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First published online 27 February 2008
doi: 10.1242/dev.009936
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1 University of Dundee, College of Life Sciences, Dow Street, Dundee DD1 5EH,
UK.
2 University of California, San Diego, Natural Sciences Building Room 6111, 9500
Gilman Drive, La Jolla, CA 92093-0380, USA.
3 Biomedical Research Foundation (SBF), Lauchefeld 31, CH-9548 Matzingen,
Switzerland.
Author for correspondence (e-mail:
j.g.williams{at}dundee.ac.uk)
Accepted 18 January 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: STAT, Dictyostelium, Tyrosine phosphatase, Stress, DIF-1
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
They are activated when diverse signalling pathways are stimulated, but the
various JAK-STAT pathways form the paradigm. In response to the binding of a
cytokine to its receptor, an associated tyrosine kinase of the JAK family
phosphorylates a STAT at a unique site near its C terminus. This leads, via
reciprocal phosphotyrosine:SH2 domain interactions, to dimerisation of the
STAT and the STAT dimers accumulate in the nucleus
(Reich and Liu, 2006).
Dictyostelium cells use STAT signalling to regulate several
aspects of their differentiation
(Williams, 2003).
Extracellular cAMP signalling activates STATa, which can function as either a
repressor or an activator of specific gene expression
(Araki et al., 1998;
Fukuzawa and Williams, 2000).
DIF-1 is a chlorinated hexaphenone that induces differentiation of one of the
prestalk cell subtypes, pstO cells
(Thompson and Kay, 2000). At
the slug stage, STATc is nuclear localised in pstO cells, where it acts to
prevent ectopic expression of a marker of pstA cell differentiation
(Fukuzawa et al., 2001).
Addition of DIF-1 to cells early in development leads to the premature
tyrosine phosphorylation, dimerisation and nuclear accumulation of STATc.
Nuclear accumulation of STATc in response to DIF-1 is regulated at the
level of nuclear export (Fukuzawa et al.,
2003). In uninduced cells, the effect of a nuclear import signal,
located near the N terminus of STATc, is negated by a DIF-1-regulated nuclear
export signal located near the centre of the protein. The balance between
import and export activity seems to be linked to the homo-dimerisation that is
triggered by phosphorylation of STATc on tyrosine residue 922. The mechanism
by which STATc becomes tyrosine phosphorylated is unknown. There are no
apparent Dictyostelium homologues of the class of tyrosine kinases
that modify metazoan STATs (Goldberg et
al., 2006). There are, however, an unusually large number of
tyrosine kinase-like enzymes that perhaps subsume their function.
In mammals, STAT1 and STAT3 are activated by specific cytokines but
hyper-osmotic stress is also an activator
(Gatsios et al., 1998).
Similarly, STATc accumulates in the nucleus rapidly when cells are subjected
to hyper-osmotic stress (Araki et al.,
2003). Tyrosine phosphorylation and nuclear localisation of STATc
are maintained for at least 30 minutes. By contrast, the fraction of STATc
protein that is tyrosine phosphorylated and nuclear localised after DIF-1
treatment reaches a sharp peak at 3-5 minutes of treatment and STATc is then
de-phosphorylated and exits the nucleus.
One likely explanation for the temporal disparity, between the stress and
the DIF-1 responses, is a difference in de-phosphorylation kinetics. In
metazoa TC45, the nuclear isoform of the T-cell protein tyrosine phosphatase
(TC-PTP), serves to de-phosphorylate STAT1 and this causes it to re-localise
to the cytoplasm (ten Hoeve et al.,
2002). TC-PTP-null cells are also defective in STAT3
de-phosphorylation but TC45 may not be the only phosphatase involved; because
the cytosolic form of PTP
de-activates STAT3 when overexpressed
(Tanuma et al., 2000). STAT5
activation is normal in TC-PTP-null cells and here there is evidence for
direct interaction of STAT5 with the non-receptor tyrosine phosphatases SHP2
(Yu et al., 2000) and with
PTP1B (Aoki and Matsuda, 2000).
