat (BIR) motifs located
at the amino-terminal and central portions of the protein, and most of
them have a carboxy-terminal RING finger motif. Both baculovirus and
Drosophila IAPs inhibit RPR-induced apoptosis in
SF-21 cells (27, 28), physically interact with RPR through
their BIR region (27), and alter its subcellular
localization to punctate perinuclear locations which coincide with IAP
localization (27). When expressed alone, RPR levels decline
rapidly in the cells undergoing RPR-induced apoptosis, but
coexpression with IAPs appears to stabilize RPR (27).
IAP homologs have also been identified in mammals. NAIP, one of the
human IAPs, is linked to the progression of spinal muscular atrophy, which involves neuronal cell death (22). Two
other mammalian IAPs, c-IAP1 and c-IAP2, bind tumor necrosis
factor receptor 2 (TNFR-2)-associated factor 2 (TRAF-2) through
their BIR domains (21). c-IAP1 also appears to be a
component of the TNFR-1 signaling complex and may exert antiapoptotic
activity by modifying signaling through TRAF-related pathways
(24). Human X-chromosome-linked IAP (X-IAP) binds
and directly inhibits two members of the caspase family,
caspase-3 and caspase-7, in vitro (10). Expression of the
most recently identified IAP homolog, human Survivin, correlates with
oncogenic transformation (1). Mammalian IAPs block
apoptosis in several mammalian cell lines induced by a
variety of stimuli (1, 6, 11, 16, 26).
In this study we examine the induction of apoptosis by HID
and GRIM in SF-21 cells. We demonstrate that baculovirus
and Drosophila IAPs inhibit HID- and GRIM-induced
apoptosis and physically interact with HID and GRIM through
their BIR motifs. We also determine the regions of HID and GRIM which
can promote apoptosis and interact with IAPs. We define a short
region at the N terminus of both proteins with proapoptotic and IAP
binding activities.
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MATERIALS AND METHODS |
Cell line and plasmid construction.
Spodoptera
frugiperda (Lepidoptera: Noctuidae) IPLB-SF-21 (SF-21) cells were
maintained in TC-100 medium (Gibco BRL, Gaithersburg, Md.) supplemented
with 10% fetal bovine serum (Intergen, Purchase, N.Y.) and 0.26%
tryptose broth as previously described (18). All the
plasmids except for the pKV-based plasmids used in in vitro binding
studies are derived from pHSP70PLVI+CAT, a plasmid expressing the
chloramphenicol acetyltransferase (cat) gene under the
Drosophila hsp70 promoter (8). Plasmids
expressing nontagged Op-iap, Cp-iap,
D-iap1, D-iap2, p35, or
cat, Flag-tagged rpr, Op-iap, or
cat, and HA(Epi)-tagged Op-iap, Op-BIR, Op-RING,
D-iap2, D-iap1-BIR, p35,
or cat were previously described (8, 27, 28). In
Flag-D-iap1-BIR, the cat from pHSP70PLVI+CAT is
substituted with Flag-epitope tag N-terminally fused to
D-iap1-BIR; in Epi-Cp-iap, Epi-D-iap1, or Epi-D-iap1-RING,
cat is replaced with HA.11-epitope tag N-terminally
fused to Cp-iap, D-iap1, or D-iap1-RING. The plasmid expressing hid cDNA (pHSP70PLVI+HID) was
made by replacing cat from pHSP70PLVI+CAT with
hid cDNA. The sequence encoding the 410-amino-acid
hid open reading frame (ORF) was amplified from pHSP70PLVI+HID by PCR with Pfu polymerase (Stratagene) and
the primers NHID (GAAGATCTACAATGGCCGTGCCCTTTTAT) plus
CHID (ATATCCCGGGTTAACGTCTCCTGCGCTTTCAT). The
resulting product was digested with BglII and
XmaI and subcloned into the BglII and
PstI sites of pHSP70PLVI+CAT to generate pHSP70PLVI+HID-ORF. In hid-Flag or hid-Epi, cat from pHSP70PLVI+CAT was replaced
with hid-ORF C-terminally fused to either a FlagHis6-tag or
an HA.11His6-tag. pHSHID-EpiHisVI+ was digested with XhoI
and religated to generate pHSHID
(38-335)-EpiHisVI+, which encodes
the first 37 and last 74 amino acids of the 410-amino-acid
hid ORF. Sequences encoding the first 37 and last 74 amino
acids of the hid ORF were amplified by PCR with
Pfu polymerase and the primers NHID plus
MCTAGHID (GGACTAGTTGCGCTCGAGGGAAGTGG) and MNHID
(GAAGATCTACAATGGCCTCGAGCAGCAGCAATAAT) plus CTAGHID
(GGACTAGTTCGCGCCGCAAAGAAGC), respectively. The resulting products were digested with BglII and SpeI
and subcloned into the BglII and SpeI sites of
pHSHID-EpiHisVI+ to generate pHSHID(1-37)-EpiHisVI+ and
pHSHID(336-410)-EpiHisVI+. Sequence encoding amino acids
2 to 53 of the 250-amino-acid cat ORF was
amplified by PCR with Pfu polymerase and the primers
NECOCAT (CGGAATTCGAGAAAAAAATCACTGG) plus CECOCAT
(CGGAATTCCGGATGAGCATTC). The resulting product was digested
with EcoRI and inserted into the EcoRI site of
