From the Department of Chemistry & Biochemistry, University of California at San Diego, La Jolla, California 92093-0359
Received for publication, July 25, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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p105, also known as NF- The NF- NF- It has long been thought that all I p105 and p100 are the precursors of NF-B1, is an atypical
I
B molecule with a multi-domain organization distinct from other
prototypical I
Bs, like I
B
and I
B
. To understand the
mechanism by which p105 binds and inhibits NF-
B, we have used both
p105 and its C-terminal inhibitory segment known as I
B
for our
study. We show here that one I
B
molecule binds to NF-
B dimers
wherein at least one NF-
B subunit is p50. We suggest
that the obligatory p50 subunit in
I
B
·NF-
B complexes is equivalent to the N-terminal p50
segment in all p105·NF-
B complexes. The nuclear localization signal (NLS) of the obligatory p50 subunit is masked by I
B
, whereas the NLS of the nonobligatory NF-
B subunit is exposed. Thus,
the global binding mode of all I
B·NF-
B complexes seems to be
similar where one obligatory (or specific) NF-
B subunit makes
intimate contact with I
B and the nonobligatory (or nonspecific) subunit is bound primarily through its ability to dimerize. In the case
of I
B
and I
B
, the specific NF-
B subunit in the complex is p65. In contrast to I
B
·NF-
B complexes, where the exposed NLS of the nonspecific subunit imports the complex to the nucleus, p105·NF-
B and I
B
·NF-
B complexes are cytoplasmic. We
show that the death domain of p105 (also of I
B
) is essential for
the cytoplasmic sequestration of NF-
B by p105 and I
B
. However,
the death domain does not mask the exposed NLS of the complex. We also
demonstrate that the death domain alone is not sufficient for
cytoplasmic retention and instead functions only in conjunction with
other parts in the three-dimensional scaffold formed by the association of the ankyrin repeat domain (ARD) and NF-
B dimer. We speculate that
additional cytoplasmic protein(s) may sequester the entire p105·NF-
B complex by binding through the death domain and other segments, including the exposed NLS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B family of transcription factors plays an important
role in a large number of cellular processes including immune response
and inflammation, cellular development, and differentiation (1-4).
This family comprises of five distinct members, p50, p65, p52, c-Rel,
and RelB, that exhibit a high degree of sequence homology at their N
termini. This region, known as the Rel homology region, is
responsible for important functions like DNA binding, dimer formation,
nuclear localization, and I
B binding (1-4).
B dimers are regulated by inhibitor I
B proteins, which include
I
B
, I
B
, I
B
, I
B
, p105 (NF-
B1), p100
(NF-
B2), Bcl-3, I
B
, and MAIL (4-21). In most cells NF-
B
remains inactive as a complex with I
B. In response to a variety of
extracellular signals, the I
B molecule is phosphorylated by I
B
kinases, which leads to the ubiquitination and subsequent degradation
of I
B by the proteosome machinery within the cell (22).
B proteins inhibit
NF-
Bs by masking the nuclear localization signal
(NLS)1 of NF-
B and thereby
sequestering them in the cytoplasm (1-4, 22). However, recent studies
have indicated that the I
B proteins differ with regard to their
regulation of NF-
B subcellular localization. The I
B
·NF-
B
complex exhibits dynamic shuttling between the cytoplasm and the
nucleus (23-27). However, despite its transient presence in the
nucleus, NF-
B remains bound to I
B
, and this prevents DNA
binding and activation of transcription. I
B
, on the other hand,
sequesters NF-
B in the cytoplasm of resting cells (26-28). The
detailed mechanism of this process is not clear. It has been suggested
that other ancillary proteins may be required for this
function.2
B subunits p50 and p52,
respectively, which are located in their N termini. Both p105 and p100
have similar structural organizations (3) (Fig.
1). The central portion of these
molecules has a glycine-rich region that has been shown to play a
critical role in processing of the precursor (29, 30). The C termini
resemble other I
B molecules that possess ankyrin repeats (AR) (14,
15, 31). These two proteins also contain a death domain immediately
C-terminal to the ankyrin repeat domain (ARD). The I
B kinase
phosphorylation sites are located further downstream within a region
called the destruction box (see Fig. 1). A separate gene also encodes
the C-terminal part of p105. This gene product, known as I
B
, has been shown to exist only in certain cell types, like mouse pre-B cells
(12) (Fig. 1).
