From the Rosenstiel Basic Medical Sciences Research Center and the Department of Biology, Brandeis University, Waltham, Massachusetts 02454
Received for publication, December 22, 2000
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ABSTRACT |
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The I The NF- The association of Rel with I Recently, I In our earlier studies we also showed that I Cell Lines and Strains--
D5 h3 T hybridoma cells and A20
mature B cells were grown in Dulbecco's modified Eagle's medium and
RPMI 1640 medium, respectively, with 10% heat-inactivated fetal bovine
serum, 50 µM Plasmids--
pGFP-p65 and pCDNA3-HA-I Immunostaining--
The procedures for immunostaining adherence
cells were the same as described previously (15). For staining
suspension cells (T and B cells), the procedures were also as described
previously (18).
Protease Digestion--
The proteases Asp-N and Lys-C were
purchased from Roche Molecular Biochemicals. Proteases were used
according to the manufacturers' specifications.
Fluorescence Microscopy--
The subcellular localization of GFP
and the immunofluorescence signals were observed by fluorescence
microscopy (Axiophot II, Zeiss) with a GFP generic filter, fluorescein
isothiocyanate, rhodamine, and DAPI filter.
The nuclear export property of IB family of proteins regulates
NF-
B-dependent transcription by inhibiting DNA binding
and localizing these factors to the cell cytoplasm. I
B
does this
by shifting the balance between nuclear import of Rel proteins and
their export from the nucleus. Here we show that, unlike I
B
,
I
B
and I
B
appear to sequester p65 or c-Rel in the cytoplasm
by inhibiting nuclear import. Furthermore, because I
B
does not
undergo nucleocytoplasmic shuttling, it cannot remove nuclear proteins
like I
B
does. We conclude that the mechanism of action differs
among I
B family members.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B1/Rel family
of transcription factors plays a central role in immune and
inflammatory responses (1). In most cell types these proteins are
sequestered in the cell cytoplasm complexed to a family of inhibitory
I
B proteins (2, 3). Cellular activation results in I
B
degradation, which leaves the DNA-binding protein free to translocate
to the nucleus and activate gene expression. Because of the widespread
effects of NF-
B activation, its localization in the cytoplasm must
be strictly maintained. I
B
-deficient mice are a striking example
of the importance of NF-
B sequestration in the cytoplasm; these mice
die of a wasting disease that has been attributed to tumor
necrosis factor-
production (4, 5). Nuclear factor-
B has also
been detected in several diseased tissues, where it has been proposed
to contribute to the pathology in part by inhibiting apoptosis (6,
7).
B
has been proposed to hide the NLS
of Rel proteins (8, 9), thereby precluding nuclear entry of the
transcription factor. In addition to hiding the NLS, association with
I
B
also inhibits DNA binding by NF-
B. Thus, association of
NF-
B with I
B ensures that NF-
B-dependent gene transcription occurs only when cells are stimulated appropriately.
B
has been shown to shuttle between the nucleus and
the cytoplasm. Nuclear entry is mediated by a nonclassical NLS located
in the second ankyrin repeat of I
B
(10-12), and nuclear export
is determined by a CRM1-dependent nuclear export sequence located in the N-terminal domain preceding the first ankyrin domain (13-15). A second nuclear export sequence has been identified at the C
terminus of I
B
, but its functional significance is unclear at
present (16, 17). The observation that cytoplasmic sequestration of
p65·RelA also required nuclear export was unexpected and led to a
reassessment of the existing sequestration model. We and others
(13-15) have proposed that the cytoplasmic location of Rel proteins by
I
B
is a dynamic process that depends on the active export of
Rel·I
B
complexes out of the nucleus.