pHSHID(1-37)-EpiHisVI+ between the end of hid-encoding
sequence and the start of the C-terminal HA.11His6-tag to generate
pHSHID(1-37)C-EpiHisVI+. pHSCAT(1-53)-EpiHisVI+ was generated by
subcloning the sequence encoding the first 53 amino acids of
cat ORF fused to C-terminal HA.11His6-tag from
pHSP70PLVI+HID(1-37)C-Epi. pHSCAT(1-53)HID(37-410)-EpiHisVI+ was
generated by subcloning the sequence encoding the amino acids 37 to 410 of hid ORF into the EcoRI site of
pHSCAT(1-53)-EpiHisVI+. Sequence encoding the amino acids 15 to 410 of hid ORF was amplified by PCR with
Pfu polymerase (Stratagene) and the primers NMUTHID (GAAGATCTACAATGGTAGCGTCGAGTTCATC) plus CTAGHID. The
resulting product was digested with BglII and
SpeI and subcloned into the BglII and
SpeI sites of pHSHID-EpiHisVI+ to generate
pHSHID(2-14)-EpiHisVI+. The plasmid expressing grim cDNA
(pHSP70PLVI+GRIM) was made by replacing cat from
pHSP70PLVI+CAT with grim cDNA. The sequence encoding
the 138-amino-acid grim ORF was amplified from
pHSP70PLVI+GRIM by PCR with Pfu polymerase
(Stratagene) and the primers NGRIM (CGGAGATCTACAATGGCCATCGCCTATTTC) plus CGRIM
(CGGACTAGTTTAGTTCTCCTTGGAGGT). The resulting product was
digested with BglII and SpeI and
subcloned into the BglII and SpeI sites of
pHSP70PLVI+CAT to generate pHSP70PLVI+GRIM-ORF. In grim-Flag or
grim-Epi, cat from pHSP70PLVI+CAT was replaced with
grim-ORF C-terminally fused to either a FlagHis6-tag or an HA.11His6-tag. Sequences encoding the first 16 or the first 37 amino
acids of the 138-amino-acid grim ORF were amplified by PCR with Pfu polymerase and the primers NGRIM plus CDGRIM
(CGACTAGTTCTGGCCAACAATTGGGC) or NGRIM plus MCTAGRIM
(CGACTAGTTGCAGCAGCTGTTGCAGTC), respectively. The resulting
products were digested with BglII and SpeI and
subcloned into the BglII and SpeI sites of
pHSHID(1-37)C-EpiHisVI+ to generate pHSGRIM(1-16)C-EpiHisVI+ and
pHSGRIM(1-37)C-EpiHisVI+, respectively. Sequence encoding the amino
acids 15 to 138 of the grim ORF was amplified by PCR with
Pfu polymerase (Stratagene) and the primers NMUTGRIM
(CGGGATCCACAATGGCCAGAAGCTATCAGC) plus CTAGRIM
(CGGACTAGTGTTCTCCTTGGAGGTGGC). The resulting product was
digested with BamHI and SpeI and subcloned into the BglII and SpeI sites of
pHSHID-EpiHisVI+ to generate pHSGRIM(2-14)-EpiHisVI+. In
pKV-HID, pKV-GRIM, and pKV-Flag-D-IAP1-BIR, sequences
encoding HID-Epi, GRIM with four additional methionines at the C
terminus for the efficient labeling, and Flag-D-IAP1-BIR were
subcloned into pKV vector (3) under control of the T7 promoter.
DNA fragmentation and viability assays.
SF-21 cells
(105 per 35-mm-diameter tissue culture dish) were
transfected with 2.5 µg of each of the indicated plasmids by Lipofectin-mediated transfection (Gibco BRL). The cells were heat shocked at 20 h posttransfection for 30 min at 42°C. DNA
fragmentation and viability assays were described previously (8,
28).
In vivo binding assay, immunofluorescence microscopy, and
immunoblot analysis.
All were performed as described previously
(27).
In vitro binding assay.
The Flag-D-IAP1-BIR,
35S-labelled HID and 35S-labelled GRIM were
obtained by in vitro transcription and translation by using a TNT
T7-coupled reticulocyte lysate system (Promega). After translation, equivalent amounts of 35S-labelled HID or
35S-labelled GRIM were incubated with Flag-D-IAP1-BIR or
empty vector (pKV) in the presence of anti-Flag affinity resin
(Eastman) in Nonidet P-40 lysis buffer (27) for 4 h at
4°C with agitation. The resin was washed five times in Nonidet P-40
lysis buffer and boiled in sodium dodecyl sulfate (SDS)-sample buffer
(18), and proteins were resolved by SDS-polyacrylamide gel
electrophoresis and visualized by autoradiography.
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RESULTS |
HID and GRIM induce apoptosis in SF-21 cells.
To
determine whether the Drosophila gene hid can
induce apoptosis in SF-21 cells, we transiently expressed
hid by transfecting the cells with a plasmid containing the
entire hid cDNA under the control of the
Drosophila hsp70 promoter and inducing expression by heat
shocking at 20 h posttransfection. As early as 1 to 2 h after
the induction of hid expression, cells began to exhibit membrane blebbing which became pronounced and was accompanied by
apoptotic body formation by 8 h after induction (data not shown). Expression of hid cDNA also induced
oligonucleosomal ladder formation 8 h after induction
(Fig. 1B, lane 2) which was
not observed in cells expressing the control cat gene (Fig.
