(Received for publication, February 8, 1995; and in revised form, August 18, 1995)
From the
The tyrosine kinase inhibitor herbimycin A was found to block
NF-B stimulation in response to interleukin-1 and phorbol
12-myristate 13-acetate in EL4.NOB-1 thymoma cells and phorbol
12-myristate 13-acetate in Jurkat T lymphoma cells. The effect appeared
not to involve inhibition of tyrosine kinase activation as neither
interleukin-1 nor phorbol 12-myristate 13-acetate induced major changes
in tyrosine phosphorylation in EL4.NOB-1 or Jurkat cells, respectively.
Herbimycin A did not interfere with I
B-
degradation, and in
unstimulated cells, it modified NF-
B prior to chemical
dissociation with sodium deoxycholate. Because herbimycin A is
thiol-reactive, we suspected that the target was the p50 subunit of
NF-
B, which has a key thiol at cysteine 62. Herbimycin A inhibited
DNA binding when added to nuclear extracts prepared from stimulated
cells, which were shown to contain high levels of p50. Incubation of
herbimycin A with 2-mercaptoethanol attenuated the effect. Herbimycin A
was also shown to react directly with p50, blocking its ability to bind
to the NF-
B consensus sequence. However, a mutant form of p50 in
which cysteine 62 was mutated to serine was insensitive to herbimycin
A. Finally, we demonstrated that the compound inhibited the expression
of interleukin-2 (an NF-
B-regulated gene) in EL4.NOB-1 cells.
These data therefore suggest that herbimycin A inhibits NF-
B by
modifying the p50 subunit on cysteine 62 in the NF-
B complex,
which blocks DNA binding and NF-
B-driven gene expression. The
results urge caution in the use of herbimycin A as a specific tyrosine
kinase inhibitor and suggest that the development of agents that
selectively modify p50 may have potential as a means of inhibiting
NF-
B-dependent gene transcription.
NF-B is a transcription factor that regulates the
expression of genes involved in the immune and inflammatory responses,
including many that code for cytokines, cell-surface receptors,
adhesion molecules, and acute-phase proteins(1) . It becomes
activated in many cell types in response to viruses, bacteria, and
stress factors as well as inflammatory cytokines such as interleukin-1
(IL-1) (
)(2) and nonphysiological stimuli such as
the protein kinase C activator phorbol 12-myristate 13-acetate
(PMA)(3) . The DNA-binding subunits of NF-
B currently
comprise five members in mammals: p50, p65 (RelA), c-Rel, p52, and RelB (4, 5, 6) . RelA, RelB, and c-Rel are capable
of transactivation, normally forming heterodimers with p50 or p52. This
results in complexes with high DNA binding affinity. The predominant
form of NF-
B in resting cells, however, is a p50-RelA heterodimer,
which is retained in the cytoplasm complexed to an inhibitor protein,
I
B. Upon stimulation with such agents as IL-1, I
B dissociates
from the NF-
B heterodimer, which translocates to the nucleus,
where it binds with high affinity to the NF-
B consensus sequence
in target genes, thereby modulating gene
expression(1, 3) . Multiple forms of I
B also
occur(6, 7, 8, 9, 10) ,
with I
B-
and I
B-
being the two most important forms
for NF-
B activation.
A model for NF-B activation involving
phosphorylation and proteolysis of I
B-
has been
proposed(11, 12, 13, 14) . The
evidence for proteolysis has come from studies demonstrating
I
B-
degradation during the activation process and from the
observation that inhibitors of chymotrypsin-like proteases block
NF-
B activation in response to diverse stimuli(14) .
