(Received for publication, June 23, 1995)
From the
Different Stat proteins are activated through phosphorylation of
unique tyrosine residues in response to different cytokines and growth
factors. Interferon- activates Stat1 molecules that form
homodimers and bind cognate DNA elements. Here we show that treatment
of permeabilized cells with 200-500 µM peroxo-derivatives of vanadium, molybdenum, and tungsten results
in the accumulation of constitutively phosphorylated Stat1
molecules. In contrast, treatment of permeabilized cells with
orthovanadate, vanadyl sulfate, molybdate, and tungstate at the same
range of concentrations does not result in the accumulation of
activated Stat1
molecules in the absence of ligand. However, these
compounds inhibit the inactivation of interferon-
-induced
DNA-binding activity of Stat1
. A 4-6-h exposure of the
permeabilized cells to orthovanadate, molybdate, and tungstate, but not
vanadyl sulfate, results in a ligand-independent activation of
Stat1
, which is blocked by the inhibition or depletion of NADPH
oxidase activity in the cells, indicating that NADPH oxidase-catalyzed
superoxide formation is required for the bioconversion of these metal
oxides to the corresponding peroxo-compounds. Interestingly,
ligand-independent Stat1
activation by peroxo-derivatives of these
transition metals does not require Jak1, Jak2, or Tyk2 kinase activity,
suggesting that other kinases can phosphorylate Stat1
on tyrosine
701.
Soluble extracellular signaling polypeptides like cytokines and
growth factors transmit their signals from cell surface to nucleus
through a number of protein
messengers(1, 2, 3) . Propagation of signals
down the pathways can be envisioned as a cascade of physical and
chemical changes in these signaling molecules. In the
IFN()-
-activated pathway, the signal is initiated by a
physical interaction of the cytokine with its receptor on the plasma
membrane that leads to structural change(s) in the engaged receptor
complex. This in turn triggers tyrosyl phosphorylation of Jak1 and Jak2
kinases resulting in the induction of their catalytic activities, and
phosphorylation of a unique tyrosyl residue on the receptor tail that
serves as a docking site for the latent Stat1 molecules, Stat1
(p91) and Stat1
(p84)(1, 3, 4) . The
immobilized Stat1 molecules are phosphorylated at tyrosine 701 by Jak
activities(1, 4, 5) . The activated Stat1
molecules form homodimers through an intermolecular SH2-domain and
tyrosyl-phosphate interactions (1, 6) . The Stat1
dimers are translocated to the nucleus and bind cognate DNA elements.
However, only the Stat1
homodimer (GAF) can activate the
transcription of the cognate genes(1, 7) .
Tyrosine
phosphorylation is a pivotal reaction that activates IFN- and many
other cytokine signals(8) . While the down-regulation of the
signals is less well understood, removal of the phosphate group from
the tyrosine residues of the activated signaling proteins could be a
potential mechanism for the termination of cytokine signals. A
SH2-domain-containing protein-tyrosine phosphatase (PTP), SH-PTP1 (also
known as PTP1C/HCP), which is preferentially expressed in hematopoietic
cells, acts as a negative regulator of cell signaling by erythropoietin (9, 10) , interleukin-3(11) ,
c-Kit(12) , and antigen receptor(13) . In contrast,
SH-PTP2 (also known as PTP1D/Syp), another SH2-domain-containing PTP,
acts as positive regulator of mitogenic signal transduction
pathways(14, 15, 16, 17) . Larner
and co-workers(18, 19) have shown that pre- but not
simultaneous treatment of reconstituted cell-free signaling systems
with PTP inhibitors blocks the formation of ISGF3 and GAF by IFN-
and IFN-
, respectively, indicating a positive role of PTP in IFN
signaling pathways. Based on the observation that pervanadate prevents
the inactivation of IFN-induced signals, the involvement of a nuclear
PTP in the down-regulation of IFN-induced gene activation has been
implicated(20) . However, the levels of Stat activation
resulting from the individual actions of IFN and pervanadate were not
distinguished(20) , although it was subsequently demonstrated
that incubation of cells with pervanadate causes IFN-independent Stat
activation(21, 22) .
