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Mol Cell Biol, March 1998, p. 1213-1224, Vol. 18, No. 3
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Distinct Domains of I
B
Regulate c-Rel in
the Cytoplasm and in the Nucleus
Ignacio
Luque1 and
Céline
Gélinas1,2,3,*
Center for Advanced Biotechnology and
Medicine,1
Department of
Biochemistry, Robert Wood Johnson Medical
School,2 and
Cancer Institute of New
Jersey,3 University of Medicine and
Dentistry of New Jersey, Piscataway, New Jersey 08854-5638
Received 20 October 1997/Accepted 5 December 1997
 |
ABSTRACT |
I
B
is a critical regulator of Rel/NF-B-mediated gene
activation. It controls the induction of NF-B factors by
retaining them in the cytoplasm and also functions in the nucleus to
terminate the induction process. In this study, we show that IB
regulates the transcriptional activity of c-Rel in the nuclear
compartment. We also demonstrate that discrete functional domains of
IB are responsible for the cytoplasmic and nuclear regulation of
c-Rel. We show that the determinants for the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin regions of IB and that the N-terminal domain of IB is required to mask the c-Rel nuclear localization signal. Importantly, IB sequences necessary to regulate c-Rel in the nucleus map to its central ankyrin
domain and to a few negatively charged amino acids that immediately
follow in the C-terminal IB PEST domain. The mapping of the
IB determinants that control the cytoplasmic and nuclear activities of c-Rel to specific regions of the molecule suggests that
IB inhibitors could be designed to antagonize Rel/NF-B activity in different subcellular compartments or at defined stages of
activation.
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INTRODUCTION |
The Rel/NF-B family of
transcription factors plays a central role in the immune, inflammatory,
and acute-phase responses and in the control of cell proliferation
(reviewed in references 5-7, 52,
68 and 72). The vertebrate
proteins p50/NF-B1, p52/NF-B2, RelA, c-Rel, and the viral
oncoprotein v-Rel belong to this family (7, 72). These
proteins share an N-terminal Rel homology domain that participates in
their DNA binding, dimerization, and nuclear localization. The
formation of Rel homodimers and heterodimers enables their binding to
the major groove of decameric B DNA sites and the regulation of gene
expression (reviewed in references 5-7,
52, 68, and
72).
Rel factors share common pathways of activation that involve their
rapid release from inhibitory IB proteins in response to various
stimuli (reviewed in references 7,
32, 52, and 72). IB factors associated with cytoplasmic Rel
dimers mask the Rel nuclear localization signal (NLS), thereby impeding
Rel translocation to the nucleus (10, 28, 77). In response
to stimuli, IB undergoes phosphorylation and rapid degradation via the 26S proteasome (1, 15, 16, 20, 43, 50, 58, 59, 62, 69,
73). These processes allow the nuclear translocation of Rel
proteins and the activation of gene expression. In turn, nuclear Rel
factors trigger the resynthesis of IB, giving rise to an
autoregulatory loop that terminates the activation process (17,
21, 46, 64, 66). The transient accumulation of newly synthesized
IB in the nucleus of stimulated cells correlates with the
repression of Rel/NF-B-dependent gene expression (3, 56).
Accumulating evidence supports a model in which the entry of IB
into the nucleus triggers the relocalization of Rel complexes from the
nucleus to the cytoplasm to terminate the activation process (4,
11, 41). The recent identification of a nuclear export signal in
IB is consistent with this model (4). In light of
these findings, it appears that IB functions as a dual regulator
of Rel proteins by controlling their activation in the cytoplasm and by
terminating their activity in the nucleus.
The N-terminal domain of IB contains serine and lysine residues
that undergo phosphorylation and ubiquitination, respectively, and that
are critical for its inducible degradation (1, 15, 16, 20, 24, 43,
50, 58, 59, 62, 69, 73). Mutation or deletion of this domain
stabilizes the inhibitor under inducing conditions (2, 15, 43,
65). A central domain of six ankyrin repeats is essential for the
interaction of IB with Rel factors (34-37). The
C-terminal 35 amino acids of IB contain a PEST region, rich in
negatively charged residues. While this portion of the protein is
dispensable for its inducible degradation and for its interaction with
Rel, phosphorylation of this portion regulates the constitutive
degradation of the protein (2, 8, 48, 51, 63, 65, 71). This
portion of IB also has been implicated in the inhibition of RelA
and c-Rel DNA binding (26, 61). Of the different Rel domains
that contact IB, the Rel NLS region appears to be the main site
for interaction with the inhibitor. Mutation of the NLS decreases the
affinity of Rel for IB and enables Rel to escape the inhibitory
function of IB (10, 28, 45, 77).
Among the different Rel/NF-B dimers, p50-RelA complexes are the most
extensively studied. Much less is known, however, about the regulation
and function of the transactivating subunit c-Rel. Studies with
transgenic mice and knockout animals demonstrated an essential role for
c-Rel in lymphocyte proliferation, in immune and inflammatory
responses, and in T-cell development (12, 42). As
c-rel is the cellular homolog of the viral oncogene
v-rel, it is not surprising to find a correlation between
the amplification and overexpression of the c-rel gene and
oncogenesis (reviewed in references 31 and
49). The activity of c-Rel therefore must be tightly
regulated.
In this study, we characterized the functional domains of IB
involved in the cytoplasmic and nuclear regulation of c-Rel. We show
that IB can function in vivo to regulate the transcriptional activity of c-Rel in the nuclear compartment. Our studies indicate that
the central ankyrin domain of IB, together with a few negatively charged C-terminal acidic amino acids, is necessary and sufficient to
regulate c-Rel in the nucleus. While the C-terminal domain of IB
is dispensable for the cytoplasmic retention of c-Rel, we show that its
N-terminal domain and ankyrin repeats are essential for this function.
We also provide evidence that the N-terminal region of IB is
required to mask the Rel NLS. Our results indicate that the
determinants for the cytoplasmic regulation of c-Rel reside in the
N-terminal and central ankyrin domains of IB, while those
necessary for nuclear Rel protein regulation map to its central ankyrin
domain and to a few acidic amino acids that immediately follow in the
C-terminal IB PEST region.
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MATERIALS AND METHODS |
Plasmids and mutagenesis.
