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(Received for publication, October 26,
1995; and in revised form, January 16, 1996) From the
In the present study, the role of the I The NF- The role
of I Following inducer mediated stimulation, I In this study we have examined the role of the I
Figure 1:
Schematic representation of human
I
Figure 2:
Dissociation of NF-
In vivo, I
Figure 3:
I
Figure 4:
Tetracycline-responsive expression of
human I
Figure 5:
In vivo association of
I
Figure 6:
Inducer-mediated degradation of
I
Figure 7:
Analysis of I
Figure 8:
Stabilization of mutant I
In this study, we examined the biochemical and functional
properties of C-terminal deletions in I Recent studies by Brown et al.(33) demonstrated that residues Ser-32 or Ser-36 were
required for TNF- Sequences within the C terminus of
I Deletion of virtually the entire PEST domain in I 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 I 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 I In view of the predominantly
cytoplasmic localization of I 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
I
Volume 271,
Number 18,
Issue of May 3, 1996 pp. 10690-10696
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
B
in Protein Degradation
and Stabilization (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B
C terminus in
NF-B/IB regulation was examined in NIH 3T3 cells
engineered to inducibly express wild type or mutated human IB
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 IB
3),
including most of the PEST domain, were dispensible for IB
function. However, deletions from aa 261 to 317 or aa 269 to 317
(IB1 and IB2 respectively), lacked the
ability to dissociate NF-B/DNA complexes in vitro and
were unable to inhibit NF-B dependent transcription. Moreover,
IB1 and IB2 mutants were resistant to
inducer-mediated degradation. Analysis of IB deletions in the
presence of protein synthesis inhibitors revealed that, independently
of stimulation, IB1 and IB2 had a
half-life four times shorter than wild type IB and the
interaction of IB1 and IB2 with p65 was
dramatically decreased in vivo as measured by
co-immunoprecipitation. Interestingly, protease inhibitors which block
inducer-mediated degradation of IB also stabilized the
turnover of IB1 and IB2. 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 IB
from a constitutive protease activity.
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 IB proteins
that bind to and mask a nuclear translocation signal within the Rel
homology domain(3, 4) . A number of IB proteins
have been identified, all of which contain multiple ankyrin repeats,
including IB(5) , IB
(6) ,
IB
(7, 8) , Bcl-3(9, 10) ,
and the precursors p105 (11) and p100(12) .B 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 IB, although IB preferentially
associates with
p65(11, 14, 15, 16, 17, 18) .
IB 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 IB 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 IB, 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 (IB)
gene, thereby establishing an autoregulatory loop in which newly
synthesized IB restores the cytoplasmic pool of latent
NF-B(1, 21, 24, 25) . B becomes
hyperphosphorylated, detectable in immunoblots as a slowly migrating
form, sensitive to phosphatase treatment(23, 24) .
Hyperphosphorylation does not impair the ability of IB 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 IB following
TNF- stimulation has been demonstrated (13) and only
proteasome inhibitors have been shown to prevent IB
degradation induced by
TNF-(27, 28, 29, 31) .
Proteasome inhibitors such as PSI and MG115 prevent IB
degradation but not IB 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 IB(32, 33) .
Substitution of these residues prevents IB phosphorylation,
ubiquitination, and degradation(13) . Similarly, C-terminal
truncation of IB has been shown to prevent inducer-mediated
degradation(32, 33, 34, 35) .
However, the function of IB C terminus remains undefined. B C
terminus in inducer-mediated degradation. NIH 3T3-derived cell lines
were generated that express human wild type or mutant IB
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
IB constitutively unstable and diminish the association of
IB with p65. Stabilization of C-terminal IB mutants with
proteasome inhibitors, suggests that in unstimulated cells, the
C-terminal domain functions to protect IB from proteasome
action.
