369
Journal of Cell Science 110, 369-378 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS9567
Nuclear localization of IκBα promotes active transport of NF-κB from the
nucleus to the cytoplasm
Fernando Arenzana-Seisdedos1, Pierre Turpin2, Manuel Rodriguez1, Dominique Thomas1, Ronald T. Hay3,
Jean-Louis Virelizier1 and Catherine Dargemont2,*
1Unité d’Immunologie Virale, Institut Pasteur, 75015 Paris, France
2Laboratoire de Transport Nucléocytoplasmique, UMR 144 Institut Curie-CNRS, 26 rue d’Ulm, 75231 Paris Cedex 05,
3School of Biological and Medical Sciences, Irvine Building, University of St Andrews, Fife KY169AL, Scotland, UK
France
*Author for correspondence (e-mail: [email protected])
SUMMARY
IκBα tightly regulates the transcriptional activity of NF-κB
by retaining it in the cytoplasm in an inactive form. In the
present work, we report that IκBα, when expressed in the
nuclear compartment, not only abrogates NF-κB/DNA
interactions and NF-κB-dependent transcription, but also
transports NF-κB back to the cytoplasm. This function of
IκBα is insured by a nuclear export sequence located in the
C-terminal domain of IκBα and homologous to the previously described export signal found in HIV-1 Rev protein
as well as in PKI (the inhibitor of the catalytic subunit of
protein kinase A). Thus, inhibition of NF-κB/DNA binding
and the consecutive efficient nuclear export of the transcription factor by IκBα could represent an important
mechanism for the control of the expression of NF-κBdependent genes.
INTRODUCTION
localization sequence (NLS), but also inhibits the ability of NFκB to bind DNA (Beg et al., 1992; Henkel et al., 1992;
DiDonato et al., 1996). IκBα is composed of a central,
protease-resistant domain containing five ankyrin repeats and
flanked by a surface exposed N-terminal extension and a
compact, highly acidic C-terminal region which is connected
to the core by a flexible linker (Jaffray et al., 1995). Mutational
analysis, as well as functional study of protease cleavage
products, reveals that, while the N-terminal region is not
essential for binding to relA, both the ankyrin repeats and
acidic domain are required for IκB activity (Blank et al., 1991;
Hatada et al., 1993; Inoue et al., 1992; Jaffray et al., 1995). In
particular, the linker region has been proposed to interact with
NF-κB (Jaffray et al., 1995).
Upon cell stimulation with various stimuli, such as tumor
necrosis factor (TNF) or interleukin 1, IκBα is phosphorylated
and degraded, thus allowing active NF-κB to translocate to the
nucleus (Henkel et al., 1993). An efficient resynthesis of IκBα
is then observed, due partially to an increase in mRNA transcription through interaction of NF-κB with the NF-κBbinding sites located in the IκBα promoter (Chiao et al., 1994;
Le Bail et al., 1993; Sun et al., 1993).
Beside its cytoplasmic expression, IκBα has also been
reported to be localized in the nuclear compartment when it is
overexpressed from a transfected vector or microinjected into
the cytoplasm (Cressman and Taub, 1993; Zabel et al., 1993).
In untransfected cells, it has been recently shown that,
following a brief exposure of HeLa cells to TNF, newly synthesized IκBα localizes to the nuclear fraction where it is found
to associate with NF-κB/rel complexes. Concomitant to the
The transcription factor NF-κB plays a major role in the
inducible expression of a number of cellular genes particularly
those involved in immune and inflammatory responses and participates in the regulation of human immunodeficiency virus
(HIV) transcription (Hay, 1993; Liou and Baltimore, 1993).
Human NF-κB is composed of a homo- or heterodimer of
proteins that belong to the multigene rel family including p50
(Ghosh et al., 1990; Kieran et al., 1990), p52 (Bours et al.,
1992; Neri et al., 1992; Schmid et al., 1991), p65/rel A (Nolan
et al., 1991; Ruben et al., 1991), c-rel (Wilhelmsen et al., 1984)
and rel B (Ryseck et al., 1992). These proteins share a very
conserved region, the rel homology domain, which contains
sequences responsible for DNA binding, dimerization and
nuclear localization (Liou and Baltimore, 1993; Verma et al.,
1995).
The transcriptional activity of NF-κB is primarily controlled
by its intracellular localization. Indeed, in most unstimulated
cells, NF-κB remains in a cytoplasmic, inactive complex
through its association with the inhibitor proteins, IκBs.
Members of the IκB family, including IκBα (Haskill et al.,
1991) or MAD-3, IκBβ (Zabel and Bauerle, 1990), Bcl-3
(Ohno et al., 1990), cactus (Geisler et al., 1992; Kidd, 1992)
and IκBγ (Blank et al., 1991; Inoue et al., 1992; Liou et al.,
1992), are characterized by both their ability to interact with
NF-κB subunits and the presence of multiple conserved
ankyrin domains. In most mammalian cells, IκBα and IκBβ
represent the two major forms of the IκB family. IκB not only
prevents nuclear translocation of NF-κB by masking its nuclear
Key words: NF-κB, IκBα, Nuclear export sequence, Transcription
regulation
370
F. Arenzana-Seisdedos and others
accumulation of newly synthesized IκBα in the nucleus, a progressive reduction of both NF-κB-DNA binding and NF-κBdependent transcription along with a profound decrease in the
amount of NF-κB subunits is observed in the nucleus
(Arenzana-Seisdedos et al., 1995). These intringuing findings
are compatible with the hypothesis that NF-κB, after interacting with newly synthesized IκBα, would be exported out of the
nucleus (Zabel et al., 1993; Arenzana-Seisdedos et al., 1995).
