(Received for publication, March 7, 1995; and in revised form, May 8, 1995)
From the
Activation of NF-
The NF- The
latent cytoplasmic NF- Recent studies have suggested
that degradation of I
EMSA was performed by
incubating the nuclear extracts (
Figure 1:
Activation of
RelA
To investigate the
underlying mechanism of this induction event, studies were performed to
test the effect of calyculin A on the fate of I Parallel EMSA studies revealed that the
kinetics of phosphorylation and degradation of I
Figure 2:
Effect of the chymotrypsin type of TPCK
protease inhibitor and the reducing agent PDTC on calyculin A-induced
phosphorylation and degradation of I
Figure 3:
Phosphorylation of I
To examine whether TNF-
Figure 4:
Effect of the proteasome inhibitor MG132
on the degradation of I
Figure 5:
Synergy between calyculin A (Cal
A) and TNF-
The nuclear expression and biological function of the
NF- A previous study has shown that activation of
NF- We have also shown that
induction of I Recent studies have demonstrated that
inhibition of I
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B by various cellular stimuli involves the
phosphorylation and subsequent degradation of its inhibitor,
I
B
, although the underlying mechanism remains unclear. In the
present study, the role of serine/threonine phosphatases in the
regulation of I
B
phosphorylation was investigated. Our
studies demonstrate that incubation of human T cells with low
concentrations (
1-5 nM) of calyculin A or okadaic
acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type
2A (PP-2A), induces the phosphorylation of I
B
even in the
absence of any cellular stimulus. This action of the phosphatase
inhibitors, which is associated with the activation of the
RelA
p50 NF-
B heterodimer, is not affected by agents that
block the induction of I
B
phosphorylation by tumor necrosis
factor alpha (TNF-
). Furthermore, the phosphorylated I
B
from calyculin A-treated cells, but not that from TNF-
-stimulated
cells, is sensitive to PP-2A in vitro, suggesting the
existence of fundamental differences in the phosphorylation of
I
B
induced by the two different NF-
B inducers. However,
induction of I
B
phosphorylation by both TNF-
and the
phosphatase inhibitors is associated with the subsequent degradation of
I
B
. We further demonstrate that TNF-
- and calyculin
A-induced I
B
degradation exhibits similar but not identical
sensitivities to a proteasome inhibitor. Together, these results
suggest that phosphorylation of I
B
, mediated through both the
TNF-
-inducible and the PP-2A-opposing kinases, may serve to target
I
B
for proteasome-mediated degradation.
B transcription factor plays a pivotal role in the
regulation of various cellular genes involved in the immediate early
processes of immune, acute phase, and inflammatory
responses(1, 2) . In addition, NF-
B has also been
implicated in the transcriptional activation of human viruses, most
notably the type 1 human immune deficiency virus
(HIV-1)(
)(3, 4, 5, 6, 7) .
NF-
B corresponds to a set of hetero- or homodimeric complexes
composed of a family of related polypeptides. In higher vertebrates,
this family includes p50, p52, RelA (previously termed p65), RelB, and
c-Rel, all of which contain an N-terminal domain of homology (Rel
homology domain,
300 amino acids; (8, 9, 10, 11, 12, 13, 14, 15) ,
reviewed in Refs. 16 and 17). In most cell types, the predominant form
of NF-
B is a heterodimer composed of p50 and RelA. This form of
NF-
B is normally sequestered in the cytoplasmic compartment by
physical association with an inhibitory protein, termed
I
B
(18, 19) . Recent studies have revealed
that I
B
specifically binds to and masks the nuclear
localization signal of RelA, thereby preventing the nuclear
translocation of the RelA
p50 NF-
B
heterodimer(20, 21, 22, 23) .
B complex can be posttranslationaly
activated by a variety of cellular stimuli, including mitogens like
phorbol esters, cytokines such as tumor necrosis factor alpha
(TNF-
) and interleukin-1, and the Tax protein from the type I
human T-cell leukemia virus (for a recent review, see (17) ).