Plasmids
A cDNA encoding the hybrid
transactivator tTA (36) , a fusion protein between the
tetracycline repressor and the HSV VP16 transactivator, was inserted
between the HindIII and BamHI sites in the expression
plasmid pREP4 (Invitrogen). In the resulting plasmid (pPREP-4-tTA), tTA
transcription is controlled by the Rous sarcoma virus promoter, and tTA
activity is inhibited by tetracycline(36) , at concentrations
ranging from 0.1 to 1.0 µg/ml. Wild type IB cDNA was
cloned downstream of the tetracycline responsive promoter CMV
(36) and inserted into the pREP9 expression plasmid
between the XhoI and BamHI sites, therefore replacing
the existing Rous sarcoma virus promoter. Mutated IB
expression vectors were created by replacing wild type IB in
pREP-9-IB(wt) plasmid with deleted or mutated versions.
Briefly, the C-terminal deletion mutants IB(1),
IB(2), IB(3), and IB(4)
were generated by inserting an artificial stop codon in the human
IB gene at amino acid positions 261, 269, 288, and 296,
respectively. Cassettes encoding IB mutants were also
inserted in the mammalian expression vector pSVK3
(Pharmacia)(37) . IB(DM) is a full-length human
IB in which serine 283 and threonine 291 were substituted for
alanine residues; in IB(3C), serine 283, threonine 291, and
threonine 299 were also substituted for an alanine residue.Generation of tTA and I
Plasmid pREP4-tTA was introduced in NIH 3T3 cells by
lipofection (Lipofectamine, Life Technologies, Inc.) according to the
manufacturer's instructions. Cells were selected beginning at 48
h in Dulbecco's modified Eagle's medium containing 10% calf
serum and 300 µg/ml hygromycin B (Boeringer Mannheim). Resistant
cells carrying the pREP4-tTA plasmid (tTA-3T3 cells) were then
transfected with the various pREP9-CMVB-expressing Cell
Lines-IB
plasmids. Cells were selected and maintained in Dulbecco's
modified Eagle's medium containing 10% calf serum, 300 µg/ml
hygromycin B, and 400 µg/ml G418 (Life Technologies, Inc.).
Colonies of resistant cells (tTA-IB) were expanded
individually or pooled together to create polyclonal population. At all
times, cell lines were maintained in the presence of tetracyline (1
µg/ml) to repress exogenous IB expression.Expression and Phosphorylation of Recombinant
I
GST-IBB fusion proteins from Escherichia coli were isolated as described
previously(37, 38) . Phosphorylation of IB
recombinant proteins (2 ng) by CKII was performed for 30 min at 30
°C with 5 units of recombinant CKII enzyme (New England Biolabs) in
a buffer containing 25 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM MnCl
, 1 mM MgCl, and 10 mM ATP.Electromobility Shift Assay
Nuclear extracts (39) were prepared from tTA-3T3 cells treated 30 min with
TNF- (0.5 ng/ml) and subjected to electromobility shift assay
using an interferon- PRDII probe as described
previously(40) . To demonstrate the specificity of the
protein-DNA complex formation, 200-fold molar excess of either a
mutated PRDII oligonucleotide (Mut) or wild type PRDII oligonucleotide
(Wt) was added to the nuclear extract before addition of the PRDII
probe. To evaluate the DNA binding inhibitory activity of IB
or the various mutants, 2 ng of recombinant IB or mutant
IB was added to the extract 10 min before the probe was
added. Similarly, CKII phosphorylated recombinant IB proteins
(2 ng) were tested for their ability to inhibit NF-B DNA binding
activity.Inhibition of NF-
Using the calcium phosphate method(41) ,
N-Tera-2 cells were co-transfected with pHIV-CAT reporter plasmid (3
µg) along with CMV-p65 plasmid (3 µg) and various pSVK3
plasmids (Pharmacia) encoding wild type or mutated IB-dependent
TranscriptionB (9
µg). In the pHIV-CAT plasmid, the chloramphenicol acetyltransferase
gene is under the control of a minimal SV40 promoter fused to one copy
of the HIV-1 enhancer. CMV-p65 is a CMV promoter-driven expression
plasmid encoding NF-Bp65. Thirty hours after transfection, cells
were stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (Sigma).