1B, lane 1). We PCR amplified the sequence encoding only the
410-amino-acid hid ORF and expressed it under hsp70 promoter
control. Transient expression of hid ORF induced cell death
to the same extent as expression of the entire hid
cDNA (Fig. 1B, lane 3). Next we generated constructs which
express hid ORF with C-terminal HA.11-His6 (hid-Epi)
or Flag-His6 (hid-Flag) tags (Fig. 1A). We tested these
constructs for induction of apoptosis and found that the
presence of the C-terminal tag did not affect the proapoptotic
function of HID (Fig. 1B, lanes 4 and 5).
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FIG. 1.
The abilities of hid and grim constructs to induce
apoptosis in SF-21 cells. (A) Schematic representation of hid
and grim constructs. Boxes represent coding regions, dashed lines
indicate deleted regions, and single lines are untranslated
sequences. Numbers on the top of the solid areas indicate amino
acid position in the full-length protein. The ability of the
construct to induce apoptosis is indicated by the plus or minus
to the left. The abilities of hid (B) and grim (C) constructs to induce
nucleolytic degradation of chromatin into oligonucleosomal ladder-sized
fragments of DNA are shown. SF-21 cells were transfected with the
control cat constructs (lanes 1 in panels B and C, and lane 9 in panel
C) or with the hid and grim constructs indicated above each lane (lanes
2 to 9 in panel B and lanes 2 to 8 in panel C). Cellular DNA was
harvested 8 h after induction and analyzed by agarose gel
electrophoresis. Size markers (in base pairs) are indicated on the
left.
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To define the region within HID that is responsible for the induction
of apoptosis, we constructed an in-frame deletion that
fuses
the first 37 and the last 74 amino acids of HID (Fig.
1A).
This
deletion, hid
(38-335)-Epi, induced apoptosis to the same
level as hid-Epi (Fig.
1B, lane 6). In order to determine whether
the
first 37 or the last 74 amino acids of HID possess proapoptotic
activity, we expressed the two regions independently [hid(1-37)-Epi
and hid(336-410)-Epi, respectively] (Fig.
1A). However, neither
of
these regions induced apoptosis (Fig.
1B, lane 8; and data
not
shown). Immunoblot analysis revealed that hid(336-410)-Epi
was
expressed at detectable levels (see below), indicating that
this region
of HID was not sufficient to induce apoptosis. On
the other
hand, hid(1-37)-Epi could not be detected by immunoblot
analysis (data
not shown), suggesting that the expressed protein
product was unstable.
To increase the stability of hid(1-37)-Epi
we generated a construct,
hid(1-37)C-Epi (Fig.
1A), which expresses
the first 37 amino acids of
HID fused to the first 53 amino acids
of CAT, an apoptotically
neutral protein, and an HA.11-His6 tag.
This construct expressed
detectable levels of protein product
(see below) and induced
apoptosis (Fig.
1B, lane 7). To rule out
any toxicity of the
fusion protein, we made a construct expressing
the first 53 amino acids
of CAT fused to amino acids 37 to 410
of HID and an HA.11-His6 tag.
This protein, CAT(1-53)HID(37-410)-Epi,
did not induce
apoptosis (data not shown). The N-terminal 37 amino
acids of
HID contain a region (amino acids 2 to 14) with similarity
to the N
terminus of RPR and GRIM (Fig.
1A). We, therefore, made
a construct in
which this region is deleted from hid-Epi, hid
(2-14)-Epi
(Fig.
1A),
and tested its ability to induce apoptosis. This deletion
mutant did not induce apoptosis in SF-21 cells (Fig.
1B,
lane
9) although it expressed detectable levels of protein product
(see below). Thus, the first 14 amino acids of HID were required
for
the induction of apoptosis, and the first 37 amino acids were
sufficient to induce apoptosis as long as they were stably
expressed.
We investigated the ability of
grim to induce
apoptosis in SF-21 cells in the same way as we did for
hid. grim cDNA,
grim ORF, and C-terminally tagged
versions of
grim (grim-Epi and grim-Flag)
(Fig.
1A) all
induced apoptosis in SF-21 cells as documented by
oligonucleosomal ladder formation (Fig.
1C, lanes 2 to 5) and
membrane blebbing (data not shown). Constructs containing only
the
N-terminal 37 or 16 amino acids of GRIM fused to the first
53 amino
acids of CAT and HA.11-His6 tag, grim(1-37)C-Epi and
grim(1-16)C-Epi
(Fig.
1A), also induced apoptosis (Fig.
1C, lanes
6 and 7)
although the construct expressing the first 53 amino
acids of CAT
fused to HA.11-His6 tag (Fig.
1A), cat(1-53)-Epi,
did not (Fig.
1C, lane 9). This suggested that the region of similarity
among RPR,
GRIM, and HID is sufficient to induce apoptosis as
long as it
is stably expressed. However, a construct in which
the N-terminal 14 amino acids of GRIM were deleted, grim
(2-14)-Epi
(Fig.
1A), still
retained proapoptotic activity (Fig.