View larger version (14K):
[in a new window]
Fig. 1.
Domain organization of p105 and
I B
. A schematic
representation of various domains in p105·I
B
with key regions
indicated by arrows is shown. Some of the constructs used in
this study, p105 (1-971), p105
N (245-971), I
B
(365-971),
and ARD-I
B
(500-971), are shown. The corresponding C-terminal
domain-deleted forms are p105
C (1-800), p105
N
C (245-800),
I
B
C (365-800), and ARD-I
B
C (500-800), and the
destruction box-deleted form is p105
DB (1-887).
In resting cells, p105 is partially processed, generating p50. The
exact mechanism of this limited processing event is not known, although
co-translational processing events have been proposed (32, 33). The
unprocessed p105 functions as an inhibitor molecule and nonspecifically
inhibits almost all NF-B subunits, including p50 (34-36). An
earlier study has shown that p105 retains itself, as well as other
NF-
B molecules, in the cytoplasm (37). The mechanism of inhibition
is, however, still unclear. The p105 molecule, like the classical I
B
inhibitors, can also undergo complete degradation in response to
signals and the sequential action of I
B kinases, ubiquitin ligases,
and the 26 S proteasome (38-41). Both the death domain and the
destruction box have been shown to be important for I
B kinase
phosphorylation (41, 42). In contrast to p105, the mechanism of p100
processing is very different. In resting cells, most of p100 remains
unprocessed, and in response to appropriate signals all of it gets
processed into p52 (43, 44).
In this study, we address the functional properties of p105
by asking the following questions. How does p105 regulate subcellular distribution of NF-B subunits? How does p105 nonspecifically inhibit
other NF-
B proteins? What is the relationship between p105·NF-
B
and I
B
·NF-
B complexes? We find that although I
B
resembles other I
Bs in exhibiting a 1:1 stoichiometry of binding to
NF-
B dimers, it has a unique specificity for NF-
B dimers that
contain at least one p50 subunit. This obligatory p50 subunit in
I
B
·NF-
B complexes is structurally equivalent to the
N-terminal p50 segment of p105 in p105·NF-
B complexes.
Interestingly, although I
B
fails to mask one NF-
B NLS, it
still retains NF-
B in the cytoplasm. We observe that the death
domain of p105 is necessary but not sufficient for this cytosolic
retention. We suggest that p105 and I
B
inhibitors may require an
as yet unknown cellular factor(s) to sequester NF-
B in the cytoplasm.
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EXPERIMENTAL PROCEDURES |
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Protein Expression and Purification from Escherichia
coli--
The cloning, expression, and purification of the NF-B
subunits has been described previously (45, 46). Full-length and truncated glutathione S-transferase-I
B
were made by
cloning into the pGEX-2T vector (Amersham Biosciences). The fusion
protein was expressed in E. coli BL21 DE3 and purified by
glutathione-agarose column chromatography following the manufacturer's
protocol (Amersham Biosciences).
Native Polyacrylamide Gel Electrophoresis--
Proteins and
protein complexes were diluted in 10 mM Tris (pH 7.5), 200 mM NaCl, 4% glycerol, and 2 mM
-mercaptoethanol. The reactions were allowed to equilibrate at room
temperature for 1 h. Native gel loading dye (50 mM
Tris, pH 7.5, 0.1% bromphenol blue, 10% glycerol, and 1.25 mM
-mercaptoethanol) was then added to each sample. 10%
native polyacrylamide gels were prepared with 0.25× Tris-borate-EDTA
buffer. The samples were loaded on the gel and run in Tris-borate-EDTA
buffer for 2 h at a constant current (3 mA). The protein bands
were visualized by Coomassie staining.
Fluorescence Polarization Competition
Assay--
Fluorescence polarization competition assays were done as
described previously (47). Briefly, varying concentrations of IB
were mixed with constant amounts of p50 homodimer pre-equilibrated with
fluorescein-labeled DNA. The competition assay binding curves were
analyzed for IC50 values, defined as the concentration of I
B
at 0.5 fractional occupancy.