Forty-eight hours after transfection, cells were harvested and lysed.
Protein extracts (100 µg) were then assayed during 4 h at 37 °C
for CAT activity.Immunoblot Analysis of I
tTA-IB
TurnoverB expressing cells cultured in
tetracycline-free Dulbecco's modified Eagle's medium
supplemented with 10% calf serum, were stimulated with 5 ng/ml
TNF- (Life Technologies, Inc.) or 10 µg/ml LPS (Sigma), either
with or without addition of 50 µg/ml cycloheximide. In some
experiments, cells were pretreated for 1 h with either 100 µM calpain inhibitor I (ICN), 40 µM MG132 proteasome
inhibitor (kindly supplied by MyoGenics Inc.)(31) , or an
equivalent volume (8 µl/ml) of their respective solvent (ethanol
and dimethyl formamide, respectively) as a control. Cells were washed
with phosphate-buffered saline and lysed in 10 mM Tris
Cl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1
mM dithiothreitol, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10
µg/ml pepstatin, 10 µg/ml aprotinin (WBL buffer). Equivalent
amounts of protein (15 µg) were electrophoresed on a 10%
SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and
IB was detected using affinity purified IB antibody
AR20(42) .Immunoprecipitation of p65 and I
Cells
were washed with phosphate-buffered saline and labeled for 60 min in
methionine-free RPMI 1640 containing 400 µCi/ml
TranB
S-label (Amersham). Cells were collected in 40 mM TrisCl, pH 7.5, 1 mM EDTA, pH 8.0, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride (TEN buffer) and
lysed in 20 mM TrisCl, pH 7.5, 200 mM NaCl,
0.5% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride (TNT
buffer). Cell lysates (300 µg) were incubated with 10 µl of
IB antibody (AR20) or p65 antibody (AR28) (43) and 30
µl of protein A-Sepharose beads (Pharmacia) for 3 h at 4 °C.
Beads were washed five times with TNT buffer and the immunoprecipitates
were eluted by boiling the beads 3 min in 1% SDS, 0.5%
-mercaptoethanol. The eluate was diluted with 1 volume of TNT
buffer and incubated overnight at 4 °C with 10 µl of
IB or p65 antibody. Beads were again washed five times with
TNT buffer and the immunoprecipitate was eluted by boiling the beads 3
min in SDS sample buffer. Eluted proteins were electrophoresed on 10%
SDS-polyacrylamide gel electrophoresis and detected by autoradiography.
The specificity of IB antibody recognition was confirmed by
competition with IB peptide (aa 2-16).
Inhibition of NF-
To investigate the role of the IBDNA Complex
FormationB C terminus
in NF-B/IB regulation, we generated a series of
C-terminal deletions of IB (Fig. 1). The IB
proteins were generated by inserting an artificial stop codon in the
human IB gene at aa 261(1), aa 269(2), aa
288(3), and aa 296(4), respectively. IB(DM)
represents full-length human IB in which the Ser-283 and
Thr-291 were substituted for alanine residues and IB(3C)
contains the S283A, T291A, and T299A substitutions (Fig. 1).
Wild type and C-terminal deletions of IB were examined for
their ability to inhibit NF-BDNA complex formation in an
electromobility shift assay. Extracts from tTA-3T3 cells stimulated
with TNF- for 30 min were analyzed for NF-B binding activity
using a 
P-labeled probe corresponding to the PRDII region
of the interferon promoter (Fig. 2). Addition of
recombinant wt IB, IB(4),
IB(3), IB(DM), and IB(3C) proteins
reduced the intensity of the NF-B/PRDII band more than 10-fold (Fig. 2, lanes 2-4, 7, and 8), whereas
addition of IB(1) or IB(2) had no affect
on NF-BDNA complex formation (Fig. 2, lanes 5 and 6). This result demonstrated that the region located
between aa 269 and 287 was important for inhibiting NF-BDNA
complex formation in vitro.