1C, lane
8) unlike the equivalent
hid construct, hid
(2-14)-Epi. These
results suggest that GRIM
harbors another region, in addition
to the 16 N-terminal amino
acids, that possesses proapoptotic
activity.
Inhibition of hid- and grim-induced
apoptosis by p35 and iaps.
To
assess the nature of the apoptotic pathway induced by hid
and grim in SF-21 cells, we cotransfected cells with
plasmids expressing hid-ORF or grim-ORF and
several known antiapoptotic genes. Compared to cells
transfected with cat gene alone, cotransfection of
hid and cat and of grim and
cat induced apoptosis in 46% (Fig. 2A) and 43% (Fig. 2B) of the cells,
respectively. Cotransfection of hid or grim with
a plasmid expressing the baculovirus gene p35, a
general caspase inhibitor, almost completely inhibited hid-
and grim-induced apoptosis (Fig. 2A and B). We also
tested members of the iap class of antiapoptotic genes for
their ability to block hid- and grim-induced
apoptosis. Baculovirus Op-iap and Cp-iap
and Drosophila D-iap1 and D-iap2
blocked apoptosis induced by hid and
grim, although Drosophila iaps were not as
effective as baculovirus iaps (Fig. 2A and B). The BIR
region of D-iap1 was sufficient for the inhibition of
hid- and grim-induced apoptosis and even
more efficient than the full-length D-iap1 (Fig. 2A and B),
which is in concordance with previously published data (15). Baculovirus and Drosophila iaps also blocked
apoptosis induced by the above-described (Fig. 1A) deletion
constructs of hid and grim as well as they
blocked the full-length constructs (data not shown). In addition, we
tested various members of the bcl-2 family (human
bcl-2, human bcl-xL,
Caenorhabditis elegans ced-9 and adenovirus E1B19K), but
they exhibited little or no ability to block hid- and
grim-induced apoptosis in SF-21 cells (data not
shown).
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FIG. 2.
Protection against hid- and
grim-induced cell death in SF-21 cells by antiapoptotic
genes. The cells were cotransfected with plasmids expressing
hid (A) or grim (B) and either cat,
p35, Op-iap, Cp-iap,
D-iap1, D-iap1-BIR, or D-iap2.
Cell viability was determined by trypan blue exclusion 8 h after
heat shock. The results shown are relative to viability of cells
transfected with the cat gene alone (set at 100% viability)
and represent at least three independent experiments for each
cotransfected combination. Standard deviations are indicated by the
error bars.
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IAPs physically interact with HID and GRIM.
Because
baculovirus and Drosophila IAPs physically interact with RPR
and inhibit RPR-induced apoptosis (27), we
investigated the possibility that baculovirus and Drosophila
IAPs also interact with HID and GRIM. To do so, we first transiently
coexpressed HID-Flag with HA-epitope-tagged versions of
baculovirus IAPs, Epi-Op-IAP and Epi-Cp-IAP, or
Drosophila IAPs, Epi-D-IAP1 and Epi-D-IAP2, and determined
if anti-Flag antibodies could coimmunoprecipitate the IAP fusion
proteins. As a negative control, we tested an HA-tagged version of the
baculovirus P35 protein, Epi-P35, which blocked HID- and GRIM-induced
apoptosis as well as the untagged version. We found that
all IAPs coprecipitated with HID (Fig.
3A, lanes 1, 3, 5, and 7), but P35
did not (Fig. 3A, lane 9), although it was efficiently expressed (Fig.
3B, lane 9). Expression of the HA-tagged and Flag-tagged constructs in
the cell lysates was confirmed for all transfections (Fig. 3B and C).
Coprecipitation of Epi-D-IAP1 with RPR-Flag served as a positive
control (Fig. 3A, lane 10), and coprecipitation of Epi-Cp-IAP
with Flag-CAT served as an additional negative control (Fig. 3A,
lane 11).
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FIG. 3.
IAPs physically interact with HID. (A) SF-21 cells were
transiently transfected with plasmids expressing HID-Flag,
RPR-Flag, or Flag-CAT and indicated HA-epitope-tagged (Epi)
genes. At 3 h after heat shock, aliquots of cell lysates were
immunoprecipitated with the M2 anti-Flag monoclonal antibody resin.
Coprecipitated HA-epitope-tagged constructs were detected by
immunoblot analysis with the rabbit anti-HA.11 polyclonal antiserum.
(B) Expression of the HA-epitope-tagged proteins was confirmed by
immunoblot analysis with the mouse HA.11 monoclonal antibody of
aliquots of the total cell lysates from panel A. (C) Expression of the
HID-Flag, RPR-Flag, and Flag-CAT was confirmed by reprobing the
membrane from panel A with the M2 anti-Flag antibody. The positions of
the Flag- or HA-epitope-tagged proteins and monoclonal
immunoglobulin G antibody are indicated by arrows on the right.
Molecular mass markers (in kilodaltons) are shown on the left.
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In order to determine which portion of IAPs possessed HID binding
activity, we tested the ability of HA-tagged BIR and RING
finger
domains to individually bind HID-Flag. Epi-Op-BIR and
Epi-D-IAP1-BIR
coimmunoprecipitated with HID-Flag (Fig.
4A, lanes 2 and 5), but
Epi-Op-RING and
Epi-D-IAP1-RING did not (Fig.
