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Mol Cell Biol, May 1998, p. 2596-2607, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Alternative Splicing Variants of I
B
Establish
Differential NF-
B Signal Responsiveness in Human Cells
Fuminori
Hirano,1,
Mirra
Chung,2
Hirotoshi
Tanaka,3
Naoki
Maruyama,3
Isao
Makino,3
David D.
Moore,2 and
Claus
Scheidereit1,*
Max-Delbrück-Center for Molecular
Medicine MDC, 13122 Berlin, Germany1;
Department of Cell Biology, Baylor College of Medicine,
Houston, Texas 770302; and
Second
Department of Internal Medicine, Asahikawa Medical College, 078 Asahikawa, Japan3
Received 25 August 1997/Returned for modification 17 October
1997/Accepted 29 January 1998
 |
ABSTRACT |
To release transcription factor NF-
B into the nucleus, the
mammalian I
B molecules I
B
and I
B
are inactivated by
phosphorylation and proteolytic degradation. Both proteins contain
conserved signal-responsive phosphorylation sites and have conserved
ankyrin repeats. To confer specific physiological functions to
members of the NF-
B/Rel family, the different I
B molecules
could vary in their specific NF-
B/Rel factor binding activities and
could respond differently to activation signals. We have demonstrated
that both mechanisms apply to differential regulation of NF-
B
function by I
B
relative to I
B
. Via alternative RNA
processing, human I
B
gives rise to different protein isoforms. I
B
1 and I
B
2, the major forms in human cells, differ
in their carboxy-terminal PEST sequences. I
B
2 is the most
abundant species in a number of human cell lines
tested, whereas I
B
1 is the only form detected in murine cells.
These isoforms are indistinguishable in their binding preferences to
cellular NF-
B/Rel homo- and heterodimers, which are distinct from
those of I
B
, and both are constitutively phosphorylated. In
unstimulated B cells, however, I
B
1, but not I
B
2, is found
in the nucleus. Furthermore, the two forms differ markedly in their
efficiency of proteolytic degradation after stimulation with several
inducing agents tested. While I
B
1 is nearly as responsive as
I
B
, indicative of a shared activation mechanism, I
B
2 is
only weakly degraded and often not responsive at all. Alternative
splicing of the I
B
pre-mRNA may thus provide a means to
selectively control the amount of I
B
-bound NF-
B heteromers to
be released under NF-
B stimulating conditions.
 |
INTRODUCTION |
Transcriptional induction of genes
driven by members of the NF-
B/Rel family of activators plays a
crucial role in many processes of the immune response and under a
number of cellular stress conditions including viral infection (2,
5, 40, 44). The activation of NF-
B through different pathways
is controlled primarily by its release from the interaction with
inhibitory I
B molecules. Five different mammalian
NF-
B/Rel proteins are known, p50, p52, p65, c-Rel,
and RelB, which form homo- and heterodimers. These are sequestered by
the I
B molecules I
B
, I
B
, and I
B
and by p105 and
p100, the precursor molecules for p50 and p52, respectively (2,
42). Best understood is the activation of NF-
B from complexes
with I
B
by diverse inducers like tumor necrosis factor alpha
(TNF-
), lipopolysaccharide (LPS), phorbol myristate acetate, (PMA)
or human T-cell leukemia virus type 1 Tax (7, 8, 38, 43).
These lead to phosphorylation at Ser 32 and 36. Phosphorylated I
B
is then polyubiquitinated and subsequently degraded by the proteasome
(3, 9, 34). In addition to the amino-terminal signal
response box containing Ser 32 and 36, signal-dependent degradation is
more efficient in the presence of the carboxy-terminal PEST sequence of
I
B
(7, 8, 38, 43), although the mechanistic need of
the latter is not understood. The high turnover of I
B
in the
resting state is also driven by the proteasome but is independent of
the amino-terminal serines and of the PEST sequence and does not
involve ubiquitin conjugation of the molecule (19).
Processing of p105 is, at least in some cell lines, signal inducible
and involves the same steps as I
B
breakdown, including
phosphorylation, ubiquitin conjugation, and degradation by the
proteasome (14, 29, 31). An endoprotease activity
initially cleaves p105 into p50 and the carboxy-terminal I
B
domain (21). A multisubunit kinase, which phosphorylates
I
B
at Ser 32 and 36 and which requires ubiquitination of one of
its subunits, has been identified biochemically (10).
Recently, two I
B
kinases which phosphorylate I
B
at Ser 32 and 36 and mediate NF-
B activation in response to TNF-
have been
cloned (24, 35).
The overlapping functions of the various I
B and NF-
B proteins
contrast with their specific physiological activities, as revealed by
gene-targeting experiments (2). These specific physiological
activities imply that functional specificity may be imposed by the
differential response of individual I
B or NF-
B/Rel proteins to
particular signaling pathways. Therefore, potential regulatory
differences between diverse I
Bs, such as I
B
and I
B
, are
of interest.
I
B
was first identified by Zabel and Baeuerle (45),
and murine I
B
was cloned with probes derived from peptides of the purified protein (37). Human cDNAs encoding I
B
were
isolated by using the two-hybrid system (20) (see below). A
notable difference between I
B
and I
B
is the basal
phosphorylation of I
B
, which is needed for interaction with
NF-
B (22). Recent reports have shown that I
B
and
I
B
have properties in common. Murine I
B
is proteolytically
degraded after stimulation with LPS or interleukin-1 (IL-1)
(37). In contrast to I
B
, I
B
is not resynthesized immediately after stimulation and is degraded with delayed kinetics compared to I
B
(37). The degradation of murine
I
B
after stimulation with TNF-
, IL-1, or human T-cell leukemia
virus type 1 Tax depends on Ser 19 and Ser 23 (12, 25).
These residues are embedded in a sequence similar to the one
surrounding Ser 32 and 36 in I
B
, and a recently identified I
B
kinase phosphorylates both proteins at these residues (13,
33). Furthermore, the carboxy-terminal PEST sequence of I
B
is needed for efficient proteolysis (16, 41). Inducible
degradation of I
B
is blocked by inhibitors of the proteasome and
possibly requires ubiquitination (12, 25). Unphosphorylated
recombinant I
B
, but not I
B
, can associate with Rel factors,
and latent cytoplasmic complexes of I
B
and Rel factors are
sensitive to phosphatase treatment. Major sites of constitutive
phosphorylation of I
B
have been mapped to Ser 313 and Ser 315, which are phosphorylated by casein kinase II (CKII) (11).
Phosphorylation at these residues is required for efficient complex
formation between I
B
and c-Rel (11). Recombinant
unphosphorylated I
B
can bind to p65 but not to c-Rel
(11). Carboxy-terminal phosphorylation by CKII of I
B
increases its affinity to NF-
B (39). Tax-induced
degradation of I
B
leads to the induction of c-Rel-containing
complexes (15), further indicating that c-Rel-I
B
complexes are physiologically important.
Newly synthesized I
B
first accumulates as an underphosphorylated
species, which forms a stable complex with NF-
B p65
(36). It has been shown that in contrast to the
phosphorylated form, underphosphorylated I
B
interacts with p65
without shielding the nuclear localization signal region. De
novo-translated, underphosphorylated I
B
may sequester
NF-
B, thereby protecting it from being withdrawn into
cytoplasmic complexes with I
B
, and it can be transported with
NF-
B into the nucleus (36).
We show here that in human cells the I
B
gene gives rise to
different gene products, which elicit differential responsiveness to
several inducers tested. Two of these, termed I
B
1 and I
B
2, have been analyzed in detail. Compared to I
B
, I
B
1 is most similar in responsiveness to inducing agents whereas I
B
2, which has a carboxy-terminal sequence substitution truncating a PEST domain,
is either degraded incompletely or inert in response to some inducers.
Both I
B
1 and I
B
2 bind with highest affinity to p65