B and C-terminal deletion mutants. Human IB
contains five internal ankyrin repeats (SWI6/ANK) involved in
the binding to NF-B molecules. At the N-terminal of IB
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
IB 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 IB(1) (aa 1-260), IB(2)
(aa 1-268), IB(3) (aa 1-287), and
IB(4) (aa 1-295) are depicted. In mutant
IB(DM), Ser-283 and Thr-291 were substituted for alanines and
in IB(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.
BDNA
complexes by recombinant IB. 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-BDNA
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
IB (lanes 2 and 9), IB(4) (lanes 3 and 10), IB(3) (lanes 4 and 11), IB(2) (lanes 5 and 12), IB(1) (lanes 6 and 13),
IB(DM) (lanes 7 and 14), or
IB(3C) (lanes 8 and 15) were added to the
extracts prior to probe addition. The recombinant IB 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).
B
is constitutively phosphorylated at the C terminus by casein kinase
II(37, 43) . Several previous reports demonstrated
that the phosphorylation level of IB influenced the ability
of IB to dissociate NF-BDNA
complexes(15, 44, 45) . However, in vitro phosphorylation of wild type or mutant IB proteins with
casein kinase II (Fig. 2, lanes 9-15) did not
modulate the capacity of IB to inhibit NF-BPRDII
DNA complex formation in vitro.Inhibition of NF-
Next, the effect of IB-dependent Gene
ExpressionB C-terminal
truncations on the inhibition of NF-B dependent gene expression
was examined. NF-B activity was detected by measuring CAT activity
derived from pHIV-CAT reporter plasmid in N-Tera-2 cells which are
deficient in NF-B activity(46, 47) . The level of
NF-B was increased in these cells by co-expressing RelA and
treating with phorbol 12-myristate 13-acetate(33) . CAT
activity was observed only when p65 was co-expressed and this activity
was inhibited by excess wild type IB expression (Fig. 3). IB(4) and IB(3)
expression also repressed NF-B dependent CAT activity, whereas
IB(1) and IB(2) expression did not reduce
NF-B dependent activity (Fig. 3). These results
demonstrated that IB(1) and IB(2) were
unable to suppress p65 dependent transcription, which correlates with
their reduced ability to dissociate NF-B complexes in
vitro.
B 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 IB (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.
Tetracycline Control of I
To
analyze IB ExpressionB deletions in stably transformed cells,
IB expressing NIH-3T3 cells were generated using the
tetracycline responsive system (see ``Materials and
Methods''). Polyclonal tTA-IB cells, cultured in the
absence of tetracycline for 72 h were examined for exogenous
IB expression (Fig. 4A); IB(wt),
IB(DM), IB(1), IB(2),
IB(3), and IB(4) proteins were detected
with apparent molecular masses of 38, 38, 31, 32, 35, and 36 kDa,
respectively (Fig. 4A, lanes 2, 4, 6, 8, 10, and 12). Wild type human IB was distinguished from the
37-kDa murine homologue, due to a slight molecular weight difference (Fig. 4A, lanes 1 and 2). The human
IB proteins were expressed in polyclonal populations at
levels ranging from equivalent to the endogenous murine IB
(IB4) to levels 20-50-fold higher than the
endogenous IB (IB1). Exogenous IB
levels were reduced when cells were cultured in the presence of
tetracycline (1 µg/ml) for more than 24 h (Fig. 4A,
lanes 1, 3, 5, 7, 9, and 11), although the degree of
repression varied between cell lines. A representative analysis of
selected individual clones of IB(wt) and IB1
clones is shown in Fig. 4B.
