(Received for publication, December 19, 1994; and in revised form, April 6, 1995)
From the
Inhibitors of phosphotyrosyl protein phosphatases, pervanadate
and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced
nuclear factor B (NF-
B) nuclear translocation in transformed
cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF).
The inhibitors also abrogate NF-
B activation by the
phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in
U-937 cells. Inhibition of NF-
B activation is not due to a general
inhibitory effect since neither pervanadate nor phenylarsine oxide
treatment affected the constitutive DNA-binding activity of the
transcription factors octamer-1 and cAMP response element-binding
protein in U-937 cells, nor did these compounds inhibit the TNF-induced
phosphorylation of proteins, viz. hsp-27, eukaryotic
initiation factor 4e, and pp19, in MRC-5 fibroblasts. Overexpression of
the protein-tyrosine phosphatase HPTP
resulted in a constitutive
nuclear NF-
B-like DNA-binding activity in REF cells. Conversely,
treatment of human protein-tyrosine phosphatase
-overexpressing
cells with phenylarsine oxide led to a loss of the constitutive
NF-
B activity. The presence of a tyrosine phosphorylation site on
the inhibitor of NF-
B (I
B-
) suggested that it could be a
target for TNF/okadaic acid-induced tyrosine dephosphorylation.
However, no tyrosine phosphorylation was detected on I
B-
from
unstimulated cells, while TNF/ okadaic acid-treated cells showed
increased phosphorylation of I
B-
exclusively at serine
residue(s). Treatment of cells with pervanadate inhibited TNF-induced
I
B-
phosphorylation and degradation, whereas the serine
protease inhibitors tosylphenylalanyl chloromethyl ketone and N
-p-tosyl-L-lysine
chloromethyl ketone prevented TNF-induced I
B-
degradation and
NF-
B nuclear translocation, but not the TNF-induced
phosphorylation of I
B-
. The data suggest that TNF and okadaic
acid induce the activation of a putative protein-tyrosine
phosphatase(s), leading to I
B-
serine phosphorylation and
degradation and NF-
B nuclear translocation.
Reversible protein tyrosine phosphorylation is a common feature
in early transmembrane signaling, affecting cellular metabolism,
proliferation, and differentiation(1, 2) . Although
the activation of growth factor receptor and non-receptor tyrosine
kinases was previously thought to initiate many cellular processes,
with PTPases ()playing a crucial but more passive role as
signal terminators, it is increasingly clear that PTPases cannot be
only regarded as antagonists of the tyrosine kinase activity. Indeed,
several PTPases are now known to act as positive regulators of a
variety of cellular processes, including proliferation, T-cell
activation, and hematopoiesis(3, 4) .
TNF is a
primary mediator of the immune and inflammatory responses(5) .
It has also been implicated in the pathogenesis of acquired immune
deficiency syndrome, the endotoxic shock response, and cachexia. Two
TNF receptors have been identified(6) , both of which lack
intrinsic phosphotransferase activity. Even so, the binding of TNF to
its receptor(s) rapidly induces the phosphorylation of several proteins (7) . The lack of receptor phosphotransferase activity and the
stringent requirement for receptor trimerization in signal initiation (6) suggest that the signal may be transduced through
association of the receptor(s) with signaling molecules. The positive
regulatory role of PTPases in the signaling systems of other cytokines, viz. IL-3, IL-4, granulocyte/macrophage colony-stimulating
factor, and interferon types 1 and 2(4, 8) , the
receptors of which are also devoid of phosphotransferase activity,
resulted in the initiation of this study to determine whether PTPases
were similarly involved in the early post-receptor events induced by
TNF. For this, the activation of the transcription factor NF-B was
chosen as an end point assay for TNF signal transduction since TNF is a
potent and rapid inducer of NF-
B (9) . Many of the
TNF-induced genes, viz. human immunodeficiency virus type 1,
major histocompatibility complex class 1 and 2 antigens, IL-2 receptor
-chain, IL-6, and IL-8, appear to require NF-
B for
transcriptional activation(10) .