CCR encodes the wild-type chicken
c-Rel protein (18). The CCR.SVNLS mutant was generated by
insertion of a double-stranded oligonucleotide
(5'-CTAGAGATCACGCCACCAAAGAAGAAAAGGAAAGTGGAGGACCCT-3') encompassing the simian virus 40 (SV40) large T antigen NLS at the unique HincII site of the chicken c-rel gene.
Mutant CCR.KO.SVNLS was derived by site-directed mutagenesis of
CCR.SVNLS to inactivate the Rel NLS (KRQR to NWLT). A 1.7-kb
EcoRI DNA fragment containing the chicken
ib gene (23) was cloned into the
EcoRI site of the pAlter-1 plasmid (Promega). IB
deletion mutants were generated by introducing stop codons at defined
positions by use of the Altered Sites mutagenesis system (Promega).
Mutants 
267, 282, 76-284, 285, 287, and 301,
respectively, lack 52, 37, 34, 34, 32, and 18 amino acids from the C
terminus of IB. Mutants 76-318 and 76-284 were generated by
deletion of the first 75 amino acids from the N terminus of full-length
IB (76-318) or of mutant 285 (76-284), followed by the
introduction of an ATG codon at position 75 by site-directed
mutagenesis. Mutations were confirmed by DNA sequence analysis with
Sequenase (U.S. Biochemical).
c-rel and ib genes were
expressed in vitro from the T7 promoter of the pAlter-1 plasmid.
Wild-type and mutant c-Rel and IB proteins were expressed in vivo
under the control of the cytomegalovirus (CMV) immediate-early promoter
of pJDCMV19SV (25) or pcDNA I/Amp (Invitrogen). Plasmid
pIL6CAT expresses the chloramphenicol acetyltransferase (CAT) reporter
gene from the interleukin-6 promoter and has three copies of the
IL6-B DNA-binding motif (54).
Cell transfection and CAT assays.
Cos-7 SV40-transformed
African green monkey kidney cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
1% penicillin and streptomycin. Cells were maintained at 37°C in an
atmosphere of 5% CO2. Transfections were carried out by a
modified calcium phosphate procedure (19). Cells (5 × 105) in 60-mm dishes were transfected with 5 µg of CMV
rel plasmid DNA in the presence or absence of wild-type or
mutant CMV ib DNA and 3 µg of pIL6CAT.
The total amount of plasmid DNA transfected (13 µg) was kept constant
by the addition of the CMV vector. The calcium phosphate precipitate
was removed after 16 to 24 h of incubation, and the cells were
washed and refed with medium containing 3% fetal bovine serum. Cell
extracts were prepared 36 to 48 h after transfection, and the
protein concentration was measured by the method of Bradford
(14). CAT activities were determined within the linear range
of the assay as described previously (29). Assays were
performed with 10 µg of total cellular protein for 20 min. Normalized
CAT activity from the average of three independent experiments is
shown.
Nuclear and cytoplasmic extracts.
Nuclear and cytoplasmic
extracts were prepared as described previously (55).
Briefly, cells were harvested and resuspended in buffer A (10 mM HEPES
[pH 7.8], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol
[DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.1% Nonidet
P-40). Extracts were incubated for 10 min on ice and fractionated by
centrifugation at 10,000 × g. Supernatants containing
the cytoplasmic fractions were recovered. The pellets were resuspended
in buffer A and centrifuged again at 10,000 × g. The
nucleus-containing pellets were lysed in 20 µl of buffer C (20 mM
HEPES [pH 7.8], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) and incubated for 20 min on ice.
Nuclear extracts were separated from cell debris by centrifugation at
10,000 × g and diluted in 80 µl of buffer D (20 mM
HEPES [pH 7.8], 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT,
0.5 mM PMSF).
Immunofluorescence.
Cells were seeded onto glass coverslips
and transfected as described above. Coverslips were harvested at
48 h posttransfection. The cells were fixed in 4%
paraformaldehyde and permeabilized in phosphate-buffered saline-0.2%
Triton X-100. Coverslips were incubated with rabbit polyclonal antibody
Ab1801, specific for the C terminus of c-Rel (45), or with
anti-p40/IB antibody (39) followed by
fluorescein-conjugated goat anti-rabbit antibodies. Coverslips were
mounted in the presence of 0.2% para-phenylenediamine (Sigma) and photographed at a magnification of ×600.
Coimmunoprecipitation assays.
Cos-7 cells were harvested at
48 h posttransfection, lysed by sonication in 50 mM Tris HCl (pH
7.5)-150 mM sodium chloride-1% sodium deoxycholate-1% Triton
X-100-10 µg of leupeptin per ml-10 mM sodium pyrophosphate-50 mM
sodium fluoride-0.5 mM sodium orthovanadate (57), and
quantitated for protein concentration by the method of Bradford
(14). Equal amounts of whole-cell extracts (400 to 600 µg)
were immunoprecipitated with anti-c-Rel antibody Ab1801 (45) or with antibody Ab1507, specific for the c-Rel NLS
(26), and protein A-Sepharose (Pharmacia). Samples were
washed extensively with ELB buffer (50 mM HEPES [pH 7.0], 250 mM
NaCl, 5 mM EDTA, 0.1% Nonidet P-40) (26) supplemented with
Complete protease inhibitor cocktail (Boehringer Mannheim Biochemicals)
and resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis.
Wild-type or mutant c-Rel and IB proteins were produced in vitro
with a TNT rabbit reticulocyte expression system (Promega) in the
presence of 35S-labeled cysteine. Translation reactions
were quantitated with a PhosphorImager. c-Rel translation reaction
mixtures (3 µl) were incubated in the presence or absence of a
threefold molar excess of wild-type or mutant IB protein for 15 min at room temperature. Reactions were immunoprecipitated with
antibody Ab1801 (45) or Ab1507 (26) and protein
A-Sepharose.
DNA-binding assays.
DNA-binding assays of in
vivo-synthesized c-Rel proteins were performed with nuclear (3 µg) or
whole-cell (20 µg) extracts from transfected cells prepared as
described for coimmunoprecipitation assays. Extracts were incubated
with 3 × 104 cpm of a 32P-labeled
IL6-B oligonucleotide probe in 12.5 mM HEPES (pH 7.9)-12% glycerol-5 mM MgCl2-60 mM KCl-0.2 mM EDTA-1 mM DTT-1
µg of bovine serum albumin per µl-1 µg of poly(dI-dC) per µl
(74) and analyzed on 5% native polyacrylamide gels. In
supershift assays, extracts were preincubated with 0.5 to 2 µl of
antisera specific for c-Rel (45) or IB (39)
prior to DNA-binding reactions.
