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INTRODUCTION |
The transcription factor NF-
B is an important regulator of
genes involved in immune and inflammatory responses, apoptosis, and cell proliferation (1, 2). The NF-B/Rel family of transcription factors are a family of proteins that homo- and heterodimerize through
a conserved Rel homology domain that consists of approximately 300 amino acids. The Rel homology domain of NF-B is responsible for
homo- and heterodimerization, DNA binding activity, and nuclear localization (3-5). A large number of stimuli can cause NF-B to
translocate from the cytoplasm to the nucleus, and activate target gene
transcription. Stimuli that can activate NF-B include proinflammatory cytokines, bacterial lipopolysaccharide, phorbol esters, okadaic acid, and viral infection (2, 6-8).
A group of inhibitory proteins belonging to the IB family regulate
NF-B activation by sequestering NF-B in the cytoplasm. IB
exerts its inhibitory effects by associating with the Rel homology
domain of NF-B proteins, effectively masking their nuclear localization signal (9-12). Although there are a number of IB proteins, IB
is the primary regulator of rapid signal induced activation of NF-B. Upon stimulation by a proinflammatory cytokine such as TNF,1 a signaling
cascade is initiated that results in the activation of the IB
kinases IKK1 and IKK2 (13-17). This leads to the rapid phosphorylation
of IB at the signal-induced phosphorylation sites, serine 32 and
serine 36 (18-21). Once phosphorylated, IB is polyubiquitinated
by the Ubc5/E3RSI
B ubiquitination enzyme
pair (22, 23) on lysine 21 and lysine 22 (24-26). Polyubiquitinated
IB is degraded by the 26 S proteasome, thus exposing NF-B's
nuclear localization signal and allowing NF-B to translocate to the
nucleus (27-29). Once in the nucleus NF-B activates transcription
of target genes including IB (6, 30). When the NF-B inducing
signal is removed the newly synthesized IB can suppress NF-B
activity by preventing it from binding to the genomic DNA, and
sequestering NF-B in the cytoplasm (31-33).
The regulation of IB is carried out mainly through
phosphorylation. Several phosphorylation sites have been identified on IB and they include the signal-induced IKK phosphorylation sites located at serine 32 and serine 36 (18-21), the constitutive CKII phosphorylation sites located in the carboxyl-terminal PEST domain (34,
35), the protein kinase C site located in Ankyrin repeat 6 (ank6) (36,
37), and a tyrosine phosphorylation site that can cause the
dissociation but not the degradation of IB from NF-B in Jurkat
T cells (38). Although extensive analysis of the
signal-dependent degradation of IB has been done,
little distinction has been made between the
signal-dependent and -independent degradation of free and
NF-B-bound IB. Therefore, we undertook a study to examine the
role of various phosphorylation sites on IB to determine their
effects on both the signal-dependent and -independent
degradation of free and NF-B-bound IB.
In this study, we characterize a full-length IB mutant that is
unable to associate with NF-B (designated as mutC) and exists as a
free molecule in the cell. We also show that distinct phosphorylation sites can directly influence the efficiency of ubiquitination and
subsequent degradation of free and NF-B-associated IB in the
presence or absence of stimuli. These results demonstrate the
complexity of IB regulation necessary to ensure that NF-B is
rapidly and specifically activated by a diverse group of stimuli.
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EXPERIMENTAL PROCEDURES |
Materials--
The proteasome inhibitor
clasto-lactacystine 
-lactone (-lactone) and the
ubiquitin hydrolase inhibitor ubiquitin aldehyde (Ubal) were purchased
from Boston Biochem Inc. Ubiquitin and cyclohexamide were purchased
from Sigma and okadaic acid was purchased from Life Technologies, Inc.
Antibodies against IB (c-21, sc-371), RelA/p65 (sc-109), and
IKK (H-470) were purchased from Santa Cruz Biotechnology. Antibodies
against the FLAG (M2) and HA (12CA5) tags were purchased from Eastman
Kodak Co. and Roche Molecular Biochemicals, respectively. TNF,
inorganic pyrophosphatase, and creatine phosphokinase were purchased
from Calbiochem.