Termination of cytokine or
growth factor signals has been recognized as an important cellular
event, particularly when a signal acts as an activator of genes that
are constitutively silent or expressed at a very low basal
levels(23) . The precise mechanism of termination of
IFN-induced signals and the consequent deactivation of IFN-stimulated
genes remained to be resolved. The down-regulation of the
IFN-stimulated gene transcription may be a consequence of either the
inactivation of transcription factors or the induction/activation of
repressor molecules or a combination of both. For example, induction of
repressors, like IRF-2 and positive regulatory domain 1 binding factor
1, has been demonstrated in the down-regulation of IFN- gene
transcription(24, 25, 26) . Some of the
IFN-stimulated genes have IRF-1/IRF-2-binding sites in their promoters,
which is similar to or same as an IFN-stimulated response
element(1, 27) ; however, a role for IRF-2 or any
other proteins in the deactivation of IFN-stimulated genes has not been
described so far.
To address the roles of PTP activities in
regulating ligand (IFN-)-dependent and constitutive activation of
Stat1 proteins, we investigated the kinetics of Stat1
activation
and deactivation in cells treated with regulators of PTPs. We present
evidence that more than one PTP activity is involved in the regulation
of both the constitutive and IFN-
-dependent signal transduction
mediated through the tyrosyl phosphorylation of Stat1
. By using
cell lines lacking Jak1, Jak2, and Tyk2, we show that none of these
kinases individually is required for the ligand-independent activation
of Stat1
molecules.
Figure 1:
Inactivation
time course of IFN--induced GAF. EMSA was performed using an
end-labeled GAS probe and WCE prepared from HeLa S3 and Namalwa cells
that were treated with IFN-
(50 units/ml) for 30 min and washed 2
times with phosphate-buffered saline followed by washing and incubation
in fresh medium for the indicated length of time minus 30 min (time
indicated on the top of the lane was counted after
the addition of IFN to the cells).
Figure 2:
Stat1 level in IFN-
-treated
cells. Western blot analysis was performed with WCE containing 10
µg of protein, derived from HeLa and Namalwa cells as described in Fig. 1. Equivalent amount of WCE prepared from 2fTGH and U3A
cells were used as positive and negative controls, respectively, for
the detection of Stat1
band. The immunoblot was probed with a
Stat1
-specific polyclonal antibody.
Inhibition of protein synthesis by cycloheximide
prolongs the induction period of IFN-induced
genes(35, 36, 37) . To determine whether
protein synthesis inhibition altered the inactivation rate of the
DNA-binding activity of Stat1, GAF activity was followed in the
presence and absence of cycloheximide for several hours after HeLa
cells were treated with IFN-
for 30 min. The rate of inactivation
of Stat1
was not markedly affected by protein synthesis inhibition (Fig. 3). A similar observation was made with Namalwa cells
(data not shown).
Figure 3:
Inactivation time course of
IFN--induced GAF in HeLa cells in the presence of cycloheximide (CHX). EMSA was performed using an end-labeled GAS probe and
WCE derived from cells that were treated with IFN-
(50 units/ml)
for 30 min and then washed as described in Fig. 1. The cells
were incubated in fresh medium with or without cycloheximide (50
µg/ml) for the indicated length of time minus 30 min. The leftmostlane represents a control of 6 h of
cycloheximide treatment.
Figure 4:
GAF activation by PTP inhibitors in intact
HeLa cells. EMSA was performed using an end-labeled GAS probe and WCE
derived from cells that were treated with 500 µM PTP
inhibitor (OV, sodium orthovanadate; VS, sodium
vanadyl sulfate; Mo, sodium molybdate; W, sodium
tungstate; F, sodium fluoride) for 30 min. Either an aqueous
solution of the compound or its peroxo-derivative (prepared by
treatment with HO
) was used for the
treatment.