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RESULTS |
c-Rel mutants that escape cytoplasmic regulation by IB.
To bypass cytoplasmic regulation by IB, c-Rel was constitutively
targeted to the nucleus by inserting the NLS of the SV40 large T
antigen 3' to the Rel homology domain of the chicken c-rel gene (38) (CCR.SVNLS; Fig.
1A). As a negative control, the Rel NLS
was inactivated by mutagenesis of CCR.SVNLS (CCR.KO.SVNLS; Fig.
1A). The effect of IB on the subcellular localization of wild-type and mutant c-Rel proteins was examined by immunofluorescence. As anticipated, c-Rel localized to the nucleus of transiently transfected Cos-7 cells, and the coexpression of the chicken IB protein led to the sequestration of c-Rel in the cytoplasm (Fig. 1B,
panels a and b) (26, 61). In contrast, CCR.SVNLS and
CCR.KO.SVNLS constitutively localized to the nucleus
irrespective of the presence or absence of IB (Fig. 1B, panels c
to f). This result demonstrated the ability of these proteins to escape
cytoplasmic regulation by IB.
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FIG. 1.
c-Rel mutants that bypass cytoplasmic regulation by
IB. (A) Structures of the CCR.SVNLS and CCR.KO.SVNLS
mutants of c-Rel. The Rel NLS and the SV40 NLS are depicted as white
and black boxes, respectively. Mutation of the Rel NLS in
CCR.KO.SVNLS is indicated by X. WT, wild type. (B) Subcellular
localization of c-Rel and c-Rel-derived mutants. Cos-7 cells
cotransfected with wild-type or mutant c-rel genes, together
with CMV or CMV ib expression plasmids, were
analyzed by indirect immunofluorescence with an anti-c-Rel antibody.
(C) Transcriptional activity of wild-type and mutant c-Rel proteins.
Cos-7 cells were transfected with CMV vector DNA or with expression
plasmids encoding c-Rel or c-Rel-derived mutants, alone or together
with an expression vector for IB. The pIL6CAT reporter plasmid,
containing three B DNA sites upstream of the interleukin-6 promoter,
was included in the transfection. The average CAT activity from three
independent experiments was normalized to that of wild-type c-Rel.
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The ability of I
B
to regulate the transcriptional activity of
c-Rel and its nuclear mutants was examined in transiently
transfected
Cos-7 cells. c-Rel, CCR.SVNLS, and CCR.KO.SVNLS transfected
alone strongly activated
B DNA site-dependent gene expression
in
comparison to that obtained with the control pJDCMV19SV vector
(Fig.
1C). This result verified that the insertion of the SV40
NLS was not
detrimental to the transcriptional activity of c-Rel.
Cotransfection
with I
B
led to a moderate decrease in the activity
of the
CCR.KO.SVNLS control, in agreement with the reduced affinity
of
I
B
for Rel proteins with mutations in the NLS (
10,
28;
data not shown). In contrast, I
B
abolished
transactivation mediated
by c-Rel and by the nuclear CCR.SVNLS protein
(Fig.
1C). The inhibition
of c-Rel-mediated transcription likely
resulted from the cytoplasmic
retention of c-Rel by I
B
(Fig.
1B).
However, the inhibition
of CCR.SVNLS activity suggested its regulation
by I
B
in the
nucleus, consistent with the colocalization of the
CCR.SVNLS and
I
B
proteins in the nuclear compartment (Fig.
1B;
see also Fig.
4). This result indicated that I
B
can regulate the
activity
of c-Rel in the nucleus and that CCR.SVNLS could be used to
map
the domains of I
B
necessary for the nuclear regulation of
c-Rel.
Mapping of C-terminal IB sequences necessary for the
cytoplasmic and nuclear regulation of c-Rel.
The C-terminal PEST
domain of IB was implicated in the inhibition of Rel DNA binding,
and the phosphorylation of serine and threonine residues in this region
was proposed to be required for this activity (26, 61). The
C-terminal sequences of IB necessary for the nuclear regulation
of c-Rel were mapped by deletion analysis (Fig.
2). The serine and threonine residues in
the PEST domain were mutated to alanine to address the role of
phosphorylation in the nuclear regulation of c-Rel (A5; Fig. 2).
Mutants were assayed for association with c-Rel and CCR.SVNLS, for
effects on their subcellular localization, and for inhibition of their DNA-binding and transcriptional activities.
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FIG. 2.
Structures and properties of IB mutants. The
structure of IB, comprised of an N-terminal domain (stippled
box), ankyrin repeats (hatched boxes), and a C-terminal region (open
box) containing a PEST domain (black box), is depicted (not to scale).
C-terminal deletion mutants 267, 282, 285, 287, 301, and
76-284 were generated by introducing stop codons. Translation start
codons were introduced at position 75 in mutants 76-318 and 76-284. Mutant A5 contains alanine substitutions for the serines at positions
287, 292, 293, and 295 and the threonine at position 300 of IB.
Mutant 287-295 has an internal deletion of nine amino acids in the
C-terminal PEST domain of IB. Mutants 285-Tail 4 Glu,
285-Tail 2 Asp, and 285-Tail 4 Ala, respectively, have Glu, Asp,
and Ala tails added to the C terminus of mutant 285. nd, not
determined.
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Mutations affecting most of the C terminus of I
B
, including the
PEST domain, had no detrimental effect on its association
with c-Rel or
CCR.SVNLS. As shown in Fig.
3, mutant
I
B
proteins
affected in this region coprecipitated with c-Rel or
CCR.SVNLS
as efficiently as wild-type I
B
(
287,
287-295,
301, and A5;
compare lanes 3 and 12 to lanes 7 to 10 and lanes 16 to
19, respectively).
Whereas c-Rel was nuclear when expressed alone in
Cos-7 cells,
its coexpression with these mutant I
B
proteins led
to their
colocalization in the cytoplasm, as seen by immunofluorescence
(Fig.
4, panels 1, 3, 4, and 11 to 18).
The deletion of all sequences
mapping 3' to the ankyrin domain of
I
B
also led to the cytoplasmic
retention of c-Rel, despite a
slight decrease in Rel protein affinity
observed in
coimmunoprecipitation assays (
285; Fig.
3, lanes
6 and 15, and Fig.
