From the Institute of Biomolecular Sciences, School
of Biomedical Sciences, University of St. Andrews, The North Haugh, St.
Andrews KY16 9TS, Scotland, United Kingdom, and
Laboratoire de
"Transport nucleocytoplasmique," Unité Mixte de Recherche
144 Institut Curie-CNRS, 26 rue d'Ulm, 75248 Paris Cedex 05, France
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ABSTRACT |
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Transcriptional activation of nuclear factor The NF- I Newly synthesized I In the present study, LMB was used as a tool to inhibit NES-mediated
nuclear protein export and evaluate the consequences for regulation of
NF- Reagents--
Leptomycin B was a gift from B. Wolff-Winiski
(Novartis) and was used at 20 nM. TNF Plasmid Construction--
The pSVB
(CLONTECH) and pRC/RSV (Invitrogen) vectors were
digested with NotI. Corresponding DNA fragments were ligated
to obtain the pRC/RSV reporter plasmid in which Cell Culture and Transfections--
HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. Before treatment with IL-1
The expression of both the 3Enhancer-kB-conA-Luc (49) and the
pRC/RSV- Western Blot Analysis--
Cells grown in 6-well plates were
harvested in 150 µl of lysis buffer (50) for Western blot analysis.
15 µl of whole cell extracts were resolved in 10% SDS-polyacrylamide
gel electrophoresis, transferred to polyvinylidine difluoride membranes
(Sigma) by electroblotting, and processed for Western blotting as
described previously (38). When indicated, membranes were stripped and processed as reported previously (29). Primary polyclonal antibodies anti-I Cell Fractionation and Immunoprecipitation--
Cells were
washed twice with Dulbecco's modified Eagle medium and once with
Buffer S (115 mM potassium acetate, pH 7.3, 25 mM Hepes, pH 7.4, and 2.5 mM MgCl2)
at 37 °C and then treated with 2 µg/ml streptolysin O (SLO) (52)
in Buffer S for 3 min at 37 °C. SLO supernatant (cytosolic extracts)
was kept on ice, cells were washed once with Buffer S, and the
resulting washing volume was mixed with SLO supernatant (cytosolic
extracts). Cells were then washed three times with Buffer S and
solubilized in Buffer T (30 mM Tris, pH 8.6, 150 mM NaCl, 2 mM EDTA, and 2% Triton X-100). This
extract corresponds to the nuclear fraction. The quality of
fractionation was controlled by Western blotting using monoclonal
antibodies against heterogeneous nuclear ribonucleoprotein C (nuclear
protein) and Indirect Immunofluorescence Analysis--
For indirect
immunofluorescence analysis, HeLa cells grown on coverslips were fixed
with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 for
10 min. Monoclonal antibodies to I Measurement of Luciferase and Transcriptional Activation of NF-
Continuous exposure of cells to TNF Leptomycin B Inhibits Signal-induced Degradation of
I Nuclear Accumulation of I
To establish the subcellular localization of I
In the absence of any cell stimulation, p65 was predominantly
cytoplasmic and was found to interact with I
In conclusion, inhibition of NES-mediated nuclear protein export leads
to the accumulation of I Leptomycin B Does Not Increase I
Simultaneous TNF Nuclear I
To determine the step of the signal-induced degradation of I
However, this observation does not exclude the possibility that other
downstream steps leading to signal-induced degradation of I The experiments reported here indicate that inhibition of I In unstimulated cells, there is clearly a requirement for the
transcription of essential NF-B
(NF-
B) is mediated by signal-induced phosphorylation and degradation
of its inhibitor, I
B
. However, NF-
B activation induces rapid
resynthesis of I
B
, which is responsible for post-induction
repression of transcription. Newly synthesized I
B
translocates to
the nucleus, where it dissociates NF-
B from DNA and transports
NF-
B from the nucleus to the cytoplasm in a nuclear export
sequence-dependent process that is sensitive to leptomycin
B (LMB). In the present study, LMB was used as a tool to inhibit
nuclear export sequence-mediated nuclear protein export and evaluate
the consequences for regulation of NF-
B-dependent
transcriptional activity. Pretreatment of cells with LMB inhibits
NF-
B-dependent transcriptional activation mediated by
interleukin 1
or tumor necrosis factor
. This is a
consequence of the inhibition of signal-induced degradation of
I
B
. Although LMB treatment does not affect the signal
transduction pathway leading to I
B
degradation, it blocks
I
B
nuclear export. I
B
is thus accumulated in the nucleus,
and in this compartment it is resistant to signal-induced degradation.