Recent evidence suggests that the multicatalytic cytosolic protease
(proteosome) may be responsible for I
B-
breakdown(15) . A role for phosphorylation was indicated in
studies demonstrating phosphorylation of I
B-
in vitro by protein kinases A and C and heme-activated kinase, which
resulted in dissociation of I
B from
NF-
B(16, 17, 18) . Furthermore, a
transient change in the electrophoretic mobility of I
B was
apparent in cytosolic extracts following exposure of cells to diverse
stimuli. Treatment of these extracts with calf intestinal phosphatase
or potato acidic phosphatase followed by Western blot analysis showed
that this modified form was converted to the unmodified
(nonphosphorylated) form (11) . Phosphorylation of
I
B-
has not been shown directly, however, nor has the protein
kinase(s) responsible for I
B phosphorylation in intact cells been
isolated. Recent evidence has suggested that I
B-
phosphorylation may tag the protein for subsequent and rapid
degradation by the chymotrypsin-like subunit of the proteosome,
indicating that both phosphorylation and proteolysis are equally
necessary for NF-
B activation(15) . Further complexity is
suggested, however, from studies demonstrating that both p50 and RelA
become phosphorylated upon activation of cells and require
phosphorylation for DNA binding and transactivation(19) .
The involvement of a tyrosine kinase in NF-B activation has
been indicated from the observation that tyrosine kinase inhibitors
such as herbimycin A and genistein inhibit NF-
B activation in
response to IL-1(20, 21, 22) . The target for
these inhibitors has not been precisely determined, however. Both
inhibitors have different mechanisms of action. Genistein is a
competitive inhibitor for tyrosine kinases(23) , while it has
been suggested that herbimycin A, through its benzaquinone moiety, can
directly modify a key thiol group on Src and Abl tyrosine
kinases(24, 25) . The possible involvement of a
Src-like tyrosine kinase was further suggested from studies
demonstrating that expression of v-src in T cells correlated
with NF-
B activation, which was sensitive to herbimycin
A(26) .
Using herbimycin A, we have attempted to clarify the
role of tyrosine kinases in the activation of NF-B in response to
IL-1 and PMA in the murine thymoma cell line EL4.NOB-1 and PMA in the
human T lymphoma line Jurkat E6.1. Our results suggest that the
inhibitory effect is consistent with a model involving the direct
covalent modification of the p50 subunit of NF-
B, rather than
inhibition of tyrosine kinase activity or other signals that lead to
I
B-
dissociation and degradation in these cells. The results
further suggest that, using herbimycin A, it is possible to modify the
p50 subunit of NF-
B in intact cells and interfere with DNA binding
and NF-
B-driven gene expression.
Experiments were also carried out with recombinant human p50 and a mutant form of this protein in which cysteine 62 was mutated to serine. Both proteins were in buffer comprising 10 mM Tris-HCl, pH 7.5, 1 M NaCl, 10 mM EDTA, 40% glycerol, 1 mg/ml nuclease-free bovine serum albumin, and 0.25 mM DTT. 11 ng of p50 or 200 ng of C62S mutant were incubated with herbimycin A (100-0.1 µM) for 2 h at 37 °C prior to assessing DNA binding. Higher amounts of mutant were required to detect DNA binding, as described previously(30) .
Figure 1:
Herbimycin A inhibits NF-B
activation by IL-1
and PMA in EL4.NOB-1 and Jurkat E6.1 cells.
Cultures of EL4.NOB-1 cells (1-5
10
/ml) were
pretreated with the indicated concentrations of herbimycin A (HbA) or medium alone for 1 h, followed by stimulation with or
without IL-1
(10 ng/ml) for 1 h (A) or PMA (100 ng/ml)
for 24 h (B). Cultures of Jurkat E6.1 cells (5
10
/ml) were similarly pretreated with herbimycin A,
followed by stimulation with PMA (100 ng/ml) for 1 h (C).
Nuclear extracts were prepared subsequent to stimulation and analyzed
for NF-
B binding activity as described under ``Experimental
Procedures.'' Open arrowheads indicate constitutive
NF-
B, and closed arrowheads indicate induced
NF-
B
DNA complexes. Unbound free probe (FP) is shown
in A, while B and C show NF-
B
DNA
complexes only.
