Received 10 October 1997/Returned for modification 11 November
1997/Accepted 20 February 1998
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INTRODUCTION |
Directional transport of proteins
through the nuclear pore complex provides a powerful regulatory
mechanism for controlling gene expression, as illustrated by the
NF-
B/Rel family of transcription factors (for reviews, see
references 3, 5, and 30).
Association of the inhibitor of B
(IB) protein with dimeric
NF-B/Rel complexes containing either c-Rel or p65 (RelA) results in
the sequestration of the Rel dimer in the cytoplasm, through masking of
the nuclear localization sequences (NLSs) within Rel proteins (4,
20, 26, 41, 65, 77). In response to a variety of extracellular
stimuli, including proinflammatory cytokines, viral infection,
bacterial lipopolysaccharide, phorbol esters, oxidants, and UV light,
IB becomes inducibly phosphorylated at serine residues 32 and 36 (9, 10, 15, 72). The recently identified protein kinase
complex, IKK (IB kinase), phosphorylates IB at these
N-terminal serine residues and targets IB for ubiquitin-dependent
degradation by the 26S proteasome (16, 48, 60, 63, 76).
Degradation of IB enables the free Rel dimer to translocate to
the nucleus and activate B-dependent gene expression. One of the
target genes of Rel proteins is the IB gene itself, resulting in
the rapid induction of newly synthesized IB protein (1, 42,
45, 70).
Several lines of evidence have led to the suggestion that newly
synthesized IB can function in the nucleus as a postinduction repressor of B-dependent gene expression. First, ectopically overexpressed IB is readily detected in the nucleus, consistent with the suggestion that IB has a nuclear function (13, 50, 77). Second, following cytokine stimulation of cells, a
significant fraction of newly synthesized endogenous IB appears
transiently in the nucleus (1). Nuclear expression of
IB correlates with inhibition of NF-B-dependent transcription
and disappearance of NF-B from the nucleus (1). Third,
brief stimulation of wild-type fibroblasts with tumor necrosis factor
alpha (TNF-) results in a transient activation of nuclear NF-B
(6). In contrast, brief stimulation of IB null-mutant
fibroblasts with TNF- results in a sustained level of nuclear
NF-B (6). Finally, IB can inhibit NF-B-dependent
transcription in the nucleus in vivo and can remove Rel proteins from
functional preinitiation complexes in vitro (73). Taken
together, these results are consistent with a model in which newly
synthesized IB proteins can enter the nucleus, displace dimeric
Rel proteins from DNA, and export Rel proteins from the nucleus to the
cytoplasm. Implicit in this model is the ability of both Rel and
IB proteins to enter the nucleus. In contrast, this model
postulates that the Rel-IB complex is exported from the nucleus
and is efficiently retained in the cytoplasm.
Nuclear import of Rel proteins is accomplished by virtue of an NLS
located at the C-terminus of the Rel homology domain. The Rel-derived
NLS is characterized by a short stretch of basic amino acids that
resembles a classical NLS typified by the NLS of the simian virus 40 (SV40) large T protein (4, 26, 29, 31, 43, 64, 77). Nuclear
import of proteins bearing such classical NLSs is accomplished by a
soluble heterodimeric protein complex consisting of a 60-kDa protein,
importin-, and a 90-kDa protein, importin-
(12, 19, 33, 40,
51, 52, 59, 74). Importin- binds to NLS-containing proteins
and, through interaction with importin-, mediates the docking of the
NLS-containing protein to nucleoporins and the subsequent translocation
of the NLS-containing protein to the nucleus (12, 19, 33-35, 37,
40, 51, 52, 59, 61, 74). Although a direct involvement of the
importin--importin- (importin--) receptor in the nuclear
import of Rel proteins has not been demonstrated, the presence of a
classical NLS within Rel proteins suggests that nuclear import of Rel
proteins is mediated by an importin---dependent pathway.
Similar to nuclear import, nuclear protein export is also a
sequence-dependent receptor-mediated process. One class of nuclear export sequences (NESs) is characterized by a cluster of five leucine
or isoleucine residues, each separated by one or two amino acid
residues (for reviews, see references 36 and
54). NESs from several proteins, including the Rev
protein of human immunodeficiency virus and the protein inhibitor of
cyclic AMP-dependent protein kinase (21, 75), have been
described. IB contains a sequence located between the ankyrin
repeat domain (ARD) and the C-terminal PEST domain which resembles the
previously described NESs in Rev and protein kinase inhibitor. The
IB-derived NES can functionally substitute for the NES in Rev and
is required for IB-mediated nuclear export of NF-B (2,
24). IB-mediated nuclear export of Rel proteins is likely
mediated by exportin 1 (CRM1), a recently identified
importin--related protein that mediates the nuclear export of
NES-containing proteins (22, 25, 56, 69).
The mechanism by which IB is able to localize to the nucleus is
not known. IB does not contain a region of basic residues that
resembles previously characterized NLSs. Thus, it has been proposed
that the small size of IB might allow passive, NLS-independent accumulation of IB in the nucleus (1, 77). We now
demonstrate that nuclear localization of IB is mediated by a
novel nuclear import sequence within the second ankyrin repeat of
IB. A region encompassing the second ankyrin repeat from IB
can functionally substitute for the classical NLS in nucleoplasmin.
ARDs from other proteins, including 53BP2 and GABP, are also able to
function as nuclear import sequences. We propose that the IB
ankyrin repeats define a novel class of cis-acting nuclear
import sequences.
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MATERIALS AND METHODS |
Construction of recombinant DNA molecules.