These results indicate that the signal-induced degradation of I
B
is mainly, if not exclusively, a cytoplasmic process. An efficient
nuclear export of I
B
is therefore essential for maintaining a low
level of I
B
in the nucleus and allowing NF-
B to be
transcriptionally active upon cell stimulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B1/Rel family
of transcription factors is implicated in regulation of the expression
of a number of cellular genes involved in immune responses,
inflammation ,and apoptosis (for recent reviews, see Refs. 1-3). In
vertebrates, the NF-
B family of proteins is composed of
transcriptionally active p65/Rel A (4, 5), c-Rel (6), or Rel B (7) and
transcriptionally silent p50/NF-
B1 (8, 9) or p52/NF-
B2 (10-12).
All NF-
B proteins share a conserved region known as the Rel homology
domain that contains the nuclear localization signal as well as the
dimerization and DNA binding functions. The NF-
B form activated by
extracellular signals is composed of p50 and p65. NF-
B
transcriptional activity is controlled by inhibitor I
B proteins that
contain ankyrin repeat domains. Association of p50/p65 with I
B not
only occludes the nuclear localization sequence of p50 and p65, leading
to cytoplasmic sequestration, but also prevents NF-
B DNA binding
activity. Several I
Bs have been described including I
B
(13), I
B
(14), I
B
(15), and Bcl-3 (16). Additionally, the
precursors of p50 (p105) and p52 (p100) possess inhibitory ankyrin
repeat domains that in isolation are known as I
B
(17-19) and
I
B
(20, 21), respectively.
B
is organized in three domains: (a) an unstructured
amino-terminal (aa 1-72) signal response domain, (b) a
central region (aa 73-242) consisting of five ankyrin repeat domains,
and (c) a carboxyl-terminal region (aa 243-317) containing
a highly acidic domain (aa 276-317) that is bound to the ankyrin
repeat domain by a protease-sensitive linker (aa 243-275) and is
protected by bound p65 (22). Both amino- and carboxyl-terminal domains
are required for the signal-induced degradation of I
B
(23, 24). After signal induction, I
B
is phosphorylated on Ser-32 and Ser-36 (25-29) by the recently described dimeric I
B kinase (IKK) (30-34). After phosphorylation, I
B
is ubiquitinated on Lys-21 and Lys-22 (35-37), which targets the molecule for degradation by the proteasome. Once I
B
is degraded, NF-
B can translocate to the nucleus,
where it induces the transcription of several genes including that of its inhibitor, I
B
.
B
is accumulated in the cytoplasm but also in
the nucleus, where it terminates NF-
B-dependent
transcription. This is accomplished by inhibition of the NF-
B/DNA
interaction and export of NF-
B back to the cytoplasm (38). The
latter function of I
B
is conferred by a leucine-rich nuclear
export sequence (NES) present in its carboxyl-terminal region (aa
265-277) (39) and homologous to the NES found in many proteins
including the human immunodeficiency virus-type 1 Rev protein and the
protein kinase A inhibitor (40-42). Such NESs constitute transferable
transport signals that are necessary and sufficient to mediate rapid
and active export from the nucleus to the cytoplasm. The nuclear
protein CRM1 (also known as exportin 1) has been recently identified as the NES receptor (43-46). CRM1 belongs to the karyopherin
family and, in particular, it shares sequence homology in the Ran-GTP binding
domain with members from this family (47). The formation of CRM1/NES
complex is facilitated by the presence of Ran in its GTP-bound form. It
has been proposed that this ternary complex is transported through the
nuclear pore complex and dissociates in the cytoplasm due to GTP
hydrolysis by Ran-GAP (43). In addition, CRM1 has been shown to be the
cellular target of the drug leptomycin B (LMB) that inhibits
NES-mediated protein export both in vivo and in
vitro (43-45, 48).
B-dependent transcriptional activity. Pretreatment of
cells with LMB inhibits NF-
B-dependent transcriptional
activation mediated by IL-1
or TNF
. This is a consequence of the
inhibition of signal-induced degradation of I
B
. Although LMB
treatment does not affect the signal transduction pathway leading to
I
B
degradation, it blocks I
B
nuclear export. I
B
is
thus accumulated in the nucleus, and in this compartment it is
resistant to signal-induced degradation. These results indicate that
the signal-induced degradation of I
B
is mainly, if not
exclusively, a cytoplasmic process. An efficient nuclear export of
I
B
is therefore essential for maintaining a low level of I
B
in the nucleus and allowing NF-
B to be transcriptionally active upon
cell stimulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, which was
obtained from the MRC Reagent Project, and IL-1
(Sigma) were used at
10 ng/ml for the indicated time. Cycloheximide (Sigma) was used at 100 µg/ml. Z-LLL-H (MG132) was a gift from F. Baleux (Institut Pasteur)
and was used at 20 µM.