Plasmids, Cell Culture, and Transfections--
cDNAs
encoding full-length p50 and full-length/truncated p105 were cloned
into a pcDNA vector (Invitrogen) containing either an N-terminal
FLAG (p50 constructs) or an N-terminal HA (p105·IB
constructs)
tag. HeLa cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics and transfected with plasmid DNA using the
LipofectAMINE Plus reagent (Invitrogen). Protein expression was checked
by Western blot.
Immunofluorescence-- HeLa cells were grown on 8-well chamber slides (Lab Tek). The cells were transfected with a total of 0.2 µg of plasmid DNA. After 24 h, the cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. The cells were then permeabilized with 0.25% Nonidet P-40 in PBS for 1 min and blocked with 5 mg/ml bovine serum albumin in PBS containing 0.1% Tween 20 at room temperature for 30 min. Fluorescent detection was done by incubating the cells with monoclonal antibody 12CA5 (against HA), M2 (against FLAG), and H-286 (against p65) in PBS containing 5 mg/ml bovine serum albumin and 0.2% Nonidet P-40 at room temperature for 2 h. The cells were washed three times with buffer containing 0.2% Nonidet P-40 in PBS and incubated with fluoresceinated secondary antibody at room temperature for 1 h. Finally, the cells were washed three times with buffer containing 0.1% Tween 20 in PBS, and the slide was mounted with Vectashield (Vector Laboratories).
Immunoprecipitation--
For immunoprecipitation from whole cell
lysates, HeLa cells were harvested 24 h post-transfection and
lysed with buffer containing 20 mM Tris (pH 7.5), 0.2 M NaCl, 1% Triton X-100, 1 mM EDTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, and protease inhibitor mixture (Sigma). Immunoprecipitation
was carried out by the protein A pull-down method. Briefly, 0.1-0.5 mg
equivalent of protein from transfected cell extract was diluted with
300 µl of lysis buffer. The appropriate antibodies were added to the extract and incubated overnight with protein A-Sepharose 4B beads (Sigma) at 4 °C. The beads were pulled down by brief centrifugation and washed three times with the lysis buffer. The immunoprecipitates were then eluted from the beads with 2× Laemmli buffer devoid of
-mercaptoethanol by heating at 95 °C for 5 min. The bound proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The bound proteins were then identified by Western blot. In vitro immunoprecipitation was carried out with a similar
protocol using purified E. coli proteins in place of whole
cell extract.
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RESULTS |
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Interactions between NF-B Dimers and I
B
--
Unprocessed
p105 functions as an inhibitor of NF-
B, although its mode of
inhibition appears to be quite different from the prototypical I
B
proteins such as I
B
and I
B
. As opposed to I
B
and
I
B
that bind through p65, p105 inhibits all NF-
B members nonspecifically. To understand the biochemical basis of NF-
B inhibition by p105, we wanted to first test how the p105 C
terminus (I
B
) binds to NF-
B dimers. We have characterized the
interactions between I
B
and various NF-
B dimers by
protein-protein gel shift assays under native conditions. To simplify
the assay we generated a truncated I
B
containing only the ARD
fused to a poly-His peptide. This construct will be referred to as
ARD-I
B
C. We observe that ARD-I
B
C binds strongly to
the p50·p50 homodimer and p50·p65 heterodimer but only weakly to
the p65·p65 and c-Rel·c-Rel homodimer (Fig.
2A). Although no stable
complexes are formed between I
B
and p65·p65 or c-Rel·c-Rel
homodimers, we do not observe free I
B
in these lanes. It is
likely that these complexes are weaker and smear during
electrophoresis. Based on these results we suggest that complexes
between I
B
and non-p50 containing NF-
B dimers are highly
unstable.
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The fact that p105 binds to all NF-B proteins and
I
B
binds only to p50 dimers (any NF-
B dimer that contains at
least one p50 subunit) suggests that the required p50 subunit in
I
B
·NF-
B complexes is likely the N-terminal p50 segment of
p105 in p105·NF-
B complexes. Thus, the p105·p50 (or p105·p65)
complex can be considered to be structurally equivalent to the
I
B
·p50·p50 (or I
B
·p50·p65) complex (Fig.