The construction
of recombinant DNA molecules was performed according to standard
techniques (66). Mutant p40 and MAD3 cDNAs were generated
from the respective cDNAs encoding either the wild-type or the
epitope-tagged proteins from phagemid single-strand DNA (66). The presence of each mutation within the respective
cDNAs was confirmed by nucleotide sequence analysis. Typically, two independent isolates of each mutant p40 or MAD3 gene were separately subcloned into expression vectors and independently assayed for function. In no cases were any differences found between independent isolates of the same mutation. The p40 and MAD3 genes were expressed in
chicken embryo fibroblasts (CEF) by using a spleen necrosis virus
(SNV)-driven retroviral vector derived from pJD214 (17) and
in COS-1 cells by using either the SNV-driven vector or a cytomegalovirus (CMV)-derived vector (9). The epitope-tagged p40 protein (LBD-p40) contains a C-terminal 18-amino-acid peptide derived from the ligand binding domain (LBD) of the platelet-derived growth factor. The LBD epitope tag consists of the sequence
EVIVVPHSLPFML. A plasmid containing a segment of DNA encoding the LBD
epitope tag and affinity-purified antipeptide sera against the LBD
epitope tag were provided by Dan Donoghue (University of California). The epitope-tagged MAD3 protein (myc-MAD3) contains a C-terminal 11-amino-acid peptide derived from the c-Myc protein. The c-Myc epitope
tag consists of the sequence MEQKLISEEDL. A modified pcDNA1 plasmid
(Invitrogen) containing a segment of DNA encoding the myc epitope tag
was provided by Gideon Dreyfuss (University of Pennsylvania). The
epitope tags did not significantly alter the localization or the
biochemical properties of the respective IB proteins.
The full-length c-Rel gene (11) was subcloned into pCMV4 as
an XbaI fragment. The CMV-derived expression vectors for p65 (RelA) and IB were obtained from Dean Ballard (Vanderbilt
University). The cDNA encoding 53BP2 was obtained from Louie Naumovski
(Stanford University). The cDNA encoding GABP was obtained from Mark
Martin (University of Missouri). The cDNA encoding Notch1 was obtained from Anthony Capobianco (University of California). The cDNAs encoding
myc-tagged nucleoplasmin core (NPc) and myc-tagged NPc-M9 were obtained
from Gideon Dreyfuss (University of Pennsylvania). The cDNA encoding
chicken muscle pyruvate kinase (PK) was obtained from Dan Donoghue. To
facilitate construction of the NPc fusion proteins, a PmlI
restriction site and a termination codon were introduced after the
nucleoplasmin open reading frame. All NPc fusion genes were constructed
by the insertion of blunt-ended fragments into the PmlI
restriction site. Some of the inserts were obtained by PCR prior to
cloning into the NPc expression vector, while other inserts were
isolated out of their respective cDNA clones by the use of specific
restriction enzyme sites. The complete nucleotide sequence of the
PCR-derived inserts was determined to confirm faithful amplification of
the cDNA. For construction of the myc-tagged PK fusion proteins, PCR
amplification of a cDNA encoding PK was used to introduce a
BamHI site upstream of codon 17 and a PmlI site
downstream of codon 524. This fragment was subcloned into the
appropriate myc-tagged expression vectors. Details of all plasmid
constructions and primer sequences are available upon request.
Cell culture and transfection.
CEF were obtained from Spafas
and grown in M199 containing 10% tryptose phosphate and 10% fetal
calf serum (FCS). DNA transfections into CEF were performed with
calcium phosphate coprecipitates as previously described
(65). The biochemical properties of the Rel or p40 proteins
were typically analyzed 4 to 5 days after transfection of CEF with the
appropriate plasmids. Monkey COS-1 cells were grown in Dulbecco's
modified Eagle's medium (DMEM) containing 10% FCS. Wild-type
(WT+/+) and mutant 3T3 fibroblasts, and primary mouse
embryo fibroblasts (MEF) lacking both the c-Rel and the p65 genes
(c-Rel/p65
/) were prepared in David Baltimore's
laboratory (California Institute of Technology) and were grown in DMEM
containing 10% donor calf serum (7, 67). Transfections into
COS-1 cells were performed on 35-mm-diameter plates by using
Lipofectamine with a total of 2 µg of plasmid DNA in accordance with
the directions from the manufacturer (GIBCO BRL). Transfections into
3T3 cells and into MEF were performed on 35-mm-diameter plates by using
LipofectaminePLUS with a total of 1 µg of plasmid DNA in accordance
with the directions from the manufacturer (GIBCO BRL). The cellular
localization and the biochemical properties of the ectopically
expressed proteins were typically analyzed 36 to 48 h after
transfection of the COS-1 cells, 3T3 fibroblasts, or MEF with the
appropriate plasmids.
Antibodies.
The following primary antibodies for detection
of the respective ectopically expressed proteins were used: rabbit
polyclonal anti-p40 (R1807), rabbit polyclonal anti-MAD3 (Santa Cruz
Biotechnology), rabbit affinity-purified anti-LBD (Dan Donoghue), mouse
monoclonal anti-myc (Sigma), rabbit polyclonal anti--galactosidase
(Chemicon International), rabbit polyclonal anti-Rel (28),
mouse monoclonal anti-c-Rel (HY87) (Henry R. Bose, Jr., University of
Texas), rabbit polyclonal anti-p65 (Santa Cruz Biotechnology), and
mouse monoclonal anti-p65 (Boehringer Mannheim). The appropriate
anti-rabbit or anti-mouse fluorescein isothiocyanate-conjugated
secondary antibody (Jackson Labs) or anti-rabbit Cy5-conjugated
secondary antibody (Jackson Labs) was used for detection of the
ectopically expressed proteins by indirect immunofluorescence. The
appropriate anti-rabbit (Amersham) or anti-mouse (New England Biolabs)
immunoglobulin G (IgG) conjugated to horseradish peroxidase was used in
conjunction with the enhanced chemiluminescence system (ECL; Amersham)
for detection of the ectopically expressed proteins by immunoblot analysis.
Indirect immunofluorescence.
Indirect immunofluorescence
assays using CEF, COS-1 cells, 3T3 fibroblasts, or MEF were conducted
on coverslips with the appropriate antisera as previously described
(28). The coverslips were mounted onto glass slides with
Mowiol containing 2.5% DABCO (Sigma).
Biochemical experiments.
