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J Biol Chem, Vol. 273, Issue 46, 30073-30076, November 13, 1998
andFrom the Department of Biology and UCSD Cancer Center, University of California San Diego, La Jolla, California 92093-0322
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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Interferon (IFN) induction of immediate-early
response genes is mediated through the signal transducers and
activators of transcription (STATs). Activation of STAT1 by IFN Interferons as well as many other cytokines and growth factors
mediate their biological effects through the induction of a set of
immediate-early response genes (1-10). This process depends on the
activation of a family of SH2 and SH3 domain containing signal
transducers and activators of transcription (STATs)
(11-16).1 Activation of
latent, cytoplasmic, or membrane-associated STAT proteins is
accomplished through their tyrosine phosphorylation (11, 15-17), which
in most cases depends on the activity of the Janus protein-tyrosine
kinases (Jaks) (18-25). IFN After its tyrosine phosphorylation, STAT1 either homodimerizes or forms
heterodimers when STAT2 is activated by IFN Here we demonstrate the activation of STAT1 by IFN Cells--
2ftgh, U3-SH2mut, and U6A cells were described
previously (26, 38). Cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum, penicillin,
and streptomycin (Irvine Scientific).
Interferons and Reagents--
IFN Whole Cell Extracts--
Following treatment, cells were washed
with PBS and lysed on the plates with lysis buffer (1 ml) containing 20 mM Hepes, pH 7.4, 1% Triton X-100, 100 mM
NaCl, 50 mM NaF, 10 mM Immunoprecipitation and Immunoblotting--
Lysates were
precleared by incubation with protein G-Sepharose for 30 min;
subsequently, lysates were incubated for 2 h with a polyclonal
antibody against the C terminus of STAT1 and protein G-Sepharose for an
additional hour. Immunoprecipitates were washed three times with
ice-cold lysis buffer, resuspended in SDS sample buffer, and resolved
on a 7.5% SDS-polyacrylamide gel (Bio-Rad). After transfer onto
polyvinylidene difluoride membrane, proteins were detected with
anti-phosphoSTAT1 (1:500; New England Biolabs) or anti-STAT1 (1:1000;
Transduction Labs) antibodies. Blots were developed with horseradish
peroxidase-conjugated secondary antibodies and enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Electrophoretic Mobility Shift Assay--
EMSAs were performed
using whole cell extracts prepared as described above and an
end-labeled oligonucleotide corresponding to the GRR sequence found in
the promoter sequence of the Fc Immunoflourescence--
Cells were seeded onto coverslips in
6-well plates and incubated overnight at 37 °C in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. After
treatment, coverslips were washed once with PBS followed by one wash
with PIPES buffer. Cells were fixed in methanol at room temperature for
6 min, and nuclei were permealized by incubating with 0.5% Nonidet
P-40/PIPES buffer for 13 min at room temperature. Coverslips were
washed three times with PBS, blocked with 10% goat serum for 35 min, and incubated with anti-STAT1 (Transduction Laboratory) for 50 min at
room temperature. Cells were rinsed four times for 5 min in PBS before
incubation with Cy3-conjugated secondary antibody for 40 min at room
temperature. After washing, coverslips were mounted onto glass slides
in 50% glycerol/PBS.
Significant progress has been made in our understanding of the
mechanisms that lead to the activation of STAT proteins through numerous cytokine and growth factor receptors. However, very little is
known about the process of nuclear translocation of STAT proteins after
their tyrosine phosphorylation. Although tyrosine phosphorylation of
STAT1 is required for its nuclear localization, it remains unclear
whether phosphorylation of Tyr701 is solely required for
STAT dimerization or whether a cytosolic transport protein utilizes
this phosphotyrosine residue as a docking site. Previous findings
suggested that the SH2 domain of STAT1 was required to recruit latent
STAT1 to activated receptors (29, 35, 36, 40-42). Consequently, the
reverse approach of introducing an Arg It has been previously shown that incubation of cells with a
combination of 1 mM hydrogen peroxide and 0.1 mM orthovanadate (H/V) results in the ligand-independent
activation of STAT proteins (43, 44). This treatment even proved
effective in the activation of STAT1 in the absence of the Jak tyrosine
kinases that are required for STAT activation (44), indicating that
this procedure effectively bypasses the need for receptor-mediated
signaling. We therefore decided to explore the possibility that this
treatment might also activate a STAT1 carrying the Arg We therefore decided to titrate the concentration of H/V to a level
where it did not affect the normal nuclear translocation of wild-type
STAT1 after stimulation of cells with IFN. It was found that a
significantly lower concentration of H/V (referred to as h/v, to
indicate 10 µM hydrogen peroxide and 5 µM
orthovanadate) did not interfere with the nuclear translocation of
wild-type STAT1 after IFN stimulation (see Fig. 3A,
lower panel) but was also unable to induce tyrosine
phosphorylation of wild-type STAT1 or STAT1-SH2mut (Fig.