NF-B is a heterodimer
comprising a 48-55-kDa DNA-binding subunit (p50 or NF-
B1)
and a 65-68-kDa transactivator (p65 or RelA)(10) . It is
sequestered within the cytosol by association with an inhibitory
protein known as I
B-
. Activation is post-translational and
results from the dissociation of the I
B-
NF-
B
complex followed by translocation of the released NF-
B into the
nucleus. Several other agents are known to activate NF-
B,
including the cytokines IL-1 and IL-2, viruses, bacterial
lipopolysaccharides, and T-cell activators(10) . Although
modification of I
B-
by phosphorylation and/or proteolysis
appears to be involved in NF-
B
activation(11, 12, 13, 14) , none of
the signaling pathways leading to activation has been fully elucidated.
Several signaling events have been implicated in the TNF-induced
phosphorylation of I
B-
, viz. activation of
phosphatidylcholine-specific phospholipase C/sphingomyelinase/ceramide,
Ras/c-Raf, and phosphatidylcholine-specific phospholipase
C/
-protein kinase C
pathways(13, 15, 16) , while recent findings
suggest that TNF-induced I
B-
degradation occurs via the
ubiquitin-proteasome pathway(17, 18) .
In this
study, we report that PTPase inhibitors, pervanadate and phenylarsine
oxide (PAO), abrogate the TNF-induced nuclear translocation of
NF-B in all transformed and primary cells tested. The inhibitors
also abrogate NF-
B activation by okadaic acid, a
phosphoseryl/threonyl phosphatase inhibitor that mimics the early
phosphorylation events induced by TNF(19) . Correspondingly, a
constitutive NF-
B-like DNA-binding activity was present in nuclear
extracts of REF cells overexpressing the receptor-like PTPase
HPTP
. Although a tyrosine phosphorylation site is present on
I
B-
(20) , it was not phosphorylated at tyrosine and
was found to undergo only serine phosphorylation in TNF- or okadaic
acid-treated cells. Pervanadate treatment inhibited both the
TNF-induced phosphorylation and degradation of I
B-
,
suggesting that PTPase activity is required for the TNF/okadaic
acid-induced phosphorylation and proteolysis of I
B-
and
NF-
B activation. Although I
B-
phosphorylation is
sufficient to induce NF-
B DNA-binding activity in
vitro(11, 12, 13) , this is not the case in vivo as the protease inhibitors TPCK and TLCK, which do not
affect the TNF-induced phosphorylation of I
B-
, effectively
prevent the TNF-induced proteolysis of I
B-
with a concomitant
loss of NF-
B nuclear translocation.
Figure 1:
PTPase inhibitors abrogate the TNF- or
okadaic acid-induced NF-B DNA-binding activity. Cells were treated
with H
O
(1 mM), orthovanadate (Van) (100 µM), pervanadate (pV) (100
µM; prepared by mixing H
O
at 1
mM with orthovanadate at 100 µM), or PAO (2.5
µM) in the absence or presence of TNF at 1.2 nM for 15 min (A, C, and D) or with
okadaic acid (OA) at 2 µM for 30 min (E). Nuclear extracts (5 µg) were analyzed for NF-
B
DNA-binding activity using
P-labeled wild-type NF-
B
sequence. The protein-DNA complexes were immunologically identified as
NF-
B or homodimers of p50(11) . Nonspecific DNA-binding
complexes (n.s.) and the unbound probe (open
arrowheads) are indicated. B, U-937 cells were treated
with various compounds as described above. Whole cell extracts (10
µg of protein) were analyzed for phosphotyrosine by
immunoblotting.
Immunoblotting with an anti-phosphotyrosine antibody
showed that pervanadate and PAO treatment resulted in a marked increase
in the phosphotyrosine content of cellular proteins, an observation
consistent with the expected inhibitory effect of these compounds on
PTPase activity (Fig.1B, compare lane1 with lanes4 and 5). However,
differences were noted between the pervanadate- and PAO-induced
patterns of protein phosphorylation, suggesting differences in the
sensitivity of cellular PTPases to the two agents. Although inhibition
of PTPase activity by HO
or orthovanadate alone
has been reported (28, 31) , this was not observed
here (Fig.1B, lanes 1-3). The effect of
pervanadate, H
O
, and orthovanadate on PTPases
was verified by PTPase activity assays using extracts from
pervanadate-treated cells, which possessed no detectable PTPase
activity against the tyrosine-phosphorylated hirudin C-terminal peptide
(residues 53-65) or gastrin N-terminal peptide (residues
1-17) as substrate, while the activity in
H
O
- or orthovanadate-treated cells was
comparable to that in untreated controls (data not shown). This
indicates that H
O
or orthovanadate alone is
significantly less effective than pervanadate as a PTPase inhibitor
when used on intact cells. The inhibition of NF-
B activation by
pervanadate was dose-dependent (25-100 µM) and
correlated with the inhibition of cellular PTPase activity (data not
shown).