Cell lysates for the
coimmunoprecipitation analysis were prepared in ELB (50 mM Tris-HCl
[pH 7.9], 250 mM NaCl, 0.1% Triton X-100, 5 mM EDTA, and 1 mM
dithiothreitol). Protease inhibitors and phosphatase inhibitors were
routinely included in the lysis buffers. The protease inhibitors used
were 1 mM phenylmethylsulfonyl fluoride; antipain, aprotinin,
leupeptin, and soybean trypsin-chymotrypsin inhibitor (5 µg/ml each);
and pepstatin (0.5 µg/ml). The phosphatase inhibitors used were 0.4 mM sodium orthovanadate and 1 mM sodium fluoride. Equivalent aliquots
of ELB cell lysates were used for coimmunoprecipitation analysis.
Immunoprecipitation of LBD-tagged p40 proteins was performed with 3 µl of affinity-purified anti-LBD serum per sample. The
immunoprecipitations were conducted in antibody excess to ensure
quantitative precipitation of the LBD-tagged p40 proteins. DNA-binding
of Rel proteins was determined by electrophoretic mobility shift assay
as previously described (65).
IB localization and expression following TNF- and CHX
treatment.
COS-1 cells (on 35-mm-diameter plates) were
cotransfected with 0.5 µg of a CMV-derived -galactosidase
expression vector and 1.5 µg of either the myc-MAD3 or the
myc-MAD3-110A3 expression vector. At 36 h posttransfection, the
transfected COS-1 cells were either refed with complete medium (DMEM
containing 10% FCS) or were cultured in complete medium containing
cycloheximide (CHX) (100 µg/ml; Sigma) and TNF- (10 ng/ml;
Chemicon International) for 4 h. The TNF- and CHX were
subsequently removed, and the transfected cells were washed three times
with DMEM and refed with complete medium. At 0, 15, 30, or 60 min
following removal of the TNF- and CHX, the chase samples were fixed
for double-label indirect immunofluorescence. ELB cell lysates of the
untreated and the TNF-- and CHX-treated samples were collected in
parallel for determination of the protein levels of the ectopically
expressed proteins.
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RESULTS |
Nuclear localization of IB requires the integrity of a
hydrophobic cluster of amino acids located within the second ankyrin
repeat.
Both the mammalian (MAD3) and the avian (p40) IB
proteins contain two clusters of hydrophobic residues that resemble
previously described NESs (14, 21, 24, 39, 75). One cluster
of hydrophobic residues (amino acids 114 to 124 in p40 [Fig.
1]) is located within the second ankyrin
repeat, while a C-terminal cluster of hydrophobic residues (amino acids
273 to 284 in p40 [Fig. 1]) is located between the ARD and the acidic
and serine-rich PEST domain. The hydrophobic cluster within the second
ankyrin repeat is highly conserved among other IB proteins, while
the C-terminal hydrophobic amino acids are unique to the IB
proteins (14, 27, 39, 46, 47, 55, 71). The C-terminal
cluster of hydrophobic amino acids of the mammalian IB protein is
required for nuclear export of NF-B following coinjection of in
vitro-synthesized IB and NF-B into Xenopus oocyte
nuclei (2). However, the role of these NES-like sequences in
the distribution of IB between the nucleus and the cytoplasm has
not been established.
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FIG. 1.
Domain organization of IB. The avian (p40) and
mammalian (MAD3) IB proteins are represented by long rectangular
boxes. The numbers to the left of each box indicate the first amino
acid of each protein, and the numbers to the right of each box indicate
the total number of amino acids in each protein. The IB proteins
contain an N-terminal regulatory domain, a central domain containing
five ankyrin repeats, and a C-terminal acidic and serine-rich (PEST)
domain. The sites of N-terminal cytokine-inducible serine
phosphorylation and the sites of constitutive serine phosphorylation
within the C-terminal PEST domain of IB are indicated by the
circled P's. The amino acid sequences of two clusters of hydrophobic
residues are indicated in the single-letter code below the rectangle
representing each IB protein. The residues relevant to the
present work are in boldface type, and the mutations introduced into
the IB proteins are indicated. The scale of this line drawing is
indicated by the length of the bar at the bottom of the figure.
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Mutant IB proteins containing amino acid substitutions within
either the region between residues 114 and 124 or the region between
residues 273 and 284 were constructed (Fig. 1). Expression vectors
coding for the wild-type and mutant IB proteins were transfected
into CEF and into COS-1 cells. The cellular distribution of the
IB proteins was determined by indirect immunofluorescence. As
previously reported (13, 50, 77), the wild-type p40 protein was predominantly nuclear (Fig. 2A and E;
Table 1) while the wild-type MAD3 protein
was distributed throughout both the nucleus and the cytoplasm (Fig. 2C
and G; Table 1) in both CEF and COS-1 cells. Alanine substitution of
leucine residues 119 and 121 and of isoleucine residue 124 in the
second ankyrin repeat of p40 (p40-114A3) or alanine substitution of the
corresponding hydrophobic residues in MAD3 (MAD3-110A3 [Fig. 1])
significantly reduced nuclear accumulation of the IB proteins
(Fig. 2B, D, F, and H; Table 1). Fusion of the classical NLS derived
from the SV40 large T protein onto the cytoplasmic p40-114A3 protein
(p40-114A3-NLS) restored the nuclear localization of the mutant
p40-114A3 protein (Table 1).
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FIG. 2.
Cellular localization of wild-type and mutant IB
proteins. CEF were transfected with SNV-derived retroviral vectors (A
to D) and COS-1 cells were transfected with CMV-derived expression
vectors (E to H) encoding either wild-type p40 (A and E), p40-114A3 (B
and F), MAD3 (C and G), or MAD3-110A3 (D and H), or COS-1 cells were
transfected with SNV-derived expression vectors encoding either
p40- AR2 (I), p40-AR2+3 (J), p40-AR4 (K), or p40-AR5 (L).