4, panels 9 and 10). In contrast, deletions
affecting the ankyrin
domain of I
B
compromised its interaction
with c-Rel (
282 and
267). These mutants failed to associate
with c-Rel or CCR.SVNLS
in coimmunoprecipitation assays (Fig.
3, lanes 4, 5, 13, and 14).
Similarly, these mutations respectively
decreased or abolished the
cytoplasmic retention of c-Rel (Fig.
4, panels 5 to 8). As anticipated,
CCR.SVNLS constitutively localized
to the nucleus, regardless of
the presence or absence of wild-type
or mutant I
B
proteins (Fig.
4, panels 19 to 36). Together, these
results indicated that the
C-terminal domain of I
B
is dispensable
for association with c-Rel
and for the retention of c-Rel in the
cytoplasm.
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FIG. 3.
Interaction of c-Rel and CCR.SVNLS with wild-type or
mutant IB. Extracts from Cos-7 cells transfected with CMV vectors
expressing c-Rel (lanes 2 to 10) or CCR.SVNLS (lanes 11 to 19),
alone (lanes 2 and 11) or together with expression plasmids for
IB (lanes 3 and 12) or IB mutants (lanes 4 to 10 and lanes
13 to 19), were immunoprecipitated with an anti-c-Rel antibody.
Complexes were resolved on 12% polyacrylamide-SDS gels and analyzed
by immunoblotting with antibodies specific for c-Rel (top panels) or
IB (bottom panels). The positions of c-Rel, CCR.SVNLS, and
wild-type or mutant IB proteins are indicated. Control
immunoblots verified that equivalent amounts of wild-type and mutant
IB proteins were expressed in the cells (data not shown).
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FIG. 4.
Subcellular localization of c-Rel and CCR.SVNLS in
the presence of wild-type or mutant IB proteins. Cos-7 cells were
transfected with expression plasmids encoding c-Rel or CCR.SVNLS,
together with control CMV vector DNA or CMV expression plasmids for
IB or IB mutants. Cells were analyzed by
immunofluorescence with anti-c-Rel or anti-IB antibodies
(). A minimum of 80 positively staining c-Rel-expressing cells were
examined. Representative fields are shown.
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I
B
sequences specifically involved in the regulation of c-Rel in
the nucleus were mapped by analyzing the effects of C-terminal
I
B
mutations on the transcriptional activity of c-Rel and CCR.SVNLS.
As expected, the transactivation potential of c-Rel was significantly
inhibited by wild-type and mutant I
B
proteins that led to its
efficient retention in the cytoplasm (
285,
287,
287-295,
301,
and A5; Fig.
5A). Similarly, some
of these mutants with deletions
or mutations affecting most of the
I
B
PEST domain strongly inhibited
the activity of the nuclear
CCR.SVNLS protein (
287,
287-295,
301, and A5; Fig.
5A).
Importantly, however, the elimination
of the entire PEST domain in
mutant
285 dramatically decreased
the inhibition of
CCR.SVNLS-mediated transactivation but had little
effect on the
inhibitory activity of I
B
mutant
285 toward c-Rel
(Fig.
5A).
Mutants with deletions affecting the ankyrin domain
showed little or no
inhibitory activity toward c-Rel or CCR.SVNLS-mediated
transcription (
267 and
282). This result agreed with their
impaired
association with c-Rel and CCR.SVNLS (Fig.
3, lanes 4, 5, 13,
and 14). The fact that mutant
285 selectively lost its
inhibitory
activity toward transcription mediated by the nuclear
CCR.SVNLS
protein suggested that the first two amino acids of the
I
B
PEST
domain are essential for regulating c-Rel in the nucleus.
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FIG. 5.
Effects of IB mutants on the transcriptional and
DNA-binding activities of c-Rel. (A) Differential effects on c-Rel- and
CCR.SVNLS-mediated transcription. Cos-7 cells were transfected with
CMV vector DNA or CMV expression plasmids encoding c-Rel or
CCR.SVNLS, alone (0) or together with expression plasmids for
IB or IB mutants. The pIL6CAT reporter plasmid was included
in the transfections. The average CAT activity from three independent
experiments was normalized to that of wild-type c-Rel. (B) Inhibition
of c-Rel and CCR.SVNLS DNA binding. Whole-cell extracts (20 µg)
from Cos-7 cells transfected with CMV expression vectors for c-Rel
(lanes 3 to 12) or CCR.SVNLS (lanes 14 to 23), alone (lanes 3 and 14) or together with CMV ib (lanes 4 and 15)
or ib mutants (lanes 5 to 11 and 16 to 22), were
analyzed in gel retardation assays. Extracts were incubated with a
32P-labeled IL6-B DNA probe and resolved in 5% native
acrylamide gels. DNA-bound complexes containing c-Rel or
CCR.SVNLS were supershifted with an anti-c-Rel antibody ()
(lanes 12 and 23). The bracket on the left indicates the position
of DNA-bound c-Rel and CCR.SVNLS complexes. The bracket on the
right indicates the position of the supershifted Rel complexes. (C)
Formation of complexes containing IB C-terminal deletion
mutants, c-Rel, and DNA. Whole-cell extracts (20 µg) from Cos-7 cells
transfected with vector DNA (lane 1) or CMV c-rel alone
(lanes 2, 6, and 9) or together with vectors expressing IB (lane
3) or IB C-terminal deletion mutant 285 (lanes 4, 7, and 10)
or 287 (lanes 5, 8, and 11) were analyzed for binding to an
IL6-B DNA probe in gel retardation assays. DNA-bound complexes were
supershifted with anti-c-Rel (lanes 6 to 8) or anti-IB (lanes 9 to 11) antisera (). Electrophoresis was carried out three times
longer than usual to allow the separation of the different complexes.
The brackets indicate the positions of DNA-bound Rel proteins
(left) and of supershifted complexes (right).
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The effects of C-terminal I
B
mutations on the DNA-binding
activity of CCR.SVNLS were analyzed in gel retardation assays
with
whole-cell extracts from transiently transfected Cos-7 cells.
As shown
in Fig.
5B, the DNA-binding activity of CCR.SVNLS was
abolished by
wild-type and mutant I
B
proteins that efficiently
inhibited its
transcriptional activity (
287-295,
301, and A5;
lanes 20 to 22).
Interestingly, however, I
B
mutant
287, which
efficiently
blocked transcription by CCR.SVNLS, was unable to
inhibit its
DNA-binding activity (Fig.
