From the
The human T-cell leukemia virus type I Tax protein activates
NF-
The human T-cell leukemia virus type I Tax protein is a potent
inducer of NF-
Recently
we have identified two serines (Ser
In the present
study we have utilized the two signal-defective I
Our evidence in support of the involvement of normal
signaling paths in activation of NF-
Our results
rule out a model wherein Tax might have activated NF-
We are grateful to A. S. Fauci for support and review
of the manuscript.
B transcription factors from preformed cytoplasmic pools,
including those pools that are retained by the I
B-
inhibitory
protein. Degradation of I
B-
is enhanced by Tax, resulting in
the liberation of some NF-
B, which then translocates into the
nucleus. Here we have investigated the mechanism by which Tax causes
degradation of I
B-
. Two I
B-
mutants defective in
extracellular signal-induced degradation of I
B-
also blocked
Tax-mediated
B-dependent transactivation when cotransfected into
Jurkat T cells. Cotransfected wild-type I
B-
or an irrelevant
mutant did not significantly effect transactivation induced by Tax. The
signal-defective I
B-
proteins are mutated at either of two
closely spaced serines in the N terminus of the protein (Ser
and Ser
). In wild-type I
B-
, one or both of
these serines are inducibly phosphorylated with extracellular stimuli,
and such phosphorylation appears necessary for subsequent degradation
and thus activation of NF-
B. These results suggest that Tax
triggers I
B-
degradation and thus NF-
B activation by a
mechanism that converges with that induced by extracellular stimulation
such as phorbol 12-myristate 13-acetate/ionomycin or tumor necrosis
factor
. A role for Tax in activating signal transduction pathways
upstream of I
B-
is implied.
B activity
(1) . NF-
B dimers, typically
heterodimers of p50 (NF-
B1) and p65 (RelA), are normally kept in
the cytoplasm by association with their inhibitors, primarily with
I
B-
but also including p100 (NF-
B2), p105
(NF-
B1)
(2) , and I
B-
(3) . Tax induces
translocation of NF-
B from the cytoplasm into the nucleus,
apparently by liberating NF-
B from several distinct cytoplasmic
complexes
(4, 5, 6, 7) . In particular,
degradation of I
B-
is enhanced in the presence of Tax,
suggesting that some NF-
B can be released by rapid turnover of
this inhibitor
(4) . I
B-
is also rapidly degraded
during physiologic activation of NF-
B by extracellular
stimuli
(2) . Such activation proceeds via signal-induced
phosphorylation, which marks I
B-
for proteolytic degradation,
presumably by proteasomes
(8, 9) . The question arises
how Tax induces degradation of I
B-
. Previous reports
suggested at least two divergent explanations. Based on observed
binding of Tax to various I
B and NF-
B
proteins
(6, 7, 10) , Tax may effect dissociation
of the inhibitor, which in turn could lead to rapid turnover of the now
uncomplexed and thus more unstable inhibitor; alternatively, Tax may
have an indirect effect, possibly by affecting physiologic signal
transduction pathways, which then lead to activation of NF-
B.
Indirect support for the latter model comes from the observation that
Tax-expressing cells have detectable amounts of phosphorylated
I
B-
(4, 5) ; in addition, the antioxidant
pyrrolidinedithiocarbamate has been noted to inhibit both normal
signal-induced as well as Tax-induced activation of NF-
B, although
the mechanism by which this occurs is unknown
(11) .
and Ser
)
in the N terminus of I
B-
that are critical to activation of
NF-
B in response to signals such as phorbol 12-myristate
13-acetate (PMA)
(
)
/ionomycin or tumor necrosis
factor
(12) . I
B-
proteins in which either of
these closely spaced serines is altered cannot be inducibly
phosphorylated at these sites nor are the mutant proteins subject to
signal-induced degradation, presumably as a consequence to the block in
phosphorylation. As expected, these I
B-
mutants also prevent
signal-induced activation of bound NF-
B. Wild-type I
B-
is tagged by induced phosphorylation at these serines to undergo rapid
proteolysis. Phosphorylation by itself is insufficient to dissociate
the complex, suggesting that degradation of I
B-
is initiated
while NF-
B is still attached to the inhibitor. In addition to
phosphorylation of the two N-terminal serine(s), signal-induced
degradation of I
B-
also requires additional sequences rich in
Pro, Glu/Asp, Ser, and Thr residues (PEST sequences) located in the
C-terminal PEST region of the inhibitor
(12) .