The ability of herbimycin A to inhibit the activation of NF-B
by PMA was in direct contrast to a previous study(20) , which
reported that PMA activation of NF-
B in 70Z/3 pre-B cells was
insensitive to the drug. 70Z/3 cells differ from EL4 cells in that the
activation of NF-
B by PMA in the cells is more rapid. We therefore
next examined another cell type in which PMA causes rapid activation of
NF-
B, the human T lymphoma line Jurkat E6.1. A 1-h treatment of
the cells with PMA resulted in a strong activation of NF-
B (Fig. 1C, lane 2), which was sensitive to
herbimycin A, with concentrations of 0.02-2 µM inhibiting this activation (lanes 3-5).
Furthermore, a similar pattern of inhibition was observed following a
24-h exposure to PMA (data not shown).
Figure 2:
Effect of herbimycin A on protein tyrosine
phosphorylation in EL4.NOB-1 and Jurkat E6.1 cells. Cultures of
EL4.NOB-1 (A) and Jurkat (B) cells (5
10
/ml) were pretreated for 1 h with 2 µM herbimycin A (HbA) or left untreated and then exposed to
medium and IL-1
(10 ng/ml) (A), PMA (100 ng/ml) (B), or PMA (10 ng/ml) and PHA (10 µg/ml) (C) for
the indicated times. Cell lysates were prepared and subjected to
SDS-polyacrylamide gel electrophoresis along with protein molecular
mass markers as described under ``Experimental Procedures.''
The proteins were transferred onto nitrocellulose membranes and stained
with Ponceau S to ascertain equal loading and position of molecular
mass markers. The blots were then probed with anti-phosphotyrosine
antibody and processed as recommended using enhanced chemiluminescence. A shows a control extract and an IL-1-treated extract from EL4
cells probed using the primary antibody in the presence of 1.5
mM phosphotyrosine (lanes 7 and 8) or 1.5
mM phosphoserine (lanes 9 and 10). Molecular
mass standards are indicated in kilodaltons. Arrowheads indicate the proteins that consistently showed enhanced
phosphorylation in response to IL-1
(A), PMA (B), or PMA + PHA (C).
Figure 3:
Herbimycin A does not prevent the
IL-1- or PMA-stimulated degradation of I
B-
and modifies
NF-
B prior to chemical dissociation with deoxycholate. Cell
cultures (5
10
/ml) were pretreated either with
medium or with 2 µM herbimycin A (HbA) for 1 h
prior to stimulation for various periods with IL-1
(10 ng/ml) for
EL4.NOB-1 cells (A) or with PMA (100 ng/ml) for Jurkat E6.1
cells (B). Cell lysates were prepared according to the method
outlined under ``Experimental Procedures'' and subjected to
SDS-polyacrylamide gel electrophoresis. Proteins were transferred to
nitrocellulose membranes and probed with anti-I
B-
antibody
according to the manufacturer's recommendations (Santa Cruz
Biotechnology, Inc.). Blots were developed as recommended for enhanced
chemiluminescence (Amersham International). No other proteins other
than those shown were detected. Corresponding molecular mass markers
are shown in kilodaltons. In C, EL4.NOB-1 cells (5
10
/ml) were left untreated (lanes 1 and 2) or were treated with herbimycin A (2 µM) (lanes 3 and 4) for 1 h; cytosolic extracts were
prepared and left untreated (lanes 1 and 3) or were
treated with deoxycholate and Nonidet P-40 (lanes 2 and 4) as described under ``Experimental Procedures.''
Samples were then assessed for NF-
B. NF-
B
DNA complexes
are presented.