Cell Culture--
HeLa cells, human embryonic kidney 293 cells,
and IB 
/ mouse embryo fibroblasts (MEF) (39) were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum in an atmosphere of 10% CO2, at 37 °C. 293 cells stably expressing Moloney gag and pol
(293gp) were maintained as above and selection was maintained with
blastacidin (20 µg/ml). TNF and -lactone were used at final
concentrations of 10 ng/ml and 10 µM, respectively, unless otherwise noted. Cyclohexamide was used at a final concentration of 75 µg/ml.
Plasmids and in Vitro Translation--
Mutants were generated by
polymerase chain reaction-based site-directed mutagenesis and confirmed
by sequencing. Wild-type (wt) murine IB, mutF (S283A, S288A,
T291A, S293A, and T296A), 3236 (S32A and S36A), M (mutF and 3236 mutations combined), mutC (T247A, S252A, T257A, S262A, and T263A), and
3236mutC (3236 and mutC mutations combined) were ligated into the
BamHI and HindIII sites of the pCMX-PL1
polylinker (40). PCMX-RelA/p65 was described previously (30). Wild-type
IB and the IB mutants were also ligated into the
pCLBabepuro retroviral vector. pCLBabepuro is a derivative of the
retroviral construct pBabepuro (41). The pCMX-PL1-IB constructs
and pCLBabepuro were cut with HindIII and EcoRI,
respectively, and then blunted with Klenow. Both were then cut with
BamHI and the resulting
BamHI/HindIII(blunt) IB fragment was
ligated into the BamHI/EcoRI(blunt) pCLBabepuro
retroviral vector.
In vitro transcription-translation was performed using the
T7 promoter contained in the PCMX-PL1 vectors. Wild-type and mutant IB proteins labeled with [35S]methionine were
produced using a wheat germ extract TNT kit as instructed by the
manufacturer (Promega). 60 µl of the TNT reaction was used for each
sample that was tested in the in vitro ubiquitination assay.
Transfections--
293 cell transfections were performed by the
calcium phosphate method (42). Briefly, DNA was mixed in 0.625 M CaCl2 and then added to an equal volume of
2 × HEPES buffer saline solution (560 mM NaCl, 50 mM HEPES, 1.5 mM
Na2HPO4, pH 7.1). The mixture was added to
approximately 106 cells in 10 ml of medium and incubated at
37 °C for 6 h (h) in 3% CO2. The medium was then
changed and the cells were incubated for 48 h at 37 °C, in 10%
CO2.
Production of Virus and Stable Pools--
Virus production for
infection of the IB / MEF cell line was performed by calcium
phosphate co-transfection of the 293gp packaging cell line with 20 µg
of the pCLBabepuro retroviral vector, containing cDNAs for
wild-type (wt) IB or the various IB mutants, and 5 µg of
the pMDG plasmid containing the vesicular stomatitis virus (VSVg)
envelope downstream of the cytomegalovirus promotor-enhancer. After
48 h, the media was removed, filtered, Polybrene added (8 µg/ml), and it was immediately used for infection. Each infection was
performed by adding 3 ml of 1:1000 diluted virus supernatant to
approximately 105 cells for 6 h. The cells were
allowed to expand for 48 h and the infected cells were selected
using 6 µg/ml puromycin (Calbiochem). In order to get equivalent
levels of expression in the mutC and 3236mutC stable pools it was
necessary to perform three rounds of infection and selection as
described above.