5B, lane 19). Mutants with
further deletions
were also unable to block CCR.SVNLS DNA binding
(
267,
282,
and
285; Fig.
5B, lanes 16 to 18). As expected,
similar data were
obtained in gel retardation assays with nuclear
extracts from
CCR.SVNLS-expressing cells (data not shown). The
analysis of
c-Rel-expressing cells was used as a control to verify
that the
insertion of the SV40 NLS did not interfere with the
inhibition of
CCR.SVNLS DNA binding by I
B
. These results indicated
that the
presence of only 2 amino acids C terminal to the ankyrin
domain is not
sufficient to inhibit c-Rel DNA binding (
287).
Moreover, our finding
that I
B
mutant
287 blocked CCR.SVNLS-mediated
transcription without interfering with its DNA-binding activity
implicates the residues at the very beginning of the I
B
PEST
domain in the specific inhibition of transcriptional activation
by
nuclear c-Rel proteins.
The observation that I
B
mutants
285 and
287 associated with
c-Rel but failed to inhibit its DNA-binding activity led us
to
investigate whether the interaction of c-Rel with DNA would
displace
them from the complex. Cells expressing c-Rel alone showed
efficient
binding to a
B DNA site probe in gel retardation assays
(Fig.
5C,
lane 2). As expected, I
B
abolished c-Rel DNA binding,
whereas
mutants
285 and
287 had no such effect (Fig.
5C, lanes
3 to 5).
Importantly, reaction mixtures containing either of these
mutants
together with c-Rel showed an additional DNA-bound complex
of retarded
mobility (Fig.
5C, compare lanes 4 and 5 to lane 2).
An anti-c-Rel
antibody supershifted both bands, indicating the
presence of c-Rel in
both complexes (Fig.
5C, compare lanes 7
and 8 to lanes 4 and 5, respectively). While an anti-I
B
antibody
had little effect on the
lower Rel-DNA complex, it supershifted
the upper complex in cells
expressing c-Rel together with I
B
mutant
285 or
287 (Fig.
5C, compare lanes 10 and 11 to lanes
4 and 5, respectively). The
formation of complexes containing
I
B
mutant
285 or
287
together with c-Rel and DNA agrees with
the inability of these mutants
to block c-Rel DNA binding.
Negatively charged residues at the beginning of the IB PEST
domain are essential for regulation of the activity of c-Rel in the
nucleus.
The acidic nature of the IB PEST domain and the
important role of amino acids immediately C terminal to the ankyrin
repeats in the inhibition of CCR.SVNLS-mediated transcription led
us to investigate the effect of acidic amino acids C terminal to
residue 285 on the nuclear regulation of c-Rel. IB mutants with
tails of aspartate or glutamate residues added to the C terminus of mutant 285 were analyzed for inhibition of c-Rel- or
CCR.SVNLS-mediated transcription in transient CAT assays
(285-Tail 2 Asp and 285-Tail 4 Glu; Fig. 2 and
6A). A neutral tail of alanines was added
as a control (285-Tail 4 Ala; Fig. 2). In agreement with the
findings described above, mutant 285 inhibited c-Rel-mediated
transcription via cytoplasmic retention but had no significant
inhibitory activity toward the constitutively nuclear CCR.SVNLS
protein (Fig. 6A). In sharp contrast, the addition of as little as two
aspartate or four glutamate residues to the C terminus of IB
mutant 285 fully restored its ability to inhibit the transcriptional
activity of CCR.SVNLS. In contrast, the neutral tail of alanine
residues had no inhibitory effect, despite the efficient association of the 285-Tail 4 Ala protein with c-Rel and CCR.SVNLS in
coimmunoprecipitation assays (Fig. 6A and data not shown).
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FIG. 6.
Effects of negatively charged residues in the C terminus
of IB on the transcriptional and DNA-binding activities of c-Rel.
(A) Effects on Rel-mediated transcription. Cos-7 cells were transfected
with vector DNA or CMV plasmids encoding c-Rel or CCR.SVNLS, alone
(0) or in combination with expression vectors for IB or IB
mutants. Reporter plasmid pIL6CAT was included in the transfections.
The normalized CAT activity from the average of three independent
experiments is shown. (B) Effects on Rel DNA binding. Shown is a gel
retardation analysis of whole-cell extracts (20 µg) from Cos-7 cells
transfected with CMV vectors expressing c-Rel (lanes 2 to 7 and 14) or
CCR.SVNLS (lanes 8 to 13), alone (lanes 2, 8, and 14) or together
with vectors encoding wild-type (lanes 3 and 9) or mutant (lanes 4 to 7 and 10 to 13) IB proteins. Proteins were assayed for binding to a
32P-labeled IL6-B DNA probe. The bracket indicates the
position of ectopically expressed Rel-DNA adducts. The arrowhead
indicates c-Rel-DNA complexes supershifted with an anti-c-Rel
antiserum () (lane 14). The lower band present in all lanes
corresponds to a DNA-bound complex of endogenous proteins.
|
|
The inhibitory activity of the
285-Tail mutants toward c-Rel and
CCR.SVNLS DNA binding was examined in gel retardation assays.
In agreement with the results described above, the addition of
two or
four acidic residues at the C terminus of
285 significantly
or
fully restored the inhibition of DNA binding by c-Rel and
CCR.SVNLS,
whereas a tail of alanine residues did not (Fig.
6B,
lanes 5 to
7 and 11 to 13). Together, these experiments indicated that
negatively
charged residues at the C-terminal end of the ankyrin domain
of
I
B
are necessary to regulate the transcriptional activity of
nuclear c-Rel proteins and their binding to DNA. Consistent with
our
deletion analyses, these assays also demonstrated that only
a few
acidic residues at this position are sufficient to regulate
the
activity of c-Rel in the nucleus.
The N terminus of IB is required to retain c-Rel in the
cytoplasm.
We complemented our study of IB by characterizing
the activity of mutants lacking its N terminus or both its N- and
C-terminal domains (mutants 76-318 and 76-284; Fig. 2). Mutants were
analyzed for their effect on the subcellular localization of c-Rel by
immunofluorescence. As expected, the coexpression of c-Rel with
wild-type IB led to its confinement to the cytoplasm (Fig.
7A, compare panels c and a). However, the
coexpression of c-Rel with N-terminal IB mutant 76-318 or 76-284 resulted in weak cytoplasmic and strong nuclear staining for c-Rel
(Fig. 7A, compare panels e and g with panel c). This result suggested
that deletion of the N terminus of IB impaired its capacity to
retain c-Rel in the cytoplasm.