homodimers and with lower affinity to heterodimers containing c-Rel or
p65. The affinity depends largely on constitutive phosphorylation at
the carboxy-terminal PEST sequences of both molecules. I
B
1, but
not I
B
2, is partially localized in the nucleus in B cells, indicating different roles of the two isoforms for modulating or
maintaining constitutive NF-
B activation. The resistance of I
B
2, the predominant form in human cells, to a number of inducers tested suggests that this isoform may serve to limit the fraction of
cytoplasmic NF-
B released into the nucleus upon cellular
stimulation.
 |
MATERIALS AND METHODS |
Cell culture.
Namalwa and IM9 cells were grown in RPMI
1640 (BRL/GIBCO)-4 mM glutamine-100 U of penicillin per
ml-100 µg of streptomycin per ml-7.5% fetal calf serum; suspension
HeLa cells were kept in Joklik modified minimum essential medium
(BRL/GIBCO)-1% nonessential amino acids, 100 U of penicillin per
ml-100 µg of streptomycin per ml-10% fetal calf serum. Jurkat
cells were grown in RPMI 1640 (BRL/GIBCO)-4 mM glutamine-100 U of
penicillin per ml-100 µg of streptomycin per ml-10% fetal calf
serum. Where indicated, cells were stimulated with 10 ng of TNF-
(Biomol) per ml, 50 ng of PMA (Sigma) per ml-5 µg of ionomycin
(Sigma) per ml, or 50 U of IL-1
(Biomol) per ml for the indicated
times.
Genomic PCR and reverse transcription-PCR.
PCR was performed
by standard methods with I
B
-specific primers and HeLa
cell-derived nucleic acids.
Library screening.
A HeLa cell cDNA library was screened
with the insert of the TRIP9 clone isolated by two-hybrid screening
(20) as a probe. Positive cDNAs were sequenced on both
strands by the dideoxynucleotide method.
DNA constructs for prokaryotic expression.
pETI
B
2
(encoding amino acids 1 to 338) was generated from
pCDM8I
B
(351/21) by PCR with the appropriate oligonucleotides with
the insert cloned into the BamHI site of pET3c (Novagen). Protein expression in Escherichia coli BL21(DE3)pLysS, and
purification was carried out as described previously (27).
DNA constructs for eukaryotic cell expression.
pECEp50,
pECEp65, pECEc-rel, and pcDNA3I
B
have been described previously
(19, 28). FLAG-tagged pcDNA1I
B
2 (amino acids 1 to
338), FLAG-tagged pcDNA1I
B
2
N (amino acids 55 to 338), FLAG-tagged pcDNA1I
B
2
C (amino acids 1 to 307), and FLAG-tagged pcDNA1I
B
2
NC (amino acid 55 to 307) were generated from
pCDM8I
B
(351/21) by PCR and cloning into the BamHI site
of FLAG-tagged pcDNA1, which contained a FLAG epitope inserted into the
HindIII and BamHI sites of pcDNA1.
FLAG-tagged pcDNA1I
B
1 was generated by cloning the I
B
1 cDNA
from pCDM8I
B
(351/1) into pcDNA1. The NF-
B reporter plasmid
2X
B-Luc contained the annealed tandem Ig
enhancer sequence 5'-CAGTTGAGGGGACTTTCCCAGATCTAGTTGAGGGGACTTTCCCAG-3' and the
45 to +83 fragment of the human immunodeficiency virus type 1 promoter (EcoRI and HindIII sites) inserted
into KpnI-HindIII of pGV-B (Toyo Ink Co.,
Tokyo, Japan).
Antibodies for immunoblots and immunoprecipitation.
We used
anti-p65 (rabbit) (Santa Cruz Biotechnology Inc., sc-109), anti-p50
(rabbit) (Rockland), anti-c-Rel (rabbit) (Santa Cruz Biotechnology
Inc., sc-272), anti-relB (rabbit) (Santa Cruz Biotechnology Inc.,
sc-226), anti-p52 (mouse) (Upstate Biotechnology Inc., 05-361),
anti-I
B
/MAD3 (rabbit) (Santa Cruz Biotechnology Inc., sc-203),
anti-FLAG (rabbit) (Santa Cruz Biotechnology Inc., sc-807),
anti-I
B
(rabbit) (Santa Cruz Biotechnology Inc., sc-969, against
the N-terminal sequence of I
B
1 and I
B
2 [N-20]),
anti-I
B
(rabbit) (Santa Cruz Biotechnology Inc., sc-945, against
the C-terminal sequence of I
B
1 [C-20]), and the antibody
against the C-terminal sequence of I
B
2 [b2(C)], raised against
the peptide VSQEERQGSPAGGSG (amino acids 324 to 338 of
I
B
2, synthesized by Eurogentec S.A.).
Preparation of cell extracts.
For whole-cell extracts, cells
were washed with phosphate-buffered saline (PBS) twice and incubated on
ice for 15 min in 20 mM HEPES (pH 7.9)-350 mM NaCl-1 mM
MgCl2-0.5 mM EDTA-0.1 mM EGTA-1% Nonidet P-40
(NP-40)-0.5 mM dithiothreitol-0.4 mM
4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride
(Boehringer Mannheim)-50 mM sodium fluoride-1 mM sodium
orthovanadate. After centrifugation at 14,000 rpm for 20 min in an
Eppendorf centrifuge, the supernatant was used as a whole-cell extract.
For preparation of cytoplasmic and nuclear extracts, the cells were
washed and resuspended in buffer A and 0.125% NP-40 was added. The
cells were left for 5 min on ice and centrifuged at 1,000 × g for 10 min. The supernatant was used as the cytoplasmic
extract, and the pellet was treated with buffer C for 15 min to yield
the nuclear extract as described previously (27).
Immunoblots.
Cell extracts (40-µg samples) were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and prior to transfer, the gels were equilibrated in
ice-cold blotting buffer (25 mM Tris-HCl [pH 8.3], 0.01% SDS, 20%
methanol). The proteins were transferred to a polyvinylidene difluoride
membrane (Millipore) as described previously (28). Western
blots were analyzed by chemiluminescence (Tropix) as described
previously (19).
Immunoprecipitation.
Antibodies (10-µl samples) were
coupled to protein A-Sepharose (Pharmacia) in PBS for 2 h. The
whole-cell extract or cytoplasmic extract was incubated for 2 h
with antisera or antibodies coupled to protein A-Sepharose. The
precipitated proteins were washed several times with ice-cold PBS,
boiled, separated by SDS-PAGE, and transferred to a PVDF membrane as
described previously (27).
Electrophoretic mobility shift assay (EMSA).
Gel retardation
assays were performed with an H2K oligonucleotide probe. DNA binding
reactions were performed in 20 µl of binding buffer [20 mM HEPES (pH
8.4), 60 mM KCl, 4% Ficoll, 5 mM dithiothreitol 1 µg of bovine serum
albumin, 2 µg of poly(dI-dC)] for 20 min at 30°C. The reaction
mixture was loaded onto a 4% nondenaturing polyacrylamide gel
containing 1× Tris-borate-EDTA (TBE). The gels were run at 250 V for
1 h, dried, and visualized by autoradiography.
Immunoprecipitation-EMSA (IP-shift) assay.
The precipitated
proteins, coupled to protein A beads, were washed several times with
ice-cold PBS and incubated in 20 µl of 0.8% deoxycholate on ice for
10 min. After centrifugation, NP-40 (final concentration, 1%) was
added to the supernatant, which was then used for EMSA.
Phosphatase treatment of cytoplasmic extracts.
Cell extract
proteins (40 µg) were incubated with 2 U of shrimp alkaline
phosphatase (United States Biochemical Co.) at 37°C for 30 min. As a
control, shrimp alkaline phosphatase was inactivated at 65°C for 30 min.
Transient transfection.
Transfection of suspension HeLa
cells was performed by electroporation with a Bio-Rad Gene Pulser.
Cells were grown at 5 × 105 cells/ml, harvested 5 to
6 h after the last feeding, and resuspended at 2 × 107 cells/ml. Then 0.4 ml of cells was mixed with 20 µg
of DNA at room temperature, electroporated at 250 V and 950 µF, and
immediately transferred to small culture flasks containing 10 ml of
prewarmed medium. At 48 h later, the transfected cells were
harvested. For reporter gene assays, HeLa cells were transfected by
electroporation with 2×
B-Luc (10 µg), p65 expression vector (5 µg), and expression vectors containing I
B
, I
B
1, or
I
B
2 (1 or 5 µg). Luciferase activities were measured with a
Luminometer Lumat LB9501 (Berthold).
 |
RESULTS |
Human I
B
is expressed as a set of differentially spliced
isoforms.
We previously isolated a human homolog of I
B
by
using the human thyroid receptor as a bait in the two-hybrid system
(20). Rescreening HeLa cell cDNA libraries with the original
yeast isolate yielded additional clones with two distinct 3' ends.
Sequence alignment with murine I
B
(37) demonstrates
that one of these encodes a protein, I
B
1, that is most similar to
the murine protein. The variant I
B
2 isoform differs from
I
B
1 by a substitution of new C-terminal and 3' untranslated
sequences, which results in truncation of the C-terminal PEST motif
(Fig. 1).