Thus, the metazoan STATs, which differ significantly in their mechanisms of
nuclear accumulation (Reich and Liu,
2006), are also heterogeneous in their modes and sites of
de-activation. The two processes are of course intimately inter-related; a
nuclear protein must actively shuttle between nucleus and cytoplasm if a
cytosolic tyrosine phosphatase is to serve to de-activate it.
The tyrosine phosphatase that catalyses de-phosphorylation of STATc is
unknown. Dictyostelium encodes three PTPs, all of which are predicted
to be non-transmembrane proteins (Howard
et al., 1992; Howard et al.,
1994; Gamper et al.,
1996). PTP1 and PTP2-null strains show only minor defects in
development, but PTP1 and PTP2 overexpressing strains develop aberrantly and
contain an altered spectrum of tyrosine phosphorylated proteins. PTP1 is a
negative regulator of STATa tyrosine phosphorylation but the failure to detect
a direct interaction between the two proteins, using a substrate-trapping form
of PTP1, suggests that PTP1 acts indirectly, at some point upstream of STATa
(Early et al., 2001).
The third phosphatase, PTP3, is divergent in several otherwise highly
conserved amino acid residues and the bacterially produced enzyme has a very
low intrinsic phosphatase activity (Gamper
et al., 1996). Analysis of a tagged version of the protein
suggests it to be partly cytosolic and partly nuclear
(Gamper et al., 1999). In the
Ax3-derived strain JH10 there are two copies of the PTP3 gene. It was possible
to disrupt one copy, but the second copy was refractory to disruption
(Gamper et al., 1996).
Antisense inhibition also proved ineffective. Although PTP3 seems to be
essential for cell viability, overexpression studies have yielded some
insights into its developmental functions. The PTP3 overexpression strain
grows slowly and forms large aggregation streams; in addition, many structures
arrest development at the mound stage and there are changes in the tyrosine
phosphorylation level of several proteins
(Gamper et al., 1996;
Gamper et al., 1999). In
parental cells, hyper-osmotic stress, which is generated by the addition of
glucose containing growth medium, induces the tyrosine phosphorylation of a
130 kDa protein. This response is greatly attenuated in the PTP3
overexpressing strain and PTP3 itself becomes serine-threonine phosphorylated
under these stress-inducing conditions.
In order to identify a PTP that downregulates STATc after DIF-1 induction,
we analysed mutants for the three Dictyostelium tyrosine
phosphatases. We show that PTP3 directly interacts with and dephosphorylates
STATc, and that STATc is the 130 kDa stress-responsive tyrosine phosphorylated
protein identified by Gamper et al.
(Gamper et al., 1996). We also
present evidence that PTP3 has a further, more significant role: as a mediator
of the activation of STATc by DIF-1 and osmotic stress.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Pang et
al., 1999). Transformants were selected at 20 µg/ml G418.
Clones were isolated by growth on a lawn of bacteria or by plating on 96-well
plates. Exponentially growing cells were harvested and washed twice in
phosphate buffer (KK2). For suspension, development cells were resuspended in
KK2 at a concentration of 1x107 cells/ml and were shaken for
4 hours at 200 rpm. For late developmental stages cells were plated on 1.5%
non-nutrient agar plates.
Immunohistochemistry
Cells in suspension culture and slugs on agar plates were fixed in absolute
methanol for 10 minutes on ice. After rehydration with phosphate-buffered
saline (PBS), cells and slugs were incubated with anti-STATc antibody (7H3),
followed by incubation with secondary antibody (Alexa488-conjugated goat
anti-mouse antibody, Molecular Probes).
Western transfer
Proteins were separated on pre-cast 4-12% polyacrylamide gels (Invitrogen)
and electro-transferred to nitrocellulose membranes. Membranes were blocked
with 5% skimmed milk in TBS-Tween (Tris-buffered saline, 0.05% Tween20) for 30
minutes and incubated with primary antibody overnight at 4°C. Signals were
detected using an HRP-conjugated goat anti-mouse antibody (Bio-Rad) with a
chemi-luminescent detection system (Pierce). The 7H3 STATc antibody and
anti-GSK3 antibody (clone 4G-1E, Upstate) were used as control.