This
was further suggested from studies involving deoxycholate-treated
cytosolic extracts, as shown in Fig. 3C. As has been
well documented, treatment of cytosolic extracts prepared from
unstimulated cells with detergents such a deoxycholate and Nonidet P-40
will reveal latent NF-B by chemically dissociating I
B from
the NF-
B complex(37) . Deoxycholate-treated cytosolic
extracts from EL4 cells incubated with herbimycin A showed decreased
DNA binding activity compared with control cells that had not been
exposed to herbimycin A (Fig. 3B, compare lanes 2 and 4). This result suggested that herbimycin A was able
to interfere with NF-
B while complexed with I
B-
in the
cytosol and that this impaired the DNA binding capability of the
p50-RelA heterodimer.
Figure 4:
Herbimycin A reacts directly in vitro with IL-1-activated NF-
B and recombinant p50. A, nuclear extracts (4 µg) from IL-1
-stimulated cells
(10 ng/ml, 1 h) (lane 2) were incubated in vitro with
250 µM herbimycin A (lane 3) or a mixture of
herbimycin A (HbA) and 2-mercaptoethanol (2-ME) (142
mM) (lane 4) for 10 min at room temperature. The
mixture of herbimycin A and 2-mercaptoethanol had been incubated for 10
min at room temperature prior to addition to extract. Extracts from
IL-1-treated cells were also incubated with vehicle (dimethyl
sulfoxide) (lane 5) or 2-mercaptoethanol (142 mM)
alone (lane 6). Binding buffer and poly(dI
dC) were then
added, followed by a 30-min incubation with radiolabeled NF-
B
probe. Extracts were analyzed for NF-
B DNA binding activity as
described under ``Experimental Procedures.'' A control
extract from cells not treated with IL-1 is also shown (lane
1). B, NF-
B
DNA complexes are presented. The
protocol employed in A was repeated with slight modifications
using nuclear extracts from EL4.NOB-1 cells treated with IL-1 (10
ng/ml, 1 h) prepared in DTT-free buffers. Lane 1 shows samples
from IL-1-treated cells subsequently left untreated. The indicated
concentrations of herbimycin A (lanes 2-5) or of the
mixture of herbimycin A and 2-mercaptoethanol (lanes
6-9) were added to extracts as indicated and left for 30 min
at room temperature. Binding buffer with poly(dI
dC) but without
DTT was then added and assayed for
NF-
B.
We
suspected that the presence of 50 mM DTT in the DNA binding
reaction buffer might react with herbimycin A, thus decreasing the
concentration of herbimycin A available to react with thiol group(s) on
NF-B. Therefore, we probed this possibility as this could account
for the higher concentration of herbimycin A required in vitro to observe an effect. This involved carrying out the binding
reaction in the absence of DTT as well as using nuclear extracts
prepared in DTT-free buffers. As DTT aids DNA binding, the overall
binding capability of IL-1-activated NF-
B was slightly diminished (Fig. 4B, lane 1) Much lower concentrations of
herbimycin A were capable of inhibiting DNA binding in the absence of
DTT, however (Fig. 4B, lanes 2-5). Prior
incubation of herbimycin A with 2-mercaptoethanol again prevented
herbimycin A from affecting NF-
B (Fig. 4B, lanes 6-9). The presence of 2-mercaptoethanol generally
increased DNA binding in all samples, as expected (Fig. 4B, lanes 6-9), with DTT having a
similar effect (data not shown). High concentrations of vehicle
(dimethyl sulfoxide) equivalent to those used in experiments with 250
µM herbimycin A had a modest inhibitory effect on the
fastest migrating complex (Fig. 4B, lane 10).
Figure 5:
Recombinant human p50, but not the C62S
p50 mutant, is susceptible to inhibition by herbimycin A. 11 ng of
human recombinant p50 (A) or 200 ng of C62S p50 mutant (B) were incubated with the indicated concentrations of
herbimycin A (HbA) at 37 °C for 2 h. Binding buffer and
poly(dIdC) were then added, and the samples were assessed for
B binding as described under ``Experimental
Procedures.'' Protein-DNA complexes are
shown.