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FIG. 1.
(Top) Schematic presentation of human I B 1 and
I B 2 compared to murine I B . The position of the peptides
used for raising the antibodies is indicated. (Bottom) Sequence
alignment of the carboxy-terminal PEST sequences of human I B 1 and
I B 2 and murine I B . a.a., amino acids.
|
|
Northern blot analysis of various human tissues with a common probe
confirms the existence of two major mRNAs of approximately
1.8 and 2.8 kb (
20). The divergence between I

B

1 and I

B

2
occurs at a consensus 5' splice site present only in the I

B

2
sequence, suggesting that the two transcripts are generated by
alternative mRNA processing. Analysis by reverse transcription-PCR
and
genomic PCR confirms the existence of two transcripts and
demonstrates
that I

B

2 retains intronic sequences extending beyond
the exon
that encodes the sixth ankyrin repeat (data not shown).
Since a poly(A)
tract is present in the I

B

1 sequence approximately
150 nucleotides downstream of the site of divergence, while the
I

B

2 sequence extends much further, the former presumably
corresponds
to the shorter transcript and the latter corresponds
to the longer
transcript. Overall, these results indicate that
the I

B

gene
encodes at least two transcripts as a
consequence of alternative
mRNA splicing.
I

B

1 and I

B

2 encode proteins of 38 and 35.5 kDa,
respectively. To investigate the protein expression of both forms, a
Western
blot analysis of Namalwa cell cytosol was performed (Fig.
2A).
The blot was probed either with an
antibody recognizing an amino-terminal
sequence common to both forms
(N-20, lane 1) or with antibodies
raised against the isoform-specific
carboxy-terminal sequences
(lanes 2 and 3). Two specific bands of 43 and 41 kDa, respectively
(I

B

1 and I

B

2), were detected. The
specificity of the I

B

1
and I

B

2 signals was confirmed by
peptide competition (lanes
4 to 6). Immunoprecipitation with the N-20
antibody and immunostaining
with the isotype-specific antibodies
further confirmed the expression
of both splicing variants (Fig.
2B).
The larger sizes of both
isoforms compared to the predicted molecular
masses suggest posttranslational
modifications of both forms. To
compare the relative amounts of
I

B

1 and I

B

2 in several
human and murine cell lines, Western
analysis was carried out with the
amino-terminal antibody (Fig.
2C). All human cell lines expressed both
isoforms (Fig.
2C, left
panel). The relative abundance varied (a
summary is given in Fig.
2C, bottom panel), but I

B

2 was the
most abundant isoform in
most human cell types. In contrast, only
I

B

1 was detected in
all the murine cell types tested (Fig.
2C, right panel). In some
human cell lines, further signals were
detected, some of which
may represent further splicing isoforms (data
not shown). We conclude
that both of the cloned I

B

transcripts
are expressed as proteins
and that I

B

2 is the predominant isoform
in human cell lines.

