From the Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0359
Received for publication, January 30, 2003
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ABSTRACT |
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INTRODUCTION |
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Although present in most cell types, NF-B dimers with transactivation potential are maintained inactive through their stable association with the inhibitor proteins I
B
and I
B
(4, 5). These I
B proteins share many properties, including domain structure and selectivity toward NF-
B binding partners. However, significant differences exist between them (69). Degradation of I
B
leads to rapid but transient activation of NF-
B (1012). However, many cellular activities, such as lymphoid cell development, endothelial and brain cell function, as well as various pathological conditions and viral infections, require sustained activation of NF-
B (1325). In each case, I
B
has been shown to be the critical mediator of persistent NF-
B activity. The mechanism underlying this persistently active NF-
B remains unclear.
In an effort to understand the source of differential NF-B activation kinetics, several groups have begun investigating the molecular mechanisms of action of the I
B
and I
B
inhibitor proteins. One clear difference has emerged involving the manner by which these two proteins regulate NF-
B subcellular localization (26). Immunofluorescence studies have revealed that I
B
·NF-
B complexes shuttle between the cytoplasm and nucleus in quiescent cells (2729). In contrast, inactive I
B
·NF-
B complexes remain exclusively in the cell cytoplasm (3032).
A mechanistic explanation for the dynamic shuttling behavior of IB
·NF-
B complexes has been described recently. X-ray structures of the I
B
·NF-
B p50/p65 heterodimer complex revealed that I
B
masks the nuclear localization signal (NLS)1 of the NF-
B p65 subunit but fails to mask the p50 subunit NLS (33, 34). Protease protection assays revealed that one NLS in the I
B
·NF-
B p65 homodimer and I
B
·NF-
B c-Rel homodimer complexes is also exposed to solvent and sensitive to protease cleavage (32).2 Nuclear import of the I
B
·NF-
B p50/p65 heterodimer complex is imparted by the free p50 NLS, whereas the one free p65 subunit NLS is sufficient to convey similar shuttling properties upon the I
B
·NF-
B p65 homodimer complex (32). Active export of the complex from the nucleus relies on nuclear export signals located in the N-terminal signal response region of I
B
and the transactivation domain of the p65 subunit (2729, 35).
In an accompanying study, we describe the x-ray crystal structure of an IB
·NF-
B p65 homodimer complex (see Ref. 43). The structure reveals that, like I
B
·NF-
B complexes, the NLS of one NF-
B p65 subunit (subunit A) is effectively masked by I
B
, whereas the second p65 subunit (subunit B) NLS is largely solvent-exposed. This observation is somewhat unexpected because the second NF-
B subunit NLS is significantly less sensitive to cleavage by proteases when bound to I
B
(32). Therefore, it remains unclear as to how I
B
·NF-
B complexes are retained within the cytoplasm of resting cells.
Recently, a small Ras-like protein was identified from a yeast two-hybrid screen as an IB
C-terminal PEST sequence-interacting factor and inhibitor of NF-
B activation (36). It was suggested that this protein, named
B-Ras, might function by inhibiting I
B
degradation. In the present study, we show that
B-Ras is a critical regulator of I
B
·NF-
B complex subcellular distribution.
B-Ras binds through a unique 47-amino acid insert between ankyrin repeats 3 and 4 of I
B
and partially masks one p65 subunit NLS in vitro and completely masks it in vivo. The conversion of serines in the I
B
PEST sequence to non-phosphorylatable residues further alters the subcellular localization properties of I
B·NF-
B complexes. These data suggest that additional factors may bind
B-Ras and I
B
·NF-
B complexes and sequester them to the cytoplasm of quiescent cells. We propose that regulated association and dissociation of
B-Ras determine the subcellular localization of I
B
·NF-
B complexes.
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EXPERIMENTAL PROCEDURES |
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ImmunofluorescenceCells (with or without LMB treatment) were fixed in 3% paraformaldehyde for 20 min at room temperature and then permeabilized with phosphate-buffered saline buffer containing 0.5% Nonidet P-40 and 0.01% sodium azide (ISB). Blocking was done using 5 mg/ml bovine serum albumin followed by incubation for 30 min with primary antibodies in ISB. Cells were then washed three times with ISB. Fluorescent-tagged secondary antibody was added in ISB at room temperature.