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FIG. 7.
Effect of IB N-terminal deletion mutants on the
subcellular localization and transcriptional and DNA-binding activities
of c-Rel. (A) Effects on subcellular localization. The subcellular
localization of c-Rel and IB proteins was assessed by
immunofluorescence analysis of Cos-7 cells expressing c-Rel alone (0)
or together with IB or its mutants 76-318 and 76-284, with
anti-c-Rel or anti-IB antibodies (). (B) Effects on
Rel-mediated transcription. Cos-7 cells were transfected with pIL6CAT
together with vector DNA or expression plasmids for c-Rel or
CCR.SVNLS, alone (0) or in combination with plasmids encoding
wild-type IB or mutant 76-318 or 76-284. The average CAT activity
from three independent experiments was normalized to that of wild-type
c-Rel. (C) Effects on Rel DNA binding. Whole-cell extracts (20 µg)
from Cos-7 cells transfected with vector DNA (lane 1) or with CMV
c-rel alone (lanes 2 and 6) or together with expression
plasmids for IB or mutant 76-318 or 76-284 (lanes 3 to 5) were
analyzed for binding to a 32P-labeled IL6-B DNA probe in
gel retardation assays. As a control, the DNA-bound c-Rel
complex was supershifted with an anti-c-Rel antibody ().
The bracket indicates the position of c-Rel-DNA complexes. The
arrowhead indicates supershifted c-Rel complexes. The lower band
present in all lanes corresponds to a DNA-bound complex of endogenous
proteins.
|
|
In spite of its decreased ability to retain c-Rel in the cytoplasm,
mutant 76-318 inhibited c-Rel-mediated transcription as
efficiently as
wild-type I
B
. The inhibition of c-Rel-induced
transcription by
wild-type I
B
resulted from the retention of
c-Rel in the
cytoplasm. However, its inhibition by mutant 76-318
occurred primarily
in the nucleus, as evidenced by the predominant
nuclear localization of
both c-Rel and mutant 76-318 (Fig.
7A,
panels e and f). Consistent with
this model, mutant 76-318 also
strongly inhibited the transcriptional
activity of the nuclear
CCR.SVNLS protein (Fig.
7B). In keeping
with these observations,
I
B
mutant 76-318 exhibited wild-type
inhibitory capacity toward
c-Rel DNA binding (Fig.
7C, lane 4). As
predicted from our analysis
of C-terminal I
B
mutants, the
deletion of both the N-terminal
and the C-terminal domains of I
B
abolished its inhibitory effect
toward c-Rel DNA-binding and
transcriptional activities (mutant
76-284; Fig.
7B and C, lane 5).
Combined, these experiments indicated
that the N terminus of I
B
is important for the cytoplasmic regulation
of c-Rel but is dispensable
for its regulation in the nucleus.
IB mutants with N-terminal deletions fail to mask the c-Rel
NLS.
The inefficient retention of c-Rel in the cytoplasm by
IB mutants with N-terminal deletions suggested that the c-Rel NLS may still be exposed when c-Rel is associated with these mutants. To
verify this possibility, complexes of 35S-labeled c-Rel
with wild-type IB or IB mutants with N-terminal deletions
were analyzed by immunoprecipitation with an antibody directed against
the c-Rel NLS (Ab1507; Fig. 8A). An
antibody specific for the C terminus of c-Rel was used as a control
(Ab1801). The antibody directed against the C terminus of c-Rel
efficiently coprecipitated IB and its N-terminal deletion mutants
(Fig. 8A, lanes 2 to 4). As anticipated, the anti-c-Rel NLS antibody Ab1507 failed to coprecipitate wild-type IB along with c-Rel due
to masking of the Rel NLS (Fig. 8A, lane 6). In contrast, IB
mutants 76-318 and 76-284 complexed with c-Rel were efficiently coprecipitated by this antibody (Fig. 8A, lanes 7 and 8). This result
showed that the Rel NLS remained exposed when associated with IB
N-terminal deletion mutants, whereas it was inaccessible to antibody
Ab1507 when complexed with wild-type IB. Consistent with this
observation, the amount of c-Rel brought down by this antibody in the
presence of wild-type IB was significantly lower than that
brought down in the presence of IB mutant 76-318 or 76-284 (Fig.
8A, compare lane 6 with lanes 7 and 8).
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FIG. 8.
The c-Rel NLS is unmasked when associated with IB
N-terminal deletion mutants. (A) Coimmunoprecipitation assay of in
vitro-translated proteins. 35S-labeled c-Rel produced by in
vitro translation was incubated in the presence of a threefold molar
excess of in vitro-translated wild-type IB (lanes 2 and 6) or
N-terminal deletion mutants of IB (lanes 3, 4, 7, and 8). c-Rel
was incubated with an equal volume of a mock translation as a control
(lanes 1 and 5). Reaction mixtures were immunoprecipitated with
antibody Ab1801 against the C terminus of chicken c-Rel (lanes 1 to 4)
or anti-c-Rel NLS antibody Ab1507 (lanes 5 to 8). Immunoprecipitated
complexes were resolved in a 17% polyacrylamide-SDS gel. The open
arrowhead indicates the position of c-Rel. Closed arrowheads indicate
the positions of wild-type and mutant IB proteins. (B)
Coimmunoprecipitation assay of in vivo-labeled proteins. Extracts from
[35S]methionine- and [35S]cysteine-labeled
Cos-7 cells transfected with CMV vector DNA (lanes 1 and 6) or CMV
vectors expressing c-Rel alone (lanes 2 and 7) or together with
wild-type or mutant IB (lanes 3 to 5 and 8 to 10) were
immunoprecipitated with antibody Ab1801 against the C terminus of c-Rel
(lanes 1 to 5) or anti-c-Rel NLS antibody Ab1507 (lanes 6 to 10).
Reaction mixtures were resolved in a 17% polyacrylamide-SDS gel.
Arrowheads indicate the positions of wild-type and mutant IB
proteins. The sizes of protein molecular mass markers are indicated on
the left in kilodaltons.
|
|
Similar results were obtained when c-Rel-I
B
complexes were
analyzed in transfected cells (Fig.