4A, lanes 3 and
6). In control lanes, the
HA-tagged version of bacterial CAT (Epi-CAT)
did not
coimmunoprecipitate with HID-Flag (Fig.
4A, lane 7), and
Epi-D-IAP1-BIR
coimmunoprecipitated with RPR-Flag as expected
(Fig.
4A, lane 8).
Expression of the HA-tagged and Flag-tagged
constructs in the cell
lysates was confirmed for all transfections
(Fig.
4B and C).
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FIG. 4.
HID interacts with the BIR region of IAPs. (A) SF-21
cells were transiently transfected with plasmids expressing HID-Flag or
RPR-Flag and indicated HA-epitope-tagged (Epi) genes. At 3 h
after heat shock, aliquots of cell lysates were immunoprecipitated with
the M2 anti-Flag monoclonal antibody resin. Coprecipitated
HA-epitope-tagged constructs were detected by immunoblot analysis
with the rabbit anti-HA.11 polyclonal antiserum. (B) Expression of the
HA-epitope-tagged proteins was confirmed by immunoblot analysis
with the mouse HA.11 monoclonal antibody of aliquots of the total cell
lysates from panel A. (C) Expression of the HID-Flag and RPR-Flag was
confirmed by reprobing the membrane from panel A with the M2 anti-Flag
antibody. The positions of the Flag- or HA-epitope-tagged proteins
and monoclonal immunoglobulin G antibody are indicated by arrows on the
right. Molecular mass markers (in kilodaltons) are shown on the left.
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To investigate the possibility that IAPs interact with GRIM, we
transiently coexpressed GRIM-Flag with HA-epitope-tagged
versions
of baculovirus IAPs, Epi-Op-IAP and Epi-Cp-IAP, or
Drosophila IAPs, Epi-D-IAP1 and Epi-D-IAP2, and
determined if anti-Flag antibodies
could coimmunoprecipitate the IAP
fusion proteins. We found that
all expressed IAPs and the BIR region of
IAPs coprecipitated with
GRIM (Fig.
5A,
lanes 1 to 4, 6 and 8), but the RING motifs of
IAPs, P35, or CAT did
not (Fig.
5A, lanes 5, 7, 9, and 10), although
they were efficiently
expressed (Fig.
5B, lanes 5 and 10). Expression
of the HA-tagged and
Flag-tagged constructs in the cell lysates
was confirmed for all
transfections (Fig.
5B and C).
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FIG. 5.
IAPs physically interact with GRIM. (A) SF-21 cells were
transiently transfected with plasmids expressing grim-Flag and
indicated HA-epitope-tagged (Epi) constructs. At 3 h after
heat shock, aliquots of cell lysates were immunoprecipitated with the
M2 anti-Flag monoclonal antibody resin. Coprecipitated
HA-epitope-tagged constructs were detected by immunoblot analysis
with the rabbit anti-HA.11 polyclonal antiserum. (B) Expression of the
HA-epitope-tagged proteins was confirmed by immunoblot analysis
with the mouse HA.11 monoclonal antibody of aliquots of the total cell
lysates from panel A. (C) Expression of GRIM-Flag was confirmed by
immunoblot analysis with the M2 anti-Flag antibody of aliquots of the
total cell lysates from panel A. The positions of the
HA-epitope-tagged proteins and GRIM-Flag are indicated by arrows on
the right. Molecular mass markers (in kilodaltons) are shown on the
left.
|
|
To determine which portion of HID and GRIM had IAP binding activity, we
tested the ability of HA-tagged HID-ORF, HID
(38-335),
HID(1-37)C,
HID(336-410) or HID
(2-14) and GRIM-ORF, GRIM(1-37)C,
GRIM(1-16)C or GRIM
(2-14) to individually bind Flag-tagged
D-IAP1-BIR.
The HID-Epi, HID
(38-335)-Epi and
HID(1-37)C-Epi coimmunoprecipitated
with Flag-D-IAP1-BIR (Fig.
6A, lanes 2 to 4), but HID(336-410)-Epi
and HID
(2-14)-Epi did not (Fig.
6A, lane 5 and 6). All of the
tested
GRIM constructs coimmunoprecipitated with Flag-D-IAP1-BIR
(Fig.
6A,
lanes 8 to 11). This included grim(1-16)C-Epi containing
only the
first 16 amino acids of GRIM and grim
(2-14)-Epi lacking
the
N-terminal amino acids of GRIM, suggesting that there are
two
independent IAP-BIR binding sites in GRIM. In control lanes,
Epi-P35 and Epi-CAT did not coimmunoprecipitate with
Flag-D-IAP1-BIR
(Fig.
6A, lanes 12 and 13). Expression of the HA-tagged
and Flag-tagged
constructs in the cell lysates was confirmed for all
transfections
(Fig.
6B and C).
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|
FIG. 6.
IAP interaction with deletions of HID and GRIM. (A)
SF-21 cells were transiently transfected with plasmids expressing
Flag-D-IAP1-BIR and indicated HA-epitope-tagged (Epi) constructs.
At 3 h after heat shock, aliquots of cell lysates were
immunoprecipitated with the M2 anti-Flag monoclonal antibody resin.
Coprecipitated HA-epitope-tagged constructs were detected by
immunoblot analysis with the rabbit anti-HA.11 polyclonal antiserum.