Immunoprecipitation and Western AnalysisCells were washed three times in phosphate-buffered saline buffer. Cytoplasmic extracts were made by lysing cells in 1% Triton X-100, 20 mM Tris-HCl (pH 7.6), 200 mM NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride (lysis buffer). Fifty µg of extract was mixed with protein A-agarose and primary antibodies and incubated at 4 °C overnight. The immunoprecipitates were washed three times in lysis buffer and eluted with SDS-PAGE buffer by heating at 100 °C for 5 min. The supernatant was separated by10% SDS-PAGE. The separated proteins in the gel were transferred to Hybond nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% milk in phosphate-buffered saline with 0.2% Tween and incubated with anti-p65 polyclonal antibody (H-286; Santa Cruz Biotechnology) for 1 h at room temperature. The membrane was washed and incubated with horseradish peroxidase-conjugated anti-rabbit Ig (Santa Cruz Biotechnology). Blots were visualized by use of the ECL reagent kit (Amersham Biosciences).
For in vitro immunoprecipitation experiments, 0.5 µg of NF-B was mixed with 4 µg of I
B in the presence or absence of 4 µg of
B-Ras in a 15 µl binding reaction. The mixture was incubated on ice for 2 h followed by dilution to 100 µl in lysis buffer. Four µl of this diluted complex was used for the immunoprecipitation reaction using 0.1 µg of the anti-p65 NLS monoclonal antibody (a generous gift from Roche Diagnostics). The immunoprecipitates were then loaded on a Western blot, as described above.
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RESULTS |
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To test whether the unique 47-amino acid insertion within the ankyrin repeat domain of IB
plays any role in determining the subcellular localization of I
B
·NF-
B complexes, we deleted this insert from I
B
(residues 152192) and observed the co-localization of this mutant (I
B
-(152192)) with NF-
B p65 homodimer in HeLa cells (Fig. 1A). We observe that both complexes are cytoplasmic in resting cells. However, in the presence of LMB, an inhibitor of nuclear export receptor CRM1, the mutant I
B
-(152192)·NF-
B p65 homodimer complex, but not the wild type complex, is predominantly nuclear (37, 38) (Fig. 1A). Co-immunoprecipitation experiments show that, like the wild type I
B
, mutant I
B
-(152192) is also associated with p65 (Fig. 1C). Our results thus demonstrate that the insert of I
B
plays a role in vivo in I
B
·NF-
B complex cytoplasmic retention.
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We have also tested whether the insert of IB
is responsible for cytosolic retention of the I
B
·NF-
B p50/p65 heterodimer complex (Fig. 1B). As in the case of the I
B
-(152192)·NF-
B p65 homodimer complex, we observe that the I
B
-(152192) protein in complex with NF-
B p50/p65 heterodimer can also enter the nucleus of resting cells. A likely explanation for this phenomenon is that when the insert of I
B
is removed, the second NLS becomes free (or more loosely bound to I
B
), enabling the complex to be actively imported into the nucleus. These results suggest that the p50 subunit in the p50/p65 heterodimer acts similarly to p65 subunit B in the NF-
B p65 homodimer, which possesses an NLS polypeptide that is primarily solvent-exposed.
The p50 Subunit NLS Regulates Nuclear Translocation of IB
·NF-
B p50/p65 Heterodimer ComplexesTo further test whether nuclear translocation of the I
B
-(152192)·NF-
B p50/p65 heterodimer complex is mediated by the free p50 NLS, we performed co-transfection experiments with a p50 subunit that lacks its NLS polypeptide (p50
NLS), p65, and I
B
-(152192) (Fig. 2). We observe that this complex is cytoplasmic in both the absence and presence of LMB, suggesting that only in the I
B
-(152192)·NF-
B p50/p65 heterodimer complex is the p50 NLS free, whereas it remains masked in the wild type I
B
·NF-
B p50/p65 heterodimer complex. These results indicate that one NF-
B NLS and the I
B
insert antagonize one another in directing subcellular localization of all I
B
·NF-
B complexes.
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B-Ras Regulates the p65 Subunit B NLS of the I
B
·NF-
B p65 Homodimer Complex in Vitro and in CellsThe above experiments suggest that the I
B
insert could mask the second (subunit B) NLS of I
B
·NF-
B complexes in resting cells. Direct contact likely requires modification of one or both of these interacting elements. The small GTPase
B-Ras was recently shown to be involved in regulation of I
B degradation (36). To determine whether
B-Ras might be involved in masking the NF-
B subunit B NLS, we have performed immunoprecipitation experiments with a monoclonal antibody directed against the p65 subunit NLS used to precipitate free p65, I
B
·NF-
B p65 homodimer binary complexes, and
BRas·I
B
·NF-
B p65 homodimer ternary complexes. In parallel experiments, I
B
·NF-
B p65 homodimer complexes were probed in the presence and absence of
B-Ras. The p65 from the immunoprecipitated complexes was then visualized by anti-p65 polyclonal antibody (Fig. 3A).
