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HEMATOPOIESIS
Translocation of the inhibitor of apoptosis protein c-IAP1 from the nucleus
to the Golgi in hematopoietic cells undergoing differentiation:
a nuclear export signal–mediated event
Stéphanie Plenchette, Séverine Cathelin, Cédric Rébé, Sophie Launay, Sylvain Ladoire, Olivier Sordet,
Tibor Ponnelle, Najet Debili, Thi-Hai Phan, Rose-Ann Padua, Laurence Dubrez-Daloz, and Eric Solary
The caspase inhibitor and RING finger–
containing protein cellular inhibitor of
apoptosis protein 1 (c-IAP1) has been
shown to be involved in both apoptosis
inhibition and signaling by members of
the tumor necrosis factor (TNF) receptor
family. The protein is regulated transcriptionally (eg, is a target for nuclear factor–␬B [NF-␬B]) and can be inhibited by
mitochondrial proteins released in the
cytoplasm upon apoptotic stimuli. The
present study indicates that an additional
level of regulation of c-IAP1 may be cell
compartmentalization. The protein is
present in the nucleus of undifferentiated
U937 and THP1 monocytic cell lines. When
these cells undergo differentiation under
phorbol ester exposure, c-IAP1 translocates to the cytoplasmic side of the Golgi
apparatus. This redistribution involves a
nuclear export signal (NES)–mediated,
leptomycin B–sensitive mechanism. Using site-directed mutagenesis, we localized the functional NES motif in the
caspase recruitment domain (CARD) of
c-IAP1. A nucleocytoplasmic redistribution of the protein was also observed in
human monocytes as well as in tumor
cells from epithelial origin when undergoing differentiation. c-IAP1 does not trans-
locate from the nucleus of cells whose
differentiation is blocked (ie, in cell lines
and monocytes from transgenic mice
overexpressing B-cell lymphoma 2 [Bcl2] and in monocytes from patients with
chronic myelomonocytic leukemia). Altogether, these observations associate
c-IAP1 cellular location with cell differentiation, which opens new perspectives on
the functions of the protein. (Blood. 2004;
104:2035-2043)
© 2004 by The American Society of Hematology
Introduction
The inhibitors of apoptosis proteins (IAPs) have been initially defined as
natural cellular inhibitors of cell death. These proteins were identified in
baculoviral genome as regulators of host-cell viability during virus
infection,1 and cellular orthologues were subsequently described in
yeast, nematodes, drosophila, and mammals. The human genome
encodes at least 8 IAPs (X-linked IAP [XIAP], cellular IAP1 [c-IAP1],
c-IAP2, melanoma IAP [ML-IAP], neuronal apoptosis inhibitory protein [NAIP], survivin, IAP-like protein 2 [ILP-2], Apollon).2 All of these
proteins have in common the presence of 1 to 3 copies of a baculovirus
IAP repeat (BIR) domain.1 These domains are essential for the
antiapoptotic properties of the IAPs, which have been attributed to the
direct binding and inhibition of caspases. XIAP binds the small subunit
of caspase-9 through its BIR3 domain3 and masks the active site of
caspase-3 and -7 through a distinct segment, which is immediately
amino-terminal to its BIR2 domain.4,5 c-IAP1 and c-IAP2 bind caspase-3
and -7 but their inhibitory effect on caspases is 2- to 3-log lower than that
of XIAP.6 Some of the BIR-containing proteins do not have clear links
with apoptosis and several members of the family have demonstrated
distinct functions including cell cycle regulation,7 protein degradation,8
and caspase-independent signal transduction.9-12
In addition to the BIR domains, several IAPs, including XIAP,
c-IAP1, and c-IAP2, contain a highly conserved carboxy-terminal
RING finger domain that confers them an enzyme 3 (E3) function
in the protein ubiquitylation process. Several proteins specifically
targeted for ubiquitylation by IAPs have been identified. At least in
vitro, XIAP and c-IAP2 direct the ubiquitylation of caspase-3 and
caspase-7,13,14 whereas c-IAP1 and c-IAP2 mediate ubiquitylation
of second mitochondria-derived activator of caspase (Smac)/
DIABLO, an antagonist of IAPs.15 c-IAP1 and c-IAP2 are also
components of the type 2 tumor necrosis factor (TNF) receptor
complex through interaction with the signaling intermediates TNF
receptor–associated factor 1 (TRAF1) and TRAF2.9 c-IAP1 could
induce the ubiquitylation of TRAF2 and participated in the
TNF-␣–mediated proteasomal degradation of TRAF2,16 and cIAP2 has been involved in the TNF-␣ signaling leading to nuclear
factor–␬B (NF-␬B) activation.17
The expression and activity of IAPs are regulated at several levels.
The transcription factor NF-␬B enhances the expression of c-IAP1,
c-IAP2, and XIAP, which may contribute to the prosurvival effect
exerted in many situations by this transcription factor.18,19 XIAP
translation can be enhanced through the use of an internal ribosomal
entry site in the 5⬘-untranslated region of its messenger RNA.20 IAPs
could regulate their own degradation through autoubiquitylation,8
whereas the IAP-interacting proteins Smac/DIABLO and Omi/
HtrA2 neutralize XIAP and possibly other IAPs when released
from the mitochondria under apoptotic stimuli.21
From the Institut National de la Santé et de la Recherche Médicale (INSERM)
U517, INSERM EPI 106, IFR100, Dijon, France; INSERM U362, Institut
Gustave Roussy, Villejuif, France; INSERM EMI00-03, Institut Universitaire
d’Hematologie, Hopital St Louis, Paris, France; The Rayne Institute, King’s
College Hospital, London, United Kingdom.
Supported by grants from the Ligue Nationale Contre le Cancer.