Figure 5: GAF activation by PTP inhibitors in permeabilized HeLa cells. EMSA was performed using an end-labeled GAS probe and WCE prepared from the permeabilized HeLa cells. The cells were permeabilized in a hypotonic buffer containing 10 µg/ml digitonin for 10 min and then added PTP inhibitor (described in Fig. 5) in the same buffer followed by an incubation for 20 min at 37 °C.
Figure 6:
Pervanadate activates the IRF-1 gene
promoter. Transient transfection assays were performed in Cos-1 cells.
24 h post transfection, the cells were treated with the indicated
compound (50 µM) for 5 h. The reference plasmid expressing
the -galactosidase activity under the transcriptional control of
Rous sarcoma virus long terminal repeat was used in the transfection
assay for normalization of luciferase activity. Each bar represents the mean of normalized luciferase activities of three
experiments.
A short term
(1-2 h) treatment of permeabilized HeLa cells with sodium
orthovanadate did not induce DNA-binding activity of Stat1 but
prevented the inactivation of IFN-
induced DNA-binding activity of
Stat1
(Fig. 7). Like orthovanadate, vanadyl sulfate,
molybdate, and tungstate also prevented the inactivation of
IFN-
-activated DNA-binding activity of Stat1
molecules (data
not shown). Moreover, longer (4-6 h) treatment of permeabilized
cells with molybdate and tungstate but not vanadyl sulfate resulted in
the ligand-independent Stat1 activation (data not shown). These results
clearly demonstrate that tyrosine dephosphorylation is a mechanism for
the inactivation of ligand-activated Stat1
.
Figure 7:
Orthovanadate inhibits the inactivation of
IFN--induced GAF in permeabilized HeLa cells. EMSA was performed
using an end-labeled GAS probe and WCE derived from the HeLa cells that
were subjected to the indicated treatments. Intact HeLa monolayers were
first treated with IFN-
(50 units/ml) for 30 min followed by
permeabilization (except the leftmostlane ) for 30
min. After 10 min of permeabilization, sodium orthovanadate (500
µM) was added to the cells in the permeabilization buffer
and the treatment continued at 37 for 20 min. The reagents were washed
off, and the cells were incubated in fresh medium for the indicated
length of time.
Figure 8:
Inhibition of NADPH oxidase activity by DPI blocks the bioconversion of orthovanadate to pervanadate
in permeabilized HeLa cells. EMSA was performed using an end-labeled
GAS probe and WCE derived from permeabilized HeLa cells. The cells were
treated with IFN- (50 units/ml) for 30 min, washed, and incubated
in permeabilization buffer for 10 min and treated with 500 µM sodium orthovanadate (OV) and/or 5 µM DPI or
ethanol (vehicle for DPI). The cells were then incubated in fresh
medium with or without DPI for 6 h.
In order to further address the mechanism of the
ligand-independent phosphorylation of Stat proteins, we determined
whether Jak family kinases are necessary. Both Jak1 and Jak2 activities
have been shown to be required for Stat1 activation by
IFN-
(1, 48, 49) , and both are
tyrosine-phosphorylated in pervanadate-treated
cells(22, 45) . The mutant cell line, U4A, which does
not contain Jak1(49) , supported the pervanadate-activated GAF
formation (Fig. 9). Similarly, Daudi cells, which do not respond
to IFN-
due to the lack of Jak2 activity (
)also induced
GAF formation in response to pervanadate. In the U1A cell line, which
lacks Tyk2 activity(50) , pervanadate-mediated Stat1
activation was observed albeit at much lower level. In U2A cells, the
absence of p48, another component of IFN signaling
pathways(51) , did not affect the action of pervanadate on
Stat1
activation (Fig. 9). U3A cells, which do not contain
Stat1 proteins(34) , served as a negative control for
pervanadate-mediated Stat1
activation. Thus, the
peroxo-derivatives of vanadium, molybdenum, and tungsten, which are
known inhibitors of PTPs, activate the phosphorylation of Stat1
or
prevent the dephosphorylation of constitutively activated Stat1
in
a ligand-independent mechanism that does not absolutely require Jak1 or
Jak2 activity.
