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Blood First Edition
Paper, prepublished
online
June
8, 2004;
DOI 10.1182/blood-2004-01-0065
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.
Running Title :
Differentiation-induced nuclear export of c-IAP1
Scientific section heading: Hematopoiesis
Stéphanie Plenchette,1 Séverine Cathelin,1 Cédric Rébé,1 Sophie Launay,1 Sylvain Ladoire,1
Olivier Sordet,1 Tibor Ponnelle,2 Najet Debili,3 Thi Hai Phan,4 Rose-Ann Padua,4,5 Laurence
Dubrez-Daloz,1 Eric Solary1
1. INSERM U517, 2. INSERM EPI 106, IFR100, 7 boulevard Jeanne d’Arc, 21000 Dijon,
France; 3. INSERM U362, Institut Gustave Roussy, 38 rue Camille Desmoulins, 94805
Villejuif, France; 4. INSERM EMI00-03, Institut Universitaire d’Hematologie, Hopital St
Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France; 5. The Rayne Institute, King’s
College Hospital, 123 Coldharobour Lane, London SE5 9NU, UK.
Research grant : This work was supported by grants from the Ligue Nationale Contre le
Cancer.
Corresponding Author :
Eric Solary, INSERM U517, IFR100, 7 boulevard Jeanne d’Arc, 21000 Dijon, France
Phone: 33 3 80 39 32 56 - Fax: 33 3 80 39 34 34 - Email: [email protected]
Key words: Hematopoiesis / differentiation / monocytes and macrophages / nuclear export /
Golgi apparatus
Word counts: Abstract: 203
Total text: 4492
1
Copyright (c) 2004 American Society of Hematology
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Summary
The caspase inhibitor and RING finger-containing protein c-IAP1 has been shown to
be involved in both apoptosis inhibition and signaling by members of the TNF-receptor
family. The protein is regulated transcriptionally, e.g. is a target for 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
CARD domain of c-IAP1. A nucleo-cytoplasmic 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 translocate from the nucleus of cells whose
differentiation is blocked, i.e. in cell lines and monocytes from transgenic mice
overexpressing Bcl-2 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.
2
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Introduction
The IAPs (inhibitors of apoptosis proteins) 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 infection1 and cellular orthologues were
subsequently described in yeast, nematodes, drosophila and mammals. The human genome
encodes at least eight IAPs (XIAP, c-IAP1, c-IAP2, ML-IAP, NAIP, Survivin, ILP-2,
Apollon).2 All these proteins have in common the presence of one to three copies of a BIR
(baculovirus IAP repeat) domain.1 These domains are essential for the anti-apoptotic
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 aminoterminal 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 All 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 degradation8 and caspaseindependent 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 domain that confers them an 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-713,14 whereas c-IAP1 and c-IAP2 mediate ubiquitylation of
Smac/DIABLO, an antagonist of IAPs.15 c-IAP1 and c-IAP2 are also components of the type
2 TNF-receptor complex through interaction with the signaling intermediates TRAF1 and
TRAF2.9 cIAP-1 could induce the ubiquitylation of TRAF-2 and participated to the TNF- -
3
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mediated proteasomal degradation of TRAF-216 and c-IAP2 has been involved in the TNFsignaling leading to 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 pro-survival 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 auto-ubiquitylation8 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
Another level of regulation of IAP functions is the modulation of their sub-cellular
location. Such a regulation has been described for XIAP whose interaction with the protein
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 nucleus
export signal (NES) located in its caspase-recruitment domain (CARD). Overexpression of cIAP1 interferes with TPA-induced differentiation of leukemic cells, a process also inhibited
by the nuclear export inhibitor leptomycin B. Altogether, these observations suggest a role for
c-IAP1 in cell differentiation.