Submitted January 8, 2004; accepted May 26, 2004. Prepublished online as
Blood First Edition Paper, June 8, 2004; DOI 10.1182/blood-2004-01-0065.
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
Reprints: Eric Solary, INSERM U517, IFR100, 7 boulevard Jeanne d’Arc,
21000 Dijon, France; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
2035
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2036
PLENCHETTE et al
Another level of regulation of IAP functions is the modulation
of their subcellular location. Such a regulation has been described
for XIAP whose interaction with the protein XIAP-associated
factor 1 (XAF1) induces its sequestration in the nucleus and
suppresses its caspase-inhibitory function.22 The present study
demonstrates that c-IAP1 is located in the nucleus of various
undifferentiated cells and migrates to the cytoplasm, more specifically to the Golgi apparatus, when these cells undergo differentiation. This redistribution of c-IAP1 involves a nuclear export signal
(NES) located in its caspase recruitment domain (CARD). Overexpression of c-IAP1 interferes with 12-O-tetradecanoylphorbol
13-acetate (TPA)–induced differentiation of leukemic cells, a
process also inhibited by the nuclear export inhibitor leptomycin B
(LMB). Altogether, these observations suggest a role for c-IAP1 in
cell differentiation.
Patients, materials, and methods
Antibodies and chemicals
We used mouse monoclonal antibodies (mAbs) directed against c-IAP1
(PharMingen, La Jolla, CA); Golgin 97 (clone CDF4; Molecular Probes,
Eugene, OR); mitochondrial HSP70 (mHSP70; Affinity BioReagent, Golden,
CO); HSC70 (Santa Cruz Biotechnology, Santa Cruz, CA); GM130 (Golgi
Matrix protein of 130 kDa; fluorescein isothiocyanate [FITC]–conjugated
antibody; Transduction Laboratories, Lexington, KY); rabbit polyclonal
Abs targeting c-IAP1 (Santa Cruz Biotechnology and R&D Systems,
Abington, United Kingdom); macrophage antigen-1 (Mac-1; phycoerythrin
[PE]–conjugated antibody; Pharmingen, Becton Dickinson, Heidelberg,
Germany); Bcl-2 (FITC-conjugated antibody; Pharmingen, Becton Dickinson); CD1a (FITC-conjugated antibody; Pharmingen, Becton Dickinson),
CD71 (FITC-conjugated antibody; Pharmingen, Becton Dickinson); poly(adenosine diphosphate-ribose) polymerase (PARP; Boehringer-Mannheim, Mannheim, Germany); XIAP (R&D Systems and Stressgen Biotech,
San Diego, CA); protein disulfide isomerase (PDI; Calbiochem, La Jolla,
CA); green fluorescent protein (GFP; Invitrogen, Cergy Pontoise, France);
and survivin (Novus Biologicals, Littleton, CO). Macrophage colonystimulating factor (M-CSF), granulocyte-macrophage colony-stimulating
factor (GM-CSF), and interleukin-4 (IL-4) were obtained from R&D
Systems; erythropoietin (EPO) was from Amgen (Thousand Oaks, CA);
TPA was from Sigma-Aldrich Laboratories (St Quentin Fallavier, France);
brefeldin A (BFA) and nocodazole were from Alexis Biochemicals (Lausen,
Switzerland); and trypsin-EDTA (ethylenediaminetetraacetic acid) was
from Gibco-BRL (Carlsbad, CA). LMB was kindly provided by Dr M.
Yoshida (Tokyo, Japan) and thrombopoietin (TPO) was kindly provided by
Kirin Brewery (Tokyo, Japan).
Cell culture and differentiation
Cell lines were obtained from the American Type Culture Collection
(ATCC, Rockville, MD) and cultured as described.23 We also tested the
previously described Bcl-2–transfected U937 and HT29 cells and HT29MTX cells.23-25 The TPA-resistant variant of U937 cells were kindly
provided by Prof P. J. Parker (London, United Kingdom).26 Monocytes
from human peripheral blood were obtained with informed consent from
healthy donors and 7 patients with chronic myelomonocytic leukemia
(CMML) and purified using an isolation kit (Miltenyi Biotec, Paris, France)
following the manufacturer’s instructions. Cells were differentiated into
macrophages or dendritic cells and checked for the expression of differentiation marker CD71 and CD1a as described.23 Peripheral blood CD34⫹
cells were cultured in liquid conditions in the presence of cytokines to
generate megakaryocytes or erythroid cells as described.27,28 The Bcl-2
transgenic mice were obtained from Irv Weismann (Stanford, CA).29 Bcl-2
overexpression in Mac-1⫹ cells of transgenic mice was verified by flow
cytometry using a FACSCalibur cytometer and Cell Quest software
(Pharmingen, Becton Dickinson). Femoral bone marrow cells were isolated
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
from 6- to 8-week-old control and transgenic FVB/N female mice and
cultured for 4 hours on plastic plates before culturing adherent cells for 6
days in the presence of 10% L929 cell–conditioned medium as source of
CSF-1. Macrophage differentiation was assessed by May-GrünwaldGiemsa staining.