2B).
The binding data presented above suggest that IB
prefers p50
containing NF-
B dimers. However, these experiments do not reveal
whether one or two molecules of I
B
bind to one molecule of
NF-
B dimer. This is important considering that Bcl-3, which contains
seven AR like I
B
, has been proposed to bind to p50·p50 and
p52·p52 homodimers in a 2:1 molar ratio (48-50). To elucidate I
B
·NF-
B binding stoichiometry, we have used two different
I
B
constructs, one is the full-length I
B
expressed as a
glutathione S-transferase fusion protein (FL-I
B
), and
the other is the previously described ARD-I
B
C. As seen in Fig.
2C (lanes 4 and 5), both of these
proteins bind p50·p50 homodimer efficiently, and the resulting
complex migrates to different positions in the native gel. In a
reaction mixture containing both I
B
s and the p50 homodimer, binding of two molecules of I
B
to the homodimer would result in a
distinct, additional complex composed of these I
B
molecules of
different lengths and p50. However, when equivalent amounts of
FL-I
B
, ARD-I
B
C, and p50·p50 homodimer were mixed, only two distinct complexes, one corresponding to the
FL-I
B
·p50·p50 complex and the other to the
ARD-I
B
C·p50·p50 complex, were seen (Fig. 2C,
lane 6). Thus, we can conclude that one molecule of I
B
binds to one p50·p50 homodimer.
The Role of the NF-B NLS in I
B
Binding--
The presence
of one free NLS is a prime reason for the nucleocytoplasmic shuttling
of the I
B
·NF-
B complex. To elucidate the status of the
NF-
B NLS(s) in its complex with I
B
, p50·p50 homodimers of
different lengths, both with and without the NLSs, were prepared, and
their binding to ARD-I
B
C was tested using native gel shift
assays. Fig. 3A shows that all
three of the p50·p50 dimers were capable of binding to
ARD-I
B
C, although the dimer with both NLSs deleted
(p50-350(
NLS)) seems to bind I
B
relatively weakly (Fig.
3A, lanes 5-7). This suggests that I
B
may
not use the NLS sequence for p50 binding, or at least this region does not seem to contribute significantly. Similar binding patterns were
also observed with FL-I
B
(data not shown).
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Co-immunoprecipitation experiments were performed to further confirm
the presence of at least one free NLS in the IB
·NF-
B complex. If the p65 NLS in the I
B
·p50·p65 complex were free, then anti-p65 NLS antibody would interact with the p65 NLS. Here, the
p50·p65 heterodimer was used because of the availability of the p65
NLS-specific monoclonal antibody. Free NF-
B p50·p65 heterodimer and the I
B
·p50·p65 heterodimer complex were incubated with
p65 NLS antibody. It was seen that the antibody was able to pull down NF-
B in both the free and I
B
complexed form (Fig.
3B, top panel). This demonstrates that the
p65 NLS in the p50·p65 heterodimer is not protected by I
B
. As a
parallel control, the I
B
·p50·p65 complex was also tested. The
p65 NLS antibody was unable to pull down NF-
B in the
I
B
·p50·p65 complex (Fig. 3B, bottom
panel). This corroborates with earlier results that have shown
that the p65 NLS is masked in the I
B
·p50·p65 complex (26, 27,
52, 53). Thus, these experiments show that at least one NF-
B NLS remains unmasked in the I
B
·NF-
B complex.
IB
·NF-
B and p105·NF-
B Complexes Are
Cytoplasmic--
The above observation that at least one NF-
B NLS
is free in the I
B
·NF-
B complex suggests that these complexes
should localize to the nucleus. The subcellular distribution of the
complex was tested by simultaneously transfecting HeLa cells with
I
B
, p50, and p65. Immunostaining shows that free p50 and p65,
which are most likely to be present as a heterodimer, are nuclear (Fig. 4A, left panel),
but co-expression of I
B
leads to cytoplasmic retention (Fig.