8B). Since the anti-I
B
antibodies available failed to recognize the I
B
mutants in
immunoblots,
analysis was performed with proteins labeled in vivo with
[
35S]methionine and [
35S]cysteine. As in
our in vitro studies, anti-c-Rel NLS antibody
Ab1507 failed to
coprecipitate wild-type I
B
(Fig.
8B, compare
lanes 3 and 8).
However, it coprecipitated I
B
mutant 76-318
along with c-Rel
(Fig.
8B, lane 9), indicating exposure of the
c-Rel NLS. Our failure to
detect the coprecipitation of I
B
mutant
76-284 with antibody
Ab1507 presumably resulted from the additive
effect of its lower
content of methionines and cysteines (which
caused its inefficient
labeling) and its decreased affinity for
c-Rel in vivo, as evidenced by
the weak signal that we observed
with antibody Ab1801 (Fig.
8B, compare
lanes 10 and 5). Nevertheless,
the data clearly indicated that the
N-terminal domain of I
B
is important for the masking of the Rel
NLS and is consequently
critical for the cytoplasmic retention of
c-Rel.
| |
DISCUSSION |
The activity of the Rel/NF-B factors is tightly regulated.
While some physiological conditions require a transient Rel/NF-B response, others need prolonged activation. Integral parts of the
transient activation process are the nuclear translocation of newly
synthesized IB and the termination of Rel/NF-B-mediated transcription (3, 11, 41). Here we show that IB can
function in vivo to inhibit the activity of c-Rel in the nucleus. As
previously described for RelA (10), the introduction of an
extra NLS into c-Rel helped to circumvent its cytoplasmic regulation by
IB. By comparing the effects of IB on the nuclear
regulation of CCR.SVNLS and on the cytoplasmic regulation of c-Rel,
we demonstrated that distinct functional domains in IB are
required for the cytoplasmic and nuclear regulation of c-Rel.
Role of the IB C terminus in the cytoplasmic and nuclear
regulation of c-Rel.
Our studies showed that the C terminus of
IB, including the highly acidic PEST region, is dispensable for
the cytoplasmic retention of c-Rel. Whereas deletion of this region did
not significantly affect the capacity of IB to regulate c-Rel in
the cytoplasm (285), our deletion studies demonstrated that an
intact ankyrin domain is necessary for interaction with c-Rel and its
cytoplasmic sequestration. An IB C-terminal mutant with a
deletion of only three amino acids in the ankyrin domain was
compromised in its interaction with c-Rel (282). Although this
mutant showed some capacity to retain c-Rel in the cytoplasm, the high
transcriptional activity that we observed when c-Rel was coexpressed
with 282 indicated that some c-Rel dimers escaped cytoplasmic
retention. This result emphasized the instability of their interaction.
Thus, our failure to detect a physical association between mutant
282 and c-Rel in coimmunoprecipitation assays presumably resulted from their reduced affinity for one another and their dissociation under in vitro experimental conditions, as observed by others (26,
60, 61). Further deletion into the ankyrin domain abolished the
interaction of IB with c-Rel both in vitro and in vivo (267). These findings also agree with those of others (26, 61).
Mutant
285, with a deletion of the entire C-terminal domain of
I
B
, was highly impaired for the inhibition of
CCR.SVNLS-mediated
transcription. This result is in contrast to its
nearly wild-type
capacity to inhibit the transcriptional activity of
c-Rel by retaining
it in the cytoplasm. The differential effect of this
mutant toward
c-Rel- and CCR.SVNLS-mediated transcription did not
appear to
result from differences in their affinity for mutant
285,
as
seen in coimmunoprecipitation assays. Moreover, several observations
argue against the possibility that the presence of a foreign NLS
altered the conformation and function of CCR.SVNLS: (i)
CCR.SVNLS
activated transcription as efficiently as wild-type
c-Rel, (ii)
its affinity for wild-type and mutant I
B
proteins was
similar
to that of c-Rel, and (iii) its DNA-binding activity was
inhibited
by I
B
as efficiently as that of c-Rel. Thus, these
results indicate
that while sequences C terminal to the ankyrin domain
of I
B
are dispensable for the cytoplasmic retention of c-Rel,
they are
essential for inhibition of its transcriptional activity in
the
nucleus.
The addition of negatively charged tails to the C terminus of mutant
285 demonstrated that as few as two acidic amino acids
C terminal to
the ankyrin domain are sufficient to completely
restore the inhibitory
activity of I
B
in the nucleus. In agreement
with this result,
mutant
287, which contains a single acidic
amino acid C terminal to
the ankyrin domain, significantly inhibited
the transcriptional
activity of the constitutively nuclear CCR.SVNLS
protein. Thus, it
appears that most of the I
B
PEST domain is
dispensable for the
inhibition of c-Rel-mediated transcription
in the nucleus.
Mechanism for the nuclear regulation of c-Rel.
Nuclear
IB factors have been shown to promote the relocalization of RelA
from the nucleus to the cytoplasm, and a nuclear export sequence was
implicated in this process (amino acids 265 to 277 of human IB;
4). However, our observation that IB inhibited the in vivo
activity of the constitutively nuclear CCR.SVNLS protein indicates
that IB must also be able to actively antagonize nuclear c-Rel
function. This antagonism may occur by blocking the transcriptional activity of c-Rel and/or by promoting the dissociation of c-Rel from
DNA. In this scenario, the relocalization of Rel dimers to the
cytoplasm would likely constitute a second step in the inhibitory process. Reports that IB can dissociate Rel-DNA complexes in vitro and block Rel-mediated transcriptional activation agree with our
data (44, 70, 76).
The addition of acidic residues to the C terminus of mutant
285
simultaneously restored its capacity to inhibit the DNA-binding
and
transcriptional activities of CCR.SVNLS. This result suggested
that
I
B
may interfere with the activity of c-Rel in the nucleus
by
simply inhibiting its binding to
B DNA sites. However, while
mutant
287 failed to inhibit DNA binding by CCR.SVNLS, it efficiently
inhibited the transcriptional activity of this protein. Importantly,
this mutant was able to form complexes with c-Rel that bound to
DNA. It
is thus tempting to speculate that the association of
this mutant with
DNA-bound c-Rel proteins might interfere with
their transcriptional
activity by preventing their interaction
with components of the
transcriptional machinery (
40,
75).