Immunofluorescence studies
Cells were fixed in paraformaldehyde (PFA; 2%) for 10 minutes at room
temperature, washed twice, saturated in phosphate-buffered saline (PBS)
containing 0.1% saponin and 5% nonfat milk, and incubated overnight at
room temperature in the presence of primary Ab diluted in PBS containing
0.1% saponin and 0.5% bovine serum albumin (BSA). After washing, cells
were incubated for 30 minutes with 488-alexa goat antirabbit or antimouse
Ab (Molecular Probes) and washed 3 times with PBS. Nuclei were stained
by Hoechst 33342 (Sigma-Aldrich). To demonstrate colocalization of
c-IAP1 with Golgin 97 or GM130, cells were first incubated with
anti–c-IAP1 Ab overnight at 4°C, then with the secondary biotinylated–
immunoglobulin (Ig; 1:100; Amersham Biosciences, Orsay, France) for 1
hour at room temperature, then with a streptavidin–texas red–conjugated
Ab (Molecular Probes; 1:2000) for 1 hour. Cells were subsequently
incubated for 1 hour at room temperature with anti-GM130–FITC (1:100)
or anti–Golgin 97 (1:100), then with FITC-conjugated antimouse Ab.
Fluorescence was preserved using the FluorSave mounting medium (Calbiochem). Analysis was performed using either a fluorescence (Nikon Eclipse
80i; Nikon, Champigny, France) or a confocal (Leica TCS SP2; Leica,
Bron, France) microscope (objective ⫻ 50; original magnification ⫻ 500).
The images were captured by a 3 CCD (charge-coupled device) color video
camera (Sony, Paris, France), digitally saved using Archimed-Pro software
(Microvision Instruments, Evry, France), and further processed using
Photoshop software (Adobe Systems France, Paris, France).
Preparation of cellular extracts and Western blot analysis
Whole-cell lysates and nuclear-free extracts were prepared as described.23
Nuclear and cytoplasmic fractions were obtained by lysing the cells in lysis
buffer (10 mM Hepes [N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic
acid], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA [ethyleneglycoltetraacetic acid], 1 mM DTT [dithiothreitol], 0.6% NP-40 [nonidet P–40]) in the
presence of the protease inhibitors. Cell lysate was centrifuged at 1200g for
10 minutes. The supernatant was carefully collected (cytoplasmic fraction
[C]) and the pellet was washed once then resuspended in lysis buffer
(nuclear fraction [N]). Further cell fractionation was performed as described.30 All fractions were stored at ⫺80°C until Western blotting
analysis, and protein concentration was measured using the Bio-Rad DC
protein assay kit (Hercules, CA). Western blot experiments were performed
as previously described.23
Trypsin digestion of microsomal proteins
Proteins from reticular/microsomal-enriched fraction were digested by
0.05% trypsin in the presence of 0.02% EDTA for 30 minutes at 37°C and
analyzed by Western blotting for c-IAP1 content.31
Plasmid constructs
Plasmid-enhanced GFP (pEGFP)–c-IAP1 was constructed by subcloning
full-length c-IAP1 cDNA (kindly provided by J. C. Reed, La Jolla, CA) into
the BglII/SalI site of pEGFP-C1 (Clontech, Palo Alto, CA). Sense and
antisense oligonucleotides corresponding to leucine-rich motif (LRM)
putative NES were as follows: LRM1 sense, 5⬘-GAT CTT TTT TGG AAA
ATT CTC TAG AAA CTC TGA GGA-3⬘; LRM1 antisense, 5⬘-GAT CTC
CTC AGA GTT TCT AGA GAA TTT TCC AAA AAA-3⬘; LRM2 sense,
5⬘-GAT CTC TCT TTC AAC AAT TGA CAT GTG TGC TTC CTA TCC
TGG ATA ATC TTT TAA-3⬘; LRM2 antisense, 5⬘-GAT CTT AAA AGA
TTA TCC AGG ATA GGA AGC ACA CAT GTC AAT TGT TGA AAG
AGA-3⬘; LRM3 sense, 5⬘-GAT CTC TGT CAC TGG AAG AAC AAT TGA
GGA GGT TGC AAA-3⬘; and LRM3 antisense, 5⬘-GAT CTT TGC AAC
CTC CTC AAT TGT TCT TCC AGT GAC AGA-3⬘ (Proligo France SAS,
Paris, France). Complementary oligonucleotides were annealed and cloned
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
DIFFERENTIATION-INDUCED NUCLEAR EXPORT OF c-IAP1
2037
in a sense orientation into the BglII site of pEGFP-C1 (Clontech). All
sequences are expressed at the C-terminus of GFP. Full-length c-IAP1
mutants (GFP–c-IAP1–LRM1*,–LRM2*, and –LRM3*) were obtained by
mutagenesis of LRM1, 2, and 3, separately or in combination (leucine were
replaced by alanine) using the Quick-Change Site-directed Mutagenesis Kit
(Stratagene, La Jolla, CA). All constructs were sequenced to ensure the
accuracy of the reading frames and the site-directed mutations.
Cell transfection
HeLa cells were transfected 24 hours after seeding using Superfect
transfection reagent (Qiagen, Valencia, CA) following the manufacturer’s
instructions. Cells were studied 24 hours after transient transfection: nuclei
were stained with Hoechst 33342 and cells were fixed with 2% PFA for 5
minutes before studying the subcellular distribution of GFP fusion protein
using a fluorescence (Nikon) or a confocal (Leica) microscope. THP1 cells
were transiently transfected using the AMAXA nucleofector kit (Amaxa,
Köln, Germany) and transfected cells were enriched by a 10-day geneticin
selection (0.7 ␮g/mL) before expansion and treatment.