The p40-114A3 protein contains alanine substitutions for leucine 119, leucine 121, and isoleucine 124 in p40. The MAD3-110A3 protein contains
alanine substitutions for leucine 115, leucine 117, and isoleucine 120 in MAD3. The p40-AR2 protein contains a deletion of amino acids 98 to 142, encompassing the second ankyrin repeat in p40. The p40-AR2+3
protein contains a deletion of amino acids 117 to 188, encompassing the
second and third ankyrin repeats in p40. The p40-AR4 protein
contains a deletion of amino acids 189 to 222, encompassing the fourth
ankyrin repeat in p40. The p40-AR5 protein contains a deletion of
amino acids 223 to 256, encompassing the fifth ankyrin repeat in p40.
The cellular localization of the proteins in transfected cells was
determined by indirect immunofluorescence with anti-p40 or anti-MAD3
serum. The cells shown are representative of more than 200 cells that
were positive for the expression of the indicated proteins (see Table 1
for quantitation).
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Similar to that in the mutant p40-114A3 protein, alanine substitution
of four hydrophobic residues within the p40 region between residues 114 and 124 (p40-114A4 [Fig. 1]) resulted in a mutant p40 protein that
was predominantly cytoplasmic in COS-1 cells (Table 1). In contrast,
neither deletion of the C-terminal 51 amino acids including the region
between residues 273 and 284 (p40-267) nor alanine substitution of
four leucine residues within the region between residues 273 and 284 of
p40 (p40-273A4 [Fig. 1]) significantly altered the cellular
distribution of p40 in COS-1 cells (Table 1 and data not shown).
As alanine substitution of hydrophobic residues within the second
ankyrin repeat of IB disrupted nuclear localization of IB,
we asked whether deletion of the second ankyrin repeat would similarly
disrupt nuclear localization of IB. Deletion of the second
ankyrin repeat of p40 (p40-AR2) or deletion of both the second and
the third ankyrin repeats of p40 (p40-AR2+3) resulted in mutant p40
proteins that were predominantly cytoplasmic in COS-1 cells (Fig. 2I
and J; Table 1). In contrast, deletion of the fourth (p40-AR4) or
the fifth (p40-AR5) ankyrin repeats of p40 had only a modest effect
on the relocalization of p40 from the nucleus to the cytoplasm in COS-1
cells (Fig. 2K and L; Table 1).
The wild-type and mutant IB proteins were expressed at equivalent
levels, as determined by anti-p40 or anti-MAD3 immunoblot analysis
(data not shown, but see Fig. 9). Furthermore, the turnover rates of
the wild-type IB and the mutant IB-A3 proteins were equivalent, as determined by pulse-chase analysis in CEF and in COS-1
cells (data not shown). Thus, the inability of the IB-A3 proteins
to accumulate in the nucleus is not due to increased turnover of these
mutant proteins. Rather, our results indicate that nuclear localization
of IB is sequence dependent and requires the integrity of
hydrophobic residues within the second ankyrin repeat. In contrast to
previous suggestions (1, 77), nuclear localization of
IB does not occur by passive diffusion through the nuclear pore.
Nuclear localization of IB is independent of p50, p52, p65
(RelA), or c-Rel.
To determine whether nuclear localization of
wild-type IB protein is dependent on the presence of endogenous
p50, p52, p65 (RelA), or c-Rel, we determined the cellular distribution
of wild-type and mutant MAD3 proteins in fibroblasts which lack these
Rel proteins. The ectopically expressed wild-type MAD3 protein was
distributed throughout both the nucleus and the cytoplasm in
WT+/+ and in fibroblasts lacking both copies of the
NFB1, p65 (RelA), NFB1 and NFB2, NFB1 and p65 genes, and
c-Rel and p65 (RelA) (p50/, p65/,
p50/p52/, p50/p65/, and
c-Rel/p65/, respectively) (Fig.
3A to F, respectively) (quantified in
Table 2). Furthermore, the mutant
MAD3-110A3 protein remained predominantly cytoplasmic when ectopically
expressed in these cell types (Table 2). Therefore, the nuclear
localization of ectopically expressed IB does not require
expression of these endogenous Rel proteins.
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FIG. 3.
Cellular distribution of IB is independent of the
p50, p52, p65 (RelA), and c-Rel proteins. WT+/+ (A),
p50/ (B), p65/ (C),
p50/p52/ (D), and p50/p65/ (E) mouse
3T3 fibroblasts or c-Rel/p65/ primary MEF (F) were
transfected with CMV-derived expression vectors coding for wild-type
MAD3. The cellular localization of the ectopically expressed MAD3
protein was determined by indirect immunofluorescence with anti-MAD3
serum. The results shown are representative of at least 100 cells that
were positive for expression of the wild-type MAD3 protein (see Table 2
for quantitation).
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Nuclear localization of newly synthesized IB requires the
integrity of hydrophobic residues within the second ankyrin
repeat.
It has previously been demonstrated that upon cytokine
stimulation of cells, a significant fraction of newly
synthesized endogenous IB appears transiently in the
nucleus (1). To determine whether nuclear localization of
newly synthesized IB requires the integrity of the second ankyrin
repeat, COS-1 cells ectopically expressing either a wild-type
epitope-tagged MAD3 protein (myc-MAD3) or a mutant epitope-tagged
MAD3-110A3 protein (myc-MAD3-110A3) were treated with TNF- for
4 h in the presence of CHX and the cellular distribution and
expression levels of the newly synthesized MAD3 proteins were analyzed
at successive time points following removal of the TNF- and CHX
(Fig. 4; Table
3). An expression vector coding for
-galactosidase was cotransfected with the respective MAD3 expression
vectors to control for transfection efficiency. TNF- treatment of
COS-1 cells expressing either wild-type myc-MAD3 or mutant
myc-MAD3-110A3 resulted in a significant decline in the abundance of
both myc-MAD3 (Fig. 4, compare lanes 1 and 2) and myc-MAD3-110A3 (Fig.