(B) Expression of the HA-epitope-tagged proteins was confirmed by
immunoblot analysis with the mouse HA.11 monoclonal antibody of
aliquots of the total cell lysates from panel A. (C) Expression of the
Flag-D-IAP1-BIR was confirmed by immunoblot analysis with the M2
anti-Flag antibody of aliquots of the total cell lysates from panel A. The positions of the HA-epitope-tagged proteins and Flag-D-IAP1-BIR
are indicated by arrows and letters as follows: a, HID-Epi; b,
HID(2-14)-Epi; c, HID(38-335)-Epi; d, HID(1-37)C-Epi; e,
HID(336-410)-Epi; f, Epi-P35; g, Epi-CAT; h, GRIM-Epi; i,
GRIM(2-14)-Epi; j, GRIM(1-37)C-Epi; k, GRIM(1-16)-Epi; l,
Flag-D-IAP1-BIR; and m, D-IAP1-BIR cross-reacting with polyclonal
HA antibody. (D) HID and GRIM bind D-IAP1-BIR in vitro.
35S-labelled in vitro translated HID and GRIM were added to
in vitro translated unlabelled Flag-D-IAP1-BIR or empty vector (pKV)
and immunoprecipitated with the M2 anti-Flag affinity resin.
Coprecipitated 35S-labelled HID and GRIM were analyzed
by SDS-polyacrylamide gel electrophoresis and autoradiography (lanes 1 to 4). Equivalence of input radiolabelled proteins is shown in lanes 5 and 6. Molecular mass markers (in kilodaltons) are shown on the left.
|
|
To demonstrate the association of HID and GRIM with D-IAP1-BIR in
vitro, we tested the ability of
35S-labelled in vitro
translated HID and GRIM to bind Flag-D-IAP1-BIR.
HID
and GRIM coimmunoprecipitated with in vitro-translated
Flag-D-IAP1-BIR
(Fig.
6D, lanes 3 and 4) but not with
products from the in vitro
transcription and translation of the vector
itself (Fig.
6D, lanes
1 and 2). Therefore, baculovirus and
Drosophila IAPs physically
interact with HID and GRIM in
vivo and in vitro, and the region
encompassing the BIR motifs of IAPs
is necessary for this protein-protein
interaction.
HID and GRIM colocalize with IAPs.
The ability of IAPs to
physically interact with HID suggested that IAPs and HID should
localize to the same subcellular location when coexpressed, as do IAPs
and RPR. To verify this, we expressed hid-Flag in SF-21 cells in
the presence or absence of HA-tagged IAPs. In the absence of IAPs, HID
localized predominantly to the cytoplasm of the cells 1 h after
induction (Fig. 7A). By 4 h after induction we were not able to detect HID within the cells (Fig. 7B),
although the expression of HID caused nuclear condensation and fragmentation in transfected cells (Fig. 7C).
Coexpression with P35 inhibited HID-induced nuclear
condensation and fragmentation, and HID was observed in the cytoplasm
of transfected cells at both 1 and 4 h after induction (data not
shown).
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|
FIG. 7.
Colocalization of IAPs and HID. SF-21 cells were
transiently transfected with plasmids expressing hid-Flag and cat
(panels A, B, and C; B and C are the same field), hid-Flag and
Epi-Cp-iap (panels D to G; E to G are the same field), Epi-Cp-iap
(panel H), hid-Flag and Epi-D-iap1 (panels I to L; J to L are the same
field), or Epi-D-iap1 (panel M). At 1 h (panels A, D, and I) or
4 h (panels B, C, E to H, and J to M) after heat shock, cells were
fixed with formaldehyde, permeabilized, and analyzed by indirect
immunofluorescence. HID-Flag was visualized with mouse M2 anti-Flag
monoclonal antibody and lissamine rhodamine-conjugated goat anti-mouse
immunoglobulin G plus immunoglobulin M antibody (panels A, B, D, E, I,
and J). HA-epitope-tagged proteins were visualized with rabbit
anti-HA.11 polyclonal antiserum and fluorescein
isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody
(panels G, H, L, and M). Nuclei were visualized by costaining with DAPI
(4',6-diamidino-2-phenylindole) (panels C, F, and K). Scale bar equals
25 µm.
|
|
Cp-IAP also blocked HID-induced nuclear condensation and fragmentation
(Fig.
7F) as well as the rapid disappearance of HID
(Fig.
7E). However,
in this case, HID displayed punctate perinuclear
localization 1 h
after induction (Fig.
7D) and more pronounced
localization 4 h
after induction (Fig.
7E). This location of HID
overlapped with the
subcellular localization of Cp-IAP (Fig.
7G).
The same punctate
perinuclear localization was observed for Cp-IAP
in the absence
of HID (Fig.
7H). Similar to Cp-IAP, D-IAP1 blocked
HID-induced nuclear condensation and fragmentation (Fig.
7K) and
rapid disappearance of HID, and showed overlapping subcellular
localization (Fig.
7J and L). Colocalization of HID and D-IAP1
was not
as evident as for HID and Cp-IAP, probably because D-IAP1
displayed
less punctate staining when compared with Cp-IAP (Fig.
7, compare H
and M).
In addition, coexpression of HID and Op-IAP resulted in
an almost identical subcellular localization pattern as that
observed
upon coexpression of HID and Cp-IAP, and reversing the
epitope
tags on HID and Cp-IAP or Op-IAP did not alter the pattern
of
subcellular localization when coexpressed or expressed individually
(data not shown).