-galactosidase
transcription is driven by the RSV promoter.
or TNF
, the medium was changed
to Dulbecco's modified Eagle medium without serum. HeLa cells were
transfected by electroporation as described previously (39). A total of
10 µg of plasmid DNA encoding the chimeric protein 4NBC (24) was
transfected in 5 × 106 HeLa cells. After
transfection, cells were seeded in four wells of 6-well plates, and
incubation continued for 24 h.
-galalactosidase plasmids was stabilized in HeLa cells using
neomycin selection. Single cell clones were obtained by limiting
dilution of the neomycin-resistant cells. The 57A cell line was
selected on the basis of TNF
-induced luciferase activity and
constitutive
-galactosidase activity.
B
or anti-p65 (C-21 and C-20) were from Santa Cruz
Biotechnology. The previously described anti-SV5 tag monoclonal
antibody (51) to detect the chimeric 4NBC protein was obtained from Dr.
R. E. Randall. Monoclonal anti-
-tubulin and horseradish
peroxidase-labeled anti-rabbit or anti-mouse antibodies were purchased
from Amersham. An enhanced chemiluminescence detection system was used
to detect specific antigen-antibody interactions (POD; Boehringer Mannheim).
-adaptin (cytosolic protein). Nuclear and cytosolic
extracts were incubated for 15 min at 4 °C and centrifuged at
10,000 × g for 10 min. Anti-p65 polyclonal antibodies
and protein G-agarose beads were added to the supernatants and
incubated for 4 h at 4 °C. Beads were then washed, boiled for 5 min in Laemmli sample buffer, and analyzed by 10% SDS-polyacrylamide
gel electrophoresis and Western blotting with anti-I
B
polyclonal antibodies.
B
(10B) and polyclonal
antibodies to NF-
B p65 (C-20; Santa Cruz Biotechnology) were applied
for 30 min, followed by a 30-min incubation with fluorescein
isothiocyanate or Texas Red (TR)-conjugated donkey anti-mouse or
anti-rabbit IgG (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
fluorescein isothiocyanate and Texas Red dyes, respectively, with a
×100 oil immersion PL APO objective. Data presented on the same figure
were registered at the same laser and multipliers settings.
-Galactosidase
Activities--
75 × 103 HeLa 57A cells for each
time point were stimulated with 10 ng/ml TNF
or 10 ng/ml IL-1
as
described previously and incubated for an additional 7 h. Cells
were lysed as reported previously (38).
-Galactosidase activity was
measured using the Galacto-Light Plus kit (Tropix) according to the
manufacturer's instructions. Both
-galactosidase and luciferase
activities were measured in a bioluminometer (Berthold). Values for
fold activation of the luciferase reporter are the average of four
separate determinations and are compared with the uninduced value. In
each case,
-galactosidase activity was used as an internal control.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B Is Inhibited by Leptomycin
B--
It has been previously reported that I
B
that is newly
synthesized in response to NF-
B activation translocates to the
nucleus, where it dissociates NF-
B from DNA and transports NF-
B
from the nucleus to the cytoplasm in a NES-dependent
process that is sensitive to LMB. To explore the role of nuclear export
in the regulation of NF-
B-dependent transcriptional
activity, LMB was used to inhibit NES-mediated nuclear protein export.
To monitor transcriptional activity, a cloned HeLa cell line (57A) was
derived that contains NF-
B-dependent luciferase and
NF-
B-independent
-galactosidase reporters stably integrated into
the genome. To activate NF-
B-dependent transcription,
HeLa 57A cells were stimulated with TNF
or IL-1
for 7 h
(Fig. 1; TNF or IL-1 conditions) or for
30 or 40 min, respectively, and further incubated for 7 h at
37 °C in the absence of stimulus (Fig. 1; TNF+chase or
IL-1+chase). Unstimulated cells were exposed to control
medium lacking activators (Fig. 1; NS). After the indicated
time, cells were lysed, and reporter activity was measured. Because
-galactosidase reporter activity does not change in response to
TNF
or IL-1
, this was used to normalize the
NF-
B-dependent luciferase activity.
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Fig. 1.