Results
TPA-induced differentiation of human monocytic cell lines
is associated with the redistribution of c-IAP1 and XIAP
from the nucleus into the cytoplasm
It has been previously shown that exposure of U937 cells to 20 nM TPA
induced their differentiation into macrophage-like cells. Cells become
adherent to the culture flask and the expression of CD11b at their plasma
membrane increases.23 We used Western blotting to analyze the expression of XIAP, c-IAP1, c-IAP2, and survivin, 4 proteins that belong to the
IAP family, in U937 cells undergoing TPA-induced differentiation
(Figure 1A). c-IAP2 could not be detected in undifferentiated U937 cells
and remained undetectable at all steps of the differentiation process (not
shown). Survivin expression was limited to the nucleus of undifferentiated cells and disappeared upon differentiation. This may be related to
the differentiation-associated cell cycle exit since this protein, which has
an evolutionarily conserved role as a mitotic spindle checkpoint protein,
is expressed mainly in dividing cells.7 The expression of XIAP and
c-IAP1 was poorly influenced by the differentiation process when
studied in whole-cell lysates (Figure 1A left). However, c-IAP1, and to a
lesser extent XIAP, progressively accumulated in nuclear-free extracts
as the cells underwent differentiation (Figure 1A right). The present
study focused on c-IAP1 redistribution.
Differentiation-associated redistribution of c-IAP1 from the
nucleus to the cytoplasm was further confirmed by Western blotting
analysis of c-IAP1 expression in TPA-treated THP1 cells (Figure
1C) and by fluorescent microscopy analysis of the 2 cell lines
(Figure 1B,D). c-IAP1 was located mainly in the nucleus of U937
and THP1 undifferentiated cells and in the cytoplasm of TPAdifferentiated cells. A kinetic analysis identified a transient diffuse
staining of the cytoplasm in the first hours of TPA treatment. As the
cells progressed toward the differentiation process, a more patchy
staining close to the nucleus was observed (see THP1 cells in
Figure 1D).
c-IAP1 colocalizes with the Golgi apparatus
of differentiated cells
To precisely determine the subcellular localization of c-IAP1 in
TPA-differentiated cells, we performed Western blot experiments
in enriched cellular fractions. Figure 2A shows that c-IAP1 is
localized in the nucleus of undifferentiated U937 cells and in the
reticular fraction of TPA-differentiated U937 cells. Thus, in
Figure 1. c-IAP1 redistribution in human leukemia cell lines undergoing
TPA-induced differentiation. U937 (A-B) and THP1 (C-D) cells were treated
for indicated times with 20 nM TPA to induce a macrophage-like differentiation.
(A,C) Western blot analysis of indicated proteins in whole-cell, cytoplasmic, and
nuclear extracts. HSC70 was used as a loading control. (B,D) Fluorescence
microscopy analysis of c-IAP1 (green), as observed using an anti–c-IAP1 mAb
(Pharmingen). Nuclei, labeled with Hoechst 33342, are stained in blue. Magnification
⫻ 300.
accordance with Figure 1A, the protein migrates from the nucleus
to the cytoplasm. A similar observation was made by comparing
cellular fractions of undifferentiated and differentiated THP1 cells
(not shown). Fluorescence microscopy experiments indicated that
c-IAP1 colocalized with Golgin 97, a Golgi matrix protein, in
TPA-differentiated THP1 (Figure 2B) and U937 (not shown) cells.
c-IAP1 also colocalized, although less precisely, with GM130, a
protein associated with the cis-Golgi (Figure 2B). Addition of
either BFA, a fungal metabolite that causes disintegration of Golgi
structure through inhibition of adenosine diphosphate (ADP)–
ribosylation factor (ARF) guanosine 5⬘-triphosphate (GTP)–
binding proteins,32 or nocodazole, a microtubule-depolarizing
agent, suppressed the patchy staining of c-IAP1 in TPAdifferentiated THP1 (Figure 2C) and U937 (not shown) cells. In the
tested conditions, BFA did not modify calnexin C subcellular
localization, indicating that the endoplasmic reticulum was not
altered (not shown). Altogether, these observations indicated that
c-IAP1 was redistributed to the Golgi apparatus in cells undergoing
differentiation.
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2038
PLENCHETTE et al
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
The differentiation-induced nuclear export of c-IAP1 involves
a nuclear export signal
To characterize the mechanisms that are responsible for the nuclear
export of c-IAP1, we first used LMB, a specific inhibitor of
exportin 1 (also known as CRM1 [chromosomal region maintenance-1]), which is the receptor for leucine-rich NES.33 Addition of
100 nM LMB for 24 hours to TPA-treated THP1 (Figure 3A) and
U937 (not shown) cells prevented the redistribution of c-IAP1. To
confirm the ability of LMB to inhibit the nuclear export of c-IAP1,
we used a construct encoding full-length c-IAP1 associated,
through its N-terminus, to GFP. This construct was transiently
expressed in HeLa and 293T cell lines, in which the transfection
rate was much higher than in leukemic cell lines. Twenty-four
hours after transfection of GFP–c-IAP1 construct in these cells, the
fluorescence was detected in both the nucleus and the cytoplasm. In
the presence of LMB, the protein accumulated in the nucleus (see
HeLa cells on Figure 3B). These results suggested that the nuclear
export of c-IAP1 involved an NES and CRM1.
A leucine-rich motif in the CARD behaves as a nuclear
export signal
A software-based search in the protein sequence of c-IAP1
identified 3 hydrophobic LRMs that were consensus sequences for
potential NESs. The first one was located in the BIR2 domain
(LRM1), the second one in the CARD (LRM2), and the last one
between the CARD and the RING domain (LRM3; Figure 4A). To
determine whether one or several of these motifs played a role in
c-IAP1 nuclear export, we cloned the sequences encoding these
motifs in a GFP-encoding vector and expressed them by transient
Figure 2. c-IAP1 is localized to the Golgi apparatus in differentiated cells.