4, compare lanes 6 and 7). Within 30 min following removal of the
TNF- and CHX, the expression levels of myc-MAD3 (Fig. 4, compare
lanes 1 and 4) and myc-MAD3-110A3 (Fig. 4, compare lanes 6 and 9) were
restored to nearly untreated levels as a result of new synthesis of the
respective IB proteins. The newly synthesized myc-MAD3 could
readily be detected in the nucleus of COS-1 cells following removal of
the TNF- and CHX (Fig. 4E; Table 3). In contrast, the newly
synthesized mutant myc-MAD3-110A3 protein did not accumulate in the
nucleus but rather was detected primarily in the cytoplasm of COS-1
cells following removal of the TNF- and CHX (Fig. 4G; Table 3).
Therefore, nuclear localization of newly synthesized IB following
cytokine stimulation requires the integrity of hydrophobic residues
within the second ankyrin repeat.
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FIG. 4.
Nuclear localization of newly synthesized IB
requires the integrity of the second ankyrin repeat. (A to H) COS-1
cells were cotransfected with CMV-derived expression vectors encoding
-galactosidase and either epitope-tagged MAD3 (myc-MAD3) or
epitope-tagged MAD3-110A3 (myc-MAD3-110A3). At 36 h
posttransfection, the transfected cells were either refed with complete
medium (A to D), or were cultured in complete medium containing TNF-
(10 ng/ml) and CHX (100 µg/ml) for 4 h. The TNF- and CHX were
subsequently removed, and the transfected cells were chased in complete
medium for 0, 15, 30, or 60 min. The localization of the
-galactosidase protein (B, D, F, and H) and either the myc-MAD3 (A
and E) or the myc-MAD3-110A3 (C and G) protein was determined by
anti--galactosidase (-gal) and anti-myc (-myc) double-label
indirect immunofluorescence, as indicated. The cellular localization of
the ectopically expressed proteins at 30 min posttreatment is shown (E
to H). The cells shown are representative of more than 25 cells that
were positive for expression of both -galactosidase and the
respective myc-MAD3 protein (see Table 3 for quantitation). (I) For
determination of the protein levels of the ectopically expressed
proteins, cell lysates of the untreated and the TNF-- and
CHX-treated samples were collected in parallel. Equivalent amounts of
each cell lysate were subjected to immunoblot analysis, and the levels
of the ectopically expressed proteins from untreated samples (lanes 1 and 6) or from samples treated with TNF- and CHX for 4 h and
subsequently chased in complete medium for 0 (lanes 2 and 7), 15 (lanes
3 and 8), 30 (lanes 4 and 9), or 60 (lanes 5 and 10) min were
determined by anti-MAD3 and anti--galactosidase immunoblots, as
indicated. The myc-MAD3 and -galactosidase proteins are indicated by
arrows. Only the relevant portions of each immunoblot are shown.
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The second ankyrin repeat in IB functions as a discrete
nuclear import sequence.
To determine if IB contains a
nuclear import sequence that can functionally substitute for a
classical NLS, the IB ARD was fused onto NPc, and the cellular
distribution of the NPc-ARD fusion protein was determined by indirect
immunofluorescence in COS-1 cells. Nucleoplasmin is normally a nuclear
protein (62), and deletion of its C-terminal bipartite NLS
prevents the nuclear localization of NPc (Fig.
5 and 6A)
(49). Fusion of the p40 ARD onto NPc (NPc-p40-ARD)
relocalized NPc to the nucleus (Fig. 5 and 6B). In contrast, the p40
ARD containing the A3 mutation (NPc-p40-ARD-114A3) was not able to
efficiently relocalize NPc to the nucleus (Fig. 5 and 6C). Similar to
the intact ARD of p40, fusion of the second ankyrin repeat of p40 onto
NPc (NPc-p40-AR2) also efficiently relocalized NPc to the nucleus
(Fig. 5 and 6D), while the A3 mutation (NPc-p40-AR2-114A3) markedly
reduced the ability of the second ankyrin repeat to relocalize NPc to
the nucleus (Fig. 5 and 6E). The second ankyrin repeat of p40 was as
efficient as the M9 nuclear import sequence for relocalization of NPc
to the nucleus (Fig. 5 and 6F). Thus, the second ankyrin repeat of
IB contains a nuclear import signal that can functionally substitute for the NLS in nucleoplasmin.
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FIG. 5.
Nuclear import function of the second ankyrin repeat
from IB proteins. Fusion proteins between NPc and either the avian
IB protein (p40), the mammalian IB protein, the mammalian
Bcl-3 protein, or the M9 nuclear import signal from hnRNP A1 are
indicated (colons show fusions). The NPc protein comprises amino acids
2 through 150 of nucleoplasmin and contains an N-terminal epitope tag
derived from the c-Myc protein. The ARD from p40, the second ankyrin
repeat from p40 (p40-AR2), the second ankyrin repeat from IB
(IB-AR2), the second ankyrin repeat from Bcl-3 (Bcl-3-AR2), or
the M9 nuclear import signal was fused onto the C terminus of NPc. The
following NPc-p40 fusion proteins were constructed with the indicated
p40-derived amino acids (in parentheses): p40-ARD (49 to 272), p40-AR2
(103 to 149), p40-AR2-N/N/C (103 to 138),
p40-AR2-N/C/C (117 to 149), and p40-AR2-N (113 to 130). The
IB-derived amino acids used to construct the NPc-IB-AR2
fusion protein were 83 to 128. The Bcl-3-derived amino acids used to
construct the NPc-Bcl-3-AR2 fusion protein were 160 to 206. The hnRNP
A1-derived amino acids used to construct the NPc-M9 fusion protein were
268 to 305. The cellular localization of each fusion protein was
determined in COS-1 cells with anti-myc IgG. Cells that were positive
for expression of the indicated fusion proteins were classified as
having predominantly nuclear staining (N), staining that was
distributed equally between the nucleus and the cytoplasm (N/C), or
staining that was predominantly cytoplasmic (C). At least 200 cells
that were positive for expression of each fusion protein were scored,
and the percentage of cells in each category is given.
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FIG. 6.