A precedent for a nuclear protein being exported after interacting with an inhibitory subunit has been shown for the
catalytic subunit of protein kinase A which leaves the nucleus
associated with its inhibitor, PKI (Fantozzi et al., 1994; Wen
et al., 1995).
In this report, we describe a new function for nuclear IκBα
on the compartimentalization and eventually the functional
activity of NF-κB. Using either microinjection of in vitro synthesized IκBα proteins in Xenopus oocyte nuclei or their overexpression in mammalian cells, we show here that, following
the disruption of NF-κB/DNA interaction by nuclear IκBα, the
NF-κB/IκBα complexes are exported out of the nucleus. The
ability of IκBα to promote efficient nuclear export of NFκB/rel complexes is conferred by a nuclear export signal (NES)
located in the C-terminal domain of IκBα (residues 265 to
277).
MATERIALS AND METHODS
Animals and cells
Female Xenopus laevis frogs were purchased from the Elevage de
Xenopes du CNRS (Montpellier, France). HeLa cells were maintained
in Dulbecco’s modified Eagle Medium supplemented with 10% foetal
calf serum.
Plasmids and mutagenesis
The 3enh-kb-CONA-Luc vector (Arenzana-Seisdedos et al., 1993)
carries a luciferase gene under the control of three synthetic copies of
the κB consensus of the immunoglobulin k-chain promotor cloned
into the BamHI site located upstream of the conalbumin transcription
start site. The CONA-Luc vector is identical except that it does not
contain the κB sequences.
DNA encoding the SV5-tagged version of IκBα was amplified by
PCR strategy using the pGEX-IκB ctag vector (Rodriguez et al., 1995)
as template and cloned into the BamHI/XbaI restriction sites of the
eukaryotic expression vector pcDNA3 (InVitrogen). Punctual
mutations were introduced into IκBα ctag using a PCR-based strategy
and pcDNA3-IκBα ctag DNA as a template. The K67-87R mutant
form of IκBα ctag, as well as p50 and p65 cloned into pcDNA3, have
been previously described (Rodriguez et al., 1996). A synthetic
oligonucleotide that specifies the IκBα (265-281) sequence was fused
to the C terminus of chicken pyruvate kinase (PK) to generate the PKIκBα(265-281) construct.
DNA transfections
For transient expression experiments, HeLa cells were trypsinized and
resuspended in Dulbecco’s modified Eagle Medium supplemented
with 10% FCS and 15 mM Hepes, pH 7.5, at 25×106 cells/ml. A 50
µl sample of DNA mix (210 mM NaCl, 10 µg of pcDNA3-IκBα ctag
DNA, 30 µg of carrier DNA) were added to 200 µl of cells before
electroporation (950 µF, 240 V, using Gene Pulser II, Bio-Rad). Cells
are subsequently cultured for 24 hours before analysis.
Immunofluorescence microscopy
For indirect immunofluorescence analysis, cells were fixed for 10
minutes with 3% paraformaldehyde and permeabilized with 0.1%
Triton X-100 for 10 minutes. Rabbit polyclonal antibody against p65
(Santa Cruz Biotechnology) and monoclonal antibody SV5-Pk
(Hanke et al., 1992) were applied for 30 minutes followed by a 30
minute incubation with fluorescein-isothiocyanate (FITC)-conjugated
donkey anti-rabbit IgG and Texas red (TR)-conjugated donkey antimouse IgG, respectively (Jackson). Coverslips were mounted in
Mowiol (Hoechst, Frankfurt, Germany). Confocal laser scanning
microscopy and immunofluorescence analysis were performed with a
TCS4D confocal microscope based on a DM microscope interfaced
with a mixed-gas argon-krypton laser (Leica Laser Technik). Fluorescence acquisitions were performed with the 488 nm and 568 nm
laser lines to excite FITC and TR dyes, respectively, with a ×100 oil
immersion PL APO objective. All the data were registered at the same
laser and multipliers settings.
Expression of proteins in vitro
Coupled transcription/translation was performed using the TNT
system in a reticulocyte lysate (Promega) supplemented with
[35S]methionine and [35S]cysteine (Amersham). Translation products
were analyzed by SDS-PAGE and autoradiography. In vitro translation products to be used for microinjection into Xenopus oocytes were
dialyzed against injection buffer (10 mM Pipes, pH 7.4, 80 mM KCl,
20 mM NaCl) and stored at −20°C. The doublet observed after the in
vitro translation of IκBα results from an additional internal translation start site.
Interaction of IκBα wild type and mutants with NF-κB p65
subunit in vitro
IκBα wild type (wt) and mutants were transcribed and translated in
vitro using reticulocyte lysate. A synthetic double strand oligonucleotide encoding the whole HIV1 NF-κB consensus sequence was
radiolabeled with [α32P]dCTP and used as a specific probe for p65DNA binding. The capacity of each IκBα derivative to prevent
binding of p65 to the specific DNA probe, was assessed by preincubating at 4°C for 15 minutes equal volumes of the synthesis reactions
of the two proteins before addition of the radiolabeled DNA probe.