We also examined subcellular localization of GRIM and found that,
in the absence of IAPs, GRIM induced nuclear condensation
and
fragmentation that were concurrent with the disappearance
of GRIM from
the cytoplasm of SF-21 cells, while the coexpression
of IAPs blocked
GRIM-induced nuclear condensation and fragmentation
and resulted in
accumulation of GRIM in punctate perinuclear locations
which coincided
with IAP localizations (data not shown).
The effect of Cp-IAP on the stability of HID.
While testing
the expression levels of the HID constructs, we found that we could
detect HID 1 h after induction (Fig.
8A, lane 1) but not by 4 h after
induction (Fig. 8A, lane 2). This finding prompted us to investigate
the stability of HID constructs under conditions when HID induced
apoptosis compared to conditions when HID-induced
apoptosis was blocked by Cp-IAP. Expression of hid-Epi,
hid(38-335)-Epi, or hid(1-37)C-Epi in SF-21 cells in the absence of
Cp-IAP induced apoptosis (Fig. 1B), and under these conditions
proteins that were present 1 h after induction (Fig. 8A, lanes 1, 5, and 9) could not be detected by 4 h after induction (Fig. 8A,
lanes 2, 6, and 10). Under conditions when Cp-IAP blocked apoptosis induced by various HID constructs, HID-Epi proteins accumulated during the 4-h period after induction, and this
accumulation coincided with the appearance of the additional bands of
higher molecular mass (Fig. 8A, lanes 3, 4, 7, 8, 11, and 12).
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|
FIG. 8.
The effect of Cp-IAP on the stability of HID. (A and B)
SF-21 cells were transiently transfected with plasmids expressing
combinations of genes indicated above the lanes of each panel and
harvested 1 h (lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19) or
4 h (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 21) after
induction. Immunoblot analysis was done with the mouse anti-HA.11
monoclonal antibody, and the arrows at the right point to the major
expected product for each of the expressed genes. Molecular mass
markers (in kilodaltons) are shown at the left.
|
|
On the other hand, expression of hid(336-410)-Epi or
hid
(2-14)-Epi did not induce apoptosis (Fig.
1B), and protein products
were stable; HID(336-410)-Epi and
HID
(2-14)-Epi accumulated during
the 4-h period following induction
when expressed with or without
Cp-IAP, and were accompanied by the
appearance of higher-molecular-mass
bands (Fig.
8A, lanes 13 to 20).
Additional bands of higher molecular
mass represent increments of 7 to
8 kDa and are probably results
of posttranslational modification. As a
control, we investigated
the stability of apoptotically neutral
protein, Epi-CAT, under
the same conditions as in Fig.
8A. The
amount of Epi-CAT decreased
approximately two- to fourfold under
conditions when HID induced
apoptosis (Fig.
8B, lanes 3 and 4),
and Epi-CAT was completely
stabilized when HID-induced
apoptosis was blocked by Cp-IAP (Fig.
8B, lanes 5 and 6).
This was in sharp contrast to proapoptotic
HID constructs which were
completely degraded by 4 h after induction
(Fig.
8A, lanes 2, 6, and 10). In additional experiments P35 was
equally efficient in
stabilizing HID proteins as Cp-IAP (data
not shown). As deletion of the
14 N-terminal amino acids abrogated
both proapoptotic activity and
instability of HID, these results
suggest that instability of HID is
conferred by the N-terminal
37 amino acids and may be linked to the
ability of these residues
to induce apoptosis.
In addition, we also found that levels of GRIM declined
when expressed individually and that coexpression with IAPs stabilized
GRIM (data not shown).
| |
DISCUSSION |
In this study, we have analyzed the nature of the apoptotic
response induced by the Drosophila genes hid and
grim in a heterologous insect cell line, SF-21. A role for
hid and grim in regulating apoptosis was
previously established in Drosophila embryos (5, 12), developing eyes (5, 12, 15), and the central
nervous system (20, 32). We have demonstrated that HID and
GRIM can induce apoptosis in a lepidopteran cell line, SF-21,
and investigated which portions of HID and GRIM possess proapoptotic
activity by generating a series of deletions. Our deletion analysis
showed that the N-terminal 37 amino acids of either HID or GRIM were sufficient to induce apoptosis if stably expressed as fusion
proteins. Furthermore, the only region of sequence similarity among
RPR, HID, and GRIM (14 N-terminal amino acids) is required for the induction of apoptosis by HID. In the case of GRIM, the
N-terminal 16 amino acids were sufficient to induce apoptosis.
The C-terminal truncations of HID and GRIM appeared to induce
apoptosis efficiently, but we cannot assess the relative
ability of mutant and wild-type proteins to induce apoptosis in
these assays because they rely on overexpression of the genes; in vivo,
the mutants may exhibit only partial activity.
Overexpression of the GRIM deletion lacking the proapoptotic
N-terminal 14 amino acids is still able to induce apoptosis, suggesting that GRIM might have two independent regions with
death-promoting abilities. Sequence analysis of amino acids
present in this GRIM deletion and comparison with other
known proapoptotic protein domains (e.g., death domain, death
effector domain, BH3, caspase or pro-caspase domains) did not
reveal any significant similarity, and therefore GRIM might
contain a novel death-inducing region. According to the mutational
analysis of RPR, deletion of RPR lacking this region of similarity
(4, 28) or site-specific mutants of the most conserved
residues of this region (4, 28) still retained proapoptotic
activity. Thus, the existence of two independent death-promoting
regions may not be unique to GRIM, and future mutational analysis of
GRIM will be required to define this novel death-inducing motif.