4
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Experimental procedures
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 (Affinity BioReagent, Golden, CO), HSC70 (Santa Cruz
Biotechnology, Santa Cruz, CA) GM130 (Golgi Matrix protein of 130 kDa) (FITCconjugated antibody, Transduction Laboratories, Lexington, KY) and rabbit polyclonal Abs
targeting c-IAP1 (Santa Cruz and R&D systems; Abington, UK), Mac-1 (PE-conjugated
antibody, Parmingen, Becton Dickinson, Heidelberg, Germany), BCL-2 (FITC conjugated
antibody, Pharmingen, Becton Dickinson,), CD1a (FITC-conjugated antibody, Pharmingen,
Beckton Dickinson), CD71 (FITC-conjugated antibody, Pharmingen, Beckton Dickinson),
PARP (poly(ADP-ribose) polymerase, Boehringer-Mannheim, Germany), XIAP (R&D
Systems and Stressgen Biotech, CA), PDI (protein disulfide isomerase; Calbiochem, La Jolla,
CA), GFP (Green Fluorescent protein, Invitrogen, Cergy Pontoise, France) and survivin
(Novus Biologicals, Littleton, CO) . Macrophage-colony stimuling factor (M-CSF),
granulocyte-macrophage colony-stimuling factor (GM-CSF) and interleukin-4 (IL-4) were
obtained from R&D systems, erythropoietin (EPO) from Amgen (Thousand Oaks, LA,
Cylag), 12-O-tetradecanoylphorbol 13-acetate (TPA) from Sigma-Aldrich laboratories (St
Quentin Fallavier, France), brefeldin A (BFA) and nocodazole from Alexis Biochemicals
(Lausen, Switzerland) and trypsin-EDTA from Gibco-BRL (Carlsbad, CA). Leptomycin B
(LMB) was kindly provided by Dr M. Yoshida (Tokyo, Japan) and thrombopoietin (TPO) by
Kirin Brewery.
Cell culture and differentiation. Cell lines were obtained from the ATCC (Rockville, MD)
and cultured as described.23 We also tested the previously described Bcl-2-transfected U937and HT29 cells and HT29-MTX cells.23-25 The TPA-resistant variant of U937 cells were
kindly provided by Pr. P.J. Parker (London, UK) 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,
5
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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.29 Bcl-2 overexpression in Mac-1+ cells of
transgenic mice was verified by flow cytometry using a FACSCalibur cytometer and the Cell
Quest software (Pharmingen, Becton Dickinson, location). Femoral bone marrow cells were
isolated from 6- to 8-week old control and transgenic FVB/N female mice and cultured for 4 h
on plastic plates before culturing adherent cells for 6 days in the presence of 10% L929 cellconditioned medium as source of CSF-1. Macrophage differentiation was assessed by MayGrundwald-Giemsa staining.
Immunofluorescence studies. Cells were fixed in paraformaldehyde (PFA; 2%) for 10 min at
room temperature, washed twice, saturated in 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% BSA. After washing, cells were incubated for 30 min
with 488-alexa goat anti-rabbit or anti-mouse Ab (Molecular Probes, Eugene, OR) 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-cIAP1 Ab overnight at 4°C, then with the secondary biotynilated-Ig (1/100)
(Amersham Biosciences, Buckinghamshire, UK) for 1h at room temperature, then with a
streptavidine-texas-red conjugated Ab (Molecular Probes) (1/2000) for 1h. Cells were
subsequently incubated for 1h at room temperature with anti-GM130-FITC (1/100) or antiGolgin 97 (1/100) then FITC conjugated anti-mouse Ab. Analysis was performed using either
a fluorescence (Nikon, Champigny, France) or a confocal (Leica, Bron, France) microscope.