(A) Western blot analysis of c-IAP1 (pAb; Santa Cruz Biotechnology) expression in
the mitochondrial (M), cytosolic (C), reticular/microsomal (R), and nuclear (N)
fractions obtained from U937 cells before (Co) and after exposure to 20 nM TPA for
72 hours. The expression of poly(ADP-ribose)polymerase (PARP), Golgin 97, and
mitochondrial HSP70 was used to assess the enrichment of each cell fraction. (B)
THP1 cells were treated with TPA for 48 hours before analyzing the expression of
c-IAP1 (red), Golgin 97 (green), or GM130 (green) by confocal microscopy (magnification ⫻ 300). (Insets) Increased magnification of Golgi labeling (magnification
⫻ 3000). (C) c-IAP1 expression in TPA-differentiated THP1 cells before (Co) and
after exposure to either brefeldin A (BFA; 5 ␮g/mL; 2 h 30 min) or nocodazole (10 ␮M;
1 h). c-IAP1 expression was observed by fluorescence microscopy (magnification
⫻700) using an anti–c-IAP1 mAb (Pharmingen). (D) Western blot analysis of c-IAP1
expression under limited proteolytic digestion of the reticular/microsomal fraction of
TPA-differentiated U937 cells. Golgin 97 and protein disulfide isomerase (PDI) are
used as positive and negative controls, respectively.
c-IAP1 is located to the cytoplasmic side of the Golgi apparatus
in differentiated cells
To determine the topologic orientation of c-IAP1 in the Golgi
compartment of differentiated cells, we isolated the microsomal
fraction from TPA-treated U937 cells and submitted this fraction
to tryptic-limited digestion before Western blotting analysis
(Figure 2D). Addition of trypsin resulted in complete digestion
of Golgin 97, which is located on the cytoplasmic side of the
Golgi apparatus, whereas the endoplasmic reticulum–lumenal
PDI was resistant to trypsin digestion. In these conditions,
trypsin completely digested c-IAP1, indicating that the protein
may be located to the external cytoplasmic side of the
Golgi apparatus.
Figure 3. c-IAP1 redistribution involves a leptomycin B–sensitive mechanism.
(A) Fluorescence microscopy analysis of c-IAP1 expression (Pharmingen mAb;
green) in THP1 cells treated with 20 nM TPA for 24 hours in the presence or absence
of 100 nM leptomycin B (LMB). Hoechst 33342 was used to stain the nuclei (blue).
(B) HeLa cells were transiently transfected with a GFP–c-IAP1 construct before
staining the nuclei with Hoechst 33342 and fluorescence microscopy analysis. When
indicated, LMB (200 nM) was added 3 hours before analysis. Magnification is ⫻ 500
for panels A and B.
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
DIFFERENTIATION-INDUCED NUCLEAR EXPORT OF c-IAP1
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LRM3 were mutated, GFP-associated c-IAP1 demonstrated a
cytoplasmic and nuclear expression similar to that of the wild-type
protein. Altogether, these results indicated that LRM2 was a
functional NES in c-IAP1.
Overexpressed c-IAP1 interferes with the
differentiation process
LMB was observed to prevent TPA-induced differentiation of THP1, as
demonstrated by studying the CD11b marker (Figure 6A), which
suggested that a nucleocytoplasmic redistribution of proteins was
required for the differentiation process. In an attempt to determine
whether c-IAP1 was one of the proteins whose nuclear export was a key
event in this process, we transiently overexpressed the GFP-tagged
LRM2 mutant of c-IAP1 in THP1 cells. The protein was located mainly
in the nucleus of transfected cells (Figure 6B) and TPA exposure failed
to increase CD11b expression in GFP-tagged cells (Figure 6C-D). In
addition, adhesion of GFP-positive cells to the culture flasks was
delayed (not shown). However, similar results were obtained when
wild-type c-IAP1 was transiently overexpressed in THP1 cells (Figure
6B-C). These results indicated that c-IAP1 overexpression could interfere with cell differentiation.
Figure 4. Identification of a potential nuclear export sequence in c-IAP1.
(A) (Top) Schematic representation of amino acid motifs in c-IAP1 protein (619 amino
acids). Leucine-rich motifs (LRMs) that could behave as nuclear export signal (NES)
are indicated (BIR indicates baculovirus IAP repeat; CARD, caspase recruitment
domain). (Bottom) Amino acid sequence of regions containing a potential LRM
(underlined). (B-C) The cDNA sequences encoding the 3 LRMs were fused to GFP in
the pEGFP-C1 vector. These constructs were transiently transfected into HeLa cells
and microscopy analyses were performed 24 hours later. Nuclei were stained with
Hoechst 33342 (magnification ⫻ 500). When indicated, LMB (200 nM) was added 3
hours before analysis.
transfection in HeLa and 293T cells. The subcellular location of
GFP fusion proteins was examined 24 hours after transfection by
conventional (Figure 4B) and confocal laser (not shown) microscopy. While GFP-associated LRM1 and LRM3 were expressed in
both the nucleus and the cytoplasm, GFP-associated LRM2 was
almost exclusively expressed in the cytoplasm (Figure 4B-C). In
addition, exposure to leptomycin B induced accumulation of the
GFP-LRM2 protein in the nucleus (Figure 4C). These results
indicated that LRM2 was the only sequence to behave as a
functional NES.
To determine whether this potential NES was functional in the
whole protein, a series of mutants were prepared in which leucine
amino acids in the LRMs were replaced by alanine residues. The
mutated constructs fused to GFP in a plasmid vector were
transiently transfected in HeLa (Figure 5) and THP1 (not shown)
cells, and their subcellular location was analyzed by fluorescence
microscopy and Western blotting 24 hours later. As shown previously (Figure 3B), overexpressed wild-type c-IAP1 in HeLa cells
demonstrated a cytoplasmic and nuclear pattern of expression.