B in NIH 3T3 cells. A, human IB was
detected by immunoblotting in extracts from polyclonal
tTA-IB(wt) (lanes 1 and 2),
tTA-IB(DM) (lanes 3 and 4),
tTA-IB(1) (lanes 5 and 6),
tTA-IB(2) (lanes 7 and 8),
tTA-IB(3) (lanes 9 and 10),
tTA-IB(4) (lanes 11 and 12) cells.
tTA-IB 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 IB and
exogenous human IB. B, individual clones of
tTA-IB(1) (lanes 1-6) and
tTA-IB(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 IB and
IB(1) are indicated.
Association of I
To determine if IB with NF-B p65 in
VivoB mutants could also associate
with p65 in vivo, co-immunoprecipitation studies were
performed with anti-p65 and anti-IB antibodies (Fig. 5). In tTA-3T3 cells, endogenous IB
co-precipitated with anti-p65 antibody (Fig. 5, lane 2)
and reciprocally, p65 co-precipitated with anti-IB antibody (Fig. 5, lane 4). IB peptide present in
excess during IB immunoprecipitation prevented subsequent p65
immunoprecipitation, demonstrating the specificity of antibody
recognition (Fig. 5, lane 5). In tTA-IB(wt)
cells, human IB was detected following two sequential
IB immunoprecipitations, and migrated just above the murine
IB band (Fig. 5, lane 8), whereas in
tTA-IB(1) cells, human IB(1) migrated
below murine IB (Fig. 5, lane 13). In both
cell lines, murine and human IB were co-immunoprecipitated
with anti-p65 antibody (Fig. 5, lanes 7 and 12) and p65 was co-immunoprecipitated by anti-IB (Fig. 5, lanes 9 and 14). The reaction was
specific since co-immunoprecipitation was abolished by the addition of
excess IB peptide (Fig. 5, lanes 10 and 15). As with wt IB, the majority of
IB(3) and IB(4) present in
tTA-IB cells was associated with p65 (Table 1).
However, the amount of IB(1) complexed to p65 was
significantly reduced compared with the total amount of
IB(1) present in the cell (Fig. 5, lanes 12 and 13). Similarly, only a small fraction of
IB(2) was associated with p65 (Table 1). These
results indicated that wt IB, IB(4),
-(3), and -(DM) (S283A,T291A) were stably associated with p65 in vivo, whereas the interaction of IB(1) and
IB(2) with p65 was unstable in vivo.
B with NF-B p65. Polyclonal tTA-3T3 (lanes
1-5), tTA-IB(wt) (lanes 6-10), and
tTA-IB(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 IB
specific antibody (lanes 3, 4, 5, 8-10, and 13-15). IB antibody recognition was competed
by the addition of excess IB 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 IB
antibody (lanes 2, 3, 7, 8, 12, and 13). Bands
corresponding to p65, murine IB, human IB(wt), and
IB(1) are indicated.
Inducer-mediated Degradation of I
To
examine the effect of C-terminal deletion on inducer-mediated
degradation of IBB, tTA-IB cells were treated with
TNF- (5 ng/ml) or LPS (10 µg/ml) and cell extracts were
analyzed for IB expression by immunoblotting ( Fig. 6and Table 1). Stimulation with TNF- or LPS
resulted in a rapid decrease and disappearance of murine and human
IB from 15 to 60 min after stimulation, followed by de
novo synthesis of IB (Fig. 6A and Table 1), as described
previously(1, 21, 24, 25) .
Inducer-mediated degradation of endogenous IB was observed in
all tTA-IB expressing cells (Fig. 6, B-F). In
response to either TNF- or LPS stimulation, IB(4),
IB(3), and IB(DM) degraded rapidly within
15-30 min (Fig. 6, B, C, F, and Table 1), whereas IB(1) and IB(2)
did not undergo inducer-mediated degradation (Fig. 6, D and E, and Table 1). To eliminate effects
associated with overexpression of exogenous IB,
tTA-IB(1) cells were cultured in the presence of
tetracycline (0.1 µg/ml) which reduced IB(1)
expression to levels equivalent to endogenous murine IB. This
experiment indicates that the region deleted in IB(2)
but present in IB(3), aa 269-287, apparently plays
a role in TNF- and LPS mediated degradation.