Activation of NF-
B by these various inducers involves the
proteolytic degradation of the inhibitor I
B
, concomitant with
the nuclear translocation of the liberated NF-
B
heterodimer(24, 25, 26, 27, 28, 29, 30) .
The biologically active nuclear NF-
B complex in turn activates the
transcription of a large set of cellular genes encoding not only
various factors involved in immune stimulation, inflammation, and cell
growth but also the NF-
B inhibitor
I
B
(24, 25, 31, 32, 33, 34) .
Thus the nuclear expression of NF-
B is subject to tight control by
an autoregulatory feedback mechanism.
B
appears to be mediated through the
proteasome complex(35, 36) . Furthermore, it is likely
that the degrading enzymes within the proteasome are constitutively
active and that the inducible degradation of I
B
is regulated
by the posttranslational modification of I
B
. Interestingly,
the degradation of I
B
, induced by various NF-
B inducers,
is preceded by the transient phosphorylation of this cytoplasmic
inhibitor(30, 36, 37, 38, 39) ,
thus raising the possibility that phosphorylation may serve to target
I
B
for subsequent degradation. In support of this proposal,
kinase activators like phorbol 12-myristate 13-acetate and phosphatase
inhibitors such as calyculin A and okadaic acid have been shown to
induce the nuclear expression of
NF-
B(40, 41, 42, 43) . In the
present study, we demonstrate that incubation of the cells with
calyculin A or okadaic acid leads to the phosphorylation of
I
B
. Furthermore, unlike TNF-
, calyculin A induces
phosphorylation of I
B
at sites that are sensitive to PP-2A.
However, as observed with TNF-
, calyculin A-induced
phosphorylation of I
B
is also associated with the subsequent
proteasome-mediated degradation of this inhibitory protein. These
findings suggest that differential phosphorylation of I
B
may
target this inhibitor for degradation.
Cell Culture and Reagents
Human Jurkat leukemic
T cells were maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 2 mML-glutamine, and antibiotics.
Calyculin A and okadaic acid were purchased from LC Laboratories
(Woburn, MA). Tosylphenylalanyl chloromethyl ketone (TPCK) and
pyrrolidinedithiocarbamate (PDTC) were from Sigma. TNF- and the
purified serine/threonine phosphatases, PP-1, PP-2A, and PP-2B, were
obtained from Upstate Biotechnology, Inc. The proteasome inhibitor
MG132 was a gift from MyoGenics Inc. (Cambridge, MA).
Immunoblotting and Electrophoresis Mobility Shift Assay
(EMSA)
Jurkat T cells were collected by centrifugation and
subjected to preparation of cytoplasmic and nuclear
extracts(44, 49) . Cytoplasmic extracts (15
µg) were fractionated by reducing 10% SDS-polyacrylamide gel
electrophoresis, electrophoretically transferred to nitrocellulose
membranes, and then analyzed for immunoreactivity with a
peptide-specific antiserum recognizing the C terminus of human
I
B
using an enhanced chemiluminescence detection system (ECL;
Amersham Corp.). For phosphatase treatment, the extracts were incubated
with 0.5 units of the indicated specific serine/threonine phosphatases
or 20 units of calf intestinal alkaline phosphatase at 30 °C for 30
min prior to immunoblotting analysis.
3 µg) with a
P-radiolabeled high affinity palindromic
B probe,
B-pd (45) followed by resolving the DNA-protein complexes
on native 5% polyacrylamide gels. For antibody ``super
shift'' assays, 1 µl of anti-peptide antiserum specifically
recognizing each of the NF-
B subunits was added to the EMSA
reaction 10 min prior to electrophoresis.