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FIG. 2.
Protein expression of I B isoforms. (A) Western
blot analysis of Namalwa cell cytosol with an amino-terminal antibody
recognizing a common epitope (N-20) or carboxy-terminal antibodies
raised against isoform-specific sequences [C-20 for I B 1 and
(C) for I B 2], as indicated (lanes 1 to 3). Specific bands at
43 and 41 kDa were absent after peptide competition (lane 4 and 5) or
probing with preimmune serum (lane 6). (B) Immunoprecipitation. Namalwa
cell cytosolic extracts were subjected to immunoprecipitation with
N-20, and the precipitates were immunoblotted with C-20 or (C)
antibodies, as indicated. (C) Equal cell equivalents of human and
murine cell lines were analyzed by Western blotting for I B
isoform expression with the amino-terminal antibody, as indicated.
(Left) Human cell lines; (right), murine cell lines; (bottom), summary
of the relative expression levels of I B 1 and I B 2. N.D., not
detectable. The signals around 29 kDa are cross-reacting proteins. The
positions of I B 1 and I B 2 are indicated by solid and open
arrowheads, respectively.
|
|
Both cellular I
B
isoforms associate predominantly with p65
homodimers but also with heteromers containing c-Rel or p65; equal
inhibition of NF-
B-dependent transcription.
To investigate
the preferential association of I
B
1, I
B
2, and I
B
with
Rel factors, cellular proteins immunoprecipitated with antibodies
against the five mammalian NF-
B/Rel subunits were blotted and
subsequently probed with I
B-specific antibodies (Fig.
3A). I
B
1 and I
B
2 associated
predominantly with p65 and only weakly with p50 or
p52, presumably through their heteromeric complexes with p65 (lanes 1, 2, 5, 11, 12, and 15). In contrast, almost no RelB and only little
c-Rel associated with I
B
1 or I
B
2 (lanes 3, 4, 13, and
14), although both Rel proteins were efficiently precipitated (Fig. 3A,
bottom). Similarly, I
B
interacted predominantly with complexes
containing p65 (lanes 6 to 10). I
B
was found to a larger extent
in complexes with p50 or p52 than were I
B
1 and I
B
2 (lanes 6 and 10). Similarly, in primary and transformed murine T cells, I
B
and I
B
(I
B
1) were associated primarily with p65 and to a
lesser extent with c-Rel (11).

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FIG. 3.
Interaction and functional interference of I B 1,
I B 2, and I B with NF- B/Rel factors (A)
Coimmunoprecipitation of I Bs with NF- B/Rel. Whole-cell
Namalwa extracts were subjected to immunoprecipitation with antibodies
(Ab) directed against p50, p65, c-Rel, RelB, or p52. (Top) Precipitates
were separated by SDS-PAGE, and the blot was sequentially probed with
antibodies against I B 1 (lanes 1 to 5), I B (lanes 6 to 10),
and I B 2 (lanes 11 to 15). Specific signals are indicated by solid
arrowheads; open arrowheads indicate residual signals from the
preceding staining. (Bottom) Precipitates from anti-Rel
immunoprecipitations were probed with anti-Rel antibodies, as
indicated. The position of precipitated immunoglobulin is indicated by
open arrowheads. (B) Coimmunoprecipitation/supershift (IP-shift)
analysis of I B-bound Rel factors. (Top) Namalwa cells; (bottom) HeLa
cells. Immunoprecipitation was carried out with whole-cell extracts and
antibodies directed against I B , I B 1, or I B 2. Bound
NF- B/Rel factors were eluted from the pellets by deoxycholate
treatment and analyzed for subunit composition by EMSA and
supershifting/inhibition with NF- B/Rel-specific antibodies.
Antibodies against I B , I B 1, or I B 2 precipitated
NF- B/Rel DNA binding activity (lanes 3, 8, and 13), which
was strongly reduced or absent when the epitope-containing peptides
were included in the precipitation reaction mixture or when the
preimmuneserum was used (lanes 2, 7, and 12). DNA binding
activity obtained from I B (lanes 2 to 6), I B 1 (lanes 7 to
11), or I B 2 (lanes 12 to 16) was challenged with anti-p65
antibody (lanes 4, 9, and 14), with anti-p65 antibody and peptide
competition (lanes 5, 10, and 15), or with anti-p50 antibody (lanes 6, 11, and 16), as indicated. In lanes 1, no protein was added. Free DNA
is not shown. Supershifted complexes are indicated (S). (C)
Phosphorylation-dependent interaction of I B 1 and I B 2 with
NF- B in HeLa cells. Whole-cell extracts were subjected to
immunoprecipitation with antibodies against I B , I B 1, or
I B 2 (lanes 1 to 3, 4 to 6, and 7 to 9, respectively). The beads
were treated with shrimp alkaline phosphatase (SAP; lanes 2, 5, and 8),
heat-inactivated phosphatase (hi SAP; lanes 3, 6, and 9), or buffer
alone (lanes 1, 4, and 7), and the supernatants were tested by EMSA
with the H2K binding-site probe. (D) I B , I B 1, and I B 2
equally inhibit NF- B reporter gene activation by p65. HeLa cells
were transfected with 2× B-Luc reporter without (open bars) or with
(solid bars) p65 expression construct. I B , I B 1, or
I B 2 expression constructs were cotransfected in increasing
amounts, as indicated. The mean values from four independent
transfections are shown.
|
|
Conversely, Rel factors bound to the three I

B molecules were
analyzed by immunoprecipitation with I

B-specific antibodies,
followed by detergent elution of the precipitated NF-

B/Rel
factors
and their identification by antibody supershifting in gel
retardation
assays (Fig.
3B). In Namalwa cells (upper panel) and HeLa
cells
(lower panel), the DNA binding complexes retrieved from an
anti-I