Substrate-trapping chromatography
Substrate trapping chromatography was performed as described previously
using GST:PTP3
CS (Gamper et al.,
1999). The samples were analysed by western transfer using
anti-phosphotyrosine antibodies, a mixture of 4G10 (Upstate) and P-Tyr-100
(Cell Signaling Technology), and the CP22 STATc phospho-specific antibody.
Co-immunoprecipitation of PTP3 and STATc
Parental Ax2 cells and cells expressing myc-tagged PTP3C649S were
starved for 4 hours in suspension culture at a cell density of
1x107 cells/ml in KK2. After treatment with DIF-1 or
sorbitol, 4 ml of cell suspension (4x107 cells) was harvested
and lysed in 1 ml TT-lysis buffer [50 mM TrisHCl (pH 8.0), 150 mM NaCl, 1.0%
TritonX-100, 50 mM NaF, 2 mM EDTA (pH 7.2), 2 mM Na-pyrophosphate, 2 mM
benzamidine, 1 µg/ml pepstatin, 1 mM PMSF and Complete EDTA-free proteinase
inhibitor cocktail (Roche)] for 10 minutes on ice. After centrifugation at
20,000 g for 10 minutes, the supernatant was collected and
incubated with 9E10 anti-myc antibody (Roche) for 1 hour at 4°C, followed
by another 2 hours incubation with Protein G-agarose (Roche). Agarose beads
were washed four times in TT-lysis buffer, and finally boiled in
1xSDS-sample buffer for 10 minutes. The samples were analysed by western
transfer using the 7H3 STATc antibody and the CP22 STATc phospho-specific
antibody.
PTP3 immunopurification and enzyme assay
PTP3 activity was measured with para-nitrophenylphosphate (pNPP) as a
substrate and as described by Montalibet et al.
(Montalibet et al., 2005).
Cells expressing myc:PTP3 were shaken in suspension for 4 hours then treated
with the various stimuli for 5 and 15 minutes. Cell suspension (3 ml;
3x107 cells) was harvested and cells were lysed in 1 ml NP40
lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1.0% Nonidet-P40 (NP-40),
2 mM EDTA, 50 mM NaF, 2 mM Na-pyrophosphate, 1 mM PMSF, 2 mM benzamidine, 1
µg/ml pepstatin, 0.4 mM TLCK and EDTA-free proteinase inhibitor cocktail
(Roche)] on ice for 10 minutes. After centrifugation at 20,000
g for 10 minutes, the supernatant was pre-absorbed with
Protein G-agarose for 30 minutes at 4°C with gentle rocking. After
pre-absorption, anti-myc antibody (9E10) was added to samples and left for 30
minutes at 4°C. Then samples were incubated with Protein G-agarose for
another 1 hour at 4°C. Beads were washed four times in NP40 lysis buffer
and then once in HEPES phosphatase buffer [20 mM HEPES (pH 6.3), 150 mM NaCl,
2 mM EDTA, 5 mM DTT, 2% Glycerol and 0.01% TritonX-100]. The phosphatase
activity on pNPP and amount of immunoprecipitated PTP3 protein was measured in
each immunoprecipitated PTP3 sample. The phosphatase reaction was carried out
with 15 mM pNPP (Sigma) in HEPES phosphatase buffer at 30°C. After 30
minutes, the reaction was terminated by addition of NaOH and the activity was
measured as the OD at 405 nm. The amount of immunoprecipitated PTP3 was
quantified from western blotting with anti-myc antibody using NIH image
software. The PTP3 activity was normalized against PTP3 protein amount. The
data are presented as the activity relative to control samples.