Activation of NF-
As
described above, the phosphatase inhibitor calyculin A induces the
nuclear expression of NF-B by Phosphatase Inhibitors
Involves Phosphorylation and Degradation of I
B
B in human T cells(43) . Using
antibody super shift assays, we analyzed the NF-
B species in the
B-binding protein complex. As shown in Fig. 1A, a
major DNA-protein complex was detected by EMSA in the nucleus of
calyculin A-treated cells (Fig. 1A, lane2). More importantly, incubation of the DNA binding
mixtures with either a p50-specific (lane4) or a
RelA-specific (lane5) antiserum resulted in the
supershift of the
B binding complex (Fig. 1A, arrowheads), suggesting the presence of both p50 and RelA in
this complex. In contrast, this NF-
B/DNA complex only slightly
immunoreacted with anti-c-Rel (lane6) and had no
immunoreactivity with either anti-p52 (lane7) or a
control preimmune serum (lane3). These results
suggest that calyculin A-induced nuclear NF-
B complex contains
predominantly the p50
RelA heterodimer.
p50 NF-
B heterodimer by calyculin A and okadaic acid
involves the phosphorylation and degradation of I
B
. A, antibody super shift analysis of calyculin A-induced nuclear
NF-
B complex. Nuclear extracts from either untreated (NT) (lane1) or calyculin A-treated (25 nM for 1
h, lanes2-7) Jurkat T cells were subjected to
EMSA using a
P-radiolabeled
B probe. To determine the
NF-
B species in the DNA-protein complex, specific antisera
recognizing different NF-
B subunits (lanes4-7) or a preimmune rabbit serum (PI) (lane3) were included in the EMSA. The major super
shifted bands are indicated by arrowheads. B, the
time course analysis of I
B
phosphorylation and degradation,
as well as NF-
B DNA binding activity. Jurkat cells were incubated
with either 25 nM calyculin A (lanes2-5) or 25 nM okadaic acid (lanes6 and 7) for the indicated time periods.
Cytoplasmic and nuclear extracts, isolated from these cells, were
subjected to immunoblotting with an I
B
-specific antiserum (upper panel) and EMSA using the
P-labeled
B
probe (lower panel), respectively. I
B
* is a modified
form of I
B
.
B
, a major
cytoplasmic inhibitor regulating the nuclear expression of the
p50
RelA NF-
B heterodimer. In untreated Jurkat cells, a
single 37-kDa form of I
B
was detected with an
I
B
-specific antiserum (Fig. 1B, upperpanel, lane1). Incubation of the cells
with calyculin A (25 nM) for 5 min led to the appearance of a
more slowly migrating I
B
species (I
B
*, lane2), which became the predominant form of I
B
after 15 min of incubation (lanes3-5). The
slower mobility of this I
B
species was apparently the result
of phosphorylation, since this form could be completely converted to
the fast migrating 37-kDa species by in vitro incubation with
calf intestinal alkaline phosphatase (data not shown). Moreover,
calyculin A-induced phosphorylation of I
B
led to the
subsequent degradation of this cytoplasmic inhibitor. Indeed, the
preexisting I
B
was almost depleted at 60 min posttreatment (Fig. 1B, upperpanel, lane5). Okadaic acid (25 nM) also induced the
phosphorylation of I
B
, albeit with delayed kinetics
(3-8 h, lanes7 and 8). These results
are consistent with the previous finding that calyculin A and okadaic
acid exhibit differential kinetics in the inhibition of cellular
phosphatases(43) .
B
correlated
with that of NF-
B nuclear expression in the cells treated with the
phosphatase inhibitors (Fig. 1B, lower panel).
Thus, activation of NF-
B by the phosphatase inhibitors is likely
mediated through the induction of I
B
phosphorylation and
subsequent degradation.
Calyculin A-induced Phosphorylation of I
Prior studies
have demonstrated that phosphorylation of IB
Is
Not Affected by Inhibitors of TNF-
Signaling
B
by various
NF-
B inducers, such as TNF-
, can be blocked by the
chymotrypsin type of protease inhibitors, such as TPCK, and reducing
agents like PDTC(30, 36, 47) , suggesting the
role of chymotrypsin-like proteases and reactive oxygen species in this
signaling pathway. To explore the signaling pathway involved in the
induction of I
B
phosphorylation by the phosphatase
inhibitors, the effect of TPCK and PDTC on calyculin A-induced
phosphorylation of I
B
was investigated. As expected, in the
absence of these agents, calyculin A induced both the phosphorylation
and degradation of I
B
(Fig. 2, lanes2-4). However, in contrast to that observed with
TNF-
(30) , preincubation of the cells with TPCK did not
affect calyculin A-induced phosphorylation of I
B
(lanes5-7), although this treatment blocked the
subsequent degradation of the I
B
-P (lanes5-7). Furthermore, neither phosphorylation nor
degradation of I
B
induced by calyculin A was affected by
preincubating the cells with PDTC (lanes8 and 9). These results suggest that induction of I
B
phosphorylation by calyculin A does not involve the signaling steps
that require the action of reactive oxygen species or chymotrypsin-like
proteases.