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We observe that both IB
·NF-
B p65 homodimer and I
B
·NF-
B p65 homodimer complexes can be precipitated by the anti-p65 NLS antibody (Fig. 3A, lanes 2 and 7, respectively). Moreover, we observe that the addition of
B-Ras inhibits the ability of this antibody to bind to I
B
·NF-
B p65 homodimer complexes but not I
B
·NF-
B p65 homodimer (Fig. 3A, lanes 3 and 8). To determine whether the insert of I
B
plays a role in NLS masking by
B-Ras, we performed similar immunoprecipitation experiments with I
B
-(152192)·NF-
B p65 homodimer complexes in the presence and absence of
BRas. We observe that the addition of
B-Ras fails to enhance NLS masking of I
B
-(152192)·NF-
B p65 homodimer complexes (Fig. 3A, lanes 4 and 5). To verify specificity of the anti-p65 NLS monoclonal antibody, we tested it against the I
B
·NF-
B p50/p65 complex. The anti-p65 NLS antibody fails to recognize and precipitate p65 in these complexes. This results from the nearly complete masking of the p65 subunit A NLS polypeptide by I
B
as observed in the I
B
·NF-
B p65 complex crystal structure (Fig. 3A, lanes 9 and 10).
To further investigate whether B-Ras mediates blockade of the p65 NLS in cells, we next co-transfected 293 cells with HA-tagged p65 together with I
B
, I
B
, or I
B
-(152192). We first confirmed that p65 associates with all three I
B proteins by co-immunoprecipitation using an antibody against the transcriptional activation domain of p65 (Fig. 3B). When the anti-p65 NLS antibody was used for immunoprecipitation, we observed that only I
B
and I
B
-(152192) were pulled down, but not wild type I
B
. These experiments suggest that in I
B
·NF-
B p65 homodimer and I
B
-(152192) complexes, at least one p65 NLS is free, whereas both p65 subunit NLS polypeptides are blocked in the I
B
·NF-
B p65 homodimer complex. We conclude that the I
B
insert is required for masking of the NF-
B subunit B NLS and cytoplasmic retention of I
B
·NF-
B complexes.
B-Ras Directly Interacts with the I
B
InsertTo identify the role of the I
B
insert in
B-Ras binding, we have tested the binding of co-transfected
B-Ras and I
B
in cells. COS cells were co-transfected with plasmids expressing both wild type I
B
and
B-Ras. We show that
B-Ras can be precipitated only when wild type I
B
is present (Fig. 4). However, the association is not observed in cells expressing I
B
-(152192) and
B-Ras. Therefore, the I
B
insert is required for association with
B-Ras. Taken together, these experiments suggest that the small GTPase
B-Ras may play an in vivo role in blocking the subunit B NLS of dimeric NF-
B by binding directly to the unique insert region of the I
B
inhibitor protein.
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The IB
PEST Functions in Cytoplasmic Retention of I
B
·NF-
B ComplexesBecause
B-Ras was identified as an I
B-interacting protein in a yeast two-hybrid screen with the I
B
C-terminal PEST used as the bait, it is important to evaluate the role of the I
B
PEST in NF-
B cytoplasmic sequestration. To test whether phosphorylation of the I
B
PEST plays any role in cytoplasmic retention of NF-
B, we mutated four phosphorylatable serines (Ser312, Ser313, Ser314, and Ser316) within this region to either alanine (PEST-Ala) or aspartic acid (PEST-Asp). Two serine residues in this region have been shown to be phosphorylated by casein kinase II, and phosphorylation at these sites is important for NF-
B binding in cells (39, 40). However, we have previously shown that phosphorylation of these serines or their conversion to phosphomimetic glutamic acid residues does not alter the stability of I
B
·NF-
B binary complexes in vitro (32).