Pervanadate and PAO also inhibited the TNF-induced NF-B
DNA-binding activity in Jurkat T-cells (Fig.1C) and
primary MRC-5 fibroblasts (Fig.1D). Both inhibitors
also abrogated NF-
B activation by okadaic acid (Fig.1E), a compound reported to mimic the early
phosphorylation events induced by TNF (19) .
Figure 2:
PTPase inhibitors do not inhibit octamer-1
and CREBP DNA-binding activities or the TNF-induced phosphorylation of
cellular proteins. A and B, nuclear extracts from the
U-937 cells used in Fig.1were analyzed for octamer-1 (Oct-1) or CREBP DNA-binding activity using P-labeled oligonucleotides encompassing the binding site
for either transcription factor. C-F, shown is the
two-dimensional gel electrophoresis of cytosolic extracts from
P-labeled MRC-5 fibroblasts treated with TNF alone or
together with pervanadate (pV) or PAO as described in the
legend to Fig.1. The major substrates for TNF-induced protein
phosphorylation (hsp-27, eIF-4e, and pp19) are
indicated.
Figure 3:
PTPase inhibitors abrogate the TNF-induced
nuclear translocation of NF-B. A-D, MRC-5
fibroblasts were immunostained with peptide antigen affinity-purified
anti-p65 antibody. The bound antibody was visualized with biotinylated
goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated avidin. E, cytosolic extracts (20 µg of protein) from U-937 cells
were incubated with labeled probe for 10 min, after which sodium
deoxycholate (DOC) was added to a final concentration of 0.5%,
followed by Nonidet P-40 at 1.2% (lanes 5-9). The
wild-type NF-
B sequence was used as the probe in all lanes except lane9, where the mutant sequence was used instead.
Nonspecific protein-DNA complexes (n.s.) and the unbound probe (open arrowhead) are indicated. The time and dosage of the
treatments were as described in the legend to Fig.1. pV, pervanadate.
Figure 4:
NF-B-like DNA-binding activity in
cells overexpressing HPTP
and inhibition of the DNA-binding
activity by PAO. A, nuclear extracts from REF cells treated
with TNF alone (lane2) or in combination with
pervanadate (pV) (lane3) or PAO (lane4) were analyzed for NF-
B-like DNA-binding activity
together with nuclear extracts from REF cells transfected with the
vector pXJ41
(lane5) or with the
vector encoding the PTPase HPTP
(lanes6 and 7). The labeled wild-type NF-
B oligonucleotide was used
as the probe in all lanes except lane7, where the
mutant sequence served as the probe. Specific protein-DNA complexes (Bands 1-3), the nonspecific protein-DNA complex (n.s.), and the unbound probe (openarrowhead) are indicated. B,
HPTP
-expressing cells were incubated with PAO for the times
indicated, after which nuclear extracts were prepared and analyzed for
NF-
B-like DNA-binding activity.
Figure 5:
Kinetics of ligand-induced IB-
phosphorylation and proteolysis and NF-
B activation. A,
immunoprecipitation of I
B-
from
P-labeled cells.
U-937 cells were treated with TNF (1.2 nM) for various lengths
of time (lanes 2-8) or with okadaic acid (OA)
(2 µM) for 15 min (lane10).
Orthophosphate-labeled MRC-5 fibroblasts were incubated with TNF at 1.2
nM for 2 min (lane12). Cells were lysed,
and I
B-
was immunoprecipitated with an antibody raised
against its C terminus (lanes 1-6 and 9-12) or with the same antibody in the presence of a 1
mg/ml concentration of the peptide against which it was generated (lane7). Preimmune serum was used instead of the
anti-I
B-
antibody in lane8.