Transcriptional activation of NF- B is
inhibited by LMB. HeLa 57A cells containing
NF-
B-dependent luciferase and NF-
B-independent
-galactosidase integrated reporters were untreated or pretreated for
30 min with LMB before incubation for 7 h with TNF
or IL-1
(TNF and IL-1) or control medium (NS).
When indicated (Chase), TNF
- and IL-1
-treated cells
were extensively washed after a 30-min incubation and were maintained
in culture for an additional 7 h in the absence of stimulation. At
the end of the incubation period, total cell extracts were analyzed for
luciferase and
-galactosidase activities. Luciferase activity was
normalized to
-galactosidase activity and reported as fold
activation relative to the enzymatic activity in unstimulated cells.
The values represent an average of four independent experiments.
Bars, S.D.
and IL-1
for 7 h induced
a 196- and 358-fold stimulation, respectively, compared with the basal
noninduced activity. After 30 min of TNF
or IL-1
incubation followed by a 7-h incubation in the absence of stimulus, the
transcriptional activity was 71- and 213-fold activation, respectively.
This results in a 64% and 41% reduction in activity compared with the
continuously activated cells. In contrast, when cells were treated with
20 nM LMB for 30 min before cell stimulation, only 51- and
102-fold activation was obtained by continuous stimulation with TNF
and IL-1
, respectively. LMB treatment thus led to a 74% and 72%
inhibition of the NF-
B transcriptional activity induced by TNF
or
IL-1
, respectively. Moreover, remaining NF-
B activities were not
modified when LMB-treated cells were transiently exposed to TNF
or
IL-1
and incubation continued in the absence of stimulus. These
results indicate that an inhibition of NES-mediated nuclear protein
export not only prevents the post-induction repression of
NF-
B-dependent transcription but also strongly represses
the initial activation of NF-
B upon cell stimulation. Similar
results were obtained with an independent lung-derived cell clone
(A549) that contains NF-
B-dependent luciferase and
NF-
B-independent
-galactosidase reporters stably integrated into
the genome (data not shown).
B
--
Activation of NF-
B transcriptional activity is
mediated by signal-induced degradation of I
B
, which allows the
released NF-
B to translocate to the nucleus. The effect of LMB on
signal-induced degradation of I
B
expression was therefore
examined. Thus, untreated HeLa or LMB-pretreated cells were exposed to
TNF
for 30 min or to IL-1
for 40 min (Fig.
2, A and B). Whole
cell extracts were analyzed by Western blotting using an anti-I
B
antibody. As expected, I
B
was rapidly degraded after TNF
or
IL-1
treatments, but pretreatment of the cells with LMB
substantially inhibited signal-induced degradation of I
B
. This
effect was not restricted to HeLa cells because TNF
-induced
degradation of I
B
was also inhibited in 293 or COS7 cells
pretreated with LMB (Fig. 2, C and D). These data
suggest that LMB inhibits NF-
B transcriptional activity by reducing
the signal-induced degradation of I
B
.
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Fig. 2.
LMB inhibits signal-induced degradation of
I B
. HeLa (A and B), COS7
(D), and 293 (C) cells were untreated or
pretreated with LMB for 90 min, as indicated. Where indicated, cells
were further stimulated for 30 min with TNF
(A, C, and
D) or for 40 min with IL-1
(B). Whole cell
extracts were analyzed by Western blotting with a polyclonal antibody
specific for I
B
and a monoclonal antibody to
-tubulin.
B
Mediated by Leptomycin B Protects
It from Signal-induced Degradation--
To understand how LMB, a drug
that blocks NES-mediated nuclear protein export, inhibits I
B
degradation, the subcellular localization of both I
B
and NF-
B
p65 was analyzed by cell fractionation and biochemical analysis as well
as indirect immunofluorescence. HeLa cells were either untreated or
pretreated with LMB 30 min before stimulation with TNF
. After 30 min, TNF
was removed, and the cells were either analyzed directly or
incubated for an additional 60 min in the absence of TNF
(chase).
I
B
levels were determined by Western blotting of whole cell
extracts. In the absence of LMB, TNF
induced I
B
degradation,
followed by resynthesis during the chase period (Fig.
3A, lanes 1-3). LMB treatment
inhibited the TNF
-induced reduction in I
B
level but had no
effect on the amount of I
B
present after the 60-min chase period
(Fig. 3A, lanes 4-6).
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Fig. 3.
