(Received for publication, October 26, 1995; and in revised form, January 16, 1996)
From the
In the present study, the role of the IB
C terminus in
NF-
B/I
B
regulation was examined in NIH 3T3 cells
engineered to inducibly express wild type or mutated human I
B
proteins under the control of the tetracycline responsive promoter.
Deletion studies demonstrated that the last C-terminal 30 amino acids
(amino acids (aa) 288 to aa 317, deleted in I
B
3),
including most of the PEST domain, were dispensible for I
B
function. However, deletions from aa 261 to 317 or aa 269 to 317
(I
B
1 and I
B
2 respectively), lacked the
ability to dissociate NF-
B/DNA complexes in vitro and
were unable to inhibit NF-
B dependent transcription. Moreover,
I
B
1 and I
B
2 mutants were resistant to
inducer-mediated degradation. Analysis of I
B
deletions in the
presence of protein synthesis inhibitors revealed that, independently
of stimulation, I
B
1 and I
B
2 had a
half-life four times shorter than wild type I
B
and the
interaction of I
B
1 and I
B
2 with p65 was
dramatically decreased in vivo as measured by
co-immunoprecipitation. Interestingly, protease inhibitors which block
inducer-mediated degradation of I
B
also stabilized the
turnover of I
B
1 and I
B
2. Based on these
studies, we propose that in the absence of stimulation, the C-terminal
domain between aa 269 and 287 may play a role to protect I
B
from a constitutive protease activity.
The NF-B/Rel transcription factors participate in the
activation of immune regulatory genes including cytokines, cell surface
receptors, and acute phase proteins, as well as the HIV-1 (
)long terminal repeat (for review, see (1) and (2) ). NF-
B/Rel proteins are present in most cell types in
an inactive cytoplasmic form, complexed to inhibitory I
B proteins
that bind to and mask a nuclear translocation signal within the Rel
homology domain(3, 4) . A number of I
B proteins
have been identified, all of which contain multiple ankyrin repeats,
including I
B
(5) , I
B
(6) ,
I
B
(7, 8) , Bcl-3(9, 10) ,
and the precursors p105 (11) and p100(12) .
The role
of IB
in the regulation of NF-
B DNA binding activity has
been extensively studied (for review, see (13) ). All
NF-
B/Rel heterodimers, as well as p65 and c-Rel homodimers can
interact with I
B
, although I
B
preferentially
associates with
p65(11, 14, 15, 16, 17, 18) .
I
B
has a half-life of 1-2 h when complexed with
NF-
B but is less stable when free in the
cytoplasm(19, 20, 21) . The short half-life
of I
B
may be due to the presence of a C-terminal domain rich
in proline, glutamic acid, serine, and threonine amino acids called the
PEST domain(5, 22) . Activating agents, such as double
strand RNA, phorbol esters, tumor necrosis factor-
(TNF-
),
interleukin-1, and lipopolysaccharide (LPS), accelerate the degradation
of cytosolic I
B
, thereby promoting release and nuclear
translocation of NF-
B/Rel
dimers(1, 2, 3, 4, 23) .
Nuclear NF-
B/Rel dimers transactivate target gene expression,
including transcriptional up-regulation of the MAD3 (I
B
)
gene, thereby establishing an autoregulatory loop in which newly
synthesized I
B
restores the cytoplasmic pool of latent
NF-
B(1, 21, 24, 25) .
Following inducer mediated stimulation, IB
becomes
hyperphosphorylated, detectable in immunoblots as a slowly migrating
form, sensitive to phosphatase treatment(23, 24) .
Hyperphosphorylation does not impair the ability of I
B
to
associate with NF-
B but represents a signal for subsequent
degradation by the proteasome
pathway(26, 27, 28, 29) . Central to
the proteasome machinery is the ATP-dependent, 26 S multisubunit
protease, which can operate in a ubiquitin-dependent or independent
fashion (30) . Ubiquitination of I
B
following
TNF-
stimulation has been demonstrated (13) and only
proteasome inhibitors have been shown to prevent I
B
degradation induced by
TNF-
(27, 28, 29, 31) .