Immunoprecipitates were analyzed by SDS-PAGE followed by
autoradiography. B, in vitro transcription and
translation of HA-tagged I
B-
. Expression vector pXJ41
(lane1) or the vector carrying the
HA-I
B-
insert (lanes 2-4) was transcribed and
translated in vitro in the presence of 2.5 mCi/ml
[
S]methionine. An aliquot (
3 ng) of the
synthesized product was treated with 40 units/ml protein phosphatase 1
or 2A (PP1 and PP2A, respectively) at 30 °C for 2
h in buffer (50 mM Tris-HCl (pH 7.0), 1 mM EDTA, 0.5
mM dithiothreitol, 0.1%
-mercaptoethanol, and 0.2 mg/ml
bovine serum albumin), after which samples were resolved by SDS-PAGE. C, levels of I
B-
in TNF-treated cells. U-937 cells
were treated with TNF, and whole cell lysates were analyzed for
I
B-
by immunoblotting using the anti-I
B-
antibody. D, NF-
B activation in TNF-treated cells. Nuclear extracts
of TNF-treated U-937 cells were analyzed for NF-
B DNA-binding
activity using the
P-labeled wild-type NF-
B sequence
as the probe. E, phosphoamino acid analysis of I
B-
from TNF- or okadaic acid-treated cells. Immunoprecipitates of
I
B-
from
P-labeled U-937 cells were resolved by
SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes.
Membranes were autoradiographed, and the portion of the membrane
containing I
B-
was cut out and processed as described under
``Materials and Methods.''
TNF treatment of U-937 cells resulted in an
overall increase in IB-
phosphorylation (Fig.5A, lanes 1-6). The induced
phosphorylation was rapid and transient, peaking at
1 min after
stimulation. The kinetics of I
B-
phosphorylation paralleled
its degradation (Fig.5C), and both events preceded
NF-
B nuclear translocation (Fig.5D). Treatment of
U-937 cells with the TNF mimetic, okadaic acid(19) , also led
to enhanced phosphorylation of I
B-
(Fig.5A, lanes 9 and 10). Similar results were obtained with
TNF-treated MRC-5 fibroblasts, although the increase in phosphorylation
was less marked (Fig.5A, lanes11 and 12). Phosphoamino acid analysis of I
B-
revealed that the basal and TNF/okadaic acid-induced phosphorylations
were exclusively due to phosphorylation of serine residue(s) (Fig.5E). No phosphorylation was detected at either
tyrosine or threonine, even upon prolonged autoradiography. Although a
conserved tyrosine phosphorylation site is present on I
B-
, it
is not phosphorylated at tyrosine and cannot be the direct target for
the putative PTPase(s) involved in NF-
B activation.
Figure 6:
PTPase inhibitors abrogate the TNF-induced
phosphorylation and proteolysis of IB-
. A,
immunoprecipitation of I
B-
from
P-labeled cells.
Orthophosphate-labeled U-937 cells were treated with TNF for 2 min (lane2) or with H
O
,
orthovanadate (Van), or pervanadate (pV) for 15 min (lanes3, 5, and 7, respectively).
Treatment with a combination of H
O
,
orthovanadate, or pervanadate and TNF was performed by preincubating
cells with either reagent for 13 min followed by TNF for an additional
2 min (lanes4, 6, and 8).
I
B-
was immunoprecipitated, and samples were resolved by
SDS-PAGE. B, immunoblot of I
B-
. U-937 cells were
treated with TNF for various intervals of time in the absence or
presence of pervanadate. Pervanadate treatments were for a total of 20
min. Whole cell extracts were prepared, and the level of I
B-
was determined by immunoblotting using the anti-I
B-
antibody.