B-
mutants
in cotransfection experiments together with Tax. We have determined
that Tax-mediated
B-dependent transactivation is potently
inhibited in the presence of these mutant proteins, while wild-type
I
B-
or an irrelevant mutant is a much less effective
inhibitor. The results support a model in which Tax affects signaling
proteins functioning at or upstream of I
B-
to effect
phosphorylation and thus degradation of the inhibitor.
I
IB-
Mutants
B-
mutants used
in this study have been described
(12) . Ser
was
changed to Gly; Ser
was changed to Ala; and Ser
was changed to Ala. The mutants are referred to as m32, m36, and
m63, respectively.
Expression Vectors
pMT2T-p65, pMT2T-Tax,
pMT2T-IB-
, pMT2T-I
B
/m32, pMT2T-I
B-
/m36,
and pMT2T-I
B-
/m63 have been
described
(4, 8, 12) . The luciferase reporter
construct, Ig-
B-Luc, which contains three repeats of the
immunoglobulin
-light chain enhancer
B site
(13) , was
kindly provided by T. Fujita.
Cells, Transfection, and Luciferase Assay
Jurkat
human T-lymphocytes and EL-4 mouse T-lymphocytes stably expressing
either the wild type or the m32 mutant of human IB-
(12) were grown in RPMI 1640 medium supplemented with 10% fetal
calf serum, glutamine, and antibiotics. Electroporation (using Gene
Pulser (Bio-Rad) at 960 microfarads, 250 V) was performed with 10
cells in 300 µl of the culture medium at room temperature.
The luciferase assay was performedas described
(14) using a
luminometer (Analytical Luminescence Laboratory).
RESULTS
Jurkat T cells were transfected with a B-dependent
reporter construct and expression vectors for p65, Tax, and/or
I
B-
(Fig. 1). The amount of I
B-
used for
cotransfection was able to significantly inhibit the p65-mediated
transactivation of the reporter (lanes1 and
2). However, the same amount of I
B-
was unable to
inhibit the Tax-mediated activation of endogenous NF-
B complexes
when assayed with the same reporter (lanes3 and
4). The results suggest that Tax neutralized the inhibitory
effects of these levels of transfected I
B-
. Tax may have
induced degradation not only of endogenous but also of exogenously
introduced I
B-
proteins.
Figure 1:
Tax can neutralize inhibitory effects
of cotransfected wild-type IB-
. Jurkat cells (10
cells) were transfected with Ig-
B-Luc (5 µg) and:
lane1, pMT2T-p65 (3 µg)/pMT2T (12 µg);
lane2, pMT2T-p65 (3 µg)/pMT2T-I
B-
(0.6
µg)/pMT2T (11.4 µg); lane3, pMT2T-Tax (2
µg)/pMT2T (13 µg); and lane4, pMT2T-Tax (2
µg)/pMT2T-I
B-
(0.6 µg)/pMT2T (12.4 µg). The cells
were harvested after 24 h, and luciferase activity was assayed. Results
are expressed as -fold induction relative to the luciferase activity
from cells transfected with Ig-
B-Luc (5 µg)/pMT2T (15 µg).
Each value represents the mean of three
experiments.