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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, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT, 0.6% NP-41) in the presence of the protease inhibitors. Cell lysate was
centrifuged at 1,200 x g for 10 min. 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. 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 min at 37°C
and analysed by Western blotting for c-IAP1 content.31
Plasmid constructs. pEGFP-c-IAP1 plasmid was constructed by subcloning full length cIAP1 cDNA (kindly provided by J.C. Reed, La Jolla, CA) into the Bgl II / Sal I site of
pEGFP-C1 (Clontech, Palo Alto, CA). Sense and antisense oligonucleotides corresponding to
leucine-rich motif (LRM) putative NES were : LRM 1 sense: 5’-GAT CTT TTT TGG AAA
ATT CTC TAG AAA CTC TGA GGA-3’, LRM 1 antisense: 5’-GAT CTC CTC AGA GTT
TCT AGA GAA TTT TCC AAA AAA-3’, LRM 2 sense: 5’-GAT CTC TCT TTC AAC AAT
TGA CAT GTG TGC TTC CTA TCC TGG ATA ATC TTT TAA-3’, LRM 2 antisense: 5’GAT CTT AAA AGA TTA TCC AGG ATA GGA AGC ACA CAT GTC AAT TGT TGA
AAG AGA-3’, LRM 3 sense: 5’-GAT CTC TGT CAC TGG AAG AAC AAT TGA GGA
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GGT TGC AAA-3’, LRM 3 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 in a sense orientation into the Bgl II site of
pEGFP-C1 (Clontech). All sequences are expressed at the C-terminus of GFP. Full-length cIAP1 mutants (GFP-cIAP1-LRM1*, -LRM2* and -LRM3*) were obtained by mutagenesis of
LRM-1, -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 h after seeding using Superfect transfection
reagent (Qiagen, Valencia, CA) following the manufacturer’s instructions. Cells were studied
24 h after transient transfection: nuclei were stained with Hoechst 33342 and cells were fixed
with 2% PFA for 5 min 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 GmbH, Köln, Germany) and
transfected cells were enriched by a 10-day geneticin selection (0,7 µg/ml) before expansion
and treatment.
8
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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, four proteins that belong to the IAP family,
in U937 cells undergoing TPA-induced differentiation (Fig. 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, that has an evolutionarilyconserved 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 (Fig. 1A, left panel). However, c-IAP1, and to a lesser
extent XIAP, progressively accumulated in nuclear-free extracts as the cells underwent
differentiation (Fig. 1A, right panel). 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 (Fig.1C) and by fluorescent microscopy analysis of the two cell lines (Fig. 1B &
D). c-IAP1 was located mainly in the nucleus of U937 and THP1 undifferentiated cells and in
the cytoplasm of TPA-differentiated cells. A kinetic analysis identified a transient diffuse
staining of the cytoplasm in the first hours of TPA treatment. As the cells progressed towards
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the differentiation process, a more patchy staining, close to the nucleus, was observed (see
THP1 cells in Fig. 1D).
c-IAP1 co-localizes with the Golgi apparatus of differentiated cells. To precisely
determine the sub-cellular 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 TPAdifferentiated U937 cells (Fig. 2A). Thus, in 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 co-localized with Golgin 97, a Golgi matrix
protein, in TPA-differentiated THP1 (Fig. 2B) and U937 (not shown) cells. c-IAP1 also colocalized, although less precisely, with GM130, a protein associated with the cis-Golgi (Fig.
2B). Addition of either brefeldin A (BFA), a fungal metabolite that causes disintegration of
Golgi structure through inhibition of ARF GTP-binding proteins,32 or nocodazole, a
microtubule-depolarising agent, suppressed the patchy staining of c-IAP1 in TPAdifferentiated THP1 (Fig. 2C) and U937 (not shown) cells. In the tested conditions, BFA did
not modify calnexin C sub-cellular 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.
c-IAP1 is located to the cytoplasmic side of the Golgi apparatus in differentiated cells.
To determine the topological 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 (Fig. 2D). Addition of
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trypsin resulted in complete digestion of Golgin 97, which is located on the cytoplasmic side
of the Golgi apparatus, whereas the endoplasmic reticulum-lumenal protein disulfide
isomerase (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.
The differentiation-induced nuclear export of c-IAP1 involves a nuclear export signal.