I B
, which was retained in the nucleus
by LMB, does not undergo signal-induced degradation. A, HeLa
cells were untreated or pretreated for 30 min with LMB. When indicated,
cells were further incubated for 30 min with TNF
(lanes 2 and 5) or for 30 min with TNF
plus a 60-min chase
(lanes 3 and 6). Whole cell extracts were
analyzed by Western blotting with a polyclonal antibody specific for
I
B
and a monoclonal antibody to
-tubulin. B, HeLa
cells were treated as indicated in A and fractionated into
cytoplasmic and nuclear fraction using SLO. Cytoplasmic and nuclear
extracts were either analyzed directly by Western blotting with an
anti-p65 antibody (upper panel) or immunoprecipitated with
an anti-p65 polyclonal antibody before analysis by Western blotting
with an I
B
polyclonal antibody (lower panel).
C, HeLa cells treated as indicated in A were
processed for indirect immunofluorescence and double stained with a
mouse monoclonal antibody to I
B
and a rabbit polyclonal antibody
to Rel A. Primary antibodies were detected with a fluorescein
isothiocyanate-conjugated anti-mouse IgG and a Texas Red-conjugated
anti-rabbit IgG. Cells were visualized by confocal laser scanning
microscopy, and photographs correspond to the accumulation of four
optical sections in one projection.
B
and the p65
subunit of NF-
B under these experimental conditions, cells were
fractionated using Streptolysin O, a bacterial toxin that permeabilizes
cells without affecting the integrity of the nuclear envelope (52-54).
Cytoplasmic and nuclear extracts were analyzed either by Western
blotting with an anti-p65 antibody or by immunoprecipitation with an
anti-p65 antibody and Western blotting with an anti-I
B
antibody
(Fig. 3B). In parallel, intact cells treated under the same
experimental conditions were processed for immunofluorescence and
analyzed using anti-p65 and anti-I
B
antibodies (Fig.
3C).
B
, whereas I
B
was localized in both the cytoplasm and the nucleus (Fig. 3B, lane 1; Fig. 3C, left panel). Treatment with LMB led to
the nuclear accumulation of I
B
and p65, although to a lesser
extent (Fig. 3B, lane 4; Fig. 3C, right panel).
This result suggests that I
B
and a fraction of NF-
B are
continuously shuttling between the nucleus and the cytoplasm, even in
the absence of cell stimulation. Upon TNF
stimulation of cells that
were not treated with LMB, I
B
was degraded in the cytoplasm with
a small fraction still present in the nucleus, and p65 was partially
translocated to the nucleus (Fig. 3B, lane 2; Fig. 3C,
left panel). The addition of TNF
to LMB-treated cells led to
the loss of I
B
from the cytoplasm without affecting the nuclear
content of I
B
. Thus, the remaining I
B
after TNF
stimulation of LMB-treated cells was exclusively nuclear. Nuclear
translocation of p65 was increased by LMB treatment but mainly resulted
in the nuclear accumulation of a transcriptionally inactive
I
B
-bound form of Rel A (Fig. 1; Fig. 3B, lane
5; Fig. 3C, right panel). In cells that were not
treated with LMB that had been exposed to TNF
but incubated for an
additional 60 min in the absence of TNF
, I
B
returned to
prestimulation levels as a result of the de novo synthesis of the protein. Binding of the newly synthesized I
B
to p65
allowed the I
B
/p65 complexes to relocalize to the cytoplasm (Fig.
3B, lane 3; Fig. 3C, left panel). Under identical
conditions, I
B
levels in LMB-pretreated cells returned to those
observed before TNF
stimulation. However, LMB treatment inhibited
the relocalization of both I
B
and Rel A to the cytoplasm (Fig.
3B, lane 6; Fig. 3C, right panel).
B
in the nucleus, where it is resistant
to signal-induced degradation. Nuclear I
B
can interact with
NF-
B and therefore prevent the DNA binding of the transcription factor. Moreover, LMB-mediated inhibition of nuclear export blocks the
transport of NF-
B/I
B
complexes back to the cytoplasm. An efficient nuclear export of I
B
is thus required to maintain a low
level of I
B
in the nucleus and allow NF-
B to be efficiently activated upon cell stimulation.
B
Synthesis--
The data
presented in Figs. 1-3 indicated that LMB inhibits signal-induced
activation of NF-
B by partitioning I
B
in the nucleus, where it
is resistant to degradation. However, it is a formal possibility that
LMB has no effect on I
B
degradation but rather stimulates the
synthesis of I
B
. To distinguish between these possibilities,
cells pretreated with LMB or untreated cells were exposed to TNF
in
the presence of cycloheximide. The expression and subcellular
localization of I
B
and p65 were analyzed by Western blotting
(Fig. 4A) and indirect
immunofluorescence using anti-p65 and anti-I
B
antibodies (Fig.