This model is consistent
with a recent report showing that I
B
competes with the basal
transcription initiation complex for binding
to NF-
B
(
70). The nuclear regulation of c-Rel by I
B
may thus
involve the inhibition of DNA contact and the disruption of c-Rel
association with basal transcription factors. I
B
mutant
285
also formed complexes with c-Rel and DNA but did not block
CCR.SVNLS-mediated
transcription. Thus, these results implicate
amino acids 285 and
286 at the beginning of the PEST domain in the
inhibition of nuclear
c-Rel-mediated transcription. Other members of
the I
B family,
namely, Bcl-3 and I
B
, have been found in
complexes with NF-
B
subunits and DNA (
13,
27,
67).
However, to our knowledge
this report is the first indication of an
indirect interaction
between an I
B
protein and DNA through its
association with c-Rel.
Although the mechanism through which I
B
inhibits c-Rel DNA
binding remains to be clarified, the absolute requirement for
acidic
residues at the beginning of the PEST domain suggests that
this region
may (i) compete with DNA for interaction with the
positively charged
DNA recognition loop of c-Rel (
26,
30,
45,
53); (ii) induce
the dissociation of c-Rel from DNA by
promoting electrostatic repulsion
between c-Rel and the negatively
charged DNA backbone, as proposed by
others (
70); or (iii) induce
a conformational change in
c-Rel that is incompatible with DNA
contact. Future studies will
undoubtedly help to address these
issues.
Role of phosphorylation of the IB PEST domain in the
inhibition of c-Rel activity.
The serine and threonine residues
found in the IB PEST domain are highly phosphorylated by casein
kinase II (8, 48, 51, 61, 63). Their mutation in chicken
IB was reported to dramatically decrease its affinity for c-Rel
and to abolish its inhibitory activity toward c-Rel DNA binding
(61). These results are in sharp contrast to ours. In our
experiments, IB mutant A5, with all of the serine and threonine
residues in the PEST domain mutated to alanines, exhibited wild-type
inhibition of c-Rel DNA binding. This mutant was as effective as
wild-type IB in interactions with c-Rel, in the cytoplasmic
retention of c-Rel, and in the inhibition of c-Rel and CCR.SVNLS
DNA binding and transcriptional activation. While the discrepancy
between our results and those of Sachdev et al. (61) may
derive from differences in experimental conditions, our assays indicate
that phosphorylation of the PEST domain is not required for the
inhibitory activity of IB. Similar findings were recently
obtained with human IB (9, 22, 70).
The N terminus of IB is important for masking of the c-Rel
NLS.
An intriguing finding from this study is that the N-terminal
domain of IB led to masking of the Rel NLS. In contrast to the
situation observed with wild-type IB, c-Rel associated with IB N-terminal deletion mutants was recognized by an NLS-specific antibody. This finding is consistent with the inability of these mutants to retain c-Rel in the cytoplasm. The Rel NLS was previously postulated to be essential for interactions with IB, since
anti-NLS antibodies abolished the association of Rel with IB
(77). Likewise, deletion or point mutations in the NLS of
various Rel family members dramatically decreased their affinity for
IB and impaired the inhibition of their DNA binding (10,
28). However, recent evidence suggests that the NLS itself is not
required for the interaction of Rel with IB. (i) A c-Rel mutant
with a deletion of its NLS efficiently interacted with IB in a
yeast two-hybrid system (60). (ii) Similarly, the NLS of the
Drosophila Rel homolog Dorsal is also dispensable for the
interaction of Dorsal with the IB inhibitor Cactus (33).
(iii) Here, we showed that the transcriptional activity of a nuclear
c-Rel protein with a mutated NLS was slightly inhibited by IB but
was not abolished (CCR.KO.SVNLS). These results further suggest
that NLS mutations only partially interfere with c-Rel protein
association with the IB inhibitor.
As suggested by others, the ankyrin domain of I
B
may contact
sequences proximal to the NLS in the tertiary structure of
Rel
(
47). According to our data, this interaction may position
the N-terminal domain of I
B
in a way that masks the Rel NLS
and
impedes interaction of the NLS with the nuclear translocation
machinery. In this context, mutations in the Rel NLS perhaps disrupt
the structure of Rel in a way that affects its optimal interaction
with
the inhibitor. Results similar to ours were recently obtained
by others
with c-Rel and with p50/NF-
B1 (
57a). Interestingly,
however, it appears that N-terminal deletions in I
B
may
differentially
affect the cytoplasmic regulation of c-Rel and RelA
(
57a,
65).
Future studies will help to clarify the basis for
these differences.
In conclusion, this study demonstrates that the determinants of
I
B
involved in the cytoplasmic and nuclear regulation of
c-Rel
map to distinct domains of the molecule. The central ankyrin
domain of
I
B
mediates interactions with c-Rel. The N-terminal
domain is
essential for the cytoplasmic retention of c-Rel, while
a few of the
acidic amino acids at the very beginning of the C-terminal
PEST domain
are necessary to regulate the activity of c-Rel in
the nucleus. The
identification of these discrete domains may
help in the design of
inhibitors to antagonize Rel/NF-
B activity
in a specific cellular
compartment or at a defined stage of activation.
| |
ACKNOWLEDGMENTS |
We thank N. Rice (ABL-NCI, Frederick, Md.) for the generous gift
of antibody Ab1507 against c-Rel, H. Bose (University of Texas, Austin)
for an ib cDNA clone and antibodies against the chicken IB protein, T. Gilmore (Boston University, Boston, Mass.) for a chicken c-rel cDNA clone, and K. Nakayama (Kanazawa
University, Kanazawa, Japan) for the pIL6CAT plasmid. We are grateful
to S. Crespo for expert assistance with the construction and
characterization of some IB mutants. We thank F. Agnès, J. Bash, C. Chen, and W.-X. Zong for fruitful discussions during the
course of this work. We also thank J. Bash, C. Chen, I. Martínez-Férez, A. Rabson, and W.-X. Zong for helpful
comments on the manuscript and N. Rice for sharing results prior to
publication.
This work was supported by a grant from the National Institutes of
Health (CA54999) and by the New Jersey Commission on Science and
Technology (C.G.). I.L. was supported by postdoctoral
fellowships from Ministerio de Educación y Ciencia
(Spain)/Fulbright Program and from the International Human Frontier
Science Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CABM, 679 Hoes
Ln., Piscataway, NJ 08854-5638. Phone: (732) 235-5035. Fax: (732)
235-5289. E-mail: gelinas{at}mbcl.rutgers.edu.
| |
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Mol Cell Biol, March 1998, p. 1213-1224, Vol. 18, No. 3
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