B
. Jurkat cells were
either untreated (NT) (lane1) or incubated
for the indicated time periods with 25 nM calyculin A in the
absence (lanes2-4) or presence of either 50
µM TPCK (lanes5-7) or 200
µM PDTC (lanes 8 and 9). For the double
treatments, cells were preincubated with either TPCK for 30 min or PDTC
for 1 h before adding calyculin A (Cal A) to the culture.
Whole-cell extracts were isolated and subjected to immunoblotting
analysis with the I
B
-specific
antiserum.
A PP-2A-opposing Kinase Appears to Mediate the
Phosphorylation of I
To further explore the biochemical
mechanism underlying the induction of IB
Induced by Calyculin A but Not That
Induced by TNF-
B
phosphorylation by
the phosphatase inhibitors, the molecular nature of the opposing
phosphatases was investigated. In this regard, both calyculin A and
okadaic acid have been shown to potently inhibit the PP-2A class of
phosphatases at low concentrations (IC
is 0.5-1.0
nM) (50) . However, inhibition of PP-1 and PP-2B by
okadaic acid requires much higher concentrations (IC
values are 60-200 nM and 10 µM,
respectively). To explore the phosphatases negatively regulating
I
B
phosphorylation, concentration-dependent induction of
I
B
phosphorylation was performed using okadaic acid as
inducer. As shown in Fig. 3A, incubation of the cells
with as low as 1 nM okadaic acid led to the appearance of the
I
B
-P (lane2). The level of I
B
phosphorylation and subsequent degradation was enhanced in a
dose-dependent manner between 1 and 50 nM (lanes2-5). A similar concentration range was observed
with calyculin A although the induction of I
B
phosphorylation
by this drug was achieved within a shorter time period (30 min, data
not shown) as compared with okadaic acid (12 h, Fig. 3A). These results indicate that induction of
I
B
phosphorylation by these phosphatase inhibitors is
probably a result of the inhibition of PP-2A but not PP-1 or PP-2B type
phosphatase(s). To further explore this possibility, protein extracts
isolated from calyculin A-treated cells were incubated in vitro with various purified protein phosphatases and then subjected to
immunoblotting with the I
B
-specific antiserum (Fig. 3B). In support of our in vivo results,
incubation of the extract with PP-2A (lane2) but not
PP-1 (lane3), PP-2B (lane5), or a
control buffer (lane1) resulted in the complete
conversion of the I
B
-P to its basal form. A similar result of
I
B
dephosphorylation was obtained with okadaic acid-treated
cell extract (data not shown). Together, these in vivo and in vitro results suggest that phosphorylation of I
B
in calyculin A- or okadaic acid-treated cells is likely mediated by a
protein kinase(s) opposing the action of the PP-2A type of
phosphatases.
B
induced by
calyculin A, but not that induced by TNF-
, appears to be
negatively regulated by PP-2A-like phosphatases. A,
concentration dependence of induction of I
B
phosphorylation
by okadaic acid. Jurkat cells were incubated with okadaic acid at the
indicated concentrations for 12 h. Whole-cell extracts were isolated
from these cells and then subjected to immunoblotting with the
I
B
-specific antiserum. I
B
and its phosphorylated
form are indicated. B and C, in vitro dephosphorylation analyses of I
B
-P. A cytoplasmic
extract isolated from either calyculin A-treated (A, 25
nM, 15 min) or TNF-
-treated (C, 10 ng/ml, 5 min)
cells was incubated in vitro with either a control buffer or
the indicated phosphatases at 30 °C for 30 min and then subjected
to immunoblotting using the I
B
-specific antiserum. CIP, calf intestinal alkaline
phosphatase.