Free DNA was resolved from DNA-protein complexes on a native 6%
polyacrylamide gel (Rodriguez et al., 1996).
Oocyte injections
Stage VI oocytes were prepared from ovaries of X. laevis females,
defolliculated as described by Gurdon and Wickens (1983), and
incubated in OR2 buffer. In vitro translation reactions (20 nl) were
injected, after dialysis without further purification, directly into the
nucleus of oocytes; the red color of hemoglobin present in the in vitro
translation mixtures was used to monitor the actual site of injection.
Moreover, each translation product was co-injected with M3, an NLSmutant of nucleolin which does not show any export ability within the
first four hours after its nuclear injection (Schmidt-Zachmann et al.,
1993). Oocytes were maintained at 20°C in OR2 and 10-12 oocytes
were microinjected for each time point in every experiment. After
different incubation times, oocytes were dissected manually in 5:1
buffer (83 mM KCl, 17 mM NaCl, 10 mM Tris-HCl, pH 7.4) supplemented with 10% glycerol. Cytoplasmic and nuclear fractions from
the same oocyte pool were homogenized separately in the same buffer.
The insoluble cytoplasmic fraction was removed by centrifugation
(12,000 g, 5 minutes). After addition of protein sample buffer, nuclear
and cytoplasmic fractions were analyzed by 12% SDS-PAGE. The
gels were subsequently fixed in 30% EtOH, 10% acetic acid, washed
and incubated for 30 minutes in an enhancer solution (1 M sodium
salicylate) before drying.
Measurement of luciferase activity in oocytes
Oocytes were lysed 18 hours after DNA injection with 25 mM Trisphosphate pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1% Triton X100, 1 mM EDTA, 15% glycerol, 1% BSA (1 oocyte/20 µl lysis
Nuclear export of IκBα
buffer). Insoluble material was removed by centrifugation (12,000 g,
5 minutes) and luciferase activity was measured in a luminometer
(Berthold). For each experimental value, 4-6 oocytes were used and
the luciferase activity was measured on the protein-equivalent of 1
oocyte. The background obtained with the lysis buffer was subtracted
from each experimental value. The luciferase activity obtained after
the injection of 3enh-kb-CONA-Luc vector together with p65 was
arbitrary considered as 100% in each experiment.
RESULTS
IκBα promotes the transport of NF-κB complexes
from the nucleus to the cytoplasm
We previously showed that nuclear accumulation of IκBα
induced by a short treatment of HeLa cells with TNF promoted
a dramatic reduction of both NF-κB binding activity and the
total amount of NF-κB protein in the nucleus (ArenzanaSeisdedos et al., 1995). In order to investigate whether the latter
phenomenom was due to an export of NF-κB out of the
nucleus, we chose an experimental model which enabled us to
monitor nuclear export in the absence of concomitant nuclear
import. For this purpose, we decided to use microinjection into
Xenopus oocyte nuclei since: (i) IκBα is not imported into the
oocyte nucleus after cytoplasmic injection (data not shown);
and (ii) this model affords the possibility of studying nuclear
export quantitatively.
To compare the kinetics of nuclear export of IκBα alone or
in association with the different NF-κB subunits, in vitro translated proteins were injected into Xenopus oocyte nuclei, and
cytoplasm and nuclei were separated manually and analyzed
by SDS-PAGE. In order to monitor the injection site and
eventual nuclear leakage, each protein was co-injected with
nucleolin M3, a NLS-mutant of chicken nucleolin which has
been previously shown to remain in the nucleus during the first
four hours after its nuclear injection but to be accumulated
completely in the cytoplasm within 24 hours (SchmidtZachmann et al., 1993).
As shown in Fig. 1a, IκBα was no longer detectable in the
nucleus 90 minutes after its injection into this compartment. In
contrast, p65/relA remained in the nucleus even 3 hours after
Fig. 1. IκBα promotes the nuclear export
of NF-κB complexes. Nuclei of X. laevis
oocytes were injected with
[35S]methionine- and [35S]cysteinelabeled proteins translated in vitro. IκBα
(a), p65-relA (b), IκBα/p65 (c), IκBα
K67-87R/p65 (d), p50/p65 (e) and
IκBα/p50/p65 (f) were co-injected with
nucleolin M3, a NLS-mutant of
nucleolin, known to not be exported
during the first 4 hours after its nuclear
injection. At different times after
injection (indicated at the top), oocytes
were dissected manually, and the nuclear
(N) and cytoplasmic (C) fractions were
analyzed by SDS-PAGE and
fluorography.
371
its nuclear injection with only a minimal fraction being
detected in the cytoplasm at this time (Fig. 1b). When the
complex p65/IκBα was introduced into the nucleus, its export
displayed kinetics similar to that observed for IκBα (Fig. 1c).