Nuclear localization of NPc upon fusion of the second
ankyrin repeat of IB. COS-1 cells were transfected with CMV-based
expression vectors encoding NPc (A), NPc-p40-ARD (B),
NPc-p40-ARD-114A3 (C), NPc-p40-AR2 (D), NPc-p40-AR2-114A3 (E), or
NPc-M9 (F). The NPc fusion proteins contain an N-terminal epitope tag
derived from the c-Myc protein. The cellular localization of the NPc
proteins in transfected cells was determined by indirect
immunofluorescence with anti-myc IgG. The cells shown are
representative of more than 200 cells that were positive for the
expression of the indicated proteins.
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The hydrophobic cluster within the second ankyrin repeat is highly
conserved among other IB family members (14, 27, 39, 46, 47,
55, 71). As a nuclear function has previously been proposed for
several other IB family members, including IB and Bcl-3
(8, 23, 55, 57, 73), we asked whether the second ankyrin
repeat of either IB or of Bcl-3 would be able to functionally
substitute for the classical NLS in nucleoplasmin. Fusion of the second
ankyrin repeat from either IB or from Bcl-3 onto NPc efficiently
relocalized NPc to the nucleus (Fig. 5). Thus, nuclear import is a
conserved property of the second ankyrin repeat from these IB
proteins.
The recently described crystal structure of the ARD-containing protein
53BP2 reveals that an individual ankyrin repeat consists of a short
N-terminal -hairpin and N- and C-terminal -helices that pack in
an antiparallel fashion (32). The -hairpin which initiates the adjacent ankyrin repeat provides critical amino acid
interactions that stabilize the previous ankyrin repeat
(32). To define the minimal structural requirements for the
nuclear import function of the second ankyrin repeat, further
truncations of the second ankyrin repeat were fused onto NPc, and their
ability to relocalize NPc to the nucleus was determined. Deletion of
either the N-terminal (NPc-p40-AR2-N/C/C) or the C-terminal
(NPc-p40-AR2-N/N/C) -hairpin reduced the ability of the
second ankyrin repeat to relocalize NPc to the nucleus (Fig. 5). A
sequence encompassing the N-terminal -helix of the second ankyrin
repeat (NPc-p40-AR2-N) was unable to function as a nuclear import
signal when fused onto NPc (Fig. 5). Thus, an extended ankyrin repeat
region which includes an N-terminal -hairpin, an N-terminal
-helix, a C-terminal -helix, and an adjacent C-terminal
-hairpin constitutes a fully functional nuclear import sequence.
The ability of NPc to form oligomers is likely a critical factor in its
ability to remain in the cytoplasm in the absence of a nuclear import
signal (49). Expression of the various NPc-IB-ARD and
the NPc-IB-AR2 fusion proteins was confirmed by immunoblot analysis
(data not shown). Fusion of the IB ARD or the second ankyrin
repeat from IB proteins onto NPc did not disrupt NPc oligomer
formation (data not shown).
The ARDs of diverse proteins contain a functional nuclear import
signal.
To determine whether nuclear import is a common property
of ARDs, the ARDs from several other ARD-containing proteins were fused
onto NPc and their cellular distribution was determined by indirect
immunofluorescence in COS-1 cells. Fusion of the first, second, and
third ankyrin repeats from the p53-associated protein, 53BP2
(53), or fusion of the ARD from GABP (44), a
transcription factor which belongs to the Ets family of proteins, onto
NPc efficiently relocalized NPc to the nucleus (Fig.
7A and 8B
and C). In contrast, fusion of the ARD from the Notch1 protein
(18, 38), a transmembrane receptor protein, onto NPc did not
efficiently relocalize NPc to the nucleus (Fig. 7A and 8D). The
localization of the NPc-ARD fusion proteins was also examined in
WT+/+, p50/, and p65/ 3T3
fibroblasts. While the NPc protein remained predominantly cytoplasmic,
fusion of the ARDs from MAD3, 53BP2, and GABP onto NPc markedly
relocalized NPc to the nucleus in all three of these cell types (Table
4). Expression of the various NPc-ARD
fusion proteins was confirmed by immunoblot analysis (data not shown). Fusion of the ARDs onto NPc did not disrupt NPc oligomer formation (data not shown). Thus, the nuclear import function of ARDs derived from several ARD-containing proteins are independent of either p50 or
p65.
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FIG. 7.
Nuclear import function of ankyrin repeat domains. (A)
The structures of fusion proteins between NPc and the ARDs from the
mammalian IB protein (MAD3), 53BP2, GABP, and Notch1 are
indicated. The NPc protein comprises amino acids 2 through 150 of
nucleoplasmin and contains an N-terminal epitope tag derived from the
c-Myc protein. The ARDs from the indicated proteins were fused onto the
C terminus of NPc. The amino acid residues from each protein that were
used to construct each of the NPc-ARD fusion proteins are indicated.
The cellular localization of the NPc-ARD fusion proteins was determined
in COS-1 cells using anti-myc IgG. (B) The structure of a fusion
protein between PK and the ARD from 53BP2 is indicated. Amino acids 17 through 524 of PK were used to construct the fusion protein, which also
contains an N-terminal epitope tag derived from the c-Myc protein. The
amino acid residues that were used to construct the PK-53BP2-ARD
fusion protein are indicated. The cellular localization of the
PK-53BP2-ARD fusion protein was determined in COS-1 cells using
anti-myc IgG. For both panels, cells that were positive for expression
of the indicated fusion proteins were classified as having
predominantly nuclear staining (N), staining that was distributed
equally between the nucleus and the cytoplasm (N/C), or staining that
was predominantly cytoplasmic (C). At least 200 cells that were
positive for expression of each fusion protein were scored, and the
percent of cells in each category is given. Colons show fusions.
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FIG. 8.
Nuclear localization of NPc or PK upon fusion of ARDs.
COS-1 cells were transfected with CMV-based expression vectors encoding
NPc-MAD3-ARD (A), NPc-53BP2-ARD (B), NPc-GABP-ARD (C),
NPc-Notch1-ARD (D), PK (E), or PK-53BP2-ARD (F). The NPc and PK
fusion proteins contain an N-terminal epitope tag derived from the
c-Myc protein. The cellular localization of the NPc and PK fusion
proteins in transfected cells was determined by indirect
immunofluorescence with anti-myc IgG. The cells shown are
representative of more than 200 cells that were positive for the
expression of the indicated proteins.