Cell Stimulation and Western Blot Analysis--
The IB
/ MEF stable pools were stimulated with 10 ng/ml TNF for the
given time points. In some cases the MEFs were pretreated with 10 µM -lactone, for 1 h, prior to stimulation. MEFs
were also treated with -lactone for the indicated times in the
absence of signal. Cells were then washed 2 times with ice-cold
phosphate-buffered saline and frozen on dry ice. Cytoplasmic extracts
were made as described previously and separated on 10 or 12%
SDS-polyacrylamide gels and transferred to 0.2-µm nitrocellulose
membranes (Schleicher & Schuell). Membranes were probed with antibody,
diluted 1:1,000 in 0.2% Tween-phosphate-buffered saline containing 5%
nonfat milk, for 4 h at 4 °C. Horseradish peroxidase-conjugated
donkey anti-rabbit sera (Amersham Pharmacia Biotech) was diluted
1:3,000 and incubated with the membranes for 1 h at room
temperature. Specific bands were then resolved by using a Renaissance
detection kit (NEN Life Science Products Inc.) as instructed by the manufacturer.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
made by the micropreparation technique (43), and gel shift analysis was
performed as described previously (44). Briefly, 5 µg of nuclear
protein extract was incubated with 0.5 µg of poly(dI-dC), on ice, for
20 min to block nonspecific DNA binding activity. A
32P-labeled oligonucleotide containing the HIV-1 long
terminal repeat B site was then added, and the mixture was incubated
at room temperature for 30 min. The resulting complexes were then
resolved on a 4% acrylamide gel, exposed to a PhosphorImager
(Molecular Dynamics), and band intensities quantitated with ImageQuant software.
In Vitro Ubiquitination Assay--
HeLa cytoplasmic extracts,
used in the in vitro ubiquitination assay, were made by
lysis in hypotonic buffer (buffer A) containing 10 mM HEPES
(pH 7.9), 1.5 mM MgCl2, 10 mM KCl,
0.5 mM dithiothreitol, and protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride and 10 µg/ml aprotinin).
Lysates were cleared by centrifugation at 14,000 rpm, for 15 min, at
4 °C. Lysates were then concentrated with a Centricon 3 concentrator
(Amicon) as instructed by the manufacturer. Protein concentration was
determined using the Bio-Rad protein assay system.
In vitro ubiquitination assays were carried out as described
(29) with the following changes. Briefly, in vitro
35S-labeled IB was incubated with 5 mg/ml HeLa
extract for 15 min at 4 °C. A reaction mixture containing 50 mM Tris (pH 7.5), 5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate, 3.5 units/ml
creatine kinase, 0.6 units/ml inorganic pyrophosphatase, 1 mg/ml
ubiquitin, 3 µM okadaic acid, 3 µM
ubiquitin aldehyde, and 10 µM -lactone was then added
and the reaction was incubated at 37 °C for 90 min. 50 mM Tris (pH 7.5) was substituted for the reaction mixture in control samples.
After the incubation, RIPA (100 mM NaCl, 20 mM
Tris, pH 8.0, 0.5% Nonidet P-40) containing 5 mM
N-ethylmaleimide was added and the samples were subjected to
immunoprecipitation (IP) by antibodies directed against IB or
RelA/p65. IPs were carried out with 1.5 µg of the appropriate
antibody for 1 h at 4 °C. Immunocomplexes were precipitated by
incubation with protein A-Sepharose (Amersham Pharmacia Biotech) for
1 h at 4 °C. The pellets were washed in RIPA buffer, SDS sample
buffer was added, and the pellets were boiled for 5 min prior to
SDS-polyacrylamide gel electrophoresis on 10% gels. Following
electrophoresis, the gels were fixed in glacial acetic acid, amplified
with 2,5-diphenyloxazole, rinsed with H2O, dried, and
exposed to a PhosphorImager for quantification.