This result indicates that IκBα promotes the export of p65 from
the nucleus to the cytoplasm when these proteins are coexpressed in the nucleus. To ensure that this effect of IκBα on
p65 export was specific and dependent on p65-IκBα complex
formation, we checked the ability of an IκBα mutant K67-87R,
affected in its ability to interact with NF-κB (Fig. 2B;
Rodriguez et al., 1996), to induce the transport of p65. As
shown in Fig. 1d, this mutant was transported as well as the
wild-type IκBα, but the export rate of p65 was greatly reduced,
indicating that the association between IκBα and p65 is
required for an efficient transport of p65 back to the cytoplasm.
The capacity of IκBα to induce the export of NF-κB
complexes was also investigated. Heterodimer p50/p65 which
represents the most abundant NF-κB complex detected in vivo
was injected alone or together with IκBα into the nucleus. In
the absence of IκBα, the amount of nuclear p50/p65 complex
remained unchanged for at least 3 hours (Fig. 1e). In contrast,
when IκBα was injected together with the heterodimer, we
could observe a complete export of IκBα, p65 and p50 within
3 hours (Fig. 1f), indicating that IκBα induces the export of
the p50/p65 NF-κB complex as well as the p65 homodimer.
Co-expression of IκBα and NF-κB in the nucleus
terminates NF-κB-dependent transcription and
results in the export of NF-κB/IκBα complexes out of
the nucleus
Physiologically, NF-κB is translocated into the nuclear compartment after IκBα degradation. Subsequently, IκBα is resynthesized and expressed in nucleus and cytoplasm. In order to
mimic this succession of events in the Xenopus oocyte, we first
injected p65 together with a molar excess of 3enh-kb-CONALuc plasmid into the nucleus. This vector carries the luciferase
gene under the control of three synthetic copies of the κB
consensus of the immunoglobulin k-chain promoter cloned
upstream of the conalbumin transcription start. Ninety minutes
later, either nucleolin M3 alone or together with IκBα was
372
F. Arenzana-Seisdedos and others
Fig. 2. IκBα interacts with p65 in the nucleus and shuts down NF-κB
induced-transcription by exporting NF-κB out of the nucleus.
(A) Nuclei of X. laevis oocytes were first injected with 3enh-kbCONA-Luc plasmid and [35S]methionine and [35S]cysteine-labeled
p65 translated in vitro. After 90 minutes, nuclei were re-injected
either with nucleolin or with IκBα plus nucleolin and analyzed 90
minutes later (left panel). Nuclei of X. laevis oocytes were injected
with 3enh-kb-CONA-Luc plasmid and [35S]methionine and
[35S]cysteine-labeled p65 translated in vitro; IκBα and nucleolin and
were analyzed after 90 minutes (right panel). At different times after
injection, oocytes were dissected manually, and the nuclear (N) and
cytoplasmic (C) fractions were analyzed by SDS-PAGE and
fluorography. (B) Analysis of p65/DNA binding in the absence (−) or
the presence of different IκBα derivatives. p65 and IκB proteins
were generated in vitro. A 103 µl sample of each IκBα reticulocyte
lysate was incubated with 1 µl of p65 preparation for 15 minutes at
room temperature and a protein-DNA-binding assay was performed
using a 32P-radiolabeled, double-stranded HIV-enhancer
oligonucleotide. (C) Nuclei of X. laevis oocytes were injected with
the indicated plasmids and in vitro translated p65 with or without in
vitro translated IκBα. Oocytes were then incubated for 18 hours and
luciferase acitivity was measured in total oocyte extracts. The value
obtained after the injection of 3 enh-kb-CONA-Luc with p65 was
arbitrary taken as 100% in each experiment.
injected into the nuclear compartment and the oocytes were
further incubated for 90 minutes. In the absence of IκBα, p65
stayed in the nucleus whereas the introduction of IκBα induced
the appearance of 50% of the p65 in the cytoplasm after 90
minutes (Fig. 2A). The same result was obtained when DNA,
IκBα and p65 were injected at the same time and oocytes
analyzed 90 minutes later (Fig. 2A). The capacity of wt IKBα
to associate with p65 was assessed. Addition of in vitro translated IκBα to p65 bound to a specific NF-κB DNA consensus
sequence disrupted the interaction of p65 with the DNA (Fig.
2B). IκBα interacted more efficiently with p65 than K67-87R
mutant as is proven by the enhanced capacity of the wt protein
to inhibit p65/DNA interaction (Fig. 2B). These in vitro results
are in agreement with the respective ability of these proteins
to both associate with p65 and block NF-κB activation in intact
cells (Rodriguez et al., 1996). These data show that IκBα can
bind DNA associated-p65 in the nucleus and transport it back
to the cytoplasm.
The efficiency of in vitro translated p65 to induce the transcription from a NF-κB dependent vector in oocytes was
analyzed by co-injecting p65 with the 3enh-kb-CONA-Luc
plasmid. Luciferase activity was measured 18 hours after
injection and results were compared to the value obtained after
coinjection of p65 and the CONA-Luc vector, which lacks κB
consensus sequences. As shown in Fig. 2C, a roughly 4-fold
increasing activity was observed with the 3enh-kb-CONA-Luc
plasmid compared to that of the control vector. When IκBα
was injected simultanously or 90 minutes later than p65, a
profound decrease in the luciferase activity was observed; the
value was reduced to the level of the NF-κB-independent
enhancer-less CONA-Luc control vector. The inhibition of NFκB dependent transcription observed 18 hours after coinjection
of p65, DNA and IκBα correlates with a reduced amount
(50%) of p65 observed as soon as 90 minutes after injection of
IκBα. The apparent total inhibition of the NF-κB dependent
transcription observed at the end of the experiment may be
explained either by a complete export of p65 or to a loss of
transcriptional activity of p65.