Treatment of U-937
cells with TPCK inhibited the TNF-induced proteolysis of IB-
(Fig.7A) and nuclear NF-
B DNA-binding activity
(data not shown) without affecting I
B-
phosphorylation (Fig.7B, compare lanes1 and 2 with lanes3 and 4). Similarly,
treatment of cells with TLCK did not affect the TNF-induced
phosphorylation of I
B-
(Fig.7B, lanes5 and 6). Although the 43-kDa I
B-
was
not detected in immunoblots of whole cell extracts, it was observed
when extracts from cells cotreated with TPCK and TNF were
immunoprecipitated for I
B-
prior to immunoblotting (data not
shown). Hence, the TNF-induced phosphorylation of I
B-
can
occur under conditions that inhibit I
B-
proteolysis, and in vivo, the phosphorylation of I
B-
may not be
sufficient to induce NF-
B activation.
Figure 7:
Serine protease inhibitors prevent the
TNF-induced proteolysis of IB-
without affecting
I
B-
phosphorylation. A, immunoblot of I
B-
.
U-937 cells were incubated with TNF, for the times indicated, in the
absence or presence of TPCK. Cells were exposed to TPCK at 25
µM for a total of 1 h. I
B-
was detected in whole
cell lysates by immunoblotting using the anti-I
B-
antibody. B, immunoprecipitation of I
B-
from
P-labeled cells. Orthophosphate-labeled U-937 cells were
treated with TNF for 2 min or pretreated with TPCK (25 µM)
or TLCK (200 µM) for 1 h prior to the addition of TNF for
2 min. I
B-
was immunoprecipitated, and samples were analyzed
by SDS-PAGE.
TNF signal transduction leads to the rapid phosphorylation of
several proteins at serine, threonine, or tyrosine
residues(7, 38) . A number of the
serine/threonine-phosphorylated proteins have been identified, and the
increase in their phosphorylation appears to result from the activation
of multiple serine/threonine
kinases(4, 7, 39, 40) and the
simultaneous inhibition of the opposing phosphatases, particularly
protein phosphatase 2A(4, 41) . Little is known of the
tyrosine kinases and phosphatases that are involved in TNF signal
transduction. Here, we investigated the role of PTPases in the
activation of the transcription factor NF-B by TNF. Inhibition of
PTPase activity leads to non-dissociation of the
I
B-
NF-
B complex and abrogation of NF-
B
nuclear translocation in TNF-treated cells ( Fig.1(A, C, and D), 3, and 4A). Notably, neither of
the PTPase inhibitors used in this study affected the TNF-induced
phosphorylation of hsp-27 or eIF-4e, indicating selectivity in the
action of the inhibitors and an early divergence in the
cytokine-induced signals leading to the phosphorylation of these
proteins from signals effecting the activation of NF-
B (Fig.2, C-F). The constitutive NF-
B-like
DNA-binding activity in cells with elevated HPTP
activity (Fig.4A) and inhibition of this DNA-binding activity
by treatment of HPTP
-expressing cells with PAO (Fig.4B) support a role for PTPases in NF-
B
activation.
IB-
has been shown to undergo an overall
increase in its phosphorylation in TNF-treated cells involving
serine/threonine residue(s)(18, 42, 43) .
However, the evidence to support this is indirect. The suggested
activation of PTPase in NF-
B induction, together with the presence
of a conserved Src-like tyrosine phosphorylation site at the N terminus
of I
B-
(20) , led us to investigate if I
B-
was dephosphorylated at tyrosine in TNF-treated cells. Phosphoamino
acid analysis showed that I
B-
is not the direct target of the
putative TNF-activated PTPase(s) as it was exclusively phosphorylated
at serine residue(s) in both unstimulated and TNF-treated U-937 cells (Fig.5E). However, treatment of U-937 cells with the
PTPase inhibitor pervanadate abolished the TNF-induced phosphorylation
of I
B-
, indicating that the I
B-
phosphorylation by
its serine kinase/phosphatase is likely to be regulated by an upstream
putative TNF-activated PTPase(s) (Fig.6A). Several
studies implicate c-Raf as a kinase that phosphorylates I
B-
in vivo(12, 16) . The putative TNF-induced
PTPase may be involved in c-Raf activation, as exemplified by the Drosophila PTPase corkscrew, which in concert with
the c-Raf homologue polehole, acts to positively transduce
signals initiated by the torso receptor protein-tyrosine
kinase(44) . On the other hand, findings by Diaz-Meco et
al.(13) suggest that a
-protein kinase C-activated
50-kDa kinase rather than c-Raf is responsible for TNF-induced
I
B-
phosphorylation. Since c-Raf and
-protein kinase C
share common components upstream in the signaling pathway, including
Ras and phosphatidylcholine-specific phospholipase
C(45, 46) , it is possible that activation of
-protein kinase C may also depend on the putative TNF-induced
PTPase.