Metabolic Labeling--
6-cm plates of the IB / MEF
stable pools were washed twice in phosphate-buffered saline and
incubated for 1 h in 2 ml of cystine- and methionine-free
Dulbecco's modified Eagle's medium supplemented with 10% dialyzed
fetal bovine serum (Life Technologies, Inc.). Labeling was performed
for 3 h with 0.5 mCi of [35S]methionine (NEN Life
Science Products Inc.) per ml. Cells were then washed three times with
complete medium and then chased for the given time points in complete
medium. After each time point the cells were washed twice with ice-cold
phosphate-buffered saline and cell pellets were frozen on dry ice for
later manipulation. Cell pellets were thawed on ice and whole cell
lysis was performed by adding 500 µl of RIPA (20 mM Tris,
pH 8.0, 100 mM NaCl, 0.2% sodium deoxycholate, 0.2%
Nonidet P-40, 0.2% Triton X-100) containing the protease inhibitors
aprotinin (10 µg/ml; Sigma) and phenylmethylsulfonyl fluoride (1 mM; Sigma). DNA was sheared by 10 passes through a 20-gauge
needle and lysates were cleared by centrifugation at 14,000 rpm for 15 min, at 4 °C. The cleared lysates were measured for trichloroacetic
acid-precipitable counts and the lysates were normalized for labeling
efficiency. Normalized lysates were precleared with protein A-Sepharose
for 1 h at 4 °C and then incubated with RelA/p65 antiserum plus
protein A-Sepharose for 4 h at 4 °C. To reduce the background,
coimmunoprecipitations that were done with the RelA/p65 antiserum were
subjected to a second round of immunoprecipitation. The RelA/p65
antibody protein A-Sepharose pellets were boiled for 10 min in RIPA
containing 100 µg of bovine serum albumin/ml and 0.5% SDS. The SDS
was diluted to 0.1% with RIPA buffer that contained 100 µg of bovine
serum albumin. A second IP was then done by adding IB antiserum
and protein A-Sepharose to isolate the RelA/p65-associated IB. To
isolate the free IB, the supernatant from the original Rela/p65
co-IP was incubated with IB antiserum and protein A-Sepharose.
After immunoprecipitation, the pellets were washed in RIPA buffer,
resuspended in SDS sample buffer, boiled for 5 min, and the eluted
proteins were separated on a 12% SDS-polyacrylamide gel.
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RESULTS |
Signal-dependent Degradation of IB--
To
investigate the signal-dependent degradation of IB
in vivo, stable pools of IB / MEFs (39) were
generated by infecting naive IB / MEFs with recombinant
retroviral vectors containing either wild type (wt) murine IB,
one of the IB mutants (Fig. 1
schematically describes IB mutations), or GFP. The mutants described in Fig. 1 represent mutations of known and possible sites of
serine and threonine phosphorylation. These mutants were chosen for
this study because they would aid in the further elucidation of the
role of serine and threonine phosphorylation in the
signal-dependent and -independent degradation mechanisms of
free and NF-B-bound IB. The IB / MEF infections were
carried out at a multiplicity of infection of much less than one to
ensure that on average, after selection, each cell of a stable pool
would have only a single copy of the integrated recombinant retroviral
vector. This kept expression levels low and as close to endogenous
expression levels as possible. Fig.
2A compares the relative
expression levels of endogenous IB in wt MEF cells to that of the
transduced IB / stable pools. It can be seen that the levels
of the IB proteins in the transduced cells are within 1-2-fold
of the endogenous IB (Fig. 2, lane 8, +/+).
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Fig. 1.
Schematic display of the
IB mutants.
MutF is a mutation of all of the carboxyl-terminal CKII
phosphorylation sites. 3236 is a mutation of the
amino-terminal IKK sites. M is a combination of the mutF
mutation and the 3236 mutation. MutC is a mutation of all
the possible phosphorylation sites in the ank6 region. 3236mutC is a
combination of the 3236 mutation and the mutC mutation.
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Fig. 2.
Signal-dependent activation of
IB / MEF stable
pools. A, IB immunoblot (IB) of the
relative expression levels of the various IB / stable pools,
an IB / GFP stable pool, and endogenous IB expression
in IB +/+ MEFs. Quantitation of the relative expression level of
IB in each cell line was done using NIH Image 1.62 software. The
results are given graphically in arbitrary units. B,
IB / MEFs were infected with a recombinant retroviral vector
containing wild type murine IB or one of the mutants. The stable
pools were treated with cyclohexamide prior to being stimulated with
TNF for 0, 5, 15, or 60 min. Following stimulation the cells were
harvested, cytoplasmic extracts made, and Western blot analysis was
performed with anti-IB sera. C, gel shift analysis of
IB / MEF stable pools stimulated with TNF for 60 min.
Three independent experiments were done. Quantification was done on a
PhosphorImager, using ImageQuant software. The values obtained were
averaged and the standard deviations were calculated.