Together, these data demonstrate that co-expression of IκBα
and NF-κB in the nucleus prevents or terminates NF-κBdependent transcription and results in the export of NFκB/IκBα complexes out of the nucleus.
Characterization of a sequence responsible for the
efficient nuclear export of IκBα
Although the export of a nuclear protein has been shown to be
primarily limited by the extent of its intranuclear interactions
(Schmidt-Zachmann et al., 1993), specific sequences responsible for the efficient export of some nuclear proteins have
recently been reported. One of these nuclear export sequences
(NES) was found in hnRNP A1 (Michael et al., 1995). Another,
Nuclear export of IκBα
373
Fig. 3. IκBα (265-281) can direct efficient pyruvate kinase export
from the nucleus to the cytoplasm. (A) Comparison of the nuclear
export sequences of PKI, Rev and IκBα. Conserved residues are
indicated by higher size letters. (B) In vitro translated 35S-labeled
pyruvate kinase and pyruvate kinase fused to the 265-281 sequence
of IκBα (depicted schematically at the top) were coinjected with
nucleolin M3 into nuclei of X. laevis oocytes. At different times after
injection (indicated at the top), oocytes were manually dissected, and
nuclear (N) and cytoplasmic (C) fractions were analyzed by SDSPAGE and fluorography.
unrelated motif was identified in PKI (the inhibitor of the
catalytic subunit of c-AMP-dependent protein kinase; Wen et
al., 1995) and in the HIV-1 Rev protein (Fischer et al., 1995)
and is characterized by critical hydrophobic residues. An
homologous sequence is also found in the C-terminal domain
of IκBα at residues 265 to 277 (Fig. 3A). In order to see if
IκBα (265-277) could promote nuclear export of a reporter
protein, we fused this sequence to chicken pyruvate kinase
(PK) to yield the protein PK-IκB(265-281) shown schematically in Fig. 3B. The export behavior of this hybrid protein was
determined after its nuclear injection into Xenopus oocytes and
was compared to the transport of PK (Fig. 3B). This experiment revealed that the presence of the (265-281) motif of IκBα
strongly enhanced the export ability of PK. At 45 and 90
minutes after nuclear injection, 59% and 80% of PK-IκBα
(265-281) had reach the cytoplasm while only 6% and 24%,
respectively, of the PK had left the nucleus during the same
incubation periods. Thus, IκBα (265-281) by itself is able to
promote efficient export of a reporter protein from the nucleus.
In order to determine whether this motif plays a role in the
export of the entire IκBα molecule, we mutated the following
hydrophobic amino acids, Leu 269, Leu 272, Leu 274, Leu 277
into alanine (Fig. 4A) and analyzed the export capacity of the
resulting mutants. Mutation of the pair of leucines 269 and 272
(mutant L12) did not affect the export ability of the resulting
protein (Fig. 4A). In contrast, replacement of leucines 274 and
277 with alanines (mutant L34) significantly impaired nuclear
Fig. 4. IκBα (265-277) is responsible for the export of the IκBα and
IκBα/p65 complex. (A) Schematic representation of mutations
introduced into the IκBα (265-277) sequence. Critical leucine
residues are depicted in higher size characters. Each in vitro
translated 35S-labeled mutant was coinjected with nucleolin M3 into
nuclei of X. laevis oocytes. At different times after injection (0, 45
minutes, 90 minutes, 180 minutes), oocytes were manually dissected,
and nuclear and cytoplasmic fractions were analyzed by SDS-PAGE
and fluorography. Fluorograms from at least two experiments were
scanned in order to determine the time necessary for the export of
50% of the injected protein (t1/2; indicated on the right). (B) 35Slabeled, in vitro translated wt IκBα or L234 mutant was coinjected
with nucleolin M3 into nuclei of X. laevis oocytes. At different times
after injection (indicated at the top), oocytes were manually
dissected, and nuclear (N) and cytoplasmic (C) fractions were
analyzed by SDS-PAGE and fluorography. (C) 35S-labeled, in vitro
translated p65/L234 complex was coinjected with nucleolin M3 into
nuclei of X. laevis oocytes. At different times after injection
(indicated at the top), oocytes were manually dissected, and nuclear
(N) and cytoplasmic (C) fractions were analyzed by SDS-PAGE and
fluorography. Both panels correspond to the same gel but with two
different exposure times.
transport of the corresponding mutant out of the nucleus (Fig.