If constitutive phosphorylation of the PEST is important for stable complex formation, then substitution of these residues to alanine could prevent cytoplasmic retention of IB
·NF-
B complexes. To test this possibility, we co-transfected HeLa cells with I
B
-PEST-Ala and p65 and observed co-localization of both proteins in the cytoplasm. However, when these cells were treated with LMB followed by immunostaining, we observed that, in a large fraction of cells expressing both of these proteins, the complex localized to the nucleus (Fig. 5A). In contrast, when the cells were co-transfected with p65 and I
B
-PEST-Asp, the complex was cytoplasmic with or without LMB treatment (Fig. 5B). This property of the I
B
-PEST-Asp mutant complex is identical to that of the wild type complex. We conclude that I
B
PEST phosphorylation is critical for cytoplasmic sequestration of I
B
·NF-
B complexes.
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We further created a mutant of IB
in which the insert is deleted within the background of phosphomimetic I
B
-PESTAsp sequence ((I
B
-(152192)-PEST-Asp). When HeLa cells were co-transfected with this mutant and wild type p65, we observed that the complex shuttled between the cytoplasm and nucleus (Fig. 5C). This profile is identical to that observed in the I
B
-(152192)·NF-
B p65 homodimer complex.
It is not immediately clear to us what role a phosphorylated IB
PEST plays in sequestering the I
B
·NF-
B complexes to the cytosol. It is possible that
B-Ras interacts with both the I
B
insert and phosphorylated PEST. It is also possible that the interaction between the I
B
PEST and
B-Ras is indirect, mediated by another bridging factor.
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DISCUSSION |
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We propose the hypothesis that B-Ras may act in concert with the I
B
insert to sequester I
B
·NF-
B complexes to the cytoplasm. Indeed, we show that the small GTPase
B-Ras is able to reduce access to the NF-
B p65 homodimer subunit B NLS in complexes between p65 and I
B
. We further present evidence that binding of
B-Ras to I
B
requires the insert within the ankyrin repeat domain of I
B
. Because this insert is unique to I
B
, these results serve to explain the specificity of
B-Ras for the I
B
·NF-
B complex.
There are several published observations which suggest that phosphorylation of the IB
PEST might play an important role in regulation of NF-
B by I
B
(7, 9, 22). The most important of these is the identification of
B-Ras through the utilization of the I
B
PEST sequence as bait in a yeast two-hybrid screen (36). Also, it has been shown that PEST phosphorylation is important for stable complex formation with NF-
B (39). We show here that PEST phosphorylation of I
B
functions in cytoplasmic retention of I
B
·NF-
B complexes. Although we do not observe a clear need for PEST phosphorylation in the interaction between I
B
and NF-
B or
B-Ras·I
B
·NF-
B complex formation, our experiments do suggest that PEST phosphorylation cooperates with
B-Ras binding and the I
B
insert in regulating cytosolic retention of I
B
·NF-
B complexes. We propose that this cooperativity might be mediated through other cellular factors or through further post-translational modification elsewhere in the complex. We favor the first possibility in light of our recent findings that I
B
is present in the cytoplasm of quiescent cells as large complexes, which include NF-
B and
B-Ras as well as other as-yet-unknown factors.3 Working together, the I
B
insert and the PEST, one NF-
B NLS, and
B-Ras represent a molecular mechanism for switching between nuclear and cytoplasmic I
B
·NF-
B complexes (Fig. 6).
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Our experiments help to explain the observation that hypophosphorylated IB
·NF-
B complexes can localize into the nucleus (7). We suggest that the hypophosphorylated form of I
B
is functionally equivalent to the I
B
-PEST-Ala protein construct used in this study.
Finally, our results serve to explain one long-standing puzzle. It has long been thought that IB
binds NF-
B dimers with a significantly higher affinity than does I
B
. This conclusion was drawn based on the respective abilities of I
B
and I
B
to inhibit NF-
B DNA binding. We have shown previously that compared with I
B
, I
B
binds NF-
B p50/p65 heterodimer and p65 homodimer with only slightly weaker affinity (32, 41, 42). In light of the I
B
·NF-
B p65 homodimer complex crystal structure and the biochemical evidence surrounding
B-Ras binding to I
B
·NF-
B complexes, we suggest that the in vivo stabilities of I
B
·NF-
B complexes are nearly equivalent to those of I
B
·NF-
B complexes. This would explain why both complexes are present in almost all cells in almost equal amounts (5).
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, MC 0359, La Jolla, CA 92093-0359. Tel.: 858-822-0469; Fax: 858-534-7042; E-mail: gghosh{at}chem.ucsd.edu.
1 The abbreviations used are: NLS, nuclear localization signal; PEST, proline, glutamic acid, serine, and threonine-rich sequence; LMB, leptomycin B; HA, hemagglutinin.
2 C. Phelps, unpublished data.
3 Y. Chen, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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