B

-immunoprecipitation
were inhibited and supershifted only
partially with an anti-p65
antibody but almost completely with an
anti-p50 antibody (compare
lanes 3 with lanes 4 and 6). In striking
contrast, DNA binding
activity derived from I

B

1 complexes was
inhibited or supershifted
completely with an anti-p65 antibody but only
faintly with an
anti-p50 antibody (compare lanes 8 with lanes 9 and
11). The same
was observed for DNA binding complexes derived from
I

B

2 (lanes
13 to 16). In no case were there any significant
differences between
Namalwa or HeLa cells. Thus, I

B

1 and
I

B

2 are associated almost
exclusively with p65-containing
heteromeric complexes and presumably
to a larger extent with p65
homodimers, whereas I

B

associates
predominantly with heteromers
containing p50.
The association of cellular I

B

activity with NF-

B depends
on constitutive phosphorylation (
22). The subunit
composition
of the NF-

B DNA binding activity released from HeLa
cell cytosol
by alkaline phosphatase treatment was similar to that
observed
with the I

B

-coprecipitated NF-

B activity.
Phosphatase, but
not heat-inactivated enzyme, released a

B
site-specific band
shift activity, which was completely
supershifted or inhibited
by anti-p65 antibody but was only very weakly
affected by anti-p50,
anti-p52, or anti-c-Rel antibodies (data not
shown). To assess
possible differences in the role of basal
phosphorylation for
the Rel/NF-

B association of both I

B

isoforms, immune complexes
of I

B

, I

B

1, or I

B

2
precipitated from HeLa cells were treated
with alkaline phosphatase and
the supernatants were tested by
EMSA (Fig.
3C). Dephosphorylation
released NF-

B activity from
both I

B

1 or I

B

2 but not
from I

B

complexes (lanes 2, 5, and
8). No DNA binding activity
was obtained with heat-inactivated
enzyme (lanes 6 and 9). Therefore,
the structural difference between
the I

B

isoforms does not affect
the phosphorylation-dependent
interaction with NF-

B.
The similar interaction of cellular I

B

1 or I

B

2 with
NF-

B suggests that they should interfere equally with
NF-

B-dependent
gene activation. When I

B

, I

B

1, or
I

B

2 was cotransfected
into HeLa cells with p65 and an NF-

B
dependent reporter, all
three inhibitors strongly reduced p65-driven
activation in an
almost identical fashion (Fig.
3D). The low-level
reporter gene
activation by endogenous NF-

B in the absence of
transfected p65
was also similarly decreased in the presence of the
three I

Bs.
Thus, I

B

2 is functional and has the same
NF-

B/Rel specificity
as I

B

1.
I
B
1 and I
B
elicit a similar signal responsiveness,
whereas I
B
2 is less responsive or even resistant to
signal-induced degradation.
The sequence difference between
I
B
1 and I
B
2 affects the PEST sequence, which plays a
role in signal-dependent degradation in human I
B
(7, 8, 38,
43). To investigate potential differences in signal inducibility,
HeLa, Namalwa, or Jurkat cells were stimulated with TNF-
,
PMA/ionomycin, or IL-1
for various times and analyzed for nuclear
NF-
B accumulation and degradation of I
B
, I
B
1, and
I
B
2 (Fig. 4). An image quantitation
of the steady-state amounts of I
B shown in the
Western blots is depicted graphically (Fig. 4, bottom panels).
TNF-
or IL-1
stimulation led to a rapid translocation
of NF-
B p50-p65 and of p65 homodimers, irrespective of the cell
type. In contrast, PMA acted rapidly in Namalwa and Jurkat cells
but only slowly in HeLa cells, indicating cell type differences in the
PMA/Ca2+ induction mechanism. Similarly, the induction of
NF-
B by PMA required 30 to 45 min and was dependent on de novo
protein synthesis in Hl60 cells but was fast in 70Z/3 cells
(18). The kinetics of nuclear translocation of NF-
B
was most often paralleled by a synchronous degradation of I
B
and
of I
B
1. The relative decrease in steady-state amounts, however,
was greatest for I
B
, closely followed by I
B
1. In contrast,
I
B
2 was much less responsive and in some cases even resisted
degradation. The identical degradation kinetics suggest that all three
inhibitors were degraded by the same mechanism. In line with this
conclusion, the delayed activation of NF-
B in PMA-stimulated
HeLa cells coincided with simultaneous degradation of I
B
and
I
B
1 at the same delayed time point, while I
B
2 seemed not to
respond (Fig. 4A). Thus, the relative amount of I
B
2 produced by
alternative splicing provides a means of limiting the fraction of
I
B
-bound NF-
B that is released after cellular stimulation.

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FIG. 4.
Different efficiencies of signal-induced proteolytic
degradation of I B 1 and I B 2. EMSA (top) and Western blots
(whole-cell extracts) (middle) are shown. (A) HeLa cells; (B) Namalwa
cells; (C) Jurkat cells. Stimulation of cells with TNF- , PMA
plus ionomycin, or IL-1 was carried out for 0, 15, 30, 90, 180, or
300 min, as indicated. The positions of I B , I B 1, and
I B 2 in the Western blots are indicated. (Bottom) Densitometric
quantitation of I B 1 (open circles), I B 2 (triangles), and
I B (dots). The steady-state amounts of the three I Bs after
increasing times of stimulation are plotted, with the amounts at 0 min
arbitrarily set at 1.0. Activated NF- B consisted of p65
homodimers and p50-p65 as determined by antibody supershifting (data
not shown).
|
|
Only I

B

was significantly and consistently resynthesized within
the experimental period (Fig.
4). Resynthesis of I

B

1 and
I

B

2 was only partially observed in PMA-stimulated Namalwa cells
(Fig.
4B). Similarly, murine I

B

1 was partially resynthesized
after initial degradation following LPS stimulation of 70Z/3 pre-B
cells (
36). The longer time required to fill up the
cytosolic
I

B

pool by de novo synthesis may therefore explain the
persistent
activation of nuclear NF-

B, as has been proposed by
Thompson
et al. (
37). In line with this, the slow and
persistent activation
of NF-

B by 2-deoxyglucose or
brefeldin A, agents that promote
the accumulation of proteins
in the endoplasmic reticulum (
30),
is caused by reduced
levels of both I

B

1 and I

B

2, but not of
I

B

, at 24 h after drug administration (data not shown).
I
B
1 and I
B
2 are basally phosphorylated but differ in
their nuclear accumulation in B cells.
Since I
B
1 and
I
B
2 differ in their PEST sequence, which is a target of
constitutive phosphorylation of I
B
and of I
B
(4,
11), we analyzed the effect of alkaline phosphatase treatment on
the electrophoretic mobility of both isoforms in SDS-PAGE (Fig. 5A). Both I
B
1 and I
B
2
revealed increased mobility after treatment with alkaline phosphatase
but not after treatment with heat-inactivated enzyme. The same
result was obtained with HeLa or Namalwa cell-derived proteins. Thus,
both I
B
isoforms are constitutively phosphorylated. This finding
is also in agreement with the phosphatase-mediated release of
NF-
B activity from both isoforms (Fig. 3C).