We next demonstrated the presence of p50
in IL-1- and PMA-treated cell extracts. Fig. 6A (lane
4) shows that nuclear extracts from IL-1-stimulated EL4.NOB-1
cells contained large amounts of p50, as indicated by specific
antibodies to p50 causing a supershift in the DNA probe. p50 was also
detected in untreated samples (Fig. 6A, lane
3). Much lower amounts of RelA were evident in the samples (Fig. 6A, lane 6), the gels requiring
prolonged exposure to reveal this subunit, hence the high levels of
NF-B apparent in samples prepared from unstimulated cells shown in
this figure. No evidence for c-Rel in the DNA-binding complexes was
obtained in nuclear extracts of either untreated or IL-1-treated EL4
cells (Fig. 6A, lanes 7 and 8).
Similarly, nuclear extracts from EL4 cells treated with PMA for 24 h
showed strong supershifts with anti-p50 and anti-RelA antibodies (Fig. 6B, lanes 4 and 6,
respectively). As in Fig. 5A, untreated EL4 cells
contained p50 (Fig. 6B, lane 3).
Interestingly, samples from PMA-stimulated EL4 cells contained much
higher levels of RelA (Fig. 6B, lane 6) than
IL-1-treated samples, indicating that PMA probably induced RelA
expression in these cells. Using nuclear extracts from Jurkat cells
treated for 1 h with PMA, it again appeared that the cells contained
high levels of p50 (Fig. 6C, compare lanes 1 and 2). RelA was also detected (Fig. 6C, lane 3), and the decrease in binding observed in the presence
of anti-c-Rel (lane 4) suggested that c-Rel was also present.
The marked effect of the antibodies on the extracts indicted that the
major protein complexes binding to the NF-
B motif were p50 and, to
a lesser extent, RelA. Taken together, these results indicated that
herbimycin A could directly modify p50 most probably at cysteine 62,
and the presence of large amounts of p50 in nuclear extracts suggested
that this would be a likely target for herbimycin A in cells.
Figure 6:
IL-1- and PMA-activated NF-
B
contains p50 and RelA subunits of NF-
B. Nuclear extracts from
unstimulated (control (C)) and IL-1
-treated (10 ng/ml, 1
h) or PMA-treated (100 ng/ml, 24 h) EL4.NOB-1 cells (A and B, respectively) and PMA-treated (100 ng/ml, 1 h) Jurkat E6.1
cells (C) were incubated with antibodies to the p50, RelA, or
c-Rel subunits of NF-
B (as indicated) for 30 min at room
temperature at O °C. Binding buffer and poly(dI
dC) were then
added, followed by a 30-min incubation with radiolabeled NF-
B
probe. Extracts were analyzed for NF-
B DNA binding activity as
described under ``Experimental Procedures.''
NF-
B
DNA complexes are presented. Open arrowheads indicate constitutive NF-
B, and closed arrowheads indicate induced NF-
B
DNA complexes. Supershifted
complexes corresponding to p50 and RelA are
indicated.
Figure 7:
Inhibition of IL-1- and PMA-induced
IL-2 production by herbimycin A in EL4.NOB-1 cells. Cultures of
EL4.NOB-1 cells (1
10
/ml) were incubated in the
presence or absence of the indicated concentrations of herbimycin A (HbA) for 1 h. Cells were then washed, resuspended in medium,
and left untreated or treated with IL-1 (5 ng/ml) (A) or PMA
(1 ng/ml) (B) for a further 24 h. Supernatants were removed
and assayed for IL-2 by enzyme-linked immunosorbent assay as described
under ``Experimental Procedures.'' Each value represents the
mean ± S.E. for three separate experiments carried out in
triplicate. *, p < 0.05;**, p <
0.01
The initial aim of this study was to explore the involvement
of tyrosine kinase(s) in the activation of NF-B by IL-1 and PMA in
T lymphocytes using the tyrosine kinase inhibitor herbimycin A. We
demonstrated that herbimycin A inhibited the activation of NF-
B by
both IL-1 and PMA in EL4.NOB-1 thymoma cells and PMA in Jurkat E6.1
lymphoma cells. The target for herbimycin A, however, appeared not to
be tyrosine kinases or indeed signals leading to I
B-
dissociation and degradation, but the NF-
B complex itself. Our
results are consistent with a model involving covalent modification by
herbimycin A of cysteine 62 on the p50 subunit of NF-
B.