All NF-B activators tested appear to induce the rapid
degradation of
I
B-
(14, 35, 43, 47, 48, 49) ,
indicating that modified/unbound I
B-
has a short life span.
In TNF-treated U-937 cells, the loss of immunoreactive I
B-
was observed at about the same time as its increase in phosphorylation,
but before NF-
B nuclear translocation (Fig.5, A, C, and D). Serine protease inhibitors, viz. TPCK and TLCK, were found to inhibit ligand-induced I
B-
degradation and NF-
B DNA-binding activity (14, 35) , suggesting that I
B-
proteolysis
does not merely serve to remove the modified/released protein from the
cytosol, but is also essential for NF-
B activation. It is also
possible that abrogation of NF-
B activation is due to inhibition
of the protease-dependent phosphorylation of I
B-
rather than
to I
B-
proteolysis itself. However, neither TPCK nor TLCK
affected the TNF-induced phosphorylation of I
B-
in U-937
cells under conditions where I
B-
proteolysis and NF-
B
activation were both effectively abolished (Fig.7). Although
the 43-kDa species of I
B-
was not observed in immunoblots of
whole cell lysates (Fig.5C, 6B, and
7A), it was detectable in lysates from cells treated with TNF
and TPCK that were enriched for I
B-
by immunoprecipitation
before immunoblotting (data not shown). This suggests that in whole
cells, I
B-
phosphorylation alone does not lead to NF-
B
activation, as implied by studies in
vitro(11, 12, 13) . Our data are in
agreement with an earlier report (18) that shows that
phosphorylated I
B-
remains tightly bound to p65. Taken
together, these data indicate that I
B-
proteolysis is
essential for NF-
B activation. Pervanadate treatment also
inhibited the TNF-induced proteolysis of I
B-
(Fig.6B), indicating that the I
B-
protease,
like the I
B-
serine kinase/phosphatase, may be regulated by
an upstream putative TNF-activated PTPase(s). Alternatively, if
I
B-
phosphorylation targets the protein for subsequent
degradation, as previously suggested(14) , the inhibition of
I
B-
proteolysis would be a direct consequence of the
inhibition of its phosphorylation. Confirmation of the functional
significance of I
B-
phosphorylation will require
site-directed mutagenesis of potential serine phosphorylation sites.
Recently, it was reported that site-directed mutagenesis of serine 32
or 36 of I
B-
resulted in a protein that failed to undergo
TNF-induced phosphorylation or degradation, and NF-
B activation
was not observed in cells expressing the mutant proteins(50) .
These findings are consistent with our observation that TNF treatment
results in the phosphorylation of I
B-
at serine residue(s) (Fig.5E). They are also consistent with our findings
that inhibition of TNF-induced I
B-
phosphorylation leads to
the concomitant loss of I
B-
degradation and NF-
B
activation (Fig.1A and 6).
Although NF-B plays
an important role in the TNF-induced transcription of several genes,
little is known of the early signaling events involved in its
activation. The present findings with PTPase inhibitors suggest that a
putative PTPase(s) activity is required for the TNF-induced serine
phosphorylation and proteolysis of I
B-
and NF-
B
activation. This is consistent with a constitutive NF-
B-like
DNA-binding activity in cells expressing enhanced HPTP
activity (Fig.4A). Signal transduction by other NF-
B
inducers may also depend on PTPase activation, as suggested by the
increased stability of I
B-
and loss of NF-
B activation
in pre-B-cells expressing the v-Abl protein-tyrosine
kinase(51) . On the other hand, NF-
B induction via the
T-cell receptor complex appears to be negatively regulated by the
PTPase CD45(52) . The present data highlight a potential role
for HPTP
as a positive regulatory element in TNF signal
transduction.