B. Polyclonal tTA-IB(wt) (A),
tTA-IB(4) (B), tTA-IB(3) (C), tTA-IB(2) (D),
tTA-IB(1) (E), and tTA-IB(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-IB(1) cells were cultured in the
presence of tetracycline (0.1 µg/ml) to reduce the level of
exogenous human IB. Endogenous murine and exogenous human
IB were detected in whole cell extracts (15 µg) by
immunoblotting using affinity purified AR20
antibody.
Intrinsic I
Since
inducer-mediated degradation of IB StabilityB does not require de
novo protein synthesis(21, 48) , the turnover
rate of IB in the absence (intrinsic stability) or presence
of stimulus (inducer mediated degradation rate) was measured in cells
treated with the protein synthesis inhibitor cycloheximide (50
µg/ml). tTA-3T3 and tTA-IB cells were stimulated with
TNF- for 2 h and levels of IB were measured and
quantified (Fig. 7). The intrinsic stability of human wt
IB was similar to that of murine IB (Fig. 7, A and B, lanes 1-6); without inducer, both
proteins had a half-life of approximately 2 h (summarized in Table 1). IB(3), IB(4), and
IB(DM) also had intrinsic stabilities similar to wt
IB (Fig. 7, C and D, lanes
1-6; Table 1). However, the C-terminal deletion in
IB(1) (Fig. 7E, lanes 1-6) and
IB(2) (Table 1) destabilized IB by
reducing IB half-life to approximately 30 min. Following
TNF- stimulation in the presence of cycloheximide, the half-life
of wt IB, IB(DM), IB(4),
IB(3), and murine IB was decreased to about 5
min (Fig. 7, A-D, lanes 7-12). In
contrast, the degradation rate of IB(2) and
IB(1) in the presence of TNF- was similar to their
respective intrinsic stabilities (Fig. 7E, lanes
7-12; Table 1). These experiments demonstrate that
deletion of the amino acid domain 269-287 desensitized
IB to TNF--mediated degradation and simultaneously
accelerated IB turnover in unstimulated cells. Similar
results were obtained when LPS was used as inducer (Table 1).
B turnover rate.
Polyclonal tTA-3T3 (A), tTA-IB(wt) (B),
tTA-IB(4) (C), tTA-IB(3) (D), and tTA-IB(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 IB were detected in whole cell extracts (15 µg) by
immunoblotting, using affinity purified AR20
antibody.
Protection of I
To determine if the reduced half-life of
IB Mutants by Protease
InhibitorsB(1) and IB(2) mutants was related to
inducer-mediated degradation and the proteasome pathway,
tTA-IB(wt), tTA-IB(1), and
tTA-IB(2) cells were pretreated for 1 h with calpain
inhibitor I or the MG132 proteasome inhibitor, both of which are known
to block inducer-mediated degradation of IB (Fig. 8).
Cells were then treated with cycloheximide for 1 h and the level of
IB remaining was determined by immunoblot and densitometric
analysis. In the presence of cycloheximide, the amount of wt
IB remaining at 60 min was approximately 65-70% of the
level at time 0; pretreatment of wt IB expressing cells with
protease inhibitors did not significantly increase the amount of
remaining wt IB (60-80%). In contrast, after
cycloheximide treatment for 1 h, the level of IB in
IB(1) and IB(2) expressing cells was
reduced 5-20% of the initial level, reflecting the rapid
degradation of the 1 and 2 deletion mutants. However, in the
presence of calpain inhibitor I or MG132, both IB(1) and
IB(2) were dramatically stabilized and did not degrade
significantly during the period of cycloheximide treatment. These
results imply that IB(1) and IB(2) have a
reduced half-life because they are constitutively degraded by proteases
active during inducer-mediated degradation.
B by
peptide aldehydes. tTA-IB(wt), tTA-IB(1), and
IB(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 IB remaining at the end of the
1-h cycloheximide treatment was determined by immunoblot analysis and
compared to the amount of IB at time 0. The amount of wt
IB (open bar), IB(2) (solid
bar), and IB(1) (hatched bar) after a 1-h
cycloheximide treatment is illustrated
graphically.