Figure 9:
Pervanadate activates Stat1 in the
cells lacking protein-tyrosine kinases involved in IFN signaling
pathways. EMSA was performed using an end-labeled GAS probe and WCE
prepared from cells that were treated with 500 µM sodium
orthovanadate (OV) or 500 µM pervanadate (PV) for 30 min or left untreated as
control.
We have demonstrated that PTP inhibitors orthovanadate and
vanadyl sulfate prevent the inactivation of IFN--induced
DNA-binding activity of Stat1
molecules in permeabilized cells.
Molybdate and tungstate having structural similarities with vanadate
inhibit PTP activity in cell-free systems(40, 52) . We
have shown that like vanadium salts, molybdate and tungstate also block
the inactivation of IFN-
-activated Stat1
molecules in
permeabilized cells. In the absence of any stimulation by extracellular
signaling proteins, Jak kinases exhibit low levels of constitutive
kinase activity in normal cells (
)and can phosphorylate Stat
molecules. Recently Heim et al.(53) reported that
overexpression of Jak and Stat in Cos cells leads to the
ligand-independent phosphorylation of Stat molecules. Orthovanadate,
vanadyl sulfate, molybdate, and tungstate (group I PTP inhibitors) do
not readily penetrate the cell
membrane(38, 39, 40, 41, 42, 43, 44) ,
but when added to permeabilized cells they inhibit the PTP activity
that dephosphorylates the ligand-activated Stat molecules but not the
constitutively activated Stat molecules. In contrast, the second group
of PTP inhibitors namely, peroxo-derivatives of vanadium, molybdenum,
and tungsten, which seem to be more potent inhibitors of PTPs than the
first group, inhibits the PTP activity that dephosphorylates the
constitutively activated Jaks and Stats. Accordingly, an accumulation
of constitutively activated Stat molecules in the absence of any ligand
is detected in the cells that are exposed to the second group of PTP
inhibitors.
Based on the observed differential sensitivity of the intracellular PTP activities involved in the Jak-Stat pathways, to the group I PTP inhibitors, we propose a simple model that explains how reversible tyrosine phosphorylation regulates cell signaling through Jak-Stat pathways (Fig. 10). According to this model, the dephosphorylation of the activated Jak molecules is catalyzed by PTP-x, which is insensitive to the micromolar concentrations of group I PTP inhibitors (orthovanadate, vanadyl sulfate, molybdate, tungstate, etc.). On the other hand, the dephosphorylation of activated Stat molecules is catalyzed by a downstream PTP activity, termed PTP-y. Unlike PTP-x, PTP-y is partially sensitive to the micromolar concentrations of group I PTP inhibitors. However, both PTP-x and PTP-y are sensitive to the micromolar concentrations of group II PTP inhibitors (peroxo-derivatives of vanadium, molybdenum, and tungsten).
Figure 10: Model for selective inhibition of PTP activities in Jak-Stat pathways by PTP inhibitors. PTP-x represents a family of PTPs (probably SH2-domain containing PTPs), which are localized at the cytokine receptor tails by protein-protein interactions. PTP-x dephosphorylates activated Jak kinases and is insensitive to micromolar concentrations (200-500 µM) of group I PTP inhibitors orthovanadate (OV), vanadyl sulfate (VS), molybdate (MoO), and tungstate (WO). PTP-y represents a family of downstream PTPs that are partly inhibited by micromolar concentrations (200-500 µM) of group I PTP inhibitors. PTP-y dephosphorylates activated Stat molecules. Both PTP-x and PTP-y are inhibited by group II PTP inhibitors pervanadate (PV), permolybdate (PMo), and pertungstate (PW). Complete inhibition, ++; partial inhibition, +-; insensitive, --.