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FIG. 5.
(A) Constitutive phosphorylation of I B 1 and
I B 2. Cytosolic extracts of Namalwa (lanes 1 to 3) or HeLa (lanes
4 to 6) cells were either untreated (lanes 1 and 4), treated with
shrimp alkaline phosphatase (SAP; lanes 2 and 5), or heat-inactivated
phosphatase (hi SAP; lanes 3 and 6) and subjected to Western blot
analysis with anti-I B (N-20) antibody. (B) Nuclear localization
of I B 1, but not of I B 2, in B cells. Whole-cell extracts
(WCE), S100 extracts (S100), or nuclear extracts (NE) of Namalwa cells
(top) or HeLa cells (bottom) were analyzed in Western blots with
antibodies against I B 1 or I B 2 as indicated. (C) Nuclear
I B 1 in B cells is bound to dimers containing p65. IP-shift
analysis (as described in the legend to Fig. 3B) with nuclear extracts
of Namalwa (lanes 1 to 4) or IM9 (lanes 5 and 6) B cells. An antibody
specific for I B 1 (C-20) precipitated an NF- B activity
which was challenged with antibodies directed against p65 or p50 (lanes
2 and 3). An antibody directed against I B 2 [ (C)] did not
precipitate NF- B (lane 4). C-20, but not (C), precipitated
NF- B activity from IM9 cells (lanes 5 and 6). (D) Nuclear
I B 1 is constitutively phosphorylated. (Left) Namalwa cells,
either untreated or stimulated with PMA for 5 h, were fractionated
into S100 extracts and nuclear extracts (NE) and immunoblotted with
anti-I B 1 (lanes 1 to 4) or anti-I B 2 antibodies (lanes 5 to
8), as indicated. (Right) Nuclear extracts of unstimulated (lanes 1 to
3) or PMA-stimulated (lanes 4 to 6) Namalwa cells were treated with
shrimp alkaline phosphatase (SAP; lanes 2 and 5) or
heat-inactivated SAP (hi SAP; lanes 3 and 6) or left untreated (lanes 1 and 4) and analyzed for I B 1 migration in a Western blot.
|
|
I

B molecules are normally confined to the cytoplasm, except for the
transient poststimulation nuclear accumulation of I

B
(
1,
46) or murine I

B

(
36). When the subcellular
distribution
of I

B

1 and I

B

2 in unstimulated HeLa cells was
compared, both
forms were detected exclusively in the cytoplasm (Fig.
5B, bottom
panel). In unstimulated Namalwa B cells however, I

B

1,
but not
I

B

2 was found to some extent in the nucleus (top panel).
Since
I

B

1 and I

B

2 are almost equally abundant in Namalwa
cells (Fig.
2C), the observed differential nuclear accumulation should
be
due to the difference in the PEST sequence of the two forms.
We analyzed NF-

B/Rel proteins bound to nuclear I

B

1 by
IP-shift analysis (Fig.
5C). From both Namalwa and IM9 B cells, the
antibody specific for I

B

1 (lanes 1 and 5) but not the antibody
specific for I

B

2 (lanes 4 and 6) precipitated an
NF-

B DNA binding
activity. This activity was completely
inhibited by anti-p65 antibody
and to a large extent also by
anti-p50 antibody (compare lane
1 with lanes 2 and 3). Thus,
nuclear I

B

1 in B cells is associated
predominantly with
p65-containing hetero- and homodimers. In contrast
to the two
B-cell lines, nonlymphoid cell lines, devoid of constitutive
NF-

B/Rel activity, contain exclusively cytoplasmic
I

B

1 and
I

B

2. As observed with HeLa cells, HL60
promyelocytes, SCH and
MKN-1 stomach carcinoma cells, and T98G
glioblastoma cells did
not have nuclear I

B

1 (data not shown).
Hypophosphorylated murine I

B

1, generated after a long
stimulation, migrates to the nucleus, presumably because it does not
shield the nuclear localization signal of p65 (
36). To
investigate
potential differences in the phosphorylation status of
nuclear
and cytosolic I

B

isoforms, subcellular extracts of
untreated
or PMA-stimulated Namalwa cells were treated with alkaline
phosphatase
(Fig.
5D, left panel). Nuclear I

B

1 revealed a faster
migration
after PMA stimulation, indicating hypophosphorylation (lanes
1
and 2), whereas cytoplasmic I

B

1 was observed as a double band
before and after stimulation (lanes 3 and 4). In contrast, I

B

2
was completely retained in the cytoplasm (lanes 7 and 8) and could
not
be detected in the nucleus after PMA stimulation (lanes 5
and 6).
Alkaline phosphatase treatment of nuclear I

B

1 before
stimulation
caused a mobility shift in extracts of nonstimulated
cells (Fig.
5D,
right panel), whereas after PMA stimulation a
weaker mobility shift was
observed (lanes 1 to 6). Thus, nuclear
I

B

1 in Namalwa cells is
constitutively phosphorylated before
and after a long stimulation but
the extent of phosphorylation
decreases after PMA treatment. Nuclear
localization of I

B

1 in
Namalwa and IM9 cells but not in HeLa
cells could be functionally
coupled to the mechanism of
maintaining constitutive NF-