4A, right panel). Thus, although the
I
B
·p50·p65 has at least one NLS free, the complex does not
localize to the nucleus.
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The subcellular distribution of p105 and IB
complexes with p50
was also tested. Immunofluorescence studies show both these complexes
to be cytoplasmic (Fig. 4B). Free p50, as expected, is
localized in the nucleus (data not shown).
To further test the ability of these complexes to be retained in the
cytoplasm, a mutant p50 with an additional NLS (p50-NLS) was made.
Cells co-transfected with p50-NLS and p105 or IB
reveal that the
complexes are retained in the cytoplasm (Fig. 4C, left panels). To test whether the p105·p50-NLS complex shuttles
between the nucleus and cytoplasm, HeLa cells were treated with the
nuclear export inhibitor leptomycin B (LMB), and the subsequent changes in localization were monitored. LMB treatment does not appear to alter
the localization of the p105·p50-NLS or the I
B
·p50-NLS complexes (Fig. 4C, right panels), implying that
these complexes do not shuttle. This result is in contrast to that
observed for I
B
·NF-
B complexes where addition of the
inhibitor confines both proteins within the nucleus (26). Thus, the
p105·NF-
B complexes seem to be similar to the I
B
·NF-
B
complexes, which are primarily cytoplasmic. Free p50-NLS, as expected,
is localized within the nucleus (data not shown).
The Death Domain Is Necessary for Cytoplasmic Retention of
NF-B--
To assess the possible role of the death domain and the
C-terminal tail containing the destruction box in the cytoplasmic retention of NF-
Bs, deletion mutants of both p105 and
I
B
were constructed. HeLa cells were co-transfected
with p50 and various truncated forms of p105 or I
B
. Subcellular
localization of these complexes was monitored by immunofluorescence. As
mentioned in the previous results section, the p105·p50 complex is
cytoplasmic (Fig. 5A,
left panel). Removal of the last ~100 residues, which includes the destruction box, p105
DB, did not alter the localization of the complex (Fig. 5A, right panel). However,
deletion of the C-terminal 171 residues (encompassing the death
domain), p105
C, localizes the complex exclusively in the nucleus
(Fig. 5A, middle panel). These results suggest
that the death domain of p105·I
B
plays a role in the retention
of NF-
B complexes within the cytoplasm.
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p50 is again retained in the cytoplasm when co-expressed with a
truncated p105, p105N (Fig. 1), where the N-terminal, DNA-binding immunoglobulin domain (residues 1-244) is deleted (Fig. 5B,
left panel). However, when both the N- and C-terminal
domains (p105
N
C) are deleted, these proteins localize to the
nucleus (Fig. 5B, right panel).
Similar results were obtained with various deletion mutants of
IB
. Co-expression of full-length I
B
and p50 lead to
cytoplasmic retention of the complex (Fig. 5C, left
panel). Deletion of the C terminus, I
B
C, leads to nuclear
localization of both proteins (Fig. 5C, right
panel). Also, a shorter construct of I
B
comprising the ARD
and all residues downstream, ARD-I
B
(Fig. 1) and the corresponding C terminally deleted form, ARD-I
B
C, showed a similar retention pattern (Fig. 5D).
It is known that NF-B dimers are nuclear proteins, and it is only in
complex with I
B that they are retained in the cytoplasm. As
described above, co-expression of p50 and death domain-deleted p105 or
I
B
leads to nuclear localization of both components. To test
whether these proteins localize independently or are present as a
complex in the nucleus, co-immunoprecipitation experiments were done.
FLAG antibody (M2) was used to immunoprecipitate FLAG-tagged p50 from
whole cell lysates that had been co-transfected with FLAG-p50 and
HA-p105 or HA-I
B
. Subsequent Western blotting was done using the
HA antibody. The results clearly show that these proteins form a
complex because the FLAG antibody was able to pull down both p50 and
p105 (or I
B
) (Fig. 5E). In all, the above experiments
suggest that the death domain of p105·I
B
is necessary for
cytoplasmic retention.
