(Received for publication, June 9, 1995; and in revised form, August 31, 1995)
From the
Nuclear factor B (NF-
B) is stored in the cytoplasm as
an inactive form through interaction with I
B. Stimulation of cells
leads to a rapid phosphorylation of I
B
, which is presumed to
be important for the subsequent degradation. We have recently reported
the establishment of a lipopolysaccharide (LPS)-dependent cell-free
activation system of NF-
B in association with the induction of
I
B
phosphorylation. In this study, we have identified a
kinase in cell extracts from the LPS-stimulated human monocytic cell
line, THP-1, that specifically binds and phosphorylates I
B
.
LPS stimulation transiently enhanced the I
B
-bound kinase
activity in THP-1 cells. Mutational analyses of I
B
and
competition experiments with the synthetic peptides identified major
phosphorylation sites by the bound kinase as Ser and Thr residues in
the C-terminal acidic domain of I
B
. Moreover, we show that
the peptide, corresponding to the C-terminal acidic domain of
I
B
, blocked the LPS-induced NF-
B activation as well as
inducible phosphorylation of endogenous I
B
in a cell-free
system using THP-1 cells. These results suggested that the bound kinase
is involved in the signaling pathway of LPS by inducing the
phosphorylation of the C-terminal region of I
B
and subsequent
dissociation of the NF-
B
I
B
complex.
NF-B consists of a family of transcriptional factors that
play a key role in the regulation of a number of immune and
inflammatory response genes(1, 2, 3) ,
including several inflammatory cytokines such as IL-8 (
)and
IL-6(4, 5, 6) . Members of the family include
p65 (RelA), RelB, c-Rel, p50 (NF-
B-1), and p52 (NF-
B-2).
NF-
B is retained in an inactive form being associated with its
inhibitors, I
B, in the cytoplasm in most types of cells (7) . Following stimulation of cells with a variety of agents, e.g. IL-1, tumor necrosis factor (TNF), phorbol myristate
acetate, and lipopolysaccharide (LPS), NF-
B is released from
I
B and is translocated to the nucleus and binds to the NF-
B
binding sites, thereby activating the transcription of a set of target
genes (1, 2, 3) .
IB family proteins
possess the ankyrin-like repeats, which are thought to interact with
the Rel homology region of NF-
B(8) . This family includes
large precursors of p50 and p52, and p105 and p100, respectively, which
contain ankyrin repeats in their C-terminal region (9, 10, 11) . Cytoplasmic I
B
,
encoded by the MAD-3 gene(12) , is thought to be a major target
in the signal transduction pathway. I
B
is rapidly
phosphorylated in vivo in response to cell stimulation (13, 14, 15, 16) and is degraded
thereafter by proteasomes (13, 14, 15, 16, 17, 18) .
Recently, while several groups have demonstrated that phosphorylation
of I
B
is not sufficient to activate NF-
B in
vivo(14, 15, 16, 17, 18, 19) ,
the inducible phosphorylation of I
B
may be required for
converting I
B
into an appropriate proteasome substrate. In
contrast, we previously demonstrated that LPS induced NF-
B
activation without a significant loss of I
B
in a cell-free
system using the monocytic cell line, THP-1(20) . Lack of
degradation of I
B
was also observed upon TNF-induced
NF-
B activation in a cell-free experiment using U937
cells(21) . These observations would imply that appropriate
phosphorylation of I
B
can activate NF-
B by dissociating
from I
B
in a signal-dependent manner.
In vitro phosphorylation of IB
by several serine/threonine
kinases, including protein kinase C and protein kinase A, prevents its
binding to NF-
B(22, 23, 24) , suggesting
that I
B
phosphorylation on specific phosphorylation sites is
sufficient for the dissociation of I
B
from NF-
B.