We
initially suspected that herbimycin A was affecting something other
than tyrosine kinases because of our observation that only a 1-h
treatment with herbimycin A was required to observe inhibition.
Previous studies have shown that optimal inhibition of Src kinase by
the drug requires prolonged exposure as part of the mechanism of
inhibition involves enhanced degradation of the
enzyme(40, 41) . In addition, the observation that
herbimycin A inhibited the activation of NF-B by PMA was
unexpected as PMA is known to have weak effects on tyrosine
phosphorylation in cells(42, 43, 44) , its
major cellular target being protein kinase C. We clearly demonstrated
that herbimycin A could inhibit the activation of NF-
B by PMA,
suggesting either that tyrosine kinases were important for PMA action
or, alternatively, that herbimycin A was inhibiting something other
than tyrosine kinases. Only minor changes in tyrosine phosphorylation
were observed in response to IL1 or PMA, with herbimycin A having a
negligible inhibitory effect. Our failure to detect major changes in
tyrosine phosphorylation in EL4 cells in response to IL-1 is in
contrast to other reports showing such changes in K562
cells(45) , human A375-C6 melanoma cells(21) , and Th2
cells(22) . The major change in tyrosine phosphorylation
demonstrated in these studies occurred in the molecular mass range
40-45 kDa. It is likely that these corresponded to p42/p44 MAP
kinases as, in another study, it was demonstrated that IL-1 increased
the phosphorylation of p42/p44 MAP kinases on tyrosine, threonine, and
serine residues(46) . The phosphorylations were likely to be
due to the activation of MAP kinase kinase, which is a dual specificity
tyrosine/serine-threonine kinase that phosphorylates MAP kinase (47) and is activated by IL-1 in fibroblasts and KB epidermal
cells(48) . IL-1 has been shown to be a poor activator of MAP
kinases in EL4 cells(46) , which is consistent with our failure
to detect major changes in tyrosine phosphorylation in the molecular
mass range for MAP kinases in these cells. The area of IL-1 signal
transduction is controversial, with no clear pathway emerging despite
intense effort(49, 50, 51) . A consensus on
the activation of serine/threonine kinases belonging to the MAP kinase
family has recently emerged, however(52, 53) ; and any
tyrosine kinase changes that occur in response to IL-1 are likely to be
on kinases in these pathways rather than there being a general increase
in tyrosine phosphorylation. The involvement of MAP kinases in
NF-
B activation is ill defined.
Previous workers have suggested
a role for tyrosine kinases in the activation of NF-B by IL-1. The
evidence, however, has been largely circumstantial, with data being
presented for tyrosine kinase changes in response to IL-1 that were
inhibited by herbimycin A or another tyrosine kinase inhibitor,
genistein(20, 21) . These results were then used as
evidence for tyrosine kinase involvement in the activation of
NF-
B, which also proved susceptible to inhibition by the
compounds. The precise identity of the tyrosine kinase involved or
indeed its substrate were not determined, although recent evidence
demonstrating that overexpression of v-src leads to NF-
B
activation has suggested a role for a Src-like tyrosine
kinase(26) . Both of the inhibitor studies suggested, however,
that the putative tyrosine kinase involved in IL-1 action must occur
upstream of I
B release and degradation. Our failure to demonstrate
inhibition of I
B degradation, coupled with the effect on latent
NF-
B, questioned signal transduction processes being the target
for herbimycin A and indicated that NF-
B complexed to I
B was
susceptible to herbimycin A inhibition.