The Death Domain Is Not Sufficient for Cytoplasmic
Retention--
Next, we wanted to test whether the death domain was
the exclusive retention signal in these IB molecules. Full-length
I
B
when expressed in HeLa cells exhibits a nucleocytoplasmic
distribution (Fig. 6A,
left panel). The death domain-truncated I
B
C, as
expected, was localized within the nucleus (Fig. 6A,
right panel). Both ARD-I
B
and ARD-I
B
C, by
themselves, were present in the nucleus (Fig. 6B). Thus, the
presence of the death domain is not sufficient for cytoplasmic
localization.
|
To further test the role of the death domain, two more constructs were
made. One containing only the death domain, p105 (800-887), and the
other with all the segments further downstream, p105 (800-971). Both
of these proteins localized within the nucleus (Fig. 6C). This suggests that neither the death domain by itself nor its presence
in the IB
molecule provides the retention signal.
The Death Domain Does Not Participate in NF-B
Binding--
Because the death domain of p105·I
B
seems to be
important for cytoplasmic localization of NF-
B, a possible model of
the complex could be one in which the death domain binds and masks the
exposed NLS of NF-
B, thereby preventing nuclear import. If this were
true, then full-length I
B
must interact with the p50·p50 homodimer with higher affinity than the death domain-truncated I
B
. Although the native gel shift assays showed that the death domain was not essential for NF-
B binding, these experiments were of
a qualitative nature. Fluorescence polarization competition assays were
done to determine the binding affinity of NF-
B for both full-length
and death domain-truncated I
B
. In a solution-based competition
assay, fixed amounts of fluorescein-labeled
B DNA bound to the
p50·p50 dimer was incubated with increasing amounts of I
B
. In
this experiment, the I
B
-dependent dissociation of the
p50·p50 dimer from DNA is accompanied by a distinct change in
fluorescence polarization. The DNA binding inhibition constant at
equilibrium was then calculated. As shown in Fig.
7, both I
B
s inhibit NF-
B DNA
binding to a similar extent. Thus, the death domain does not seem to
play a major role in NF-
B binding to I
B
.
|
Limited Processing of p105: a Possible Model of the
Cytoplasmic Complex--
In addition to testing the localization of
p50 complexes with the full-length and truncated forms of p105, we have
also expressed just p105 and its truncated forms in HeLa cells and
tested their individual subcellular distribution. p105 is present
exclusively in the cytoplasm (Fig.
8A, left
panel), whereas the C-terminally truncated p105, p105C, was
exclusively nuclear (Fig. 8A, right panel). The
N-terminally truncated p105, p105
N, also localized to the cytoplasm
(Fig. 8B, left panel). The corresponding
C-terminal deleted p105, p105
N
C, was nuclear (Fig. 8B,
right panel). Localization patterns of p105 and its
derivatives are thus identical to that observed previously for the
p105·p50 complexes in HeLa cells, expressing both p105 (or its
derivatives) and p50.
|
p105 is known to undergo limited processing to generate p50 molecules.
To test whether the other truncated forms of p105 were also capable of
similar processing, Western analysis was done using HA monoclonal
antibody. We observe that like full-length p105, its truncated forms
were also processed in a limited manner within the cell. The appearance
of an additional band of lower molecular weight indicates a processed
band, derived from the C-terminal processing of the intact protein. The
lengths of the processed products vary according to the truncations of
p105. Cells transfected with the C-terminally truncated form, p105C, show a processed band corresponding to full-length p50, and cells transfected with the smaller, N-terminally truncated form, p105
N and
p105
N
C, generate a shorter p50 (Fig. 8C). On the other
hand, in cells transfected with both truncated p105 and
p50, no processed p50 band is detected. This observation corroborates
with that seen earlier for full-length p105 and p50 (41). These results lead us to suggest that p105 is first processed in a limited manner and
that the resulting complex formed between the unprocessed p105 and the
processed p50 is identical to the one in which both proteins are
expressed within the same cell.