To characterise the mechanisms that are responsible for the nuclear export of c-IAP1, we first
used leptomycin B (LMB), a specific inhibitor of exportin 1 (also known as CRM1), which is
the receptor for leucine-rich NES.33 Addition of 100 nM LMB for 24 hours to TPA-treated
THP1 (Fig. 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, though 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 Fig. 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 three hydrophobic, leucine-rich motifs
(LRM) that were consensus sequences for potential NES. 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) (Fig. 4A). To determine whether one or several of
these motifs played a role in c-IAP1 nuclear export, we cloned the sequences encoding these
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motifs in a GFP-encoding vector and expressed them by transient transfection in HeLa and
293T cells. The sub-cellular location of GFP-fusion proteins was examined 24 hours after
transfection by conventional (Fig. 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 (Fig. 4B & C). In
addition, exposure to leptomycin B induced accumulation of the GFP-LRM2 protein in the
nucleus (Fig. 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 (Fig.5) and THP1 (not shown) cells and their sub-cellular location was
analyzed by fluorescence microscopy and Western blotting 24 hours later. As shown
previously (Figure 3B), over-expressed wild-type c-IAP1 in HeLa cells demonstrated a
cytoplasmic and nuclear pattern of expression. Mutations in either LMR1 or LMR3 or both
did not affect the cellular distribution of the protein whereas all the LRM2 mutants
accumulated in the nucleus (Fig. 5A). For unknown reasons, LRM2 mutant was less
expressed that wild-type protein and other mutants. These observations were confirmed by
immunoblotting the nuclear and cytoplasmic fractions of transiently transfected HeLa (Fig.
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 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.
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Overexpressed c-IAP1 interferes with the differentiation process. LMB was observed to
prevent TPA-induced differentiation of THP1, as demonstrated by studying CD11b marker
(Fig. 6A), which suggested that a nucleo-cytoplasmic 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 (Fig. 6B) and TPA exposure failed to increase CD11b
expression in GFP-tagged cells (Fig. 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 (Fig. 6B & C). These results
indicated that c-IAP1 overexpression could interfere with cell differentiation.
The nucleo-cytoplasmic redistribution of c-IAP1 is observed in several differentiation
pathways. c-IAP1 was observed to be present mainly in the nucleus of 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 (Fig. 7A) as well as erythroblasts and megakaryocytes obtained by ex vivo
differentiation of CD34+ cells (Fig. 7B).27,28. As previously observed in TPA-differentiated
cells (Fig. 1B), c-IAP1 demonstrated a punctuated expression in the perinuclear zone and colocalized with Golgin 97 in macrophages (Fig. 7C) and dendritic cells (not shown) obtained
by differentiation of normal peripheral blood monocytes. A differentiation-associated
redistribution of c-IAP1 from the nucleus to the cytoplasm was also observed in nonhematopoietic cells, i.e. in HT29 human colon carcinoma cells undergoing partial
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differentiation when grown at confluence.34 c-IAP1 also demonstrated a cytoplasmic
expression in the well-differentiated, mucus secreting HT29/MTX clone (Fig. 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 in the same
conditions (Fig. 7E). We have previously shown that Bcl-2 overexpression prevented TPAinduced differentiation in U937 human leukaemia 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 in similar conditions
(Fig. 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 in similar conditions (Fig. 7F). We also cultured peripheral blood mononuclear
cells from seven patients with chronic myelomonocytic leukemia (CMML) in the presence of
M-CSF or the GM-CSF/IL-4 combination for 6 days, respectively. 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 cIAP1 nucleus export (For example, see Fig. 7G).
Discussion
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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 CD4035 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 protein in ubiquitinylation may contribute to both
functions, e.g. 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 (NLS) and NES. The size of c-IAP1 prevents it from passively diffusing
through nuclear pores38 and we did not identify any classical NLS in the protein, which
suggests that the protein either is carried by a co-factor 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
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changes,41 or proteolytic cleavage.42 So far, we did not detect any post-translational
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 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 cIAP1 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 co-expressed 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 a LMB-sensitive
mechanism whereas its alternative isoform survivin- Ex3 is driven to the nucleus by a Cterminal 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
16
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XIAP protein level remains 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, i.e. overexpressed human NAIP (neuronal apoptosis inhibitory protein) 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 BIRcontaining protein dBRUCE was proposed to bind to and to degrade caspases involved in
drosophila spermatogenesis.51 We have shown recently 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
-independent
(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 to move from the cytoplasm to other cell
compartments in leukemic cells undergoing differentiation but were not identified to associate
with the golgi apparatus.