B
and I
B
,
unlike I
B
, do not shuttle via the CRM1 pathway. Specifically, the
subcellular distribution of GFP-I
B
or GFP-I
B
in yeast was
not affected by a mutation in the CRM1 gene, and leptomycin B (LMB) treatment did not alter the location of these proteins in
transiently transfected mammalian cells. In addition, we did not detect
an association between CRM1 protein and either I
B
or I
B
in
a yeast two-hybrid assay (15). In this paper we demonstrate that
cytoplasmic retention of Rel proteins by I
B
and I
B
involves sequestration rather than tilting the balance of nuclear import and
export as is the case with I
B
. Furthermore, although newly synthesized I
B
can enter the nucleus, it cannot restore nuclear Rel proteins to the cytoplasm. These observations suggest that I
B
and I
B
function differently from I
B
.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and antibiotics.
COS cells were cultured in Dulbecco's modified Eagle's medium with
10% newborn calf serum and antibiotics. Yeast strain W303 and its
transformants were generally grown in synthetic medium with the
appropriate amino acid and nitrogen base supplement.
B
have
been described previously (15). pGFP-cRel contains full-length murine
cRel in frame after GFP. pCDNA3-Myc-I
B
and
pCDNA3-Myc-I
B
were made by inserting full-length murine
I
B
and I
B
cDNA, respectively, in frame behind a c-Myc
tag (MEQKLISEEDL). Yeast galactose-inducible plasmid encoding GFP-p65
and copper-inducible HA-I
B
(pCuI
B
) have been described
previously (15). The copper-inducible HA-I
B
was made by replacing
the I
B
gene with a murine I
B
full-length gene in the same
vector. All plasmids used in this study were confirmed by sequencing,
and expression of proteins was verified by immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
is essential for
cytoplasmic location of Rel proteins. However, I
B
and I
B
,
which are not nucleo-cytoplasmic shuttling proteins, can also
effectively localize Rel proteins to the cytoplasm. One possibility was
that cytoplasmic retention by I
B
/
may be mediated by export
determinants in the Rel proteins. To test this possibility, we
coexpressed green fluorescent protein (GFP)-tagged Rel proteins with
I
B
or I
B
in COS cells and assayed the location of Rel
proteins by GFP fluorescence. Both p65 and c-Rel (data not shown) were located in the cytoplasm in the presence of I
B
(Fig.
1A, left panel). However,
these complexes did not translocate to the nucleus when the cells were
treated with LMB, an inhibitor of CRM1-mediated nuclear export
(Fig. 1A, right panel). Therefore, CRM1 was not involved in
determining the subcellular location of these complexes. Similar
results were obtained with I
B
. As expected, I
B
-associated p65, or c-Rel (data not shown), was predominantly nuclear in
LMB-treated cells (Fig. 1A, top row). Thus, cytoplasmic
retention by I
B
and I
B
may involve true sequestration
rather than a balance between import and export as is the case with
I
B
.
View larger version (39K):
[in a new window]
Fig. 1.
Rel·I B
complexes shuttle continuously, but
Rel·I
B
complexes do
not. A, GFP-p65 was transiently transfected with
HA-I
B
, Myc-I
B
, or Myc-I
B
into COS cells. Half the
cells were then treated with LMB (10 ng/ml) for 3 h. Untreated
(left panel) or LMB-treated cells (right panel)
were fixed for fluorescent visualization. Green fluorescence
shows a GFP-p65 signal. The red fluorescence shows an I
B
signal from rhodamine-conjugated antibodies against either the HA tag
or the Myc tag. Blue fluorescence shows DAPI staining of
nuclei. B, A20 B cells were settled on specially
treated coverslips (Fisher). Half of these were treated with LMB (100 ng/ml) for 45 min. Cells with or without LMB treatment were fixed and
permeabilized for immunostaining. Green fluorescence shows
endogenous I
B
(first row) and I
B
(second
row) detected by fluorescein isothiocyanate-conjugated antibodies
against anti-I
B
and anti-I
B
respectively. Representative
results are shown from one of three independent experiments.