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TNF stimulation of the IB / MEF stable pools in the
presence of the translational inhibitor cyclohexamide show that, compared with wild type IB, the 3236 and M mutants were
significantly more stable (Fig. 2B). The M mutant shows no
change in expression over the time course while the 3236 mutant starts
to show a slight reduction at the 60-min time point. The mutF mutation
gives IB partial resistance to TNF-dependent
degradation but is less stable than the 3236 and M mutants. The mutC
mutant shows no increase in stability and, in fact, is significantly
less stable than wt IB. The 3236mutC mutation confers an increase
in the stability of mutC containing IB, but is significantly more
unstable than the 3236 mutation by itself.
The rate of IB degradation shown in Fig. 2B was
further examined by measuring the NF-B DNA binding activity
following TNF stimulation. The mutF mutation shows an approximate
50% reduction in DNA binding activity when compared with wt IB
(Fig. 2C). The 3236 and M mutations reduce the gel shift
activity to what is observed in unstimulated control cells.
Interestingly, cell pools containing either the mutC or 3236mutC
mutations show no significant reduction in NF-B DNA binding
activity. This is most likely due to the fact that the mutC
mutation, which is a substitution of all five serine and threonine
residues in the ank6 region of IB to alanine, disrupts the
association of IB to NF-B. Fig. 3, A (in vitro) and
B (in vivo), show that both mutC and 3236mutC do
not associate with RelA/p65. It was recently shown that the ank6 region
of IB makes a critical contact to NF-B (11, 12). Since the
mutC mutation disrupts IBs association to NF-B and blocks any
potential phosphorylation of ank6, it is possible that phosphorylation
of ank6 plays a critical role in IB's association to NF-B. We
conclude that (i) IKK phosphorylation is necessary for
signal-dependent degradation of both free and
NF-B-associated IB, and (ii) that CKII phosphorylation is
necessary for efficient signal-dependent degradation of
NF-B-associated IB.
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Fig. 3.
Association of wild type and mutC containing
IB mutants to
RelA/p65 in vitro and in vivo.
A, in vitro association assay.
35S-Labeled IB, mutC, or 3236mutC were mixed with
uninduced cytoplasmic extract as described under "Experimental
Procedures." The sample was then immunoprecipitated with
anti-IB or anti-RelA. The IB IP shows input amount of
labeled IB species. The RelA IP shows the amount of the different
IB species that associated to the RelA/p65 present in the
cytoplasmic extract. B, in vivo association
assay. The wild type, mutC, and 3236mutC IB / stable pools
were labeled with [35S]methionine for 3 h. The cells
were washed and then lysed in RIPA buffer. A RelA IP was performed and
then the precipitated material was subjected to a second round of IP
with anti-IB sera to reduce the background. This was undertaken
to examine at RelA/p65-associated IB in these cell lines.
IB IPs were also done to show that IB is being expressed
and labeled in the mutC and 3236mutC stable pools.
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Next, in vitro ubiquitination assays were performed to
determine the signal-induced ubiquitination profiles of both free and NF-B-associated IB mutants. A representative gel is shown for each experiment. Quantitation was done on a PhosphorImager, using ImageQuant software, and the resulting histograms display the percentage of the total labeled IB in the reaction that was shifted. By graphing the percentage of the total IB shifted we
were able to correct for any loading differences between samples. Therefore, the histograms give a more accurate representation of the
respective levels of ubiquitination on the various IB molecules.
When examining a pool of both RelA/p65-associated and free IB it
appears that IB containing the mutF mutation is ubiquitinated
slightly, but reproducibly, more efficiently than wt IB (see Fig.
4A, lanes 1 and 2).
The level of ubiquitination of the other IB mutants in Fig.
4A is consistent with the rate of degradation and the gel
shift results presented in Fig. 2, B and C. The
3236 and M mutants have levels of ubiquitination just above background
(Fig. 4A, lanes 3 and 4).
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Fig. 4.
In vitro and in vivo
signal-dependent ubiquitination of the
IB mutants. All
experiments in this figure were done under stimulated conditions. The
HeLa cytoplasmic extracts used in the in vitro
ubiquitination assays were activated with a reaction mixture that
contained 3 µM okadaic acid. Control samples, designated
by "C," were performed exactly like the wt sample except the
reaction mixture was replaced by 50 mM Tris (pH 7.5).