4A). Export inhibition was increased even more when
combined mutation of leucine residues 272, 274 and 277 to
alanine were made (mutant L234; Fig. 4A and B); about 60
minutes were necessary for the export of 50% of the injected
L234 whereas 90% of wt IκBα was exported within less than
374
F. Arenzana-Seisdedos and others
A
B
Fig. 5. Mutation in the NES impairs the ability
of IκBα to promote nuclear export of NF-κB
in HeLa cells. (A) HeLa cells non-transfected
(a-h) or transfected with a tagged version of wt
IκBα (i-p) were cultured in the presence of
serum (a,e,i,m), then washed and treated for 90
minutes with 100 µg/ml cycloheximide in the
absence of serum and then 45 minutes with
100 µg/ml cycloheximide and 10 ng/ml TNF
(b,f,j,n). These drugs were then removed and
cells were incubated for 90 minutes in the
absence of serum (c,g,k,o) or with 100 µg/ml
cycloheximide in the absence of serum
(d,h,l,p). (B) HeLa cells transfected with a
tagged version of either wt IκBα (a-f) or L234
(g-l) cultured in the presence of serum (a,d,g,j)
were washed and treated for 90 minutes with
100 µg/ml cycloheximide in the absence of
serum and then 45 minutes with 100 µg/ml
cycloheximide and 10 ng/ml TNF (b,e,h,k).
These drugs were then removed and cells were
incubated for 90 minutes in the absence of
serum (c,f,i,l). Cells were subsequently fixed,
permeabilized and double stained with a
polyclonal antibody against p65 and a tagspecific monoclonal antibody followed by a
FITC-conjugated anti-rabbit IgG and a Texas
red-conjugated anti-mouse IgG. Cells were
visualized with confocal laser scanning
microscope and photographs correspond to the
accumulation of 4 optical sections in one
projection.
Nuclear export of IκBα
45 minutes. As reported previously for PKI and HIV 1 Rev
proteins, hydrophobic residues also appear to be critical for the
export activity of the NES. The time necessary for the export
of 50% of a 30-40 kDa protein lacking any functional NES
(bovine mosaic virus protein, Promega) after its injection into
the Xenopus oocyte nucleus has been measured to be between
1 and 2 hours (data not shown). The existence of such NESindependent transport (Schmidt-Zachmann et al., 1993) can
explain the incomplete inhibition of the nuclear export of L34
and L234 mutant proteins (Fig. 4). Taken together, these data
demonstrate that the IκBα (265-277) region constitutes a
nuclear export signal responsible for the fast transport of IκBα
out of the nucleus and able to confer an efficient nuclear export
ability on a reporter protein.
In order to analyze the role of the IκBα(265-277) region in
the export of the complex p65/IκBα, the ability of the L234
export mutant to interact with NF-κB as well as its effect on
the export of NF-κB were analyzed. L234 mutant was found
to be as efficient as wt IκBα both to associate with and prevent
p65/DNA interaction (Fig. 2B). Microinjection of the
L234/p65 complex into the nucleus revealed that the complex
was greatly impaired in its ability to be exported out of the
nucleus (Fig. 4C; compare with Fig. 1c); 50% of p65 stayed in
the nucleus 90 minutes after its injection whereas more than
95% of the p65/wt IκBα was exported within the same period.
Coinjection of p50 with L234 led to a complete retention of
p50 in the nuclear compartment (data not shown). Thus the
IκBα(265-277) sequence is essential for the rapid export of the
NF-κB/IκBα complex although we cannot formally exclude
that an additionnal export sequence would account for the
remaining export of the p65/L234 complex.
The IκBα export mutant shows a reduced capacity,
compared to the wt protein, to promote nuclear
export of NF-κB in mammalian cells
In order to confirm the observations made in X. laevis oocytes
and to gain more physiological relevance, it was crucial to
confirm in somatic cells that IκBα promotes the nuclear export
of NF-κB and that the IκBα (265-277) sequence is responsible for this function. For this purpose, HeLa cells were transfected with the tagged version of either wt or L234 IκBα and
their respective abilities to accumulate in the nucleus and
promote NF-κB subcellular redistribution in TNF-activated
cells were assessed. The subcellular localization of exogenous
IκBα as well as endogenous p65 in different conditions were
followed by indirect immunofluorescence using a polyclonal
antibody against p65 and a tag-specific monoclonal antibody.
Before stimulation with TNF, p65 was detected, as expected,
in the cytoplasm of either untransfected or transfected cells (Fig.
5A,a,i and B,a,g). In agreement with previous observations
(Cressman and Taub, 1993; Zabel et al., 1993), overexpressed
wt IκBα-tag localized both in the nucleus and the cytoplasm of
transfected cells (Fig. 5A,m and B,d) and similar cellular distribution was observed for the L234 mutant (Fig. 5B,j). To facilitate the complete degradation of the pool of tagged, ectopic
IκBα proteins, cells were treated before stimulation with the
protein synthesis inhibitor cycloheximide for 90 minutes. and
cycloheximide was maintained during the 45 minutes of TNF
induction. This experimental condition promoted translocation
of p65 in the nucleus (Fig. 5A,b,j and B,b,h) concomitantly with
the degradation of either wt or L234 IκBα (Fig. 5A,n and B,e,k).