B activity
in B cells.
Role of the amino-terminal and carboxy-terminal domains of I
B
for NF-
B/Rel interaction, DNA binding inhibition, and basal
phosphorylation.
In both I
B
and NF-
B-p105, an acidic
sequence, which is part of the PEST sequence, is required for a
high-affinity interaction with NF-
B (17). I
B
has two acidic regions, one after the third ankyrin repeat and one as
part of the PEST sequence (20, 37). To address the role of
the carboxy-terminal PEST sequence in the interaction with NF-
B,
FLAG-tagged wild-type I
B
or I
B
mutants lacking either
amino-terminal, carboxy-terminal, or both sequences (
N,
C, and
NC) were cotransfected with p65 into HeLa cells (Fig.
6A). Immunoprecipitation with an anti-p65
antibody and blotting with an anti-FLAG antibody revealed that none of the sequences before the first and after the sixth ankyrin repeat is
required for binding to NF-
B (lanes 1 to 5). This is in contrast to I
B
, which requires carboxy-terminal sequences for
high-affinity interaction (19). Despite equal affinities
under immunoprecipitation conditions, marked differences were observed
in the ability of the I
B
mutants to inhibit the DNA binding of
NF-
B (Fig. 6B). Whereas wild-type I
B
and I
B
N
effectively inhibited DNA binding of p65 homodimers and heterodimers of
transfected p65 and endogenous p50 (compare lane 3 with lanes 4 and 5),
I
B
C inhibited only p65 dimers but not heteromeric NF-
B
(lane 6). Surprisingly, I
B
NC was even unable to inhibit either
homodimeric or heterodimeric NF-
B (lane 7). Since I
B
C
and I
B
NC efficiently bound to NF-
B (Fig. 6A), a folding
problem in these mutants is unlikely. None of the I
B
mutants was
able to inhibit the DNA binding of transfected p50 homodimers (data not
shown). I
B
or I
B
N, but not I
B
C, was able to
inhibit the DNA binding of transfected p50-c-Rel (data not shown).
Thus, efficient DNA binding inhibition with less favored NF-
B
heteromers requires the presence of the carboxy-terminal sequences of
I
B
.

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FIG. 6.
(A) Interaction of I B deletion mutants with p65 in
transfected HeLa cells as shown by immunoprecipitation. p65 was
transfected alone (lane 1) or together with wild-type I B 2 or
I B 2 mutants lacking either the sequences before the first ankyrin
repeat ( N) or after the sixth repeat ( C) or both ( NC) (lanes 2 to 5). All constructs were equally well expressed (data not shown).
After immunoprecipitation with an anti-p65 antibody, coprecipitated
I B molecules were detected by immunoblotting. (B) DNA binding
inhibition assay. Whole-cell extracts of mock-transfected (M) HeLa
cells (lane 2), cells transfected with p65 alone (lane 3), or cells
cotransfected with p65 and either wild-type I B 2, I B 2 N,
I B 2 C, or I B 2 NC (lanes 4 to 7) were subjected to EMSA.
Lane 1 contains DNA probe assayed alone. The position of DNA complexes
containing p65 homodimers and p50-p65 is indicated. (C) I B 2 is
constitutively phosphorylated in the carboxy-terminal domain. S100
extracts of HeLa cells that were either mock transfected (lanes 1 to
3), transfected with FLAG-tagged I B 2 (lanes 4 to 6),
I B 2 N (lanes 7 to 9), or I B 2 C (lanes 10 to 12) were
left untreated (lanes 1, 4, 7, and 10) or incubated with shrimp
alkaline phosphatase (SAP; lanes 2, 5, 8, and 11) or heat-inactivated
enzyme (hi SAP; lanes 3, 6, 9, and 12) and blotted with anti-FLAG
antibody.
|
|
The ability of I

B

to interact with NF-

B depends on its
constitutive phosphorylation (
22), and recombinant
underphosphorylated
I

B

1 is impaired in its ability to inhibit DNA
binding of p65
or c-Rel (
11,
36,
39). When adding
bacterially expressed
I

B

2 to transfected NF-

B, we
observed that only p65 homodimers,
but not p65-p50 or c-Rel-p50, were
partially inhibited from binding
to DNA (data not shown).
Unphosphorylated bacterial I

B

2 is thus
similar to transfected
I

B


C (Fig.
5B). In addition, I

B

1 was
shown to be basally
phosphorylated in the carboxy-terminal PEST
sequence at residues Ser
313 and Ser 315 (
11), both of which
are conserved in
I

B

2 (there is one more CKII site in the PEST
sequence of
I

B

2 at Ser 325). Both transfected FLAG-tagged I

B
and
I

B

2

N revealed an increased mobility in SDS-PAGE after
phosphatase
treatment but not after incubation with inactivated
phosphatase
(Fig.
6C, compare lanes 4 to 6 with lanes 7 to 9). In
contrast,
I