Lambda phosphatase treatment and 2D gel electrophoresis
Beads bearing immunoprecipitated PTP3 were washed twice in lambda
phosphatase buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EGTA, 2 mM
DTT, 0.2% NP-40 and 2 mM MgCl2). The samples were incubated with or
without 100 U of lambda phosphatase (NEB) for 30 minutes at 30°C. PTP3
proteins were eluted from the beads with 2D sample buffer (Destreak
rehydration solution, GE Healthcare). The eluates were applied to first
dimension strips (Immobiline drystrip 8 cm pH 6-10, GE Healthcare). After
electrophoresis and equilibration for 15 minutes, the strips were run in a
second dimension on a 4-12% gradient gel (Invitrogen).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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In this construct, myc:PTP3, the coding region of PTP3, bearing a myc tag
at its N terminus, is under the control of a semi-constitutive actin promoter.
Interestingly, the developmental phenotype in cells transformed with myc:PTP
(PTP3OE cells) resembles that observed for null mutants in DIF-1 biosynthesis
and signalling (Thompson and Kay,
2000; Austin et al.,
2006; Huang et al.,
2006; Zhukovskaya et al.,
2006); the slugs are long and frequently fragment along their
length (Fig. 1A). When such
slugs are analysed immunohistochemically, STATc nuclear accumulation is
greatly reduced relative to the parental strain
(Fig. 1B). Western transfer
analysis using an antibody (CP22) directed against the site of modification at
tyr922 of STATc confirms there to be only negligible amounts of activated
STATc in the PTP3 overexpressing strain
(Fig. 1C). The slug splitting
phenotype is unlikely to be due to an effect of PTP3 on STATc, because the
STATc null strain does not show such a phenotype
(Fukuzawa et al., 2001); it
presumably reflects an effect of PTP3 overexpression on some other target.
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Conditions that decrease PTP3 activity cause increased STATc tyrosine phosphorylation
Substrate-trapping forms of PTPs contain point mutations that prevent the
de-phosphorylation reaction, thereby stabilising interaction with their
substrates (Flint et al.,
1997). Such constructs are routinely used as dominant inhibitors
of PTP activity in vivo. myc:PTP3CS is a myc tagged version of PTP3 and also
bears a serine substitution of the catalytically essential cysteine residue
(C649) (Gamper et al., 1996).
The related mutant myc:PTP3CS bears a deletion of an N
terminus-proximal region of PTP3. This deletion, of 244 amino acids, removes
several polyN tracts, and results in an increased level of expression of the
protein (data not shown).
When either of these two mutant constructs is expressed in
Dictyostelium, under the control of the semi-constitutive actin 15
promoter, STATc shows an increased basal and induced level of tyrosine
phosphorylation (Fig. 3A; data
not shown for myc:PTP3CS). Consistent with its higher level of expression,
myc:PTP3CS exerts a stronger stimulatory effect on the level of STATc
tyrosine phosphorylation than the full-length PTP3CS protein. Ectopic
activation of STATc is also manifest at the slug stage when cells express the
substrate-trapping form of PTP3. In parental slugs, STATc is nuclear enriched
in pstO cells, while in cells expressing myc:PTP3CS (PTP3CSOE cells) STATc is
nuclear enriched in cells throughout the slug
(Fig. 1B).
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CS, which contains the same deletion of
N terminal sequences and cysteine to serine substitution as myc:PTP3CS
but is fused to GST rather than to myc. Parental and STATc-null cells were
treated with DIF-1 or sorbitol, cell extracts were subjected to affinity
chromatography and replicate eluate samples were analysed by western transfer.
As reported previously (Gamper et al.,
1996), an osmotic stress-induced protein of 130 kDa
(Fig. 4A) and a 60 kDa protein
(data not shown) are detected when a phosphotyrosine antibody is used to probe
the blot. The 130 kDa protein is also tyrosine-phosphorylated when cells are
exposed to DIF-1 but less strongly than by sorbitol
(Fig. 4A). The band of 130 kDa
is absent in the chromatographic eluates from STATc null extracts
(Fig. 4A). The above data suggest that the 130 kDa protein is STATc and this was confirmed using a phospho-specific STATc antibody to probe the western transfer. Two proteins are detected, a major species running at the position of STATc and a faster running minor species (Fig. 4A). Comparison with the STATc-null eluates indicates that the upper band is STATc, while the lower band is cross-reacting GST-PTP3 adventitiously eluted from the affinity column.