Mutations in either LRM1 or LRM3 or both did not affect the
cellular distribution of the protein, whereas all the LRM2 mutants
accumulated in the nucleus (Figure 5A). For unknown reasons,
LRM2 mutant was less expressed than wild-type protein and other
mutants. These observations were confirmed by immunoblotting
the nuclear and cytoplasmic fractions of transiently transfected
HeLa (Figure 5B) and THP1 (not shown) cells with an anti-GFP
Ab. These experiments indicated that overexpressed wild-type
c-IAP1 was detected mainly in the cytoplasmic fraction. When
leucine residues in LRM2 were mutated, GFP-associated protein
was located in the nucleus. When leucine residues in LRM1 or
Figure 5. LRM2 is the functional nuclear export signal in c-IAP1. (A) Fluorescence microscopy analysis of HeLa cells transfected for 24 hours with constructs
encoding wild-type or mutated GFP–c-IAP1 (nuclei were stained with Hoechst
33342). Leucine residues in LRMs (LRM1*: Leu250, Leu254, Leu257; LRM2*:
Leu468, Leu472, Leu476, Leu483; LRM3*: Leu556, Leu558, Leu562, Leu565) were
replaced by alanine residues using site-directed mutagenesis. *Mutated constructs
(magnification ⫻1000). (B) Western blot analysis of GFP expression in nuclear (N)
and cytoplasmic (C) extracts from HeLa cells transfected 24 hours before with
indicated constructs.
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
PLENCHETTE et al
Figure 6. Overexpressed c-IAP1 interferes with TPA-induced THP1 cell
differentiation. (A) Flow cytometry analysis of CD11b membrane expression
in THP1 cells treated with 20 nM TPA for indicated times (h) in the presence or
absence of 100 nM leptomycin B (LMB). Gray histograms indicate treated
cells; and white histograms, untreated cells. (B) Western blot analysis of GFP
expression in nuclear (N) and cytoplasmic (C) extracts from THP1 cells
transfected with wild-type (wt–c-IAP1) and LRM2-mutated (c-IAP1–LRM2*)
GFP–c-IAP1. (C) Flow cytometry analysis of CD11b expression in GFPpositive THP1 cells transfected with pEGFP empty vector (vector) or wild-type
(wt–c-IAP1) or LRM2-mutated (c-IAP1–LRM2*) GFP–c-IAP1 constructs. Cells
were treated with 20 nM TPA for indicated times (h). Gray histograms indicate
treated cells; and black lines, control untreated cells. (D) Fluorescence
microscopy analysis of THP1 cells transfected with pEGFP empty vector
(vector) or LRM2-mutated GFP–c-IAP1 construct (c-IAP1–LRM2*). Cells were
incubated with 20 nM TPA for 48 hours and labeled with an anti-CD11b Ab
(red). Nuclei were stained with Hoechst 33342 (blue). Magnification ⫻ 600.
The nucleocytoplasmic redistribution of c-IAP1 is observed
in several differentiation pathways
c-IAP1 was observed to be present mainly in the nucleus of the
CD34⫹ progenitor, in both the nucleus and the cytoplasm of
peripheral blood monocytes, and exclusively in the cytoplasm of
macrophages and dendritic cells obtained by ex vivo differentiation
of monocytes (Figure 7A) as well as erythroblasts and megakaryocytes obtained by ex vivo differentiation of CD34⫹ cells (Figure
7B).27,28 As previously observed in TPA-differentiated cells (Figure
1B), c-IAP1 demonstrated a punctuated expression in the perinuclear zone and colocalized with Golgin 97 in macrophages
(Figure 7C) and dendritic cells (not shown) obtained by differentiation of normal peripheral blood monocytes. A differentiationassociated redistribution of c-IAP1 from the nucleus to the
cytoplasm was also observed in nonhematopoietic cells (ie, in
HT29 human colon carcinoma cells undergoing partial differentiation when grown at confluence).34 c-IAP1 also demonstrated a
cytoplasmic expression in the well-differentiated, mucus-secreting
HT29/MTX clone (Figure 7D).25
c-IAP1 does not translocate from the nucleus when cell
differentiation is inhibited
The redistribution of c-IAP1 observed in parental U937 cells
when undergoing TPA-induced differentiation was not identified
in a TPA-resistant U937 cell clone treated under the same
conditions (Figure 7E). We have previously shown that Bcl-2
overexpression prevented TPA-induced differentiation in U937
human leukemia cells.23 The nucleocytoplasmic redistribution
of c-IAP1 observed in TPA-treated parental cells transfected
with an empty vector was not identified in Bcl-2–overexpressing
U937 cells treated under similar conditions (Figure 7E). To
confirm this latter observation, we tested the differentiation of
bone marrow monocytes obtained from control and transgenic
FVB/N mice overexpressing Bcl-2 in Mac-1⫹ cells. Whereas
c-IAP1 translocation to the cytoplasm was observed in control
cells induced to differentiate into macrophages by ex vivo
culture in the presence of CSF-1–containing medium, no
redistribution of the protein could be detected in cells from
transgenic FVB/N mice cultured under similar conditions
(Figure 7F). We also cultured peripheral blood mononuclear
cells from 7 patients with chronic myelomonocytic leukemia
(CMML) in the presence of M-CSF or the GM-CSF/IL-4
combination for 6 days. Cell differentiation was assessed
morphologically and confirmed by studying cell phenotype
using CD71 and CD1a to identify macrophages and dendritic
cells, respectively. Mononuclear cells from these patients failed
to differentiate, and this blockade in cell differentiation was
associated with a lack of c-IAP1 nucleus export (see Figure 7G
for example).