PTP-x and PTP-y may represent two different families of PTPs rather
than two individual enzymes. Recently a SH2-domain-containing PTP,
SH-PTP1 has been identified as phosphatase that inactivates Jak2 in
erythropoietin signaling pathway(9, 10) . The binding
site of SH-PTP1 has been identified in the cytoplasmic domain of the
erythropoietin receptor(9) . The binding is mediated through an
interaction of the SH2 domain of SH-PTP1 and a unique phosphotyrosyl
residue (Tyr-429) of erythropoietin receptor(9) . Association
of SH-PTP1 with other cytokine receptors that activate Jak2, like
interleukin-3, has been reported (11) . To prevent spurious and
nonspecific dephosphorylation of proteins and regulate the availability
of specific substrates, subcellular localization of the intracellular
PTPs through the interaction of adhesive protein-domains would be a
potential mechanism to downregulate the receptor-associated or
receptor-intrinsic protein-tyrosine kinase activities(54) .
Accordingly, we assume that SH-PTPs represent the PTP-x family in our
model. It is interesting to note that recombinant SH-PTP1 is not
sensitive to micromolar concentrations of group I PTP inhibitors ()suggesting this or a similar PTP as a candidate for PTP-x.
We have shown that group I PTP inhibitors are converted to group II
inhibitors inside the cells with the exception of vanadyl sulfate. It
has previously been suggested by Grinstein and colleagues that
orthovanadate is converted to pervanadate inside the cells through a
superoxide anion-mediated reaction(32) . We confirmed this
observation and showed that molybdate and tungstate are also converted
to their peroxo-derivatives in vivo through
superoxide-mediated reaction. Like many other transition metals,
vanadium, molybdenum, and tungsten form peroxo-compounds in their
highest oxidation states(55) , which may explain why vanadyl
sulfate (VOSO) in which vanadium is in oxidation state of
+4 does not form pervanadate either when treated with hydrogen
peroxide in vitro or through superoxide anion-mediated
reaction in permeabilized cells. Notably, in the group I PTP inhibitors
that are converted to peroxo-derivatives, the transition metals are in
their highest oxidation states.
The peroxo-derivatives of these transition metals, classified as group II PTP inhibitors, have a broad spectrum of action. Treatment of cells with pervanadate results in the accumulation of tyrosyl phosphate in many cellular proteins depending on the cell types. This results from the inhibition of PTP activities, which not only dephosphorylate nonenzyme proteins, e.g. Stats, but also activate protein-tyrosine kinases, e.g. Jaks(45) . Thus, the group II PTP inhibitors indirectly activate protein-tyrosine kinase activities by preventing the dephosphorylation of unique tyrosyl residues that positively regulate the protein-tyrosine kinase activity.
We have shown that cell lines
lacking individual Jak kinases, namely Jak1, Jak2, and Tyk2 can support
the spontaneous phosphorylation of the unique tyrosyl residue 701 of
Stat1 molecules in a ligand-independent fashion. These data
indicate that Stat1
phosphorylation at tyrosine 701 is not a
Jak-specific catalysis. This has also recently been demonstrated by
others in different experimental contexts(53, 56) .
The pervanadate-mediated activation of Stat1
molecules in
Jak-minus cell lines rules out the possibility that the constitutive
activation of Stat1
is due to the action of IFNs secreted by the
cells or present in the serum-containing medium. In general the PTPs
having 10-1000-fold higher specific activities than the
protein-tyrosine kinases in vitro are expected to prevent the
accumulation of phosphate on tyrosyl residues of many cellular proteins
resulting from the constitutive activities protein-tyrosine
kinases(57) . Thus, a strong inhibitory action of group II PTP
inhibitors leads to the accumulation of phosphate on the tyrosyl
residue of cellular proteins including Stats.
PTP activities play crucial roles in the regulation of constitutive and ligand-dependent signal transduction through Jak-Stat and other pathways. Although we have exploited the differences in relative potencies of different PTP inhibitors to address the roles of multiple PTP activities in Jak-Stat pathways, molecular identification of these PTPs (PTP-x and PTP-y) will be necessary to comprehend the complex regulation of cytokine signal transduction pathways.