A minimal complex formed between p105 and p50 seems to require just the
dimerization domain of p50. Therefore, the smallest p105 derivative,
p105N
C, can be partially processed into a truncated p50
comprising of only the dimerization domain (p50
N), and this can
associate with unprocessed p105
N
C. Such a complex would be
equivalent to the complex between the p50 dimerization domains and the
ARD-I
B
C. Indeed, native gel shift assays show that the
dimerization domain of p50 (p50
N-376 and p50
N-363) can form a
stable complex with ARD-I
B
C (Fig. 8D). However, the
p105
N
C·p50
N complex is not cytoplasmic because the death
domain is absent. In contrast, the p105
N·p50
N complex is cytoplasmic.
These results indicate that in addition to the death domain,
p105·NF-B or the equivalent I
B
·NF-
B complex formation
is necessary for cytoplasmic retention. In the retention process, only
the N-terminal domain (residues 1-244) and the C-terminal region
(residues 885-971) of p105 are dispensable.
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DISCUSSION |
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Earlier studies using competition assays have shown that IB
preferentially inhibits p50·50 homodimer (18, 31, 51). In the present
study we have performed direct binding assays to demonstrate that
I
B
interacts stably with both p50·p65 and p50·p50 dimers but
not with p65·p65 or c-Rel·c-Rel dimers. This suggests that I
B
may have a preference for p50 containing NF-
B dimers. We and others
have previously shown that I
B
interacts strongly with p65
containing dimers (23, 24, 26). The x-ray structure of
I
B
·p50·p65 heterodimer showed that the p65 subunit makes extensive contacts with I
B
(52, 53). Thus, a central theme seems
to govern all I
B·NF-
B binding wherein the stability
of the interaction is dictated by the association of an I
B molecule with one specific NF-
B subunit. The second subunit, which we refer
to as the nonspecific subunit, associates with the specific subunit
through its dimerization domain. This is illustrated in Fig.
9A. The nature of the specific
subunit is directly correlated to the nature of the binding I
B
molecule. For I
B
, p65 serves as the specific subunit. I
B
also exhibits similar dimer specificity, requiring p65 as the primary
binding partner. On the other hand, in I
B
, the specific subunit
appears to be p50. In p105·NF-
B complexes, by the very nature of
the molecule, the docking occurs intramolecularly with the N-terminal
p50 arm serving as the specific subunit. It is possible that in
I
B·NF-
B complexes the nonspecific subunit can be exchanged with
other nonspecific subunits, and this may dictate the cellular pool of
active NF-
Bs.
|
Sequestration of NF-B in the cytoplasm can be considered to be one
of the most simplistic ways by which I
B molecules exhibit their
inhibitory function. They can do this by blocking the NLS of NF-
B
dimers. However, both I
B
and I
B
, upon binding to the
specific NF-
B dimers, mask only the NLS of the specific NF-
B subunit. As expected therefore, I
B
·NF-
B complexes are not
truly cytoplasmic but dynamic nucleocytoplasmic (23-26). We observe
that in the I
B
·p50·p65 complex the p65 NLS is free. Although
no structural evidence exists to confirm that the NLS of the specific
subunit is completely masked, two lines of evidence indicate this to be most likely. First, we show that when both NLSs are removed the affinity of the p50·p50 homodimer for I
B
appears to be reduced, and second, we show that the NLS of the nonspecific p65 subunit of the
I
B
·p50·p65 complex is free. Therefore, reduced binding affinity of p50·p50 homodimer for I
B
is likely due to the
removal of the NLS of the p50 subunit that makes specific contacts with I
B
.
The domain architecture of the C-terminal inhibitory domain of p105 is
different from that of IB
and I
B
. p105 does not contain a
PEST sequence in its C-terminal tail as in I
B
and I
B
, but
it does contain a death domain. The PEST sequence in I
B
and
I
B
is important for NF-
B binding. In case of I
B
, we
observe that the death domain does not play any role in NF-
B binding, suggesting that this domain is not involved in masking the
exposed NLS of the nonspecific NF-
B subunit in p105·NF-
B or
I
B
·NF-
B complexes. The death domain, however, seems critical for cytoplasmic retention. When this domain is removed from
p105·I
B
, the subcellular localization of the
truncated molecules in their free form or their complexes with NF-
B
change completely, from cytoplasmic to nuclear. This observation is
consistent with earlier studies showing that removal of C-terminal 191 residues alters the localization from the cytoplasm to the nucleus
(37). However, although this clearly suggested the role of the
C-terminal domain of p105, this large truncation also removed most of
the last AR. Therefore, it was not clear whether removal of part of the
ARD contributed to altered localization of p105. Interestingly, the death domain, by itself, does not contain any retention signal because
we see that the free death domain localizes to the nucleus. Full-length
or N-terminally truncated I
B
, both of which contain the death
domain, are also primarily nuclear.