52
Lastly, we have shown previously that the post-mitochondrial
pathway to apoptosis remained functional in TPA-differentiated U937 cells, indicating that
17
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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 catalyses 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 TRAF2 16 and 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 over-expressing cells, in TPA-resistant
U937 cells and in monocytes from patients with CMML that do not respond to M-CSFinduced differentiation. Bcl-2 and related proteins were involved in the regulation of
differentiation, e.g. in restricting lineage determination during hematopoietic differentiation.55
The ability of over-expressed 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 sub-cellular 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
18
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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.
19
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Acknowledgments. The author thanks M. Yoshida for kindly providing LMB, JC Reed for
c-IAP1 cDNA, PJ 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-Roupie for fruitful discussions.
24
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Figures legends
Figure 1: c-IAP1 redistribution in human leukaemia 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, cytoplamic 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, labelled with Hoechst 33342, are stained in
blue.
Figure 2 : c-IAP1 is localized to the Golgi apparatus in differentiated cells. A - Western
blot analysis of c-IAP1 (pAb Santa Cruz) 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 h. 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 h before analyzing the expression of cIAP1 (red), Golgin 97 (green) or GM130 (green) by confocal microscopy. Inserts: increased
magnification of Golgi labeling. C - c-IAP1 expression in TPA-differentiated THP1 cells
before (Co) and after exposure to either brefeldin A (BFA 5µg/mL, 2h30) or nocodazole (10
µM, 1h). c-IAP1 expression was observed by fluorescence microscopy 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.
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 h, in the presence or absence of 100 nM leptomycin B
25
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(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 h before
analysis.
Figure 4: Identification of a potential nuclear-export sequence in c-IAP1. A – Upper:
schematic representation of amino-acid motifs in c-IAP1 protein (619 amino-acids). Leucinerich motifs (LRMs) that could behave as nuclear export signal (NES) are indicated (BIR:
baculovirus IAP repeat; CARD: caspase-recruitment domain). Lower: amino-acid sequence of
regions containing a potential LRM (underlined). B & C - 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 analysis were performed 24 h later. (nuclei were
stained with Hoechst 33342). When indicated, LMB (200 nM) was added 3 h before analysis.
Figure 5: LRM2 is the functional nuclear export signal in c-IAP1. A - Fluorescence
microscopy analysis of HeLa cells transfected for 24 h 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. *
indicates mutated constructs B - Western blot analysis of GFP expression in nuclear (N) and
cytoplasmic (C) extracts from HeLa cells transfected 24 h before with indicated constructs.
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).
Grey histograms : treated cells. 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
26
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cytometry analysis of CD11b expression in GFP-positive THP1 cells transfected with pEGFP
empty vector (vector) or wild-type (wt-c-IAP1) or LRM2-mutated (c-IAP1-LRM2) GFP-cIAP1 constructs. Cells were treated with 20 nM TPA for indicated times (h). Grey lines:
treated cells. 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 labelled
with an anti-CD11b Ab (red). Nuclei were stained with Hoechst 33342 (blue).
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 MCSF or GM-CSF/IL-4, respectively. Upper panels : c-IAP1 alone (green); lower panels : cIAP1 (green) + Hoechst 33352 labeled nuclei (blue). B - Fluorescence microscopy analysis of
c-IAP1 (mAb, Pharmingen) (green) in CD34+ cell-derived erythoblasts (Ery) and
megacaryocytes (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 in A. D - Fluorescence microscopy analysis of c-IAP1 (pAb,
Santa Cruz) in HT29 cells studied before (control) and after (confluent) reaching confluence
in culture, and in a methotrexate-resistant, well-differentiated derivative cell clone (HT29MTX). E - Fluorescence microscopy analysis of c-IAP1 (pAb, Santa Cruz) in control (Co),
Bcl-2 over-expressing (Bcl2) and TPA-resistant U937 cells exposed for 72 h to 20 nM TPA.