We have demonstrated that baculovirus and Drosophila iaps
physically associate with and block apoptosis induced by
Drosophila proteins HID and GRIM. The region encompassing
the BIR motifs of IAPs is both necessary and sufficient for this
interaction. In the case of baculovirus IAPs, the RING finger motif is
also important for antiapoptotic activity (8, 14) but its
role is not understood at this point. The N-terminal 37 amino acids of
HID and GRIM are sufficient to bind IAPs. We have not attempted to
further define this region of HID. In the case of GRIM, we have found
that the N-terminal 16 amino acids are sufficient to induce
apoptosis if stably expressed, suggesting that this limited region of similarity between Drosophila inducers of
apoptosis is important in the regulation as well as signaling
of these proteins. However, a GRIM deletion lacking the N-terminal 14 amino acids still induces apoptosis and associates with IAPs
indicating the presence of two distinct apoptosis-inducing
motifs within GRIM. Physical association of IAPs with HID, GRIM, and
RPR provides a possible mechanism by which IAPs can inhibit
apoptosis induced by these Drosophila inducers of
apoptosis. Through this interaction, IAPs may physically
block access of HID, GRIM, and RPR to downstream effector proteins, or
alternatively, this interaction may displace IAPs from binding as
inhibitors to effector proteins such as caspases.
Physical interaction of HID and GRIM with Cp-IAP or D-IAP1 results
in localization of HID and GRIM to the same cellular location as IAPs.
As HID and GRIM show predominantly diffused cytoplasmic staining in the
absence of IAPs, this localization is IAP directed. In SF-21 cells,
overexpression of IAPs leads to their localization to the punctate
perinuclear subcellular locations. However, that subcellular
localization is more pronounced for baculovirus IAPs than for
Drosophila IAPs, whose localization appears to be less punctate. We also do not exclude the possibility that HID might affect
colocalization of HID and D-IAP1 as colocalization of HID and
D-IAP1 is more punctate in its appearance than localization of
D-IAP1 alone. IAPs have the same effect on subcellular localization of
RPR (27). Therefore, the ability of IAPs to redirect RPR, HID, and GRIM to a different subcellular location and potentially block
the access of these inducers of apoptosis to downstream effectors may be a component of the mechanism by which IAPs inhibit induction of apoptosis by these proapoptotic proteins.
The rapid decline in the levels of HID following induction in SF-21
cells and its accumulation and relocalization in the presence of IAPs
are similar if not identical to the behavior of RPR (27) and
suggest an additional level of complexity in their regulation. The
N-terminal 37 amino acids of HID were sufficient to confer instability
to a fusion protein, while the C-terminal portion of HID lacking
the terminal 37 amino acids was stable. Since the N-terminal 37 amino
acids were sufficient to initiate apoptosis, the rapid decline
in HID levels may either be associated with activation of
apoptosis or due to proteolytic signaling by the N-terminal
residues themselves. The former is likely to be the case since
expression of P35, a caspase inhibitor which does not bind to HID, was
able to block the degradation of HID. In the case of RPR, we
demonstrated that aspartate-specific caspases are not directly
responsible for RPR degradation (27), suggesting that
another protease may be activated in the process of induction of
apoptosis. The rapid degradation of apoptotic inducers such as
HID and RPR would allow precision control of the concentration of
free inducers and finer tuning of the cellular environment. The binding
of IAPs to inducers and their cellular relocalization would
then serve to buffer the effect of a rapid rise in inducer concentrations which is known to precede apoptosis.
In addition to physically interacting with RPR, HID, and
GRIM, IAPs also associate with another Drosophila
proapoptotic protein, Doom (13), with mammalian TRAF-2
(21, 24), a protein implicated in TNF-induced signaling
pathways (21, 24), and with some mammalian members of the
caspase family of apoptotic effectors (10). None of these
proteins appear to contain sequences related to the N-terminal 16 amino
acids of RPR, HID, and GRIM. The ability of IAPs to physically interact
with a variety of inducers of apoptosis and to block
apoptosis induced by diverse stimuli places IAPs in a central
position as sensors and inhibitors of death signals that proceed
through a number of different pathways. Being at this position, IAPs
would serve as cellular checkpoints allowing apoptosis to ensue
only after the levels of inducers rise above the threshold determined
by IAP levels.
| |
ACKNOWLEDGMENTS |
We thank Hermann Steller (Massachusetts Institute of Technology)
for hid cDNA, John M. Abrams (University of Texas) for
grim cDNA and Somasekar Seshagiri for helpful discussions.
This work was supported in part by Public Health Service grant AI38262
from the National Institute of Allergy and Infectious Disease to L.K.M.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Genetics & Entomology, The University of Georgia, 413 Biological
Sciences Building, Athens, GA 30602-2603. Phone: (706) 542-2294. Fax:
(706) 542-2279. E-mail: miller{at}bscr.uga.edu.
| |
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Mol Cell Biol, June 1998, p. 3300-3309, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