A, the in vitro signal-dependent
ubiquitination assays were carried out as described under
"Experimental Procedures." The IB IP was carried out to
examine total ubiquitination of both free and NF-B-associated
IB. Quantitation was done on the PhosphorImager, using ImageQuant
software. The percentage of labeled IB shifted by ubiquitination
was calculated by dividing the shifted counts by the total count. This
calculation corrects for any differences in the input amounts of
labeled IB. Three independent samplings were performed and the
average of the three values obtained is depicted graphically.
B, the RelA IP was done to isolate the labeled IB that
had become associated to NF-B present in the activated HeLa extract.
Everything else is as was done for A. C, the
supernatant from the RelA IP done in B was subjected to a
second round of IP with anti-IB sera. This isolated the free
IB in the in vitro ubiquitination assay. D,
in vivo signal-dependent ubiquitination. The
IB / MEF stable pools were treated with -lactone for
1 h and then stimulated with TNF for 15 min. Cytoplasmic
extracts were made and an IB immunoblot was performed.
Ub, ubiquitin.
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The data with the mutC mutants (Fig. 4A, lanes 5 and
6) represents the levels of ubiquitination on free IB
molecules, since mutC does not associate with NF-B (Fig. 3,
A and B). MutC alone is ubiquitinated very
efficiently (lane 5), even better than wt (Fig. 4A,
lane 5, compared with lane 1, respectively). The
mutC3236 mutant shows a reduction in the level of ubiquitination
compared with mutC alone, but the level of ubiquitination of 3236mutC
is significantly higher than IB containing just the 3236 mutation (Fig. 4A, lane 6, compared with lane 3, respectively). These data demonstrate that free IB does undergo
signal-induced phosphorylation and ubiquitination and that serine 32 and serine 36 play a significant role in this process.
Fig. 4B displays the ubiquitination of the RelA/p65
associated pool of IB. The mutF mutation reduces the amount of
signal-dependent ubiquitination below the level seen on wt
IB when it is in association with NF-B (Fig. 4B, lanes
1 and 2). This reduction in ubiquitination by mutF of
only NF-B-associated IB explains why IB is partially stabilized and why a reduction in the DNA binding activity of NF-B
is observed in the IB / MEF mutF stable pool (Fig. 2, B and C).
In contrast to the NF-B-bound IB (Fig. 4B, lanes 1 and 2), the signal-induced ubiquitination of only the free
pool of IB reveals that mutF is ubiquitinated slightly more
efficiently than the wt (Fig. 4C, lanes 1 and 2).
The 3236 and M mutants give large reductions in the amount of
ubiquitination observed (Fig. 4C, lanes 4 and 5).
Ubiquitination of mutC and 3236mutC, in the in vitro
ubiquitination assay, could only be observed by direct anti-IB immunoprecipitation (Fig. 4A, lanes 5 and 6)
because these mutants do not associate to RelA (Fig. 3A).
The in vitro ubiquitination assay data (Fig. 4,
A-C) correlates well with the stability and gel shift
observations made in the IB / MEF stable pools (Fig. 2,
B and C).
The in vitro ubiquitination results were confirmed by
performing in vivo ubiquitination assays in the stably
transduced IB / MEF stable pools. The IB / MEF
stable pools were first treated with -lactone, a potent and specific
inhibitor of the proteasome, and then stimulated with TNF (Fig.
4D). The ubiquitination patterns obtained in this experiment
are similar to the corresponding in the in vitro assays. Wt,
mutF, 3236, and M represent NF-B associated IB (Fig. 4D,
lanes 1-4, respectively) and give ubiquitination profiles that
match those obtained for RelA/p65-associated IB in the in
vitro assay (Fig. 4B, lanes 1-4). Wt IB is
efficiently ubiquitinated in the presence of signal, mutF causes a
decrease in ubiquitination, and 3236 and M reduce ubiquitination to
almost background levels (Fig. 4D, lanes 1, 2, 3, and
4, respectively).