375
Thus, by allowing massive degradation of preexisting pools
of exogenous IκBα, we could accurately assess the respective
capacities of wt or the mutant protein resynthesized after
removal of both TNF and cycloheximide (Fig. 5A and B, Chase
column), to promote nuclear export of NF-κB. Following chase
of TNF and cycloheximide, both wt (Fig. 5A,o and B,f) and
L234 IκBα (Fig. 5B,l) were resynthesized and detected again
in the nucleus. In cells transfected with wt IκBα, accumulation
of this protein in the nucleus led to both a clear reduction of
the nuclear content and relocalization of p65 to the cytoplasm
(Fig. 5A, compare k to j, and B, compare c to b). Maintaining
cells in the presence of cycloheximide after removal of TNF
during the chase period determined the persistance of p65 in
the nucleus, likely as a consequence of the inhibition of IκBα
resynthesis (Fig. 5A,l and p). It should be noted that given the
long exposure of cells to cycloheximide required to permit
complete induced degradation of IκBα-tag proteins in transfected cells, p65 export resulting from resynthesis and nuclear
accumulation of endogenous IκBα in control, untransfected
cells, was delayed and not observed at the time used in these
experiments (Fig. 5A).
In contrast with the substantial reduction of nuclear p65 due
to accumulation of exogenous wt IκBα-tag in this compartment, nuclear expression of L234 IκBα resulted in a modest
decrease in p65 in the nucleus (Fig. 5B, compare i to h). This
result is in agreement with the reduced capacity of this mutant
to promote the nuclear export of p65 in X. laevis oocytes
despite its ability to bind p65 as efficiently as the wild-type
protein, either in vitro (Fig. 2B) or in intact cells (data not
shown).
Together, those data validate the results obtained in the X.
laevis oocyte model and show that following nuclear accumulation of IκBα in mammalian cells, the transport of p65 from
the nucleus to the cytoplasm is largely dependent on the
presence of the functional nuclear export sequence located in
the C-terminal domain between residues 265 and 277 of the
IκBα protein.
DISCUSSION
Nuclear import of transcription factors has been shown to be
an essential and in some cases (reviewed by Vandromme et al.,
1996), regulated step for the activation of transcription.
However, whether nuclear export of transcription factors could
be involved in the termination of transcription remains poorly
investigated. Although it has been demonstrated previously
that nucleo-cytoplasmic shuttling is a general phenomenom
limited primarily by intranuclear interactions (SchmidtZachmann et al., 1993), few cellular functions have so far been
shown to depend on such a transport process. In particular, two
RNA-binding proteins, hnRNP A1 and HIV-1 Rev, have been
reported to promote mRNA export from the nucleus through
short protein sequences (referred to as nuclear export signal,
NES) responsible for efficient and rapid nuclear export
(Michael et al., 1995; Fischer et al., 1995). In the present work,
we report that IκBα, when expressed in the nuclear compartment, not only abrogates NF-κB/DNA interactions and NF-κBdependent transcription, but also promotes active transport of
this transcription factor back to the cytoplasm. This new
function for IκBα is insured by a NES located in the C-
376
F. Arenzana-Seisdedos and others
terminal domain of IκBα which is homologous to the HIV-1
Rev NES.
Down-regulation of NF-κB-dependent transcription
Nuclear injection of IκBα into the oocyte nucleus induces inhibition of the NF-κB-dependent transcription initialized by the
coinjection of p65 with an NF-κB-dependent luciferase
gene.This is in keeping with our original finding that coexpression of NF-κB and IκBα in the nucleus of transiently TNFinduced mammalian cells abrogates NF-κB dependent transcription (Arenzana-Seisdedos et al., 1995). Beside this
observation, the possibility that nuclear IκBα regulates NF-κB
transcriptional activity has been proposed by other authors in
different experimental models. It has recently been speculated
that glucocorticoids could induce inflammatory effects through
enhanced synthesis and nuclear accumulation of IκBα. Indeed,
it has been shown that, in vitro and in vivo, glucocorticoids
activate IκBα transcription in an NF-κB-independent manner.
Upon TNFα stimulation, glucocorticoids promote a faster
resynthesis of IκBα and consequently a more efficient interaction between newly synthesized IκBα and newly released
nuclear p65, leading to an inhibition of NF-κB activity. It has
been suggested based on these data that in addition to the
capacity of newly synthesized IκBα to immobilize NF-κB in
the cytoplasm, newly synthesized IκBα could be imported into
the nucleus and bind nuclear NF-κB (Scheinman et al., 1995;
Auphan et al., 1995). Moreover, IκBα has also been found in
nuclei of thymocytes from transgenic mice expressing the p65
gene under the control of the murine lck promoter. In these
cells, accumulation of the transgene p65 product correlates
with an enhanced expression of IκBα both in the cytoplasmic
and nuclear compartments (Perez et al., 1995). The presence
of IκBα in the nucleus of thymocytes from transgenic animals
may account for the surprising lack of constitutive nuclear NFκB DNA-binding activity despite a significant and permanent
expression of p65 in these cell nuclei.