We then evaluated the potential
inhibitory effects of signal-defective IB-
mutants on
Tax-mediated activation. Mutations at either Ser
or
Ser
in I
B-
have recently been shown to prevent
phosphorylation that normally occurs at these sites; the two mutant
proteins (m32 and m36) are also not degraded in response to signals
because of the loss in phosphorylation
(12) . In the same series
of experiments reported previously, wild-type I
B-
and a
mutant in which Ser
(m63) was altered were fully
signal-responsive. As shown in Fig. 2, wild-type and all mutant
I
B-
s were equally effective in inhibiting p65-mediated
transactivation of the
B-dependent reporter construct, displaying
quantitatively similar dose-dependent effects. By contrast,
differential inhibitory effects were evident when the same
I
B-
s were tested for their ability to inhibit PMA plus
ionomycin-mediated transactivation of the
B-dependent reporter in
Jurkat T cells (Fig. 3). As expected, the signal-defective
mutants were much more potent in their inhibitory effects than the
wild-type form or the irrelevant mutant; this is true over a wide range
of concentrations (0.6 and 6 µg are shown). At the higher
concentration even the wild-type I
B-
can partially overcome
the signal-induced activation, presumably because the cellular capacity
to degrade I
B-
in response to signals is limited.
Figure 2:
Inhibitory effects of wild type
(wt) and mutant (m32, m36, and m63)
IB-
on transactivation by p65. Jurkat cells were transfected
with Ig-
B-Luc (5 µg) and pMT2T-p65 (3 µg) with or without
the indicated amounts of pMT2T-driven I
B-
expression vectors
(wild type or mutants). Total amount of DNA was kept constant (20
µg) by adding appropriate amounts of the empty expression vector
pMT2T. Cells were harvested after 24 h and analyzed as described in
Fig. 1.
Figure 3:
Effects of wild type (wt) and
mutant IB-
on
B-dependent transactivation by PMA plus
ionomycin. Jurkat cells were transfected with Ig-
B-Luc (5 µg)
with or without the indicated amounts of pMT2T-driven I
B-
expression vectors (wild type, m32, m36, or m63). The total amount of
DNA (20 µg) was kept constant by adding appropriate amounts of the
empty pMT2T expression vector. After 16 h, cells were stimulated with
PMA (20 ng/ml) and ionomycin (2 µM) for 8 h and harvested.
Results are presented as described in Fig.
1.
A
similar set of experiments was performed to test the inhibitory effects
of the various IB-
s on cells that were activated for
NF-
B by cotransfection of Tax rather than by stimulation with
PMA/ionomycin (Fig. 4). The Tax-mediated
B-dependent
transactivation was inhibited in ways indistinguishable from that seen
with extracellular stimulation. The signal-defective mutants were
significantly more potent in inhibiting Tax activation than the
wild-type or the irrelevant mutant (cf. lanes2 and 5 with lanes3 and 4). As
seen before with PMA/ionomycin stimulation, high concentrations of
wild-type I
B-
(or the irrelevant mutant) partially inhibited
transactivation (lanes6 and 9); however,
even at this high concentration the signal-defective mutants were
significantly more effective (lanes7 and
8). The data indicate that alterations at Ser
or
Ser
prevent Tax-mediated activation of NF-
B.
Figure 4:
Effects of
wild type (wt) and mutant IB-
on Tax-mediated
B-dependent transactivation. Jurkat cells were transfected with
Ig-
B-Luc (5 µg) and pMT2T-Tax (2 µg) with or without the
indicated amounts of pMT2T-driven I
B-
expression vectors
(wild type, m32, m36, or m63). Total amount of DNA (20 µg) was kept
constant by adding appropriate amounts of the empty pMT2T expression
vector. Cells were harvested after 24 h and analyzed as described in
Fig. 1.
We
also examined the effects of wild-type or m32 IB-
on
Tax-mediated activation in mouse EL-4 cells that stably expressed these
human I
B-
s (see Ref. 12). The exogenously expressed human
I
B-
s are present at a level comparable with that of the
endogenous mouse I
B-
. Thus approximately equal amounts of
endogenous NF-
B are inhibited and bound by the endogenous mouse
and by the exogenous human I
B-
. The
B-dependent
transactivation of the transiently transfected reporter was
significantly more inhibited in cells harboring the signal-defective
mutant than in cells with the wild-type form (Fig. 5). Again this
is true with both PMA/ionomycin-induced activation of NF-
B as well
as with activation via cotransfected Tax. We had shown previously that
PMA/ionomycin activation does not allow activation of NF-
B
complexes bound by the signal-defective form
(12) . It is thus
significant that the relative inhibition due to the presence of the m32
mutant was the same regardless of whether NF-
B was activated by
Tax or by extracellular signals.