Proteasome inhibitors such as PSI and MG115 prevent I
B
degradation but not I
B
hyperphosphorylation, illustrating
that these two events are independent(29, 31) . Recent
studies demonstrate that phosphorylation of the N-terminal serine
residues Ser-32 and/or Ser36 is the signal that leads to rapid
inducer-mediated degradation of I
B
(32, 33) .
Substitution of these residues prevents I
B
phosphorylation,
ubiquitination, and degradation(13) . Similarly, C-terminal
truncation of I
B
has been shown to prevent inducer-mediated
degradation(32, 33, 34, 35) .
However, the function of I
B
C terminus remains undefined.
In this study we have examined the role of the IB
C
terminus in inducer-mediated degradation. NIH 3T3-derived cell lines
were generated that express human wild type or mutant I
B
proteins under the control of a tetracycline responsive
promoter(36) . We demonstrate that C-terminal deletions from aa
269 to 287 abolish inducer-mediated degradation by rendering
I
B
constitutively unstable and diminish the association of
I
B with p65. Stabilization of C-terminal I
B
mutants with
proteasome inhibitors, suggests that in unstimulated cells, the
C-terminal domain functions to protect I
B
from proteasome
action.
Figure 1:
Schematic representation of human
IB
and C-terminal deletion mutants. Human I
B
contains five internal ankyrin repeats (SWI6/ANK) involved in
the binding to NF-
B molecules. At the N-terminal of I
B
are two phosphorylation sites (Ser-32 and Ser-36), shown previously to
play a role in inducer-mediated degradation(32, 33) .
A region rich in proline, serine, threonine, and glutamic acid, the
PEST domain, spans aa 264-317; the C-terminal region of
I
B
between aa 251 and 317 is expanded below the schematic to
show the one-letter amino acid sequence. The C-terminal ends of the
deletions I
B
(
1) (aa 1-260), I
B
(
2)
(aa 1-268), I
B
(
3) (aa 1-287), and
I
B
(
4) (aa 1-295) are depicted. In mutant
I
B
(DM), Ser-283 and Thr-291 were substituted for alanines and
in I
B
(3C), Thr-299 was also substituted for alanine. The
C-terminal region involved in degradation (aa 279-287) is
indicated in bold letters. The boundary aa 279 was determined
in (35) ; the boundary aa 287 was determined in this
study.
Figure 2:
Dissociation of NF-B
DNA
complexes by recombinant I
B
. Nuclear extracts from tTA-3T3
cells (5 µg) stimulated for 30 min with TNF-
were incubated
with
P-labeled probe (0.2 ng) corresponding to the
interferon-
PRDII region(55) . The NF-
B
DNA
complex was visualized on native 5% polyacrylamide gel (lane
1). The specificity of the complex formation was tested by adding
a 200-fold molar excess of unlabeled wild type or mutated PRDII double
stranded DNA to the reaction, prior to labeled probe addition (data not
shown, see ``Materials and Methods''). Recombinant wt
I
B
(lanes 2 and 9), I
B
(
4) (lanes 3 and 10), I
B
(
3) (lanes 4 and 11), I
B
(
2) (lanes 5 and 12), I
B
(
1) (lanes 6 and 13),
I
B
(DM) (lanes 7 and 14), or
I
B
(3C) (lanes 8 and 15) were added to the
extracts prior to probe addition. The recombinant I
B
proteins
were either untreated (lanes 2-8) or phosphorylated in vitro with recombinant casein kinase II prior to addition
to the electromobility shift assay reactions (lanes
9-15).