-induced phosphorylation
of I
B
is also opposed by the action of the PP-2A type of
phosphatases, in vitro dephosphorylation assays were performed
using a protein extract isolated from TNF-
-stimulated cells and
the various serine/threonine phosphatases as well as a control
nonspecific alkaline phosphatase, calf intestinal alkaline phosphatase (CIP, Fig. 3C, lane2). As
previously observed (49) , the more slowly migrating
phosphorylated form of I
B
(I
B
-P) (Fig. 3C) was readily dephosphorylated by calf
intestinal alkaline phosphatase (lane6). However,
incubation of the extracts with each of the serine/threonine protein
phosphatases tested had no effect on this form of I
B
-P (lanes3-5), which was in sharp contrast to the
dephosphorylation result obtained with calyculin A-induced
I
B
-P (see Fig. 3B). While the molecular
nature of the physiological phosphatases opposing the action of
TNF-
-induced protein kinases remains unknown, these findings
indicate that induction of I
B
phosphorylation by calyculin A
and TNF-
may involve different protein kinases.
A Proteasome Inhibitor Exhibits a Differential Inhibitory
Effect on Calyculin A- and TNF-
Proteasome inhibitors, including MG132 (MyoGenics),
have been recently shown to inhibit TNF--stimulated I
B
Degradation
-induced degradation of
I
B
, suggesting the involvement of the multicatalytic
proteasome complex in this degradation event(35) . To
investigate whether the same type of proteases is also involved in
calyculin A-induced degradation of I
B
, the effect of MG132 on
I
B
degradation was examined by immunoblotting (Fig. 4, upper panel) in cells treated with either calyculin A (lanes2-5) or TNF-
(lanes7-11). Preincubation of the cells for 1 h with 25
µM MG132 completely blocked calyculin A-induced
degradation of I
B
(Fig. 4, upper panel;
compare lanes2 and 3 with lanes4 and 5) as well as the nuclear expression of
NF-
B, as determined by a parallel EMSA (lower panel).
Under the same conditions, MG132 also strongly inhibited the
degradation of I
B
in cells treated with TNF-
(Fig. 4, upper panel; compare lanes7 and 8 with lanes9 and 10).
However, in contrast to the observation with calyculin A, MG132 failed
to completely block the degradation of I
B
induced by
TNF-
. Indeed, even in the presence of MG132, TNF-
still
stimulated slow but significant loss of the I
B
-P (Fig. 4, upper panel; compare lane9 with lane10). Remarkably, along with the
degradation of I
B
-P, a small peptide (
20 kDa) appeared
in the cells and was detected in the immunoblotting with the
anti-peptide antiserum reacting with the C terminus of I
B
(lane10, I
B
-C). This peptide was
apparently the C-terminal degradation product of I
B
since it
could also be detected by a different antiserum specific for the
C-terminal peptide of I
B
(data not shown). Moreover, the
generation of this degradation product was likely mediated through a
protease insensitive to MG132, since the appearance of this small
peptide was not affected even in the presence of higher amounts of
MG132 (50 µM, lane11, and 75
µM, data not shown). Parallel EMSA studies revealed that
MG132 also failed to completely block TNF-
induction of the
nuclear expression of NF-
B (Fig. 4, lower panel;
compare lanes7 and 8 and lanes9-11).
B
. Jurkat T cells were either
untreated (lanes1 and 6) or incubated with
50 nM calyculin A (Cal A) (lanes2 and 3) or 10 ng/ml TNF-
(lanes7 and 8) for the indicated time intervals. For the double
treatments, cells were preincubated for 1 h with the indicated amount
of MG132 and then further incubated for the indicated time periods in
the presence of calyculin A (lanes4 and 5)
or TNF-
(lanes9-13). Whole-cell extracts
were analyzed by immunoblotting with the peptide-specific antiserum
recognizing the C terminus of I
B
. I
B
-C is likely a
degradation product containing the C terminus of I
B
. This
small fragment was also detected with a different antiserum recognizing
the C terminus of I
B
(data not
shown).