MutC and 3236mutC represent free IB in the IB / MEF
stable pools (Fig. 3B), and they also give ubiquitination
patterns in vivo (Fig. 4D, lanes 5 and
6) that correlate with those obtained in vitro
(Fig. 4A, lanes 5 and 6). MutC alone is
ubiquitinated very efficiently and the 3236mutC mutation gives a
reduction in the amount of ubiquitination but the level of
ubiquitination seen is significantly higher than that seen with just
the 3236 mutant (Fig. 4D, compare lanes 3 and
6). These data show that free IB is ubiquitinated in
the presence of signal even when it is lacking its
signal-dependent phosphorylation sites. It is likely that signal-independent degradation mechanisms that are involved in removing
free IB from the cell are contributing to the ubiquitination of
the mutC containing mutants in these experiments.
From the in vitro and in vivo ubiquitination data
we conclude that phosphorylation of serine 32 and serine 36 by the IKK
is necessary for efficient signal-dependent ubiquitination
and degradation of free and NF-B-associated IB. We also
conclude that the constitutive CKII phosphorylation of the carboxyl
terminus of IB enhances the extent of ubiquitination of free
IB (Fig. 4C, lane 2), but reduces the ubiquitination
of NF-B associated IB (Fig. 4, B and D, lane
2).
Signal-independent Turnover of IB--
To investigate
whether or not the constitutive or signal-dependent
phosphorylation sites are involved in the signal-independent turnover
of NF-B-associated IB, pulse-chase experiments were performed on the wt, mutF, and 3236 IB / MEF stable pools (Fig. 5, A-C). Compared with
the half-life of wt IB, the mutF mutation gives a 2-fold increase
in IBs half-life. The 3236 mutation has no effect on basal
turnover, resulting in a half-life very similar to that of wt IB
(compare A and C). These results demonstrate that
only the carboxyl-terminal CKII phosphorylation sites play a
significant role in the basal turnover of NF-B-associated IB.
The inducible phosphorylation sites (serine 32 and 36) have no effect
on the basal turnover of NF-B-bound IB.
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Fig. 5.
Signal-independent turnover of
NF-B-bound
IB.
A-C, pulse-chase analysis was performed to determine the
half-life of NF-B-associated IB under signal-independent
conditions. The stable pools were metabolically labeled with
[35S]methionine for 3 h, washed, and then chased for
0, 60, 120, or 240 min. A RelA IP was performed and then the
precipitated material was subjected to a second round of IP with
anti-IB sera to reduce the background. This isolated the
RelA/p65-associated IB in these cell lines. Quantitation was done
on the PhosphorImager, using ImageQuant software. The data were
converted into percent relative intensity by assigning the zero time
point of each time course as 100% and assigning the remaining time
points a percentage that correlates to the fraction of its signal. The
percent relative intensities were then graphed against time, in
minutes, and half-lives were calculated.
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Several studies have been performed to evaluate the role of
phosphorylation in the degradation of free IB in the absence of
stimulation (35, 45, 46). It has been shown that the mutF mutation or
the removal of the carboxyl terminus of IB will increase the
stability of free IB by approximately 2-fold (35, 46). Here we
look at the effects of the 3236 mutation on the basal turnover of free
IB. Unstimulated IB / MEF stable pools expressing
either the mutC mutant or the 3236mutC mutant (Fig.
6, A and B) were
analyzed. As has been shown (Fig. 3B) mutC and 3236mutC do
not associate to RelA/p65, thus all the IB is in a free state in
the IB / MEF stable pools. Free IB is at least 5 times
more rapidly degraded in the absence of signal when compared with
NF-B-bound IB (compare Fig. 5A and 6A).
Interestingly, the 3236 mutation in the mutC background increases the
rate of signal-independent turnover of free IB by about 3-fold
(Fig. 6, A and B).
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Fig. 6.
Signal-independent turnover of free
IB. A
and B, everything was done the same as for Fig. 5 except
these samples were directly IP with anti-IB because they do not
associate to RelA/p65 in these cell lines. A nonspecific
(ns) band comes down in the IB IP that migrates
directly above the IB band. The standard deviations for these
samples were negligible and are not resolved at the given graphical
resolution.