In vivo, IB
is constitutively phosphorylated at the C terminus by casein kinase
II(37, 43) . Several previous reports demonstrated
that the phosphorylation level of I
B
influenced the ability
of I
B
to dissociate NF-
B
DNA
complexes(15, 44, 45) . However, in vitro phosphorylation of wild type or mutant I
B
proteins with
casein kinase II (Fig. 2, lanes 9-15) did not
modulate the capacity of I
B
to inhibit NF-
B
PRDII
DNA complex formation in vitro.
Figure 3:
IB
mediated repression of
NF-
B dependent transcription. N-Tera-2 cells were co-transfected
with pHIV CAT reporter plasmid (3 µg) along with the NF-
B p65
expression plasmid CMV-p65 (3 µg) and various SVK3-based plasmids
expressing wild type or mutant I
B
(9 µg) as indicated. At
30 h after transfection, cells were stimulated with phorbol
12-myristate 13-acetate and CAT activity was analyzed at 48
h.
Figure 4:
Tetracycline-responsive expression of
human IB
in NIH 3T3 cells. A, human I
B
was
detected by immunoblotting in extracts from polyclonal
tTA-I
B
(wt) (lanes 1 and 2),
tTA-I
B
(DM) (lanes 3 and 4),
tTA-I
B
(
1) (lanes 5 and 6),
tTA-I
B
(
2) (lanes 7 and 8),
tTA-I
B
(
3) (lanes 9 and 10),
tTA-I
B
(
4) (lanes 11 and 12) cells.
tTA-I
B
cells were cultured in the presence (lanes 1, 3,
5, 7, 9, and 11) or absence (lanes 2, 4, 6, 8, 10, and 12) of tetracycline (1 µg/ml). Arrows indicate bands corresponding to endogenous murine I
B
and
exogenous human I
B
. B, individual clones of
tTA-I
B
(
1) (lanes 1-6) and
tTA-I
B
(wt) (lanes 7-10) were grown in the
presence (lanes 1, 3, 5, 7, and 9) or absence (lanes 2, 4, 6, 8, and 10) of tetracycline (1
µg/ml). Bands corresponding to wt I
B
and
I
B
(
1) are indicated.
Figure 5:
In vivo association of
IB
with NF-
B p65. Polyclonal tTA-3T3 (lanes
1-5), tTA-I
B
(wt) (lanes 6-10), and
tTA-I
B
(
1) (lanes 11-15) were
metabolically labeled with [
S]methionine for 1
h. Cell lysates were immunoprecipitated with p65 specific antibody (lanes 1, 2, 6, 7, 11, and 12) or I
B
specific antibody (lanes 3, 4, 5, 8-10, and 13-15). I
B
antibody recognition was competed
by the addition of excess I
B
peptide (2 µg) to the
reaction (lanes 5, 10, and 15). Immunoprecipitates
were collected on protein A-Sepharose beads, washed stringently, and
boiled in 1% SDS, 0.5%
-mercaptoethanol. Supernatants were
collected and immunoprecipitated again with p65 antibody (lanes 1,
4-6, 9-11, 14, and 15) or with I
B
antibody (lanes 2, 3, 7, 8, 12, and 13). Bands
corresponding to p65, murine I
B
, human I
B
(wt), and
I
B
(
1) are indicated.
Figure 6:
Inducer-mediated degradation of
IB
. Polyclonal tTA-I
B
(wt) (A),
tTA-I
B
(
4) (B), tTA-I
B
(
3) (C), tTA-I
B
(
2) (D),
tTA-I
B
(
1) (E), and tTA-I
B
(DM) (F) cells were stimulated with TNF-
for 0 (lane
1), 15 (lane 2), 30 (lane 3), 60 (lane
4), 90 (lane 5), or 120 min (lane 6). Prior to
stimulation, tTA-I
B
(
1) cells were cultured in the
presence of tetracycline (0.1 µg/ml) to reduce the level of
exogenous human I
B
. Endogenous murine and exogenous human
I
B
were detected in whole cell extracts (15 µg) by
immunoblotting using affinity purified AR20
antibody.