4B).
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Fig. 4.
LMB does not increases I B
synthesis.
A, HeLa cells were treated as described in the Fig. 3
legend, but cycloheximide was added together with TNF
or for the
last 30 min in unstimulated cells. Whole cell extracts were analyzed by
Western blotting with a polyclonal antibody specific for I
B
and a
monoclonal antibody to
-tubulin. B, HeLa cells treated as
indicated in A were processed for indirect
immunofluorescence and double stained with a mouse monoclonal antibody
to I
B
and a rabbit polyclonal antibody specific for p65. Primary
antibodies were detected with a fluorescein
isothiocyanate-conjugated anti-mouse IgG and a Texas
Red-conjugated anti-rabbit IgG. Cells were visualized by confocal laser
scanning microscopy, and photographs correspond to the
accumulation of four optical sections in one projection.
and CX treatments did not affect signal-induced
I
B
degradation and nuclear translocation of p65 (Fig. 4A,
lane 2; Fig. 4B, left panel). However, CX abolished the
de novo synthesis of I
B
and relocalization of p65 to
the cytoplasm that occurs after TNF
treatment and an additional 1-h
incubation in the absence of TNF
(Fig. 4A lane 3; Fig.
4B, left panel). These data confirm that the expression of
newly synthesized I
B
in the nucleus is responsible for the
nuclear export of NF-
B to the cytoplasm. Treatment of cells with LMB
and CX in the presence or absence of TNF
stimulation did not
substantially modify the subcellular distribution of I
B
and p65
observed previously in cells treated only with LMB (Fig. 4B,
right panel). I
B
was still detected in LMB- and CX-treated
cells stimulated with TNF
, and when the incubation was continued in
the absence of TNF
, the amount of I
B
in LMB- and CX-treated
cells was only slightly decreased (Fig. 4A, lanes 4-6).
Thus, LMB treatment does not induce I
B
synthesis but rather
protects I
B
from signal-induced degradation as a consequence of
its nuclear sequestration.
B
Is Not Accessible to Signal-induced
Modification--
To confirm that LMB treatment protects I
B
from
signal-induced degradation through its accumulation in the nucleus and
not through an inhibition of the TNF
signal transduction cascade, a
fusion protein containing the amino- and carboxyl-terminal regions of
I
B
fused to Escherichia coli
-galactosidase (24)
was used. Because this protein contains the necessary sequence
information from I
B
, it undergoes signal-induced degradation in
response to TNF
. However, because it does not contain the ankyrin
repeats from I
B
, it does not translocate to the nucleus and is
localized exclusively in the cytoplasm, even in the presence of LMB
(data not shown). Thus, in LMB-treated cells transfected with a plasmid expressing 4NBC, the endogenous I
B
will be located in the
nucleus, whereas 4NBC will be located in the cytoplasm. HeLa cells were transiently transfected with the 4NBC-encoding plasmid, treated with
LMB or control medium, and then exposed to TNF
and CX or control
medium. After 60 min, the levels of 4NBC and endogenous I
B
were
determined by Western blotting. The combined action of TNF
and CX
induced the degradation of both endogenous I
B
and exogenous 4NBC,
although, as expected, to varying degrees (Fig.
5A, I
B
, 90% degraded;
4NBC, 58% degraded). In the presence of LMB, the extent of TNF
- and
CX-induced degradation of 4NBC was unaltered (60% degraded), whereas
I
B
was inhibited (46% degraded, Fig. 5A), indicating
that LMB does not directly affect the TNF
signal transduction
cascade. Moreover, in vitro kinase assays using
immunopurified IKK
and IKK
from HeLa cell extracts showed that
the TNF
-induced IKK activity was not affected by the LMB treatments
(data not shown).
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Fig. 5.
Nuclear I B
is not a substrate for
signal-induced modifications required for degradation. A,
HeLa cells transfected with the plasmid encoding the 4NBC chimera were
preincubated in control medium (lanes 1 and 2) or
with LMB (lanes 3 and 4) for 60 min and further
treated with TNF
plus CX (lanes 2 and 4) for
60 min. Total cell extracts were analyzed by Western blotting with the
SV5 Pk tag monoclonal antibody and a polyclonal antibody specific for
p65. The same membrane was stripped and reprobed with an anti-I
B
antibody. Levels of I
B
and 4NBC were determined by densitometry.