F - May-Grunwald-Giemsa staining (MGG) and fluorescence microscopy analysis of c-IAP1
(pAb, Santa Cruz) (c-IAP1) in bone-marrow monocytes from control (Co) and Bcl-2
transgenic (Bcl2) mice, cultured for 3 days in the presence of CSF-1-containing medium. G Peripheral blood mononuclear cells obtained from patients with chronic myelomonocytic
27
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leukemia (CMML) were cultured for 6 days in the presence of M-CSF or GM-CSF/IL-4.
Upper panel : fluorescence microscopy analysis of c-IAP1; lower panels : flow cytometry
analysis of CD71 and CD1a membrane expression. White histograms: CMML patient. Grey
histograms: healthy donor. – One representative of 7 studied patients is shown.
28
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Figure 1
B
c-IAP1
A
Cytoplasm
Whole-cell
XIAP
57
Survivin
17
HSC70
70
0 6 12 24 48
overlay
0 6 12 24 48
nuclei
70
c-IAP1
Hours
0
C
Whole-cell
Nuclei
Hours
48
Cytoplasm
70
70
c-IAP1
HSC70
0 2
7
24 48
0
2
7
24 48
0
2
7 24 48
Hours
overlay
nuclei
c-IAP1
D
0
2
7
24
Hours
29
48
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Figure 2
A
c-IAP1
70
M C R
N
M C
Co
R N
TPA 72h
PARP
116
Golgin 97
97
mHSP70
70
M C
B
C
R N
Golgin 97
overlay
c-IAP1
GM130
overlay
c-IAP1
Co
BFA
D
Nocodazole
Microsomal fraction
Trypsin
-
+
c-IAP1
70
Golgin 97
97
PDI
55
30
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Figure 3
A
c-IAP1
TPA
TPA
+
LMB
B
c-IAP1
c-IAP1
+
LMB
31
nuclei
overlay
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Figure 4
A
LRM 1
NH2
BIR 1
RING
BIR 2 BIR 3
LRM 2
LRM 3
COOH
CARD
LRM 1:
242
RHFP NC P FLE NSL E TLR F S I SN
LRM 2:
464
N R MA L F Q Q L T C V L P I L D N L L K A 485
LRM 3:
550
T E V D S G L S L E E Q L R R L Q E E R T C 571
B
GFP
GFP / Hoescht
GFP
GFP / Hoescht
LRM1
LRM2
LRM3
C
LRM2
LRM2
+ LMB
32
263
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Figure 5
A
B
HeLa
GFP
Hoescht
Wild-type
LRM 1*
LRM 2*
LRM 3*
LRM 1*2*
LRM 1*3*
LRM 2*3*
LRM 1*2*3*
33
Overlay
N C
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Figure 6
A
0
24
36
Co
LBM
00 101 102 103 00 101 102 103 00 101 102 103
CD11b
B
wt c-IAP1
N
C
vector
C-IAP1-LRM2*
C
N
C
wt c-IAP1 c-IAP1-LRM2*
0
24
48
00 101 102 103 00 101 102 103 00 101 102 103
CD11b
GFP
c-IAP1-LRM2*
vector
D
34
nuclei
CD11b
overlay
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Figure 7
A
Mo
B c-IAP1/nuclei
DC
c-IAP1
M
Ery
c-IAP1/nuclei
CD34+
Meg
D
HT29
Control
Confluent
HT29-MTX
overlay
c-IAP1
c-IAP1
C
E. U937/TPA
-
G
c-IAP1
Co
MGG
/CSF-1
c-IAP1
F. Mouse M
Bcl-2
Monuclear
cells
M-CSF
Bcl-2
GM-CSF
+
IL-4
F
CD71
35
TPA-Resistant
c-IAP1
Golgin 97
Co
CD1a
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Prepublished online June 8, 2004;
doi:10.1182/blood-2004-01-0065
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
Stephanie Plenchette, Severine Cathelin, Cedric Rebe, 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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