However, none of these kinases tested so far in vitro appears
to be responsible for the activation of NF-
B in
vivo(25, 26) . To our knowledge, the I
B
kinase, which is responsible for the phosphorylation of I
B
in vivo, has not yet been biochemically identified and
characterized. In this study, we have identified a kinase in
LPS-stimulated human monocytic cell line (THP-1) extracts that
specifically binds and phosphorylates I
B
. Moreover, we have
identified the acidic domain in the C-terminal region of I
B
as the phosphorylation target sites for this kinase. A peptide
substrate for the bound kinase, corresponding to the acidic domain,
inhibited the LPS-mediated NF-
B activation in a cell-free
system(20) , suggesting that phosphorylation of I
B
at
the acidic domain by the kinase is critical for NF-
B activation.
The GST fusion protein expression vectors were transformed into E. coli, JM109. Protein induction and purification were as described(30) . The amount of purified protein was estimated by the Bio-Rad protein assay.
For in vitro kinase
reaction, the partially purified kinase was incubated with a substrate
in 20 µl of the same kinase buffer containing 5 µCi of
[-
P]ATP (Amersham) in the presence or
absence of inhibitors for 5 min at 30 °C. The reaction was
terminated by adding EDTA.
For the in vitro kinase reaction in a cell-free system, postnuclear
fraction (40 µg of protein) was incubated in the kinase buffer with
[-
P]ATP at 30 °C for 5 min in the
presence or the absence of LPS (20 µg/ml) as described
previously(20) .
Figure 1:
Protein
kinase(s) specifically bound to IB
. A, GSH-Sepharose
beads conjugated with 2.5 µg of GST-I
B
(lanes 3 and 4) or equimolar of GST (lanes 1 and 2) were incubated with (lanes 2 and 4) or
without (lanes 1 and 3) the cytosolic extracts from
LPS-stimulated THP-1 cells. After extensive washing, kinase reaction
was performed in the presence of [
-
P]ATP as
described under ``Materials and Methods.'' Reaction products
were analyzed by 10% SDS-PAGE and autoradiography. B,
transient activation of I
B
-bound kinase by LPS stimulation in
THP-1 cells. Cytosol extracts were prepared from LPS-stimulated THP-1
cells at the indicated times, and the bound kinase activity was
determined by a binding/kinase reaction with a GST-I
B
protein
as a substrate. Reaction products were separated by 10% SDS-PAGE, and
the relative density of I
B
phosphorylation was analyzed by an
imaging analyzer. Data shown represent the mean ± S.D. of three
independent experiments.
To determine the region(s) of IB
that interacted with the kinase(s), several truncated mutants of
GST-I
B
fusion proteins were expressed (Fig. 2A), and the ability of the kinase(s) to
phosphorylate these mutated I
B
was tested in a binding/kinase
assay. As shown in Fig. 3A, mutant del-4, which lacks a
72-amino acid N-terminal region, or del-5, which lacks both N-terminal
region and 3 ankyrin repeats, was still phosphorylated by the partially
purified bound kinase(s). In contrast, the deletion of the C-terminal
35 amino acids (del-10) but not of 25 amino acids from C-terminal
(del-9) abolished the phosphorylation of I
B
by the kinase(s).
These data demonstrated the interaction of the acidic domain within the
C-terminal region of I
B
with the bound kinase. To further
directly delineate the I
B
phosphorylation sites, the effects
of truncations of I
B
were also examined in an in vitro kinase reaction using a partially purified kinase. As shown in Fig. 3B, mutant del-9 was phosphorylated by the bound
kinase to a similar level as wild type I
B
. However, when
del-10 or del-1 mutant was used as a substrate, no phosphorylation was
detected. Furthermore, GST-I
B
mutant K, in which six Ser/Thr
residues in the C-terminal acidic domain were changed to Ala (Fig. 2B), was not phosphorylated by the kinase (Fig. 3B). These data indicate that the major
phosphorylation sites by the bound kinase resided within the C-terminal
acidic domain of I
B
.
Figure 2:
Schematic diagram of the human
IB
and construction of deletion mutants (A) and
amino acid-substituted mutant (B) of GST-I
B
fusion
protein. A, five ankyrin repeats are indicated by dotted
boxes. Acidic domain in the C-terminal region was indicated by the cross-hatched box. A solid line represents the
portion present in GST-I
B
mutants with the amino acid number
at the end. B, partial amino acid sequences of wild type and
mutant K are shown with the C-terminal end of deletion mutants 9 and 10
by arrows. The position of the synthetic peptide AR (residues,
279-303) is indicated by a cross-hatched bar above the
sequence.