B, HeLa cells were untreated (lanes 1 and
2) or pretreated for 60 min with MG132 (lanes 3 and 4), LMB (lanes 5 and 6), or both
MG132 and LMB (lanes 7 and 8). After a 15-min
incubation with TNF
(lanes 2, 4, 6, and 8),
whole cell extracts were analyzed by Western blotting with a polyclonal
antibody specific for I
B
and a monoclonal antibody to
-tubulin. C, HeLa cells were pretreated with MG132
(lanes 1-4) and LMB (lanes 3 and 4)
for 90 min and further incubated for 30 min with TNF
(lanes
2 and 4). Whole cell extracts were analyzed by Western
blotting with a polyclonal antibody specific for I
B
and a
monoclonal antibody to
-tubulin. SUMO-1- and ubiquitin-conjugated
forms of I
B
are indicated (B).
B
that is prevented by the localization of I
B
in the nucleus, untreated HeLa cells or HeLa cells treated with the proteasome inhibitor MG132 were exposed to TNF
or control medium for 15 min.
This experimental condition allows the accumulation of a more slowly
migrating phosphorylated form of I
B
(Fig. 5B, lanes 1-4). To confirm that this form corresponded to phosphorylated I
B
, the same blot was stripped and reprobed with an antibody specifically recognizing I
B
phosphorylated at Ser-32 (data not shown). In contrast, I
B
degradation was not observed when cells were pretreated with LMB, and the TNF
plus MG132-mediated
accumulation of the phosphorylated form of I
B
was severely
reduced (Fig. 5B, lanes 5-8). These data indicate that
nuclear I
B
is not accessible to signal-induced phosphorylation.
B
could also be affected; thus, any effect of LMB on I
B
ubiquitination was evaluated. HeLa cells were pretreated with the
proteasome inhibitor MG132 and then treated with either LMB, TNF
, or
a combination of LMB and TNF
. As expected, MG132 treatment prevented
TNF
-induced degradation and allowed the accumulation of slowly
migrating multi-ubiquitinated forms of I
B
(29) (Fig. 5C,
lane 2). In the presence of MG132, LMB, and TNF
, the amount of
ubiquitinated I
B
was strongly reduced but not abolished (Fig. 5C, lane 4) and probably corresponds to modification of the
remaining cytoplasmic I
B
. Thus, nuclear sequestration of I
B
by LMB prevents its proper phosphorylation and subsequent
ubiquitination after signal induction. The conditions described above
did not alter the levels of the SUMO-1 modified form of I
B
(50).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
nuclear export not only prevents the post-induction repression of
NF-
B-dependent transcription but also strongly represses
the initial activation of NF-
B upon cell stimulation. Indeed,
nuclear I
B
appears to be resistant to signal-induced
phosphorylation and degradation, and this results in nuclear
accumulation of transcriptionally inactive I
B
/NF-
B complexes.
Nuclear export of I
B
and many other proteins that shuttle between
the nucleus and the cytoplasm is mediated by a leucine-rich NES that is
recognized by CRM1/exportin 1 (43-46). In our experiments, nuclear
export of I
B
was inhibited by the drug LMB. LMB specifically
targets CRM1 by blocking its interaction with the NES (45). Although
the inhibition of CRM1 by LMB is highly specific, it was important to
rule out the possibility that LMB might be interfering with the signal
transduction pathways that lead to NF-
B activation. To address this
point, we used a lacZ fusion protein (4NBC) containing the amino and
carboxyl termini of I
B
. When this protein is expressed in cells,
it is unable to translocate to the nucleus but undergoes signal-induced degradation in response to agents such as TNF
and IL-1
(24). Signal-induced degradation of the 4NBC protein was unaffected by LMB
(Fig. 5A), indicating that LMB does not inhibit the signal transduction pathway that leads to I
B
degradation. It is also clear that LMB does not inhibit transcription in a nonspecific fashion
because the activity of the integrated RSV-driven lacZ reporter was
unaffected by the presence of LMB (Fig. 1). The conclusion from these
experiments is that in HeLa cells, signal-induced phosphorylation and
degradation of I
B
occurs exclusively in the cytoplasm. One possibility to explain this restriction is that an essential component of the signal transduction pathway that leads to I
B
phosphorylation cannot gain access to the nucleus. It appears that the
I
B
kinases (IKK
and IKK
) are present in a large signaling
complex that may contain upstream kinases such as NF-
B inducing
kinase (55, 56) and scaffolding proteins such as NEMO (57). It has yet to be determined if this large complex can be imported into the nucleus. The alternative argument would be that the signal modification machinery has access to the nucleus, but that the nuclear I
B
is
in some way refractile to modification. Mechanisms to achieve this
could include prior covalent modification of I
B
to a form that is
no longer recognized by the IKK signaling complex. It has recently been
demonstrated that I
B
is modified by the small ubiquitin-like
protein SUMO-1 (50). Because this protein is linked to the same lysine
residues that are used for ubiquitination of I
B
, this renders the
SUMO-1 modified form of I
B
resistant to signal-induced
degradation. Although the known proteins that are substrates for SUMO-1
modification have been detected in the nucleus or are involved in
nuclear transport (58), we have no evidence to support the notion that
nuclear I
B
detected in the presence of LMB is resistant to
signal-induced phosphorylation because it is modified by SUMO-1. It is
also possible that I
B
could interact with a nuclear protein that
occludes the region in I
B
containing residues Ser-32 and Ser-36,
thus protecting it from signal-induced phosphorylation.