Figure 7:
Analysis of IB
turnover rate.
Polyclonal tTA-3T3 (A), tTA-I
B
(wt) (B),
tTA-I
B
(
4) (C), tTA-I
B
(
3) (D), and tTA-I
B
(
1) (E) cells were
treated with cycloheximide (50 µg/ml) alone (lanes
1-6) or stimulated with TNF-
(5 ng/ml) in the presence
of cycloheximide (lanes 7-12) for 0 (lanes 1 and 7), 15 (lanes 2 and 8), 30 (lanes 3 and 9), 60 (lanes 4 and 10), 90 (lanes 5 and 11), and 120 min (lanes 6 and 12). Endogenous murine and exogenous
human I
B
were detected in whole cell extracts (15 µg) by
immunoblotting, using affinity purified AR20
antibody.
Figure 8:
Stabilization of mutant IB
by
peptide aldehydes. tTA-I
B
(wt), tTA-I
B
(
1), and
I
B
(
2) expressing cells were treated for 1 h with calpain
inhibitor I (100 µM) or MG132 proteasome inhibitor (40
µM). Ethanol and dimethyl formamide, which are solvents
for calpain inhibitor I and MG132, respectively, were added to the
cells as controls. Cells were then treated with cycloheximide for 1 h.
The percentage of exogenous I
B
remaining at the end of the
1-h cycloheximide treatment was determined by immunoblot analysis and
compared to the amount of I
B
at time 0. The amount of wt
I
B
(open bar), I
B
(
2) (solid
bar), and I
B
(
1) (hatched bar) after a 1-h
cycloheximide treatment is illustrated
graphically.
In this study, we examined the biochemical and functional
properties of C-terminal deletions in IB
with respect to
intrinsic protein stability, inducer-mediated degradation, dissociation
of NF-
B
DNA complexes and association with p65 (RelA) in
vitro and in vivo. Our results demonstrate that: 1) the
C-terminal end of I
B
from aa 288 to 317 which includes most
of the PEST domain is apparently dispensable for function since
I
B
(
3) and I
B
(
4) behave like wt
I
B
; 2) deletion of the region between aa 269 and 287
(I
B
2) abolishes responsiveness to TNF-
and
LPS-mediated degradation; 3) I
B
1 and I
B
2
mutants have a reduced intrinsic stability (t
15-30 min) and are constitutively degraded by
proteases that are inhibited by calpain inhibitor I and MG132; and 4)
the domain between aa 269 and 287 is required for dissociation of
NF-
B
DNA complexes in vitro, for strong interaction
with p65 in vivo, and for efficient repression of NF-
B
dependent transcription.
Recent studies by Brown et al.(33) demonstrated that residues Ser-32 or Ser-36 were
required for TNF--mediated phosphorylation and degradation of
I
B
, while Brockman et al.(32) further
showed that S32A or S32A/S36A substitutions fully protected
I
B
from HTLV-1 Tax mediated degradation. Furthermore, Chen et al.(13) demonstrated that S32A/S36A substitutions
and to a lesser extent S32A substitution, prevented TNF-
induced
ubiquitination of I
B
. These studies thus support a model in
which I
B
is phosphorylated on residues Ser-32 and/or Ser-36
in response to multiple inducers and these phosphorylation events
target I
B
for ubiquitination and subsequent degradation by
the proteasome (13) .