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PK is a protein of cytoplasmic origin, and fusion of a classical NLS
onto PK localizes PK to the nucleus (43, 49). To determine
whether an ARD generally directs nuclear localization, the ARD of 53BP2
was fused onto PK and the cellular localization of the PK-53BP2-ARD
fusion protein was determined. As expected, PK was predominantly
cytoplasmic when ectopically expressed in COS-1 cells (Fig. 7B and 8E).
Fusion of the 53BP2 ARD onto PK significantly localized PK from the
cytoplasm to the nucleus (Fig. 7B and 8F). Expression of the PK-53BP2
protein was confirmed by immunoblot analysis (data not shown). Thus,
the 53BP2 ARD can functionally substitute for a classical NLS from a
normally nuclear protein and also specify the nuclear import of a
normally cytoplasmic protein.
Hydrophobic residues within the nuclear import sequence of IB
are required for association with p65 (RelA) but not c-Rel.
The
identification of a discrete nuclear import sequence within IB
indicates that nuclear localization of both IB and Rel proteins
are dependent upon cis-acting sequences within the respective proteins. However, both the p65-IB and the
c-Rel-IB complexes are sequestered in the cytoplasm (4, 20,
26, 65, 77). Cytoplasmic sequestration of these Rel-IB
complexes indicates that both the Rel NLS and the IB nuclear
import sequence are functionally inactive in the Rel-IB complex.
The p65 NLS has previously been shown to be masked in the context of
the p65-IB complex (4, 26, 77). However, the mechanism
by which the IB nuclear import sequence is functionally
inactivated within either the p65-IB or the c-Rel-IB
complex is not known.
We first examined the ability of the wild-type p40 and the p40-114A3
proteins to associate with p65 by coimmunoprecipitation analysis (Fig.
9). COS-1 cells were cotransfected with
CMV-driven expression vectors encoding p65 and either a wild-type
epitope-tagged p40 protein (p40-LBD) or a mutant epitope-tagged
p40-114A3 protein (LBD-p40-114A3). The p65 protein was not detected in
anti-LBD immunoprecipitates when singly transfected into COS-1 cells
(Fig. 9, upper panel, lane 1) but was readily detected from COS-1 cells cotransfected with wild-type LBD-p40 (Fig. 9, upper panel, lane 2).
Furthermore, p65 was not detected in anti-LBD immunoprecipitates when
cotransfected with the LBD-p40-114A3 protein (Fig. 9, upper panel, lane
3).
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FIG. 9.
Coimmunoprecipitation analysis of wild-type and mutant
IB proteins. COS-1 cells were mock transfected (lane 4),
transfected with a CMV-derived expression vector encoding p65 (RelA)
(lanes 1 to 3), or transfected with a CMV-derived expression vector
encoding c-Rel (lanes 5 to 7). CMV-derived expression vectors encoding
either the wild-type LBD-p40 (lanes 2 and 6) or the LBD-p40-114A3
protein (lanes 3 and 7) were included in some transfections. Cell
lysates were subjected to immunoprecipitation with affinity-purified
anti-LBD rabbit IgG followed by immunoblot analysis with either
anti-p65 mouse IgG (top panels, lanes 1 to 3), or anti-c-Rel mouse IgG
(top panels, lanes 4 to 7). Cell lysates were also subjected to direct
immunoblot analysis with anti-p65 rabbit serum (middle panels, lanes 1 to 3), with anti-c-Rel rabbit serum (middle panels, lanes 4 to 7), or
with anti-p40 rabbit serum (bottom panels, lanes 1 to 7). The p65
(RelA), c-Rel, and p40 proteins are indicated by arrows. Only the
relevant portions of each immunoblot are shown.
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As an independent measure of the ability of wild-type or mutant
IB proteins to associate with p65, we examined the ability of
wild-type and mutant IB proteins to inhibit the DNA-binding activity of p65. Coexpression of MAD3 with p65 inhibited DNA-binding by
p65 (Fig. 10, lane 3). In contrast, the mutant MAD3-110A3 protein was
markedly reduced in its ability to inhibit DNA-binding by p65 or the
endogenous DNA-binding activity from COS-1 cells (Fig. 10, compare
lanes 3 and 4). Similarly, wild-type p40 inhibited DNA binding by p65,
while the mutant p40-114A3 protein was markedly reduced in its ability
to inhibit DNA binding by p65 in COS-1 cells (data not shown). The
steady-state levels of p65 and of the IB proteins within the
respective cell lysates were approximately equivalent, as determined by
immunoblot analysis (data not shown). The IB-A3 proteins were
also markedly deficient for inhibition of NF-B-dependent luciferase
gene expression relative to the wild-type IB proteins in cells
treated with TNF- or cotransfected with an expression vector
encoding the Tax protein of human T-cell leukemia virus type 1 (data
not shown). Taken together, these results show that the mutant
IB-A3 proteins are markedly reduced in their ability to associate
with or to inhibit the DNA binding of p65. Our results suggest that
hydrophobic residues within the second ankyrin repeat of IB
participate in critical amino acid contacts between IB and p65.
In contrast to p65, c-Rel was readily detected in -LBD
immunoprecipitates from COS-1 cells transfected with the mutant
LBD-p40-114A3 protein (Fig. 9, upper panel, lane 7). The c-Rel protein
was also able to associate with the p40-114A3 protein in the
Saccharomyces cerevisiae two-hybrid system (data not shown).
Furthermore, coexpression of either wild-type p40 (Fig.
10, lane 8) or p40-114A3 (Fig. 10, lane
9) with c-Rel inhibited DNA binding by c-Rel. Similar to the MAD3-110A3
protein, the p40-114A3 protein was markedly reduced in its ability to
inhibit the endogenous DNA-binding activity in COS-1 cells (Fig. 10,
compare lanes 8 and 9). The steady-state levels of c-Rel and of the p40
proteins within the respective cell lysates were approximately
equivalent, as determined by immunoblot analysis (data not shown).