Figure 3:
The bound kinase phosphorylates the
C-terminal acidic domain of IB
. A, the effects of
mutations of I
B
on its phosphorylation by the bound kinase in
a binding/kinase reaction. Partially purified I
B
-bound kinase
from THP cells was incubated with GSH-Sepharose beads conjugated with
2.5 µg of wild type (wt) GST-I
B
or equimolar of
deletion mutants. After an extensive washing, a kinase reaction was
performed in the presence of [
-
P]ATP as
described under ``Materials and Methods.'' Reaction products
were analyzed by 10% SDS-PAGE and autoradiography. B, the
effects of mutations of I
B
on its phosphorylation in an in vitro kinase reaction. Partially purified
I
B
-bound kinase from THP-1 cells was incubated with 0.5
µg of wild-type GST-I
B
protein or equimolar mutated
GST-I
B
proteins in the presence of
[
-
P]ATP as described under ``Materials
and Methods.'' The reaction products were analyzed by 10% SDS-PAGE
and autoradiography.
Figure 4:
Determination of the phosphoacceptor sites
within the acidic domain of IB
by the bound kinase. A, competition experiments with various kinase substrates in
an in vitro kinase assay. Partially purified
I
B
-bound kinase was incubated with 0.5 µg (9 pmol) of the
wild type GST-I
B
as a substrate in 20 µl of the kinase
buffer containing [
-
P]ATP in the presence
of a 300-fold molar excess (2.7 nmol) of AR peptide of I
B
,
CKII substrate, or MAPK substrate. The results are expressed as a
percentage of GST-I
B
phosphorylation without a peptide
competitor. Data shown represent the mean ± S.D. of three
independent experiments. B, effects of amino acid
substitutions of AR peptide of I
B
on its phosphorylation by
the kinase. Partially purified I
B
-bound kinase was incubated
with 3 nmol of wild type (wt) or mutated AR peptide in the
kinase buffer containing [
-
P]ATP. The
reaction products were analyzed by 15% SDS-PAGE in Tris-Tricine buffer.
The results are expressed as a percentage of phosphorylation of wild
type peptide. Data shown represent the mean ± S.D. of three
independent experiments.
To determine more precisely the phosphoacceptor
sites on the acidic domain of IB
, we prepared a series of
mutated peptides in four Ser/Thr residues within the critical region of
peptide AR and tested their capacities to be phosphorylated by the
bound kinase. As shown in Fig. 4B, when the Ser-293 was
replaced by Ala, phosphorylation of the peptide by the bound kinase was
significantly reduced. The substitution of Ser-288 or Thr-291 to Ala
decreased the phosphorylation by 20-30%, whereas that of Ser-283
retained a similar level of phosphorylation as the wild type protein.
These data suggest that Ser-293 was a major phosphoacceptor site and
that Ser-288 and Thr-291 were minor phosphorylation sites.
Figure 5:
Phosphoamino acid analysis of IB
phosphorylated in the cell-free system after LPS stimulation. After
kinase reaction with a postnuclear fraction (100 µg of protein) in
a cell-free system in the presence of LPS (20 µg/ml) for 5 min,
I
B
was immunoprecipitated with anti-I
B
antibody and
separated by SDS-PAGE, then subjected to phosphoamino acid analysis as
described under ``Materials and Methods.'' The direction of
migration for the first (1st) and second (2nd)
dimensions are indicated. The(-) and (+) signs show the
orientation of the electrophoresis. The (O) and (F)
indicate the origin and the forward in the chromatography,
respectively. The positions of phosphoserine (S),
phosphothreonine (T), and phosphotyrosine (Y) are
indicated.