B

2

C did not show a mobility shift (lanes 10 to 12).
We conclude,
therefore, that I

B

2 (like I

B

1) is
constitutively phosphorylated
at its carboxy-terminal PEST sequence.
 |
DISCUSSION |
One possibility to account for specific physiological activities
of NF-
B/Rel transcription factors is differential regulation of
I
B proteins. In this study, we show that transcripts generated from
the human I
B
gene are alternatively processed, yielding products
which are distinct in their signal responsiveness. Two isoforms,
I
B
1 and I
B
2, are found in human cells, whereas the murine
cell lines tested express only I
B
1. I
B
2 differs from I
B
1 by a substitution that replaces the carboxy-terminal PEST sequences with intron-derived sequences. We have analyzed the functional consequence of this difference in protein structure for
binding specificity and affinity to NF-
B Rel proteins, for signal-responsive degradation, basal phosphorylation, nuclear localization, and inhibition of NF-
B-dependent transcription.
All human cell lines tested expressed I
B
1 and I
B
2, the
latter often as the predominant form, whereas murine cells expressed only I
B
1. We cannot fully exclude the possibility that I
B
2 is expressed in murine cells at a low level or is confined to specific
cell types. We detected I
B
1 but not I
B
2 in murine liver,
lung, lymph node, thymus, ovary, and brain by Western blotting (data
not shown). It is possible that the I
B
2 carboxy-terminal sequence
is posttranslationally modified in murine cells and thus not recognized
by the antibody. Alternatively, splicing variants in murine cells may
contain less highly conserved intron-derived sequences not recognized
by the antibody directed against the human protein. Immunodepletion and
precipitation studies indicate that murine cells may contain additional
I
B
isoforms distinct from I
B
1 and I
B
2 (data not
shown). As a further possibility, specific functions of I
B
splicing isoforms in human cells could be compensated for in murine
cells by other modifications of I
B proteins yet to be identified.
We have previously shown that I
B
mRNA is expressed at some level
as two distinct signals (1.8 and 2.8 kb) in several human tissues analyzed, such as pancreas, kidney, skeletal muscle,
liver, lung, placenta, brain, and heart tissues (20). Based
on the lengths of the cloned cDNAs, I
B
2 should represent the
longer of the two transcripts. The N-terminal anti-I
B
antibody
recognized further signals in the transformed cell lines (Fig. 2C),
suggesting the existence of still further isoforms. Several additional,
independently isolated cDNA clones lacking an internal coding segment
are being investigated further.
Our results suggest that I
B
2 binds to the same cellular
NF-
B/Rel hetero- and homodimers as I
B
1, which are
predominantly p65 homodimers and heterodimers containing p65. In
contrast, I
B
interacted predominantly with heterodimers
containing p50, such as p50-p65. Heterodimers that were bound
with lower affinity, however, such as c-Rel containing dimers,
are possibly bound more efficiently by I
B
1 than by I
B
2. p65
homodimers were observed by EMSA after cellular stimulation and
degradation of I
B
1 and/or I
B
2 and are probably liberated
from complexes with I
B
(Fig. 4). Preferential association with
p65 homodimers was also evident when the affinity of I
B
to
NF-
B was weakened by truncation of the carboxy terminus. In DNA
binding inhibition assays (Fig. 6B), only p65 homodimers, not p65
heterodimers, were affected. Therefore, we assume that the
carboxy-terminal sequences are needed to increase the binding affinity
of I
B
to NF-
B whereas the specific recognition of
NF-
B is achieved by the ankyrin repeat domain. As observed for
I
B
(17), the binding affinity of I
B
may depend
on negatively charged residues contained in the PEST sequence. Basal
phosphorylation by CKII in the PEST sequence increases the negative
charge and could therefore enhance the interaction strength of I
B
with less favored heterodimers containing c-Rel (11). Band
shift assays are more stringent than immunoprecipitations under mild
detergent conditions, possibly due to a competition between
NF-
B-DNA and NF-
B-I
B complex formation. This can
provide the explanation why some interactions seen in one assay
(precipitation) may not be detected in the other (band shift).
Different stringencies in the experimental conditions may also
explain conflicting results obtained by Chu et al.
(11) and Suyang et al. (36). The former study reported that unphosphorylated I
B
can inhibit the DNA binding of p65 homodimers, whereas the latter reported that p65 forms a
complex with I
B
that still binds to DNA. Recombinant, underphosphorylated I
B
cannot bind to c-Rel and inhibit its DNA
binding (11). In this study, we showed that a
carboxy-terminal deletion mutant of I
B
can bind to p65 homodimers
and inhibit DNA binding but cannot inhibit the DNA binding of
heterodimers. We propose that the affinity of I
B
to
NF-
B/Rel dimers decreases from p65 homodimers to p65
heterodimers or c-Rel heterodimers and that stable binding to the
latter requires the negatively charged carboxy-terminal sequences of
I
B
, which are most optimal after phosphorylation.
Previously, it has been reported that I
B
and I
B
elicit
distinct responses to different inducers of NF-
B and that
I
B
is degraded only in response to LPS or IL-1 but not in
response to TNF-
or PMA (37). In I
B
-deficient
mice, constitutive NF-
B activity was observed only in cells of
hematopoietic origin, not in fibroblasts (6).
Furthermore, NF-
B could still be stimulated by TNF-
in fibroblasts, presumably involving degradation of I
B
(6). We show that TNF-
, PMA/Ca2+
ionophore, and IL-1 can all equally induce the degradation of I
B
and I
B
1 in three different human cell lines. The extent of
responsiveness varied among cell lines and stimuli, but the fact that
time points of the onset of proteolysis are the same for I
B
and
I
B
1 suggests that both are degraded by the same mechanism. It is
therefore possible that both are phosphorylated by the same kinase(s)
and degraded by a ubiquitin-dependent proteasome pathway (12,
25). In contrast, I
B
2 was barely degraded. This refractory
response is consistent with the truncated PEST sequence in I
B
2,
since this motif promotes efficient inducible degradation of I
B
.
Thus, I
B
2 can be seen as a naturally occurring dominant negative
I
B
isoform whose expression could generate a state of NF-
B
unresponsiveness to a variety of inducing agents.
An interesting and unexpected observation is the constitutive nuclear
accumulation of I
B
1 in Namalwa and IM9 B cells which was not
found in five different nonlymphoid cell lines tested. In these
experiments, equal cellular ratios of cytoplasmic and nuclear fractions
were used and the same fractions were analyzed for I
B
1 and
I
B
2. Since I
B
2 was not found in the nucleus, leakage or
cross-contamination of the fractions can be excluded. Nuclear I
B
was associated predominantly with p65, whereas the main constitutive
NF-
B activity in B cells consists of c-Rel-p50 (23, 26,
27). It is therefore possible that nuclear I
B
1 in B cells
masks p65 which might have been activated constitutively by the same
mechanism as c-Rel-p50. Nuclear I
B
has also been detected
recently in mature murine WEHI 231 B cells (32). It is
therefore likely that B lymphocytes in general contain nuclear I
B
. Consistent with the model proposed by Suyang et al. and Phillips and Ghosh (32, 36), the nuclear accumulation of
I
B
could not only account for the prolonged NF-
B
activation after stimulation of non-B cells with agents such as LPS but
could also directly contribute to the constitutive Rel activity in B
cells.
The fact that I
B
2 is localized exclusively to the cytoplasm
indicates that the specific carboxy-terminal sequences of I
B
1 are
required for nuclear accumulation. Since both I
B
1 and I
B
2, are constitutively phosphorylated and contain the CKII phosphorylation sites at Ser 313 and Ser 315 (11), it is unlikely that
hypophosphorylation of I
B
1 but not of I
B
2 accounts for
their differential nuclear uptake. The latter mechanism has been
proposed for nuclear translocation of I
B
1 after LPS stimulation
of 70Z/3 pre-B cells (36). Therefore, mechanisms other than
basal phosphorylation may account for nuclear uptake of I
B
1 in
human cells. These may include the association and import with
(modified) Rel factors or the presence of a conditional nuclear import
signal in I
B
1.
 |
FOOTNOTES |
*
Corresponding author. Mailing address:
Max-Delbrück-Center for Molecular Medicine MDC,
Robert-Rössle-Str. 10, 13122 Berlin, Germany. Phone:
49-30-9406-3816. Fax: 49-30-9406-3866. E-mail: scheidereit{at}mdc-berlin.de.
Present address: Second Department of Internal Medicine, Asahikawa
Medical College, Nishikagura 4-5-3, 078 Asahikawa, Japan.
 |
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Mol Cell Biol, May 1998, p. 2596-2607, Vol. 18, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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