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To determine whether or not ubiquitination plays a role in the basal
turnover of NF-B-associated IB, the IB / MEF stable pools were treated with the proteasome inhibitor -lactone in the
absence of signal. Unfortunately, the prolonged treatments with
-lactone necessary to see the accumulation of ubiquitinated IB
bound to NF-B in the absence of signal seems to activate the NF-B
pathway (data not shown). This is most likely caused by the tremendous
stress that the cell experiences when its proteasomes are inactivated
for prolonged periods of time. Consequently, no concrete conclusions
could be made about the role of ubiquitination in the basal turnover of
NF-B-associated IB.
The signal-independent ubiquitination of free IB is quite robust
(Fig. 7, lanes 2-4) which may
explain the rapid basal turnover of free IB when compared that of
NF-B associated IB (110 and 550 min, respectively). Free IB
is very unstable in the absence of signal (Fig. 6A) and
a detectable pool of ubiquitinated mutC and 3236mutC is observed in the
absence of any stimulation and in the absence of a proteasome inhibitor
(Fig. 7). The 3236 mutation in the mutC background does not reduce
signal-independent ubiquitination of free IB (Fig. 7, lane
4). Therefore, phosphorylation of serine 32 and serine 36 is not
necessary for efficient signal-independent ubiquitination and
degradation of free IB.
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Fig. 7.
Signal-independent ubiquitination.
IB immunoblot of the given untreated stable IB / MEF
pools in the absence of stimuli or proteasome inhibitor. Ub,
ubiquitin.
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We have previously shown that the carboxyl-terminal CKII sites are
necessary for the efficient basal turnover of free IB (35, 46).
Here we conclude (i) that efficient basal turnover of
NF-B-associated IB requires the carboxyl-terminal CKII
phosphorylation and (ii) that IKK phosphorylation (serine 32 and 36)
plays no role in the basal turnover of free or NF-B associated
IB.
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DISCUSSION |
IB functions as the primary regulator of NF-B in both
stimulated and unstimulated cells. To accomplish this, IB itself must be a tightly regulated protein. NF-B activity is positively and
negatively regulated through IB phosphorylation and
NF-B-dependent synthesis of IB, respectively.
Mechanisms that ensure proper NF-B activity must exist to regulate
signal-dependent degradation of free and NF-B-associated
IB as well as the signal-independent turnover of free and
NF-B-associated IB. These mechanisms also must allow newly
synthesized IB the opportunity to enter the nucleus in order to
remove NF-B from the DNA and/or inhibit further activation upon the
removal of signal (33). One way that the cell can easily and
efficiently regulate the multiple states and fates of IB is
through phosphorylation. Phosphorylation seems to be involved in almost
all aspects of IBs regulation. We demonstrate when and how some
of the different phosphorylation sites on IB can influence
ubiquitination, degradation, and the overall stability of free and
NF-B-associated IB in the presence or absence of NF-B
inducing stimuli. Our conclusions about the role of phosphorylation and
ubiquitination in the regulation of signal-dependent and
-independent degradation of free and NF-B-associated IB are
summarized in Fig. 8.
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Fig. 8.
Role of phosphorylation and ubiquitination in
IB degradation
(summary). The role of phosphorylation and ubiquitination in the
regulation of signal-dependent and -independent degradation
of free and NF-B-associated IB. CKII phosphorylation
(CKII P) is necessary for efficient basal degradation of
both free and NF-B-associated IB. Ubiquitination is involved
in the basal turnover of free IB but it is still unclear whether
or not ubiquitination plays a role in the basal turnover of
NF-B-associated IB. Signal-dependent degradation
of free IB only requires IKK phosphorylation (IKK P)
while efficient signal-dependent degradation of
NF-B-associated IB requires both CKII P and IKK P. Signal-dependent degradation of free and NF-B-associated
IB takes place in a ubiquitin-dependent manner.
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B-bound I
*
§¶ and
TOP
ABSTRACT
INTRODUCTION