To assess the
physiological role of the IB
-bound kinase(s) in the signaling
pathway of LPS, we examined in a cell-free system the inhibitory
activity of the bound kinase substrate, AR peptide of I
B
, as
well as other inhibitory peptides. As shown in Fig. 6A,
LPS-mediated NF-
B binding was significantly inhibited by addition
of AR peptide of I
B
. In contrast, neither a CKII substrate
nor a MAPK substrate affected the NF-
B activation. Furthermore, AR
peptide of I
B
also blocked an inducible phosphorylation of
I
B
(Fig. 6B) in the same assay. These results
strongly suggest that the bound kinase is essential for the
LPS-dependent activation of NF-
B in THP-1 cells through an
inducible phosphorylation of the C-terminal acidic domain of
I
B
.
Figure 6:
The substrate of IB
-bound kinase
blocks NF-
B activation as well as inducible phosphorylation of
I
B
in response to LPS stimulation in a cell-free system. A, plasma membrane-enriched and cytosolic fractions were
incubated at 30 °C for 10 min in the absence (lane 2) or
presence (lanes 1, 3, 4, and 5) of
LPS (20 µg/ml) without (lanes 1 and 2) or with 15
nM of CKII substrate (lane 3), MAPK substrate (lane 4), or peptide AR of I
B
(lane 5),
respectively. Then, EMSA was performed using NF-
B binding site of
the human IL-8 gene promoter as a probe. B, kinase reaction
was performed in a cell-free system with a postnuclear fraction (40
µg of protein) in the absence (lane 1) or presence (lanes 2, 3, 4, and 5) of LPS (20 µg/ml) without (lanes 1 and 2) or with 15 nM of AR peptide
of I
B
(lane 3), MAPK substrate (lane 4),
and CKII substrate (lane 5). I
B
was
immunoprecipitated with anti-I
B
antibody and analyzed by 10%
SDS-PAGE.
In this study, we have identified a protein kinase in the
cytosolic fraction of a human monocytic cell line, THP-1, that can
specifically bind and phosphorylate IB
. The bound kinase is
serine/threonine kinase, and its activity was transiently increased by in vivo stimulation with LPS in intact THP-1 cells, indicating
that the I
B
-bound kinase is located downstream of the LPS
signaling cascade.
Mutational analysis of the IB
substrate
revealed that the I
B
-bound kinase phosphorylated the Ser/Thr
residues within the C-terminal acidic domain of I
B
. This
notion is supported by the competition experiment with the synthetic
peptide AR corresponding to the C-terminal acidic domain of
I
B
(Fig. 4A). Incomplete competition by AR
peptide for I
B
phosphorylation in Fig. 4A may
be due to lower affinity of AR peptide to the kinase compared with a
full-length I
B
. It is possible that the synthetic peptide AR
may be somehow sterically different from native phosphorylation sites
and/or that some other parts may affect the interaction with the bound
kinase in addition to actual phosphoacceptor sites. Moreover, we have
demonstrated here that AR peptide was a substrate for the bound kinase
and that the peptide significantly suppressed LPS-mediated NF-
B
activation in a cell-free system. Previously, we found a correlation
between an inducible phosphorylation of I
B
and the activation
of NF-
B in response to LPS without apparent degradation of
I
B
in the same system(20) . Furthermore, we show here
that the substrate for the bound kinase also blocked the inducible
phosphorylation of I
B
in a cell-free system. Taken together,
these findings demonstrate that in a cell-free system using a monocytic
cell line, THP-1, the I
B
-bound kinase phosphorylates the
C-terminal acidic domain of I
B
in response to LPS
stimulation, leading to dissociation of I
B
from NF-
B.
IB
is rapidly phosphorylated by cell stimulation, followed
by degradation in
vivo(13, 14, 15, 16, 17) .