Discussion
Two main functions have been assigned to c-IAP1. The protein is
involved in the signaling induced by engagement of several
members of the TNF receptor family including TNF-R2,9 CD40,35
and the lymphotoxin-␤ receptor.36 c-IAP1 was also described as an
endogenous inhibitor of apoptosis through direct binding to the
active sites of caspase-3 and -7.37 The recently described role of the
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BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
DIFFERENTIATION-INDUCED NUCLEAR EXPORT OF c-IAP1
2041
Figure 7. c-IAP1 redistribution is a differentiation-associated
event in various cell types. (A) Fluorescence microscopy
analysis of c-IAP1 (mAb; Pharmingen) in peripheral blood CD34⫹
cells and monocytes (Mo) obtained from healthy donors and in
macrophages (M⌽) and dendritic cells (DC) obtained from monocytes cultured for 6 days in the presence of M-CSF or GM-CSF/
IL-4, respectively. The top panels show c-IAP1 alone (green); the
bottom panels, c-IAP1 (green) ⫹ Hoechst 33352–labeled nuclei
(blue). (B) Fluorescence microscopy analysis of c-IAP1 (mAb;
Pharmingen; green) in CD34⫹ cell–derived erythoblasts (Ery) and
megakaryocytes (Meg). Nuclei were labeled simultaneously with
Hoechst 33352. (C) Colocalization of Golgin 97 (green) and
c-IAP1 (red) in macrophages derived from peripheral blood
monocytes as described for panel A. (D) Fluorescence microscopy analysis of c-IAP1 (pAb; Santa Cruz Biotechnology) in HT29
cells studied before (control) and after (confluent) reaching confluence in culture and in a methotrexate-resistant, well-differentiated
derivative cell clone (HT29-MTX). (E) Fluorescence microscopy
analysis of c-IAP1 (pAb; Santa Cruz Biotechnology) in control
(Co), Bcl-2–overexpressing (Bcl-2), and TPA-resistant U937 cells
exposed for 72 hours to 20 nM TPA. (F) May-Grünwald-Giemsa
staining (MGG) and fluorescence microscopy analysis of c-IAP1
(pAb; Santa Cruz Biotechnology; Bcl-2) in bone marrow monocytes from control (Co) and Bcl-2 transgenic (Bcl-2) mice, cultured
for 3 days in the presence of CSF-1–containing medium.
(G) Peripheral blood mononuclear cells obtained from patients
with chronic myelomonocytic leukemia (CMML) were cultured for
6 days in the presence of M-CSF or GM-CSF/IL-4. The top panels
show fluorescence microscopy analysis of c-IAP1; the bottom
panels, flow cytometry analysis of CD71 and CD1a membrane
expression. White histograms indicate CMML patient; and gray
histograms, healthy donor. One representative of 7 studied patients
is shown. Magnification: ⫻ 700 (A-C, E, G) and ⫻ 500 (D, F).
protein in ubiquitylation may contribute to both functions (eg, by
regulating the cellular level of the adaptor molecule TRAF216 or
the apoptosis inducer Smac/DIABLO).15 These functions imply a
cytoplasmic localization of the protein. We show here that c-IAP1
is present almost exclusively in the nucleus of the studied
undifferentiated cells, translocates to the cytoplasm when these
cells undergo differentiation, and localizes mainly to the Golgi
apparatus in differentiated cells.
Several arguments suggest a translocation of the protein rather
than a degradation followed by a synthesis in a distinct cellular
compartment. First, c-IAP1 mRNA (not shown) and protein levels
remain stable along the differentiation. Secondly, by immunofluorescence analysis, the protein was identified in the nucleus of
undifferentiated cells, in the cytosol of cells undergoing differentiation, and in the Golgi of differentiated cells, suggesting a migration.
Third, we have identified a functional NES in the protein. The
nuclear transport of proteins through nuclear pores requires the
presence of specific signals such as nuclear localization signals
(NLSs) and NESs. The size of c-IAP1 prevents it from passively
diffusing through nuclear pores38 and we did not identify any
classical NLSs in the protein, which suggests that the protein is
either carried by a cofactor or enters the nucleus using a nonconventional mechanism of active import. On the other hand, the protein
export may involve an NES-mediated, LMB-sensitive mechanism.
The retention of c-IAP1 in the nucleus of undifferentiated cells
suggests inhibition of this NES-mediated export. Activation of
such an active export mechanism has been shown in other proteins
to require an event that promotes their interaction with CRM1 such
as phosphorylation,39 monoubiquitylation,40 conformational
changes,41 or proteolytic cleavage.42 So far, we did not detect any
posttranslational modification of the protein in cells undergoing
differentiation. The location of the functional NES of c-IAP1 in its
CARD, a motif involved in protein-protein interactions, could also
indicate a role for an unidentified protein partner in the modulation
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2042
BLOOD, 1 OCTOBER 2004 䡠 VOLUME 104, NUMBER 7
PLENCHETTE et al
of c-IAP1 translocation. Such a protein partner has been identified
for XIAP, which can be retained in the nucleus through interaction
with XAF1.22
We have observed that c-IAP1 was located in the Golgi
apparatus of differentiated cells. Another IAP, referred to as
BRUCE in mice and Apollon in humans, also localizes to the Golgi
compartment and the vesicular system.43 In addition to being
related to IAPs through a BIR motif, this giant protein is an
ubiquitin-conjugating enzyme (E2).44 However, the functions of
both Apollon and c-IAP1 in the Golgi apparatus remain to be
elucidated. Proteins that accumulate in the Golgi structure can be
secreted.45 We have observed that c-IAP1 did not accumulate in
cholesterol- and sphingomyelin-enriched fractions of differentiated
cells (data not shown), suggesting that the protein did not associate
with microdomains described in the secretory pathway.46 c-IAP1
was shown to alter the cellular distribution of coexpressed reaper
and grim drosophila proteins in mammalian cells, suggesting that
c-IAP1 could modulate signaling pathways by sequestration of
proteins in cell compartments.47
c-IAP1 is not the only IAP to translocate from the nucleus to the
cytoplasm. The main isoform of survivin, a single-BIR–containing
IAP survivin involved in the control of the mitotic spindle
checkpoint,7 can be exported from the nucleus through an LMBsensitive mechanism, whereas its alternative isoform survivin⌬Ex3 is driven to the nucleus by a C-terminal NLS.48 In the present
study, we have observed that XIAP was also exported from the
nucleus in U937 cells undergoing differentiation, together with
c-IAP1. Of importance, survivin is ubiquitylated and degraded
when cells exit the cell cycle, whereas c-IAP1 and XIAP protein
levels remain stable along the differentiation process, which
suggest that cellular redistribution of these latter proteins may be
essential in their regulation.