These observations were confirmed in mammalian cells by investigating
the shuttling dynamics of endogenous Rel·IB complexes. Endogenous
proteins in mature B (A20) and mature T (D5 h3) cell lines (data not
shown) were visualized by staining fixed, permeabilized cells with
anti-I
B
, or anti-I
B
, antibodies in the presence or absence
of LMB to block nuclear export. In untreated cells both I
Bs were
predominantly cytoplasmic (Fig. 1B, left panel). A 1-h LMB
treatment induced considerable nuclear translocation of I
B
but
not I
B
(Fig. 1B, right panel). Because most
of the cellular I
B is associated with Rel proteins, we concluded
that Rel·I
B
complexes shuttled continuously, but Rel·I
B
complexes did not. Lack of Rel·I
B
shuttling is consistent with
sequestration being the major mechanism of cytoplasmic retention by
I
B
.
We found more direct evidence for differences in interaction between
IB
or I
B
and p65 through partial proteolysis assays. p65
protein was expressed by transient transfection in BOSC 23 cells in the
presence of HA-I
B
or Myc-I
B
. The p65·I
B complex was
immunoprecipitated from whole cell extract with anti-I
B
antibody
or anti-I
B
antibody and digested with different proteases. The
p65 fragments were detected using antibodies directed against the N or
C terminus of p65 to estimate the cut site from one or the other end of
p65. Only two of seven proteases showed significant differences in the
pattern of p65 fragments generated in the presence of I
B
or
I
B
.
p65 alone generated one major fragment when treated with Asp-N of ~28
kDa when assayed from the C terminus (Fig.
2, lanes 5 and 6);
this corresponds to a cut site located 293 amino acids from the
N terminus (Fig. 2, top). In the p65·IB
complex, two bands of approximately equal intensity were seen (Fig. 2, lanes 1 and 2), whereas in the p65·I
B
complex the
faster mobility (23 kDa) band was enhanced. Therefore, cutting at
residue 293 was reduced in the p65·I
B
complex compared
with p65 alone, allowing the detection of the cut site at residue 360 (which was not evident with p65 alone). This is presumably because of
the protection of the p65 NLS by I
B
, which lies close to residue
293 between residues 301 and 304. Cutting at 293 was further inhibited
in the p65·I
B
complex as shown by a relative increase in the
intensity of the 23-kDa compared with the 28-kDa band. These
observation suggest that the region around residue 293, including the
NLS, is more protected in the p65·I
B
complex.
|
p65·IB
and p65·I
B
complexes were also probed using the
protease Lys-C and p65 antibodies directed against the N terminus. Increased cutting at the residue 425 site was evident in the I
B
complex compared with the I
B
complex (Fig. 2, lanes 8 and 11). These observations also support the
interpretation that I
B
and I
B
interact differently with
p65. We suggest that the p65 NLS is better hidden by I
B
than by
I
B
.
The simplest interpretation of the experiments described above was that
Rel·IB
complexes did not enter the nucleus because the nuclear
localization sequences in both proteins were very effectively hidden in
the complex. Therefore, the question of nuclear export did not arise.
However, the question remained that if any Rel·I
B
complexes
formed in the nucleus, would I
B
be able to bring the complex out
to the cytoplasm? Such a situation may occur at the end of cell
stimulation when Rel proteins are already nuclear and new I
Bs are
synthesized to terminate NF-
B-dependent gene expression.
We addressed this question in a yeast model.