Taken together, these results show that hydrophobic residues within the
second ankyrin repeat of IB are not critically required for
association of IB with c-Rel.
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FIG. 10.
Inhibitory properties of wild-type and mutant IB
proteins. COS-1 cells were mock transfected (lanes 1 and 6) or were
transfected with CMV-derived expression vectors encoding either p65
(RelA) (lanes 2 to 5) or c-Rel (lanes 7 to 10). CMV-derived expression
vectors encoding wild-type MAD3 (lane 3), mutant MAD3-110A3 (lane 4),
wild-type p40 (lane 8), or mutant p40-114A3 (lane 9) were included in
some transfections. Cell lysates were analyzed for proteins that bound
to a 32P-labeled oligonucleotide containing a palindromic
B site. A 100-fold excess of the unlabeled palindromic B
oligonucleotide was included in some DNA-binding reaction mixtures
(lanes 5 and 10). The DNA-binding reaction mixtures were
electrophoresed through a 5% nondenaturing polyacrylamide gel. The
positions of the respective Rel-DNA complexes and unbound DNA are
indicated by arrows. The endogenous Rel DNA-binding activity in COS-1
cells is denoted by an asterisk.
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DISCUSSION |
The IB protein normally sequesters dimeric Rel proteins in
the cytoplasm in unstimulated cells (4, 20, 26, 65, 77).
Upon cellular stimulation, IB becomes inducibly phosphorylated and degraded, enabling the dimeric Rel complex to translocate to the
nucleus (9, 10, 15, 16, 48, 60, 63, 72, 76). In activated
cells, the cellular pool of IB is rapidly replenished and newly
synthesized IB enters the nucleus and exports the dimeric Rel
complex to the cytoplasm (1, 2, 42, 45, 70). The small size
of IB and the absence of a discernible classical NLS in IB
have led to the previous suggestion that IB might enter the
nucleus by passive diffusion (1, 77). Our results now
demonstrate that nuclear localization of newly synthesized IB
does not occur by passive diffusion. Rather, nuclear localization of
IB is specified by a novel nuclear import sequence within the
second ankyrin repeat.
The second ankyrin repeat of IB is the predominant nuclear import
sequence in the context of the full-length IB protein, as either
alanine substitution of hydrophobic residues within the second ankyrin
repeat or deletion of the second ankyrin repeat markedly relocalizes
IB to the cytoplasm. However, the other ankyrin repeats within
the ARD of IB might also contribute to the nuclear localization
of the full-length IB protein. In particular, neither mutations
within the second ankyrin repeat nor deletion of the second ankyrin
repeat fully restricts IB to the cytoplasm. Furthermore, deletion
of the fourth or the fifth ankyrin repeat of IB partially
relocalizes IB to the cytoplasm. Finally, similar to fusion of
the second ankyrin repeat, fusion of the third, fourth, or fifth
ankyrin repeats of IB onto NPc also relocalizes NPc from the
cytoplasm to the nucleus (64a). Therefore, although the
second ankyrin repeat of IB is the predominant nuclear import
sequence, the other ankyrin repeats may also contribute to the nuclear
localization of the full-length IB protein.
The hydrophobic residues within the second ankyrin repeat are highly
conserved among other IB family members (14, 27, 39, 46, 47,
55, 71). The ability of the second ankyrin repeat from IB,
IB, or Bcl-3 to each specify nuclear import of NPc suggests that
nuclear localization of these IB family members might be mediated by
a nuclear import function encoded within their second ankyrin repeat.
To further define the sequence requirements for the nuclear import
function of the second ankyrin repeat of IB, and by analogy with
the 53BP2 crystal structure (32), we initially chose an extended ankyrin repeat region that would encompass the N-terminal -hairpin and the two -helices of the second ankyrin repeat, and
the N-terminal -hairpin of the third ankyrin repeat. Deletion of
either -hairpin reduced the nuclear import function of this extended
ankyrin repeat region. Furthermore, an 18-amino-acid sequence
encompassing just the N-terminal -helix was not sufficient to
mediate nuclear import. Our results suggest that a fully functional ankyrin repeat-derived nuclear import sequence is comprised of an
N-terminal -hairpin, two -helices, and the -hairpin of the adjacent ankyrin repeat. The 53BP2 crystal structure shows that amino
acid interactions between adjacent -hairpins, and between a
conserved histidine residue within the N-terminal -helix and the
backbone of the -hairpin from the adjacent ankyrin repeat provide
interactions necessary for the stability of an ankyrin repeat
(32). Our results are consistent with the notion that the
ability of the second ankyrin repeat to function as a nuclear import
sequence critically requires amino acid interactions that maintain the
structural integrity of that ankyrin repeat.
The ability of the ARDs from other ARD-containing proteins, such as
53BP2 and GABP, to functionally substitute for a classical NLS
suggests that nuclear import is not a unique property of the IB
ARD. However, nuclear import is not a general property of all ARDs, as
the ARD from Notch1 is a poor nuclear import sequence. Thus, the
ability of ARDs to specify nuclear import is not equivalent among all
ARD-containing proteins. The precise structural determinants of
individual ankyrin repeats within an intact ARD which are necessary for
nuclear import function will need to be identified to understand how
specific ARDs specify nuclear import.
At least two mechanisms can be envisioned to understand how the ARD
mediates nuclear localization of IB. One possibility is that
nuclear import of IB occurs via a piggyback mechanism, in which
the IB-derived nuclear import sequence mediates association with
another protein that contains a classical NLS, and subsequent nuclear
import of IB occurs via the importin---mediated import pathway. If nuclear import of IB is accomplished via such a piggyback mechanism, our results strongly suggest that members of the
Rel transcription factor family are not the carrier protein. First,
significant nuclear accumulation of ectopically expressed IB
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B
Is Mediated by the
Second Ankyrin Repeat: the I
Columbia, Columbia, Missouri
65212,1 and
Division of Biology, California
Institute of Technology, Pasadena, California 911252
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Abstract
Introduction