The substrate-trapping results show that PTP3 and STATc interact in vitro. In order to determine whether they also interact in vivo, immunoprecipitation was performed on cells expressing the myc tagged, substrate-trapping form of PTP3. Cells were treated with DIF-1 or sorbitol and extracts were immunoprecipitated with a myc antibody and analysed by western transfer using a STATc antibody (7H3) that recognizes total STATc, and using CP22, the phospho-specific STATc antibody. The substrate-trapping form of PTP3 co-immunoprecipitates with tyrosine-phosphorylated STATc, confirming an in vivo interaction (Fig. 4B).
DIF-1 and hyper-osmotic stress treatment inhibit cellular PTP3 activity
In order to determine whether sorbitol or DIF-1 affect the activity of
PTP3, we immunoprecipitated the myc tagged form of PTP3 from control and
induced cells and determined its enzymatic activity
(Fig. 5). This was previously
analysed using growth medium as the stressor and, under non-reducing
conditions, stress induced a 2.5- to 5-fold reduction in PTP3 activity
(Gamper et al., 1999). We find
that, under similar conditions, exposure to sorbitol results in a 40% decrease
in PTP3 activity at both 5 and 15 minutes of treatment. DIF-1 produces a 20%
reduction in PTP3 activity after 5 minutes of treatment but the activity
reverts to its initial level by 15 minutes. These changes mirror the
activation kinetics of STATc after the two different treatments (bottom of
Fig. 5). Pervanadate
irreversibly modifies the active site cysteine residue of PTPs. Therefore, as
a control, we also analysed immunoprecipitates from pervanadate-treated cells.
They display negligible PTP3 activity (Fig.
5). As would be predicted from this observation, pervanadate
induces extremely high levels of tyrosine phosphorylation of STATc in the
absence of any other added inducer (Fig.
3B).
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). To
determine whether this also holds true for DIF-1, cells expressing
myc:PTP3CS were induced with DIF-1 and analysed by western transfer
using a myc antibody. On 2D gels, the uninduced myc:PTP3CS protein
migrates as a series of irregular spots
(Fig. 6). This suggests some
level of pre-existent phosphorylation. On addition of DIF, there is a shift in
the spot distribution, with an increase in the proportion of more acidic,
putatively phosphorylated, spots. Pre-incubation with lambda phosphatase
causes the tagged protein to migrate as a mass at a more alkaline pI. Lambda
phosphatase de-phosphorylates serine, threonine and tyrosine residues but PTP3
does not become tyrosine phosphorylated after DIF-1 treatment (data not
shown). Hence, by elimination, these observations indicate a change in
serine-threonine phosphorylation status. In addition, stress, a known inducer
of serine threonine phosphorylation of PTP3, causes a similar pI shift as
DIF-1 and lambda phosphatase has the same, counteracting effect (data not
shown). These results suggest the existence of multiple serine-threonine
phosphorylation states for PTP3, with an elevation in the level of
modification after DIF-1 addition. |
DISCUSSION |
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SUMMARY
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DISCUSSION
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).
Identification of PTP3 as the protein tyrosine phosphatase responsible for
de-activation of STATc is important, therefore, because it provides a specific
function for this otherwise rather enigmatic enzyme. Our results further
suggest that PTP3 is itself subject to direct biochemical regulation, induced
by DIF-1, and that it forms part of the signalling mechanism that determines
the tyrosine phosphorylation and nuclear localisation status of STATc
(Fig. 7). The evidence for this
derives from a combination of genetic and biochemical observations. Overexpression of PTP3 inhibits tyrosine phosphorylation and nuclear accumulation of STATc in response to DIF-1 or osmotic stress. Conversely, inhibition of PTP3, using substrate-trapping mutants of PTP3 as dominant-negative forms, causes constitutive STATc activation; thus, if PTP3 is rendered inactive, DIF-1 and stress become dispensable for activation. The dominant-negative construct also exerts a striking effect on normal development; now rather than being nuclear enriched predominantly in the pstO region, STATc becomes enriched in nuclei throughout the slug. These observations suggest that activation of STATc by DIF-1 and stress is bought about by the inactivation of PTP3 but leave open the possibility of additional regulation at the level of the tyrosine kinase. Because we do not have access to the STATc tyrosine kinase, we cannot test its involvement directly, but the fact that cells transformed with the dominant-negative constructs show little if any activation of STATc by DIF-1 argues against the kinase playing the dominant regulatory role.