B-dependent genes.
Low-level transcription of these genes does not take place simply as a
consequence of NF-
B-independent transcription, because I
B
overexpression effectively abolishes the activity of a
NF-
B-dependent reporter in unstimulated
cells.2 Thus, it appears that
the cell has evolved a highly dynamic system to provide for continued
low-level transcription of NF-
B-dependent genes. This
homeostatic mechanism requires the continuous proteasome-mediated breakdown of I
B
, which generates a stream of free NF-
B that can translocate to the nucleus. Once in the nucleus, NF-
B activates NF-
B-dependent genes, including that of I
B
. After
transport to the cytoplasm, I
B
mRNAs are translated, and the
free I
B
is directed to the nucleus, where it interacts with
DNA-bound NF-
B and dissociates the DNA-protein complex. By virtue of
the presence of a NES in I
B
(39), NF-
B/I
B
complexes are
recognized by CRM1, which mediates nuclear export (45). At this point, equilibrium is reestablished. Thus, rather than having a simple on-off
switch, the cell can delicately alter the NF-
B transcriptional response by varying the rate at which I
B
is turned over. The most
extreme perturbation of this equilibrium comes after exposure of the
cells to agents such as TNF
or IL-1
. In this situation, cytoplasmic I
B
is completely degraded, and a massive pulse of NF-
B is released into the nucleus to initiate high-level
transcription of NF-
B-dependent genes. However, the same
mechanism is used to bring the system back into homeostasis (38, 39). A
remarkably similar homeostatic mechanism seems to operate to control
the level of p53 within the cell. In this case, the product of the hdm2 gene targets p53 for ubiquitin-mediated proteasomal
degradation, and disruption of this interaction during the damage
response leads to the accumulation of p53. Nuclear translocation of p53 activates transcription of the hdm2 gene, and the newly
synthesized protein enters the nucleus, where it terminates
p53-dependent transcriptional activation. hdm2 also
contains a NES, and this is used to export the p53/hdm2 complex to the
cytoplasm using the same pathway that is used for nuclear export of
I
B
. Inhibition of hdm2-mediated export revealed that nuclear
export of hdm2 is required to accelerate the degradation of p53 (59).
In the case of both I
B
and p53, ubiquitin-mediated proteasomal
degradation occurs in the cytoplasm, even though proteasomes are found
in both compartments. However, proteasomal components are distributed differentially between the nucleus and the cytoplasm (60, 61), suggesting that nuclear and cytoplasmic proteasomes may have unique properties. The advantage to the cell of these homeostatic mechanisms to control NF-
B- and p53-dependent transcription is that
they are both highly sensitive to perturbation, and they can provide a
finely tuned response to external signals.
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ACKNOWLEDGEMENTS |
---|
We thank Lesley Thompson for performing IB
kinase assays, Magali Prigent for help with the immunofluorescence
studies, Prof. S. Bhakdi for the SLO, and Novartis for the LMB.
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FOOTNOTES |
---|
* This work was supported by the Medical Research Council, the Association de Recherche contre le Cancer, and the EU Biomed II program (Rocio II).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Pfizer Central Research.
¶ To whom correspondence should be addressed. Tel.: 44-1334-463396; Fax: 44-1334-462595; E-mail: rth{at}st-and.ac.uk.
2 M. S. Rodriguez, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor
B;
TNF, tumor necrosis factor;
IL-1
, interleukin 1
;
LMB, leptomycin B;
CX, cycloheximide;
NES, nuclear export sequence;
aa, amino acid(s);
IKK, I
B kinase;
SLO, streptolysin O;
SUMO-1, small
ubiquitin-like modifier 1.
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REFERENCES |
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