Calyculin A and TNF-
In view of the differential induction of I Exhibit Synergistic Action in
the Induction of I
B
Degradation and Activation of
NF-
B
B
phosphorylation by TNF-
and calyculin A, we next investigated
whether these two inducers had synergistic action on the induction of
I
B
degradation and nuclear expression of NF-
B. For these
studies, Jurkat T cells were stimulated with either TNF-
alone (Fig. 5, lanes2-6) or TNF-
together with calyculin A (lanes8-12).
Degradation of I
B
and nuclear expression of NF-
B were
detected by immunoblotting (upper panel) and EMSA (lower
panel), respectively. As expected, incubation of the cells with
TNF-
led to the transient appearance of the I
B
-P and the
gradual degradation of this inhibitor (Fig. 5, upper panel,
lanes2-6), which was concomitant with the nuclear
expression of NF-
B (lower panel). Under these conditions
(10 ng of TNF-
/7
10
cells/ml), TNF-
stimulation for 30 min led to the degradation of only about 50% of the
I
B
molecules (compare lane1 with lane6). Remarkably, costimulation of the cells with both
TNF-
and calyculin A markedly enhanced the rate of I
B
degradation (upper panel, lanes8-12).
Significant loss of I
B
was detected as early as 10 min after
stimulation (lane10), and the entire intracellular
pool of I
B
was almost depleted at 30 min (lane12). Furthermore, the enhanced rate of I
B
degradation was associated with the more rapid and remarkably higher
level of NF-
B nuclear expression (Fig. 5, lower
panel, compare lanes2-6 with lanes8-12). Of note, under the same conditions,
depletion of I
B
by calyculin A alone required at least 60 min
(see Fig. 1B, upper panel). Thus, the
phosphatase inhibitor calyculin A and the cytokine TNF-
have
synergistic activity in the induction of I
B
degradation and
NF-
B nuclear expression.
in the induction of I
B
degradation and
NF-
B nuclear expression. Jurkat cells were incubated with either
TNF-
(10 ng/ml) alone (lanes2-6) or
TNF-
together with 25 nM calyculin A (lanes8-12) for the indicated time periods. Cytoplasmic
and nuclear extracts were subjected to immunoblotting with the
I
B
-specific antiserum (upper panel) and EMSA using
the
P-labeled
B probe (lower panel),
respectively.
B transcription factor are tightly regulated through its
cytoplasmic retention by the ankyrin-rich inhibitor
I
B
(20, 21, 22, 23) .
Activation of NF-
B by various cellular stimuli involves the
proteolytic degradation of I
B
and the concomitant nuclear
translocation of the liberated NF-
B
heterodimer(24, 25, 26, 27, 28, 29, 30) .
Although the biochemical mechanism underlying the degradation of
I
B
remains unclear, it appears that degradation of
I
B
induced by various mitogens and cytokines occurs in
association with the transient phosphorylation of
I
B
(30, 36, 37, 38, 39) .
In the present study, we have demonstrated that the serine/threonine
phosphatase inhibitors, calyculin A and okadaic acid, also induce the
phosphorylation and subsequent degradation of I
B
. Induction
of I
B
phosphorylation by okadaic acid can be achieved at low
concentrations (1-5 nM), at which PP-2A, but not other
phosphatases including PP-1 and PP-2B, is able to be
inhibited(48) . These findings suggest that the action of
okadaic acid, and probably also calyculin A, is mediated through the
inhibition of the cellular PP-2A type of phosphatases. In support of
this notion, the I
B
-P produced in both calyculin A- and
okadaic acid-treated cells can be readily dephosphorylated in vitro by PP-2A but not by PP-1 or PP-2B. This result further suggests
that phosphorylation of I
B
may be directly mediated by a
PP-2A-opposing protein kinase. However, from our current studies, we
are not certain whether the activation of the I
B
kinase is a
direct result of PP-2A inhibition or is mediated through an indirect
mechanism that, for example, may involve the activation of the upstream
signaling molecules.