We have previously shown that export-dependent cytoplasmic
localization of p65 by IB
can be recapitulated in yeast (15). To
test the properties of I
B
, we coexpressed I
B
and GFP-p65 from galactose-inducible promoters in wild type or Crm1p-deficient (crm1-1) yeast strains. GFP-p65 was located in the cytoplasm
under these conditions in both strains (data not shown), correlating closely with the observations in mammalian cells (Fig. 1). In contrast,
when GFP-p65 and I
B
were coexpressed in crm1-1 cells, the complex remained in the nucleus (15). To compare the ability of
I
B
and I
B
to remove nuclear p65, we expressed GFP-p65 using a galactose-inducible promoter, followed by either I
B
, or
I
B
, from a copper-inducible promoter. A 3-h induction with
galactose was followed by growth in glucose to suppress GFP-p65
transcription. In cells that did not contain I
B expression vectors,
GFP fluorescence was strictly nuclear. Even when cells contained either
I
B
or I
B
expression plasmids, GFP fluorescence was largely
restricted to the nucleus, although whole cell expression was observed
in ~15% of the cells (Fig. 3,
middle and bottom rows, left
panel). Cytoplasmic expression under these conditions was most
likely due to basal I
B
, or I
B
, expression from the
copper-inducible promoter. Induction of I
B
with copper for 2 h resulted in a significant redistribution of GFP-p65 to the cytoplasm,
indicating that the newly synthesized I
B
exported nuclear GFP-p65
to the cytoplasm (Fig. 3, middle row, right
panel). This was mediated by Crm1p because it did not occur in the
crm1-1 strain that contains a mutated CRM1 gene
(data not shown). In contrast, there was little redistribution of
GFP-p65 after secondary induction of I
B
(Fig. 3,
bottom row, right panel). The small increase in whole
cell GFP-p65 expression was probably because of residual GFP-p65
translation during I
B
induction, which resulted in its
cytoplasmic sequestration. These observations indicate that I
B
cannot remove nuclear Rel proteins to the cytoplasm.
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DISCUSSION |
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We found that p65 or c-Rel associated with IB
or I
B
were retained in the cytoplasm, although these I
Bs did not shuttle via the CRM1 pathway. We suggest that I
Bs, unlike I
B
,
sequester rather than shuttle Rel proteins, which implies that there is no available NLS in the Rel·I
B
(or I
B
) complexes to
induce nuclear entry. Conversely, Rel·I
B
complexes must have an
available NLS to shuttle. We hypothesize that the Rel and not the I
B
component provides the functional NLS of a Rel·I
B complex. Thus,
I
B
or I
B
must hide the Rel NLS more effectively than
I
B
. Evidence in favor of this idea was obtained from partial
proteolytic studies of p65·I
B complexes.
The sequestration mechanism is based on the lack of an effect of
leptomycin B or a mutated CRM1 gene in Rel protein localization by
IB
. Alternatively, these results could indicate that
Rel·I
B
complexes shuttled by a CRM1-independent pathway. To
test this theory, we generated nuclear Rel·I
B
complexes
and determined whether they could reach the cytoplasm by an
unidentified pathway. As shown in Fig. 3, I
B
-mediated GFP-p65
export was inefficient compared with I
B
. We conclude that
I
B
is not an export chaperone like I
B
. Consequently,
I
B
cannot efficiently down-regulate nuclear Rel proteins to
restore the resting state of the cell. These results highlight the
functional differences between I
B
and I
B
.
Cheng et al. (19) showed that substituting IB
for the
I
B
gene compensated for the most obvious defects in
I
B
/
mice. They concluded that
I
B
and I
B
were functionally similar and that regulation of
expression accounted for most of the phenotype of I
B
-deficient
mice. Our contrasting conclusion regarding the mechanism of I
B
and I
B
function is not at odds with the biological results.
Clearly, if sufficient I
B
is synthesized in a cell, it can retain
Rel proteins in the cytoplasm, albeit by a mechanism different from
I
B
. The biological results show that retention of Rel proteins by
either mechanism is good enough to rescue lethality. That
I
B
is a less efficient nuclear export chaperone than I
B
may
be manifest under conditions that were not directly assayed, such as
during an immune response or chronic inflammation. We suggest that
control of such situations may require the active export-dependent reduction of NF-
B activity.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Rosenstiel
Basic Medical Sciences Research Ctr., Brandeis University, 415 South St., Waltham, MA 02454. E-mail: sen@brandeis.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.C000916200
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ABBREVIATIONS |
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The abbreviations used are:
NF-B, nuclear factor-
B;
NLS, nuclear localization signal;
GFP, green
fluorescent protein;
LMB, leptomycin B;
HA, hemagglutinin;
DAPI, 4,6-diamidino-2-phenyllindole.
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