Several groups argued that phosphorylation of I
B
is not
sufficient for NF-
B activation, based on their findings that
proteasome inhibitors blocked the activation of NF-
B with
accumulation of the phosphorylated form of I
B
(15, 16) and that the phosphorylated form of
I
B
could be coimmunoprecipitated with p65 in several cell
lines including Hela cells(14, 15) . Recently, Brown et al.(34) demonstrated that two Ser residues in the
N-terminal region of human I
B
are important for its
degradation in a mouse T cell line, EL-4. On the other hand, a recent
study showed that phosphorylation states of I
B
were
remarkably different in each cell type(14) . Thus, we speculate
that the discrepancy between these studies and our finding in a
cell-free system using THP-1 cells may be ascribed to the difference in
the cell types and stimuli. In addition, DiDonato et al.(16) reported that I
B
was phosphorylated at
multiple sites after TNF
stimulation in Hela cells. Therefore, the
function of I
B
might be differentially regulated by multiple
phosphorylation sites. It was shown that the C-terminal region of
I
B
, containing a PEST-like motif, was also important for a
signal-dependent proteolysis in vitro and in vivo in
addition to the N-terminal region(34, 35) . However,
the role of phosphorylation within the C-terminal acidic domain in an
inducible proteolysis remains to be elucidated.
It is known that
IB associates with Rel family proteins using its ankyrin repeat.
The C-terminal acidic region of I
B
was recently shown to be
involved in an inhibitory activity for DNA binding of c-Rel or
p65(28, 36) . Here, we show that the peptide substrate
for the bound kinase blocked NF-
B activation in a cell-free
system. Ernst et al.(28) , however, reported that a
synthetic peptide, corresponding to the C-terminal acidic domain, by
itself could not inhibit DNA binding activity. Similarly, we observed
that the peptide substrate for the bound kinase did not affect
NF-
B binding activity in EMSA when nuclear extracts from
LPS-stimulated THP-1 cells were used as a source of NF-
B. (
)These findings exclude the possibility that the acidic
domain peptide directly inhibits the DNA binding activity of NF-
B
in a cell-free assay. In addition, these studies demonstrated that
NF-
B made contact with two sites of I
B
, ankyrin repeats
and an acidic domain. Thus, it is likely that phosphorylation within
this acidic region of I
B
may change its affinity for
NF-
B.
Mutational analysis of GST-IB
and the substrate
for the bound kinase revealed that Ser-293 within the acidic domain is
a major phosphoacceptor site by the bound kinase and that Ser-288 and
Thr-291 are minor ones. The sequence motifs of these phosphorylation
sites are distinct from those of the proline-directed protein kinases,
including MAPK, Jun kinase, and
p38(37, 38, 39) , which were recently shown
to be activated by LPS, IL-1, or TNF stimulation in several cell lines.
In addition, the recognition sites of the bound kinase are also
distinct from protein kinase C acceptor sites, although several groups
reported the involvement of protein kinase C in LPS-signaling pathway (40) and the capacity of protein kinase C to phosphorylate
I
B
in vitro, resulting in a dissociation from the
NF-
B
I
B
complex. The major phosphorylation site,
Ser-293, is distinct from a CKII acceptor site since it lacks an acidic
residue at a third position of the C-terminal side, which is
essentially required for CKII recognition(33) . On the other
hand, one of the minor phosphorylation sites, Thr-291, belongs to a
CKII acceptor site. We found that CKII substrate could not inhibit
I
B
phosphorylation at the same concentration at which the
bound kinase substrate blocked it. At this moment, we cannot exclude
the possibility that the acidic domain of I
B
is more
physiological substrate for CKII or its related molecules than the
typical CKII substrate. Photoaffinity labeling by 8-azido-ATP of a
partially purified kinase fraction suggested that the molecular mass of
the I
B
-bound kinase is 42 kDa.
However, further
efforts will be required for the purification and molecular cloning of
the kinase.
NF-B plays a central role in the regulation of gene
activation of inflammatory cytokines such as IL-6 and TNF
.
Previously, our group demonstrated that the activation of NF-
B is
indispensable for the gene activation of human IL-8(6) , which
is a member of the leukocyte chemotactic cytokine(41) . In this
study, we have demonstrated that a peptide substrate for the
I
B
-bound kinase blocked the activation of NF-
B in a
cell-free system. This finding suggests that specific peptides
regulating the signaling pathway to induce NF-
B activity, such as
the I
B
-bound kinase substrate, can target NF-
B and
possibly be developed as a novel class of an anti-inflammatory drug.