B by okadaic acid can be partially inhibited by the antioxidant
cysteine, suggesting the involvement of reactive oxygen species in this
induction process(42) . However, our present study is not in
full agreement with these previous findings. We have demonstrated that
induction of the phosphorylation as well as subsequent degradation of
I
B
by calyculin A (25 nM) is not appreciably
inhibited by PDTC, a potent antioxidant that blocks TNF-
-induced
degradation of I
B
and nuclear expression of
NF-
B(25, 50) . This discrepancy is likely due to
the much higher concentration (2 µM) of the phosphatase
inhibitors used in the previous study. At this concentration, both
calyculin A and okadaic acid will nonspecifically inhibit most types of
the serine/threonine phosphatases(48) , which may in turn
trigger additional, perhaps antioxidant-sensitive, signaling pathways.
In support of this proposal, okadaic acid has been shown to synergize
the action of H
O
only at high concentrations (2
µM)(42) . Furthermore, a more recent study using a
low concentration of calyculin A and okadaic acid has revealed that
activation of NF-
B by these phosphatase inhibitors is indeed
insensitive to antioxidants(43) .
B
phosphorylation by calyculin A is not
inhibited by TPCK, a potent inhibitor of the chymotrypsin type of
proteases that blocks TNF-
-induced I
B
phosphorylation(30, 36) . Together, these findings
suggest that induction of I
B
phosphorylation by the
phosphatase inhibitors may either bypass the signaling steps that are
sensitive to TPCK and antioxidants or involve a signaling pathway that
is different from that triggered by TNF-
. Our data favor the
latter possibility. We have shown that the I
B
-P produced in
TNF-
-treated cells can not be dephosphorylated in vitro by PP-2A, although under the same conditions this phosphatase
proves to be efficient in removing the phosphate from calyculin
A-induced I
B
-P. Thus, phosphorylation of I
B
induced
by these two different NF-
B inducers may be mediated by distinct
kinases. However, a more conclusive answer to this question awaits the
precise determination of the phosphorylation sites within these two
types of I
B
-P.
B
degradation by certain proteasome inhibitors
leads to the accumulation of the
I
B
-P(36, 37, 38, 39) ,
thus raising the possibility that phosphorylation of I
B
may
target this inhibitor for degradation by the proteasome. In this
regard, one potential model may involve phosphorylation serving as a
molecular trigger to alter the conformation of I
B
, thus
facilitating the action of the constitutively active proteases.
According to this scenario, phosphorylation of I
B
on
different sites can cause distinct conformational changes, which may in
turn lead to the differential sensitivities of I
B
to the
cellular proteases. Indeed, this outcome has been observed in our
experiments. Specifically, calyculin A-induced degradation of
I
B
can be completely blocked in the presence of 25 µM MG132, suggesting the involvement of solely the MG132-sensitive
proteases in this degradation process. In contrast, TNF-
-induced
I
B
degradation appears to involve additional proteases that
are insensitive to MG132. In the presence of up to 75 µM of MG132, partial degradation of I
B
still occurs in
TNF-
-stimulated cells, which leads to the accumulation of a small
degradation product (
20 kDa) containing the C terminus of
I
B
. Of course, it remains a possibility that calyculin A and
TNF-
both induce I
B
kinases and up-regulate cellular
proteases, which act coordinately to eliminate I
B
.
Nevertheless, from our current studies, we cannot conclude that
phosphorylation targets I
B
for degradation. Studies are in
progress to precisely map the phosphorylation sites within the
I
B
-P, which will allow us to determine the biological
importance of phosphorylation in the degradation of I
B
.
, tumor necrosis factor
; PP-1, PP-2A, and PP-2B,
phosphatase type 1, 2A, and 2B, respectively; TPCK, tosylphenylalanyl
chloromethyl ketone; PDTC, pyrrolidinedithiocarbamate; EMSA,
electrophoresis mobility shift assay; I
B
-P, phosphorylated
I
B
.
We thank Dr. Warner Greene for the
IB
-specific antisera and MyoGenics, Inc. for providing us
with the proteasome inhibitor MG132.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.