B with respect to
intrinsic protein stability, inducer-mediated degradation, dissociation
of NF-BDNA complexes and association with p65 (RelA) in
vitro and in vivo. Our results demonstrate that: 1) the
C-terminal end of IB from aa 288 to 317 which includes most
of the PEST domain is apparently dispensable for function since
IB(3) and IB(4) behave like wt
IB; 2) deletion of the region between aa 269 and 287
(IB2) abolishes responsiveness to TNF- and
LPS-mediated degradation; 3) IB1 and IB2
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-BDNA complexes in vitro, for strong interaction
with p65 in vivo, and for efficient repression of NF-B
dependent transcription.-mediated phosphorylation and degradation of
IB, while Brockman et al.(32) further
showed that S32A or S32A/S36A substitutions fully protected
IB 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 IB. These studies thus support a model in
which IB is phosphorylated on residues Ser-32 and/or Ser-36
in response to multiple inducers and these phosphorylation events
target IB for ubiquitination and subsequent degradation by
the proteasome (13) .B also play a role in inducer-mediated
degradation(33, 34, 35) . Whiteside et
al.(35) demonstrated that IB deletion from aa
279-317 abolished TNF- and LPS-mediated
degradation(35) . Our deletion studies also demonstrate that
IB deleted from aa 269-317 (IB2) had a
similar phenotype, whereas IB deleted from aa 288-317
had a phenotype indistinguishable from wt IB, as measured in
several functional assays. Therefore, based on these two studies, a
C-terminal domain involved in IB 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 IB
activity(37) . Residues Glu-284, Asp-285, or Glu-286 were
previously identified as being critical for the dissociation of
NF-BDNA 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 IB(37) . B(3)
did not alter IB intrinsic stability or responsiveness to
inducer-mediated degradation, since this mutant behaved like wt
IB 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 IB stability from t
B from aa
269 to 287 may strengthen interaction with p65 in vivo.
Biochemical characterization of the domain structure of IB
demonstrated that IB 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
IB was bound to p65, suggesting that this region directly
interfaced with p65 and was thus masked in the IB-p65
complex.B(1) and (2) but not wt IB. Chen et al.(13) showed that deletion of the C terminus did
not prevent IB ubiquitination since the 243-317
mutant could still be ubiquitinated in vitro. We therefore
propose that in unstimulated cells, the C terminus protects
IB from constitutive proteasome-mediated degradation via p65
interaction. Upon stimulation, phosphorylation at the N terminus
abolishes protection by the C terminus and targets IB for
ubiquitination and degradation.B, the biological significance
of NF-BDNA complex dissociation by IB is not yet
understood, although IB has previously been identified in the
nucleus(49) . Furthermore, in vitro transcription
studies using purified NF-B proteins demonstrated that addition of
recombinant IB to the transcription reactions inhibited
NF-B dependent transcription(17, 38) . These
experiments suggest that a novel nuclear role for newly synthesized
IB may be to directly inhibit NF-B dependent gene
expression by dissociating NF-BDNA transcription complexes.
This idea is supported by the recent observation that following
induction de novo synthesized IB protein transiently
appeared in the nucleus and negatively regulated NF-B dependent
transcription(50) .B(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
IB regulation of NF-B activity will require further
characterization of the kinase activity involved in signal induced
phosphorylation of IB.
), tumor necrosis
factor-; LPS, lipopolysaccharide; CAT, chloramphenicol
acetyltransferase; aa, amino acid(s); wt, wild type.
We thank Drs. M. Gossen and H. Bujard for the
tetracycline responsive plasmids and helpful information about
selection of cells, as well as Dr. A. Cochrane for helpful discussions.
We are grateful to MyoGenics Inc. for MG132. We also thank Normand
Pepin for excellent technical assistance.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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