We observe that free p105, as in the p105·p50 complex, is
also cytoplasmic. However, p105 does not exist in its free form in
cells. The molecule is partly processed generating p50, and this
processed p50 forms a dimer with the precursor p105. p105·p50 complex
formation is necessary for cytoplasmic sequestration of both p50 and
p105. Our results show that limited processing does not produce an
exactly 1:1 molar ratio of p105. It is thus possible that excess
unprocessed p105 associates with other NF-Bs such as p65 and c-Rel,
and the p105·NF-
B complex is retained in the cytoplasm. Cellular
localization of truncated p105 is dependent upon the presence or
absence of the death domain. Our results suggest that the
three-dimensional scaffold formed because of the association of p105
and p50 (or another NF-
B subunit), in addition to the death domain,
is required for the cytoplasmic retention of the complex. Association
between p105 and p50 (or any other NF-
B subunit) requires only the
dimerization domain of p50 and the ARD of I
B
. However, the NLS of
the nonspecific subunit is still exposed to the solvent. How is the
complex then retained in the cytoplasm? We do not yet know the precise
mechanism of cytoplasmic sequestration of this complex.
Earlier work in our laboratory has shown that
IB
·NF-
B complexes are cytoplasmic, although even here, as in
I
B
·NF-
B complexes, one NLS is mostly solvent-exposed. Recent
studies lead us to believe that I
B
·NF-
B complexes interact
with additional cellular proteins to mask the exposed NLS, thereby
sequestering them in the cytoplasm.2 It is possible that
p105 employs a similar mechanism, but not necessarily the exact same
mechanism. Thus, a bridging factor, which requires the death domain and
the NF-
B scaffold, could be involved in masking the NLS. The death
domain has been shown to confer various homotypic and heterotypic
interactions in a large number of signaling pathways (54). It may thus
play a major role in binding to the bridging factor. Consistent with this model, an earlier study demonstrated the inaccessibility or the
reduced accessibility of the C-terminal tail in p105 by using
antibodies raised against the C-terminal (37). This study also showed
that the NLS of p105 is masked. Fig. 9B thus summarizes a
likely sequence of events based on the above results. Expression of
p105 or both p105 and p50 results in a p105·p50 complex that is
retained in the cytoplasm. As noted earlier, the N-terminal domains and
the last ~100 residues of p105 do not seem to be a part of the
retention signal. Thus, a plausible model could be one in which a
composite surface formed by the coming together of the relevant regions
of the protein complex can interact with a putative, cytoplasmic
protein that leads to its docking. This could then explain the
inability of the p105·NF-
B or the I
B
·NF-
B complex to
move to the nucleus even with an additional NLS. We conclude that the
I
B family proteins employ diverse mechanisms for cytoplasmic
sequestration of NF-
B dimers. Elucidating each of these mechanisms
in detail is imperative for understanding their cellular function and
participation in unique signal transduction pathways.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. M. Yoshida at the University of Tokyo for providing LMB. We acknowledge Amanda Fusco for aid in performing the fluorescence polarization experiment (Fig. 7) and Tom Huxford and Rashmi Talwar for critically reading the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from NCI, National Institutes of Health and Human Frontier Science Program (to G. G.).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: Dept. of Chemistry & Biochemistry, University of California at San Diego, 9500 Gilman Dr.,
La Jolla, CA 92093-0359. Tel.: 858-822-0469; Fax: 858-534-7042;
E-mail: gghosh@ucsd.edu.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M207515200
2 S. Malek, Y. Chen, and G. Ghosh, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NLS, nuclear localization signal; AR, ankyrin repeat; ARD, ankyrin repeat domain; LMB, leptomycin B; HA, hemagglutinin; PBS, phosphate-buffered saline.
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