Figure 5:
B-dependent transactivation
stimulated by PMA plus ionomycin (A) or by Tax (B) in
EL-4 cells stably expressing wild type (wt) or m32 mutant
human I
B-
. EL4 cells (10
cells) stably expressing
human I
B-
(either wild type or m32 mutant) were transfected
with: A, Ig-
B-Luc (20 µg); or B,
Ig-
B-Luc (10 µg) plus pMT2T-Tax (10 µg). For A,
half of the cells were not stimulated, and the other half was
stimulated with PMA (20 ng/ml) plus ionomycin (2 µM) 16 h
after electroporation; cells were harvested 8 h later. Results are
expressed as -fold induction of stimulated over unstimulated cells. For
B, cells were harvested 24 h after electroporation, and
results are expressed as -fold induction of cells expressing Tax over
those that do not (these cells were transfected with 10 µg of the
empty pMT2T expression vector instead). Representative data of two
independent experiments are shown.
The level of activation that
remains with the m32 mutant cells is due to release from the
endogenous, signal-responsive NF-B
I
B-
complexes;
these complexes are about equal in amount to the complexes inhibited by
the signal-defective mutant. Unlike the transiently transfected cells
that can express extremely high levels of I
B-
, resulting in
substantial levels of free, unbound I
B-
,
(
)
the permanently transfected cells contain lower levels of
the exogenously introduced form. Therefore the reduced level of induced
transactivation seen in the m32 cells is consistent with a block to
activation of signal-defective mutant-bound complexes regardless of
whether NF-
B was activated by Tax or with PMA/ionomycin.
DISCUSSION
IB-
proteins that contain a mutation at either
Ser
or Ser
effectively inhibit Tax-mediated
B-dependent transactivation; wild-type I
B-
or an
irrelevant mutant does not. We demonstrated this with transient
transfection experiments into Jurkat T cells as well as with EL-4 T
cell lines stably transfected with mutant and wild-type I
B-
.
Ser
and Ser
are critical also to activation
of NF-
B via extracellular signals; they are phosphorylated in
wild-type I
B-
in response to extracellular signals, and
alterations at either site stop signal-induced phosphorylation and
degradation of the respective mutants
(12) . Since we have
demonstrated previously that Tax is capable of inducing degradation of
I
B-
as well
(4) , our data indicate that Tax-mediated
activation of NF-
B is convergent with activation through
extracellular signals. This provides evidence for a model in which Tax
induces phosphorylation of I
B-
at the serine sites whose
phosphorylation is required also for signal-induced degradation of the
inhibitor.
B by Tax is consistent with
prior more indirect evidence cited in the Introduction. On the other
hand, these results are less easily reconciled with a recent report by
Munoz et al.(15) , which suggests that Tax-induced
turnover of I
B-
is not critical to Tax-mediated activation of
NF-
B. It is possible, however, that induced turnover is necessary
but not sufficient to observe optimal Tax-induced NF-
B activity in
cells. It has also been shown that Tax can activate NF-
B from
cytoplasmic complexes that are inhibited by the I
B-like p105 and
p100 precursor proteins, instead of
I
B-
(6, 7, 15) ; the mechanism by which
Tax activates via the precursor complexes is not known.
B via
dissociation of I
B-
-inhibited complexes merely through
physical association with these complexes; for example, Tax might have
bound to proteins competitively, preventing proper association and thus
inhibition by I
B-
, as hypothesized for
I
B-
(10) . Contrary to such a view our results indicate
that Tax must utilize normal physiologic signaling paths to cause
degradation of the I
B-
inhibitor. It remains to be shown
precisely how Tax accomplishes this. It is possible that Tax may induce
phosphorylation/degradation through direct binding to the cytoplasmic
complexes, for example by promoting or targeting the activity of an
I
B-
kinase in this way. Alternatively, Tax may stimulate a
signaling component acting further upstream in the cascade of signals
that transduces an extracellular signal from the membrane to the
cytoplasmic NF-
B complexes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.