Finally, additional evidence supporting a role for nuclear
IκBα in the regulation of NF-κB-dependent transcription has
been provided by a recent study performed in IκBα knock-out
mice (Beg et al., 1995; Klement et al., 1996). These reports
reveal that fibroblasts from IκBα deficient mice do not display
constitutive accumulation of transcriptionally active nuclear
NF-κB. In these cells, IκBβ would be responsible for retention
of NF-κB in an inactive cytoplasmic form. However, upon a
short exposure to TNF, fibroblasts from IκBα-deficient mice
display long-lasting NF-κB nuclear expression similar to that
observed in normal fibroblasts treated with cycloheximide. In
both cases, lack of IκBα synthesis and its eventual nuclear
accumulation, could account for the persistance of NF-κB
DNA-binding activity. Together these data converge to indicate
that following its nuclear translocation, IκBα releases NF-κB
from DNA and consequently down-regulates NF-κBdependent transcription. Furthermore, in addition to IκBαmediated inhibition of both NF-κB DNA-binding and NF-κBdependent transcription, nuclear accumulation of IκBα in
HeLa cells is concomitant with a dramatic decrease in the
nuclear content of the p50 subunit of the NF-κB complex
induced by either TNF or IL1 (Arenzana-Seisdados et al.,
1995). This observation led us to the hypothesis that nuclear
IκBα promotes the transport of NF-κB back to the cytoplasm.
Nuclear export of IκBα/NF-κB complexes
Although most nuclear proteins are potential shuttling proteins
(Schmidt-Zachmann et al., 1993), sequences responsible for
efficient nuclear export (NES) have only recently been identified. The present report shows that nuclear export of IκBα
observed after its injection into X. laevis oocyte nuclei is due
both to the lack of its nuclear retention and to a NES located
in the C-terminal domain. Indeed, fusion of the motif corresponding to a 17mer of IκBα (265-281) to pyruvate kinase
strongly increased the export rate of the latter protein. This is
in agreement with the observed capacity of IκBα region (263281) encompassing the putative NES sequence to confer on
Rev RNA-binding domain full nuclear export capacity (Fritz
and Green, 1996). However, these experiments were not
designed to investigate the role played by IκBα NES in either
the nuclear export of the entire IκBα or to induce the transport
of NF-κB from the nucleus to the cytoplasm. The results herein
reported show that combined mutations of leucine residues 274
and 277 (L34) or 272, 274 and 277 (L234) impaired the ability
of the corresponding mutants to be transported out of the
nucleus. The reduced export of L234 protein correlated with a
reduced capacity, as compared to wt IκBα, to promote the
transport of NF-κB back to the cytoplasm. The capacity of
IκBα to induce rapid NF-κB nuclear export in an NESdependent manner in mammalian HeLa cells underlines the
physiological relevance of the export mechanisms observed in
X. laevis oocytes.
Two different NES have been described so far; one is present
in hnRNP A1 (Michael et al., 1995) which is also involved in
the nuclear import of this protein. The other one is a leucinerich sequence (Fig. 4A) in which leucine residues have been
shown to be critical for targeting proteins out of the nucleus.
This motif is found in HIV-1 Rev, PKI and TFIIIA although its
functional role has only been demonstrated for Rev and PKI
(Fischer et al., 1995; Wen et al., 1995). The NES identified in
IκBα belongs to the latter type (Fig. 4A) and shares the same
functional properties. It is worth noting that the efficient export
capacity conferred on a nuclear protein by an NES appears to
be critical for the export of the protein itself, and also for the
export of other macromolecules complexed with the protein
carrying the functional NES. Indeed, nuclear export of Rev,
hnRNP A1 and putatively TFIIIA is crucial for the transport of
associated RNAs out of the nucleus. In addition, the NES of
PKI is only exposed when PKI is associated with the catalytic
subunit of the c-AMP-dependent protein kinase and is
therefore involved in the export of the PKI-bound catalytic
subunit rather than PKI alone. The recent discovery that NES
sequences of PKI, Rev and more recently IκBα (Fritz and
Green, 1996) can interact, in yeast, with the human nucleoporin-like protein hRIP (Fritz et al., 1995; Bogert et al., 1995)
opens the possibility that this protein promotes nuclear export
of c-AMP-dependent protein kinase, unspliced or single slicedHIV RNAs and NF-κB, respectively, through a common
nuclear export pathway.
In conclusion, apart from the role of IκBα in maintaining
NF-κB in an inactive form in the cytoplasm, our results support
a model in which NF-κB activity is also regulated by IκBα in
the nuclear compartment. Indeed, the abilities of IκBα to
localize to the nucleus and to dissociate NF-κB from DNA
should be considered as critical events in the down-regulation
Nuclear export of IκBα
of NF-κB-dependent transcription. Furthermore, we provide
evidence that IκBα, after complexing NF-κB in the nucleus,
promotes the export of this transcription factor back to the
cytoplasm. Such a process would avoid the persistence of
residual NF-κB complexes in the nucleus and would therefore
contribute to the complete down-regulation of NF-κBdependent transcription.
We are grateful to Drs Marion Schmidt-Zachmann and Françoise
Bachelerie for many helpful and stimulating discussions and Drs
Daniel Louvard, Bruno Goud, Jean Salamero, Hermann Bujard and
Susan Michelson for critical reading of the manuscript. This work was
supported by grants from ATIPE-CNRS, the Fondation Pour la
Recherche Medicale, the Ligue contre le Cancer, the Agence
Nationale pour la Recherche sur le Sida, The Medical Research
Council and the European Communities Concerted Action (Project:
Rocio II). MSR and PT are supported by a fellowship from Sidaction
(France) and Ministère de l’Education Nationale, respectively.
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(Received 12 November 1996 – Accepted 26 November 1996)