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observed a 35% decrease in PTP-PEST activity in vivo when they treated cells
with TPA, which activates PKC to phosphorylate a serine residue (S39). They
are presumably sufficient to tip the phosphorylation-dephosphorylation
equilibrium in favour of net phosphorylation. In addition, there may be
multiple spatially separated pools of PTP3 [as reported by Yudushkin et al.
(Yudushkin et al., 2007) for
PTP1B], with a tightly DIF-1 and sorbitol-regulated pool functioning to
de-phosphorylate STATc.
We do not know the precise mechanism whereby phosphorylation acts to
inhibit phosphatase activity. In principle it could be the direct result of a
conformational change induced by phosphorylation. However, the stress-induced
reduction in PTP3 activity is not observed when a reducing agent is included
in the extraction buffer (Gamper et al.,
1999). This suggests that the effect of PTP3 phosphorylation is
indirect and that it might involve selective oxidation of the phosphorylated
forms by reactive oxygen species (ROS). ROS, such as
H2O2, are believed to be generated in a localized manner
when growth factor or antigen receptors bind their ligands. They direct
reversible oxidation of the active site cysteine in PTPs, inhibiting their
enzymatic activity (reviewed by Tonks,
2005), and they are known to affect Dictyostelium
development (Bloomfield and Pears,
2003). This could explain why the apparent inhibition engendered
by DIF-1 and stress is observed only under non-reducing conditions. How does
phosphorylation favour oxidation? After cells are exposed to stress PTP3
accumulates in endosome-like structures
(Gamper et al., 1999). If
these contain a relatively high concentration of ROS, this might explain why
the phosphorylated PTP3 population becomes selectively inactivated.
Although it was known that medium-induced stress induces an increase in
total serine-threonine phosphorylation of PTP3
(Gamper et al., 1999), we have
now shown that phosphorylation of PTP3 also increases after DIF-1 treatment.
This is, to our knowledge, the first example of DIF-1-induced serine-threonine
phosphorylation. Is this likely to be a more general mechanism for
ligand-induced STAT activation? The paradigmatic mechanism of STAT activation
is via the regulated activation of a tyrosine kinase family member. There is,
to our knowledge, no demonstration of ligand-induced STAT activation mediated
via an inhibitory effect on a specific PTP. However, PTP activity is
negatively regulated, in other signalling contexts, by ROS.
H2O2 activates both STAT1 and STAT3 in murine
fibroblasts and rat vascular smooth muscle cells
(Simon et al., 1998;
Madamanchi et al., 2001), and
STAT3 is activated by H2O2 in human lymphocytes
(Carballo et al., 1999). In two
of the studies, activation could in principle be explained by concurrent JAK
stimulation (Simon et al.,
1998; Madamanchi et al.,
2001). However, the fact that pervanadate was in itself a potent
stimulator of STAT activation led two of the groups involved to posit an
additional inhibitory effect of H2O2 on an unidentified
PTP (Carballo et al., 1999;
Madamanchi et al., 2001).
Another, more fully documented case of STAT regulation by ROS derives from a
study of virus infection. When respiratory syncytial virus (RSV) infects
cells, there is a burst of ROS production and STAT1 and STAT3 are activated
(Liu et al., 2004). The
pathway seems to involve ROS inhibition of PTP activity, but the presumptive
PTP target is again unknown.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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5% of STATc
protein in the cell is recovered by the co-IP procedure.