Because of these findings,
our efforts next turned to NF-B itself. The mechanism of action of
herbimycin A with regard to tyrosine kinases involves the covalent
modification of a thiol group on target kinases such as
pp60
(24, 25, 38) .
The evidence for this initially came from the observation that
thiol-containing compounds such as 2-mercaptoethanol prevented the
inhibitory effect of herbimycin A on pp60
(25) and, more recently, from a direct demonstration that
herbimycin A covalently modifies both Src and Abl(24) . The
chemical moiety on herbimycin A that has been implicated in this
modification is a benzaquinone group that is highly
thiol-reactive(38) . Because the p50 subunit of NF-
B has
been shown to contain a key thiol on cysteine 62 that, if mutated to
serine or oxidized, greatly reduces DNA binding(30) , we
suspected that this may have been the target for herbimycin A. This was
supported by experiments in which herbimycin A proved inhibitory when
added directly to nuclear extracts from activated cells, an effect
blocked by first treating herbimycin A with 2-mercaptoethanol or DTT.
In these in vitro experiments, higher concentrations of
herbimycin A were needed than in intact cells. Similar differences have
been shown by other workers, whereby experiments demonstrating a direct
effect on tyrosine kinases in vitro require concentrations in
the 17-175 µM range(24, 25, 38) , whereas inhibition
of tyrosine kinases in intact cells generally involves submicromolar
concentrations(40, 54) . It is possible that
herbimycin A becomes concentrated inside cells. Comparing in vitro experiments with those in vivo is somewhat difficult,
however, as conditions inside the cell under which herbimycin A reacts
with thiol groups will differ from those pertaining in vitro.
This has also been postulated by Fukazawa et al.(24) as an explanation for concentration differences with
regard to tyrosine kinases. They suggest that the target kinases in
their native environment may be more accessible to the drug than in
vitro. This may also apply to p50.
We also found that the
ability of recombinant human p50 to bind DNA probe containing the
NF-B consensus sequence was blocked, while the C62S mutant was
unaffected. p50 has three cysteines at positions 62, 119, and 273,
which would be potential targets for herbimycin A. Mutagenesis studies
have shown that only cysteine 62 is important for DNA binding since the
C62S mutation decreased the affinity for the
B site
10-fold(30) . We found that the C62S mutant of p50 was
insensitive to herbimycin A. These in vitro data, coupled with
the detection of large amounts of p50 in EL4 and Jurkat cells, led us
to conclude that the most likely target for herbimycin A in EL4 and
Jurkat cells was cysteine 62 on p50. We were unable to carry out
experiments that would demonstrate adduct formation between herbimycin
A and p50 in intact cells. Previous experiments on herbimycin A and
tyrosine kinases have used metabolically labeled herbimycin A of low
specific activity and immune complexes from v-src-transformed
NIH3T3 fibroblasts or K562 cells, which express high levels of
p210
(25) . Analogous experiments on p50 in
EL4 or Jurkat cells would be difficult to perform. The amount of p50 in
the cells is likely to be very low compared with the tyrosine kinases
in the aforementioned study, and the low specific activity of
herbimycin A would further lower the detection limit of this approach.
Finally, we found that herbimycin A blocked IL-1- and PMA-induced
IL-2 in EL4 cells over a concentration range identical to that which
inhibited NF-B activation. The ability of herbimycin A to
interfere with p50 is therefore likely to have consequences for
NF-
B-driven gene expression as NF-
B has been shown to have a
central role in the induction of IL-2 by IL-1(39) .
In
conclusion, the results presented here therefore urge caution in the
use of herbimycin A as a specific tyrosine kinase inhibitor. They
further suggest that the ability to selectively modify p50 may have
potential as a means of inhibiting NF-B-dependent gene
transcription.