Two IAPs have previously been shown to interfere with cell
differentiation (ie, overexpressed human NAIP prevents neuronal
differentiation of PC12 cells,49 whereas transgenic mice overexpressing XIAP in their lymphocytes demonstrate altered T-cell maturation).50 Here, we show that overexpression of either c-IAP1 or its
NES-mutated protein interferes with TPA-induced differentiation
of human leukemic cells. How these IAPs interfere with cell
differentiation remains unidentified. The first function assigned to
c-IAP1 has been its ability to interact with and to inhibit caspases.38
These enzymes have been involved in cell differentiation processes, both in humans23,27 and drosophila.51 Obviously, caspase
activity must be carefully controlled when associated with cell
differentiation to prevent cell death by apoptosis and it is attractive
to speculate that IAPs play a role in this control. In accordance with
this hypothesis, the BIR-containing protein dBRUCE was proposed to bind to and degrade caspases involved in drosophila
spermatogenesis.51 We have recently shown that a limited activation of several caspases was required for the differentiation of
human peripheral blood monocytes into macrophages, whereas
their differentiation into dendritic cells did not depend on caspase
activation.23 Since the nucleocytoplasmic translocation of c-IAP1
was observed in both caspase-dependent (macrophages) and -inde-
pendent (dendritic cells) pathways of differentiation, the negative
control of caspase activity may not be the function of c-IAP1
shuttling. In addition, caspases have been observed moving from
the cytoplasm to other cell compartments in leukemic cells
undergoing differentiation but were not identified to associate with
the Golgi apparatus.52 Finally, we have shown previously that the
postmitochondrial pathway to apoptosis remained functional in
TPA-differentiated U937 cells, indicating that redistributed c-IAP1
did not prevent caspase activation by cytochrome-c released from
the mitochondria.
Another function assigned to c-IAP1 and other IAPs containing
a RING finger motif is ubiquitylation of proteins. c-IAP1 catalyzes
its own ubiquitylation in vitro,8 promotes ubiquitylation of the
apoptosis inducer Smac/DIABLO,15 and modulates cell response to
TNF-␣ through the regulation of the intracellular level of TRAF216
and NF-␬B essential modulator (NEMO).53 Preliminary studies
suggest that c-IAP1 down-regulation decreases the proliferation
rate of THP1 cells (data not shown). Together with the role of the
ubiquitin/proteasome pathway in the regulation of cell cycle, this
observation could indicate a connection between c-IAP1 redistribution and growth inhibition in cells undergoing differentiation.
The nucleocytoplasmic traffic of proteins modulates cellular
functions.54 Accordingly, LMB prevents TPA-induced differentiation of U937 and THP1 cells. However, the role of the inhibition of
c-IAP1 translocation in LMB-induced inhibition of cell differentiation remains to be determined. On the other hand, inhibition of the
differentiation process correlates with a lack of nuclear export of
c-IAP1, as observed in Bcl-2–overexpressing cells, in TPAresistant U937 cells, and in monocytes from patients with CMML
that do not respond to M-CSF–induced differentiation. Bcl-2 and
related proteins were involved in the regulation of differentiation
(eg, in restricting lineage determination during hematopoietic
differentiation).55 The ability of overexpressed Bcl-2 to prevent the
translocation of c-IAP1 could be a nonspecific consequence of its
influence on the differentiation process. However, we cannot rule
out that Bcl-2 directly interferes with the translocation of c-IAP1,
since the protein is expressed at multiple cellular sites including the
nuclear outer membrane56 and the nucleus.57
Altogether, the present study suggests that c-IAP1 functions
may be regulated in part by the subcellular location of the protein
that is present mainly in the nucleus of various types of undifferentiated cells and translocates to the cytoplasm when these cells
undergo differentiation. Ongoing studies may indicate whether this
redistribution of c-IAP1 plays an active role in the differentiation
process or confers specific functions to differentiated cells.
Acknowledgments
The author thanks M. Yoshida for kindly providing LMB; J. C.
Reed for c-IAP1 cDNA; P. J. Parker, T. Lesuffleur, and J. Breard for
cell lines; Irving Weismann and Eric Lagasse for the MRP8BCL-2
transgenic mice; and B. Goud, S. Khochbin, O. Hermine, and M.
Fontenay for fruitful discussions.
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2004 104: 2035-2043
doi:10.1182/blood-2004-01-0065 originally published online
June 8, 2004
Translocation of the inhibitor of apoptosis protein c-IAP1 from the
nucleus to the Golgi in hematopoietic cells undergoing differentiation: a
nuclear export signal-mediated event
Stéphanie Plenchette, Séverine Cathelin, Cédric Rébé, Sophie Launay, Sylvain Ladoire, Olivier
Sordet, Tibor Ponnelle, Najet Debili, Thi-Hai Phan, Rose-Ann Padua, Laurence Dubrez-Daloz and
Eric Solary
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