Sequences within the C terminus of
IB
also play a role in inducer-mediated
degradation(33, 34, 35) . Whiteside et
al.(35) demonstrated that I
B
deletion from aa
279-317 abolished TNF-
and LPS-mediated
degradation(35) . Our deletion studies also demonstrate that
I
B
deleted from aa 269-317 (I
B
2) had a
similar phenotype, whereas I
B
deleted from aa 288-317
had a phenotype indistinguishable from wt I
B
, as measured in
several functional assays. Therefore, based on these two studies, a
C-terminal domain involved in I
B
degradation is located
between aa 279 and 287: the sequence MLPESEDEE (outlined in bold in Fig. 1). This sequence contains one of the constitutive
casein kinase II phosphorylation sites SEDE identified
previously(37, 43, 51) , but mutation of the
Ser-283 and Thr-291 sites did not affect I
B
activity(37) . Residues Glu-284, Asp-285, or Glu-286 were
previously identified as being critical for the dissociation of
NF-
B
DNA complexes(51) . Given the number of acidic
amino acids in this short segment, it appears that the functional
activity of this part of the C-terminal domain relates to its highly
acidic nature. In fact, triple mutation of Ser-283, Thr-291, and
Thr-299 increases the intrinsic stability of I
B
(37) .
Deletion of virtually the entire PEST domain in IB
(
3)
did not alter I
B
intrinsic stability or responsiveness to
inducer-mediated degradation, since this mutant behaved like wt
I
B
in several biochemical and functional assays (summarized
in Table 1). However, deletion of the region adjacent to the PEST
domain between aa 269 and 287 decreased I
B
stability from t
Based on our observations and the recent study of
Sachdev et al.(53) demonstrating that C-terminal
mutations in chicken pp40 decreased the interaction with
p65(53) , we conclude that the region of IB
from aa
269 to 287 may strengthen interaction with p65 in vivo.
Biochemical characterization of the domain structure of I
B
demonstrated that I
B
contains a highly structured central
domain that is resistant to proteolysis and flexible N- and C-terminal
extensions that are sensitive to proteolytic digestion(54) .
The C-terminal region was protected from proteolysis up to aa 275 when
I
B
was bound to p65, suggesting that this region directly
interfaced with p65 and was thus masked in the I
B
-p65
complex.
Inducer-mediated degradation is inhibited by peptidyl
aldehydes such as MG132 and calpain inhibitor
I(13, 28) . In this study, we show that these
inhibitors also dramatically increased the intrinsic stability of
mutant IB
(
1) and (
2) but not wt I
B
. Chen et al.(13) showed that deletion of the C terminus did
not prevent I
B
ubiquitination since the
243-317
mutant could still be ubiquitinated in vitro. We therefore
propose that in unstimulated cells, the C terminus protects
I
B
from constitutive proteasome-mediated degradation via p65
interaction. Upon stimulation, phosphorylation at the N terminus
abolishes protection by the C terminus and targets I
B
for
ubiquitination and degradation.
In view of the predominantly
cytoplasmic localization of IB
, the biological significance
of NF-
B
DNA complex dissociation by I
B
is not yet
understood, although I
B
has previously been identified in the
nucleus(49) . Furthermore, in vitro transcription
studies using purified NF-
B proteins demonstrated that addition of
recombinant I
B
to the transcription reactions inhibited
NF-
B dependent transcription(17, 38) . These
experiments suggest that a novel nuclear role for newly synthesized
I
B
may be to directly inhibit NF-
B dependent gene
expression by dissociating NF-
B
DNA transcription complexes.
This idea is supported by the recent observation that following
induction de novo synthesized I
B
protein transiently
appeared in the nucleus and negatively regulated NF-
B dependent
transcription(50) .
The kinase activity responsible for
inducer-mediated phosphorylation of Ser-32 and/or Ser-36 in the
N-terminal signal response domain has not been identified. In light of
the identification of casein kinase II as the activity responsible for
constitutive phosphorylation at the C-terminal end of
IB
(37, 43) , it is a possibility that CKII
also phosphorylates at positions Ser-32 and/or Ser-36 which are
consensus CKII sites. However, at present no in vivo evidence
for CKII phosphorylation within the signal response domain has been
obtained. The resolution of the signaling events involved in
I
B
regulation of NF-
B activity will require further
characterization of the kinase activity involved in signal induced
phosphorylation of I
B
.