1, A and B,
lanes 5). Surprisingly, however, although the exposure of
cells to this low concentration of h/v did not cause tyrosine
phosphorylation of STAT1 or STAT1-SH2mut by itself, it promoted the
subsequent activation of STAT1-SH2mut by IFN
or
IFN
through its tyrosine phosphorylation involves members of the Jak
tyrosine kinases. In addition, STAT2 is activated by IFN, and,
together with STAT1 and p48/ISGF3, forms the transcription factor
complex ISGF3. Previous findings suggested that the STAT1-SH2 domain, which is required for the homo- or heterodimerization of STAT1, also
participates in the recruitment of STAT1 to the IFN-receptors, because
mutations in the SH2-domain abolished STAT1 activation by IFN.
Furthermore, STAT2 was reported to be required for the activation of
STAT1 by IFN. We were able to induce STAT1 tyrosine phosphorylation
by IFN/
in the absence of STAT2 or a functional STAT1-SH2 domain.
In contrast, IFN was unable to cause tyrosine phosphorylation of
STAT1-(SH2:Arg 
Gln). Interestingly, although STAT1 was found in the
nucleus in STAT2-deficient cells, the nuclear accumulation of the
tyrosine phosphorylated SH2-mutant STAT1 was impaired. In summary, our
results indicate that the SH2 domain of STAT1 is not required for its
ligand-dependent activation by IFN/. Moreover,
tyrosine phosphorylation is not sufficient to target STAT1 to the
nucleus; rather, dimerization appears to play a critical role in the
subcellular distribution of STAT1.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
initiation of STAT1 tyrosine
phosphorylation requires the activity of Jak1 and Jak2 (18, 19),
whereas IFN/ mediates STAT1 and STAT2 activation through the
kinases Jak1 and Tyk2 (18, 25). Activation of STAT1 by IFN/ was
also reported to depend on the presence of STAT2 (26, 27), implying a
sequential activation of STAT proteins through the IFN/ receptor.
More recently, Li et al. demonstrated that STAT1 and STAT2
are prebound to the inactive IFNAR2c chain and that this association
requires the N-terminal region of STAT2 (28).
/ to translocate to
the nucleus where site-specific binding to enhancer elements leads to
gene activation (29, 30). Nuclear import is often controlled by binding
of a karyophilic protein that contains a single or bipartite nuclear
localization signal to the nuclear pore, with subsequent import in a
GTP hydrolysis-dependent manner. Although the dependence of
STAT1 nuclear import in response to IFN on the GTPase activity of
Ran/TC4 has been demonstrated (31), no nuclear localization signal has
been identified in any of the STAT proteins. Tyrosine phosphorylation
of STAT1 is an absolute prerequisite for its nuclear translocation and
its ability to bind DNA (32, 33). The SH2 domain of STAT1 has been
implicated in the activation as well as in the dimerization process
(29, 30, 34-36), because mutations in the SH2 domain of STAT1 also prevent it from becoming tyrosine phosphorylated (34, 37). This defect
in the ability to activate an SH2 domain-mutated STAT1 (STAT1-SH2mut)
made it thus far impossible to determine whether dimerization is
required for nuclear translocation of STAT1 or whether the
phosphorylated tyrosine residue serves as a binding site for a
potential transport protein.
/, but not
IFN, in the absence of a functional STAT1-SH2 domain; furthermore, we show STAT1 activation by IFN/ in STAT2-deficient cells.
Importantly, whereas the lack of STAT2 did not affect the nuclear
localization of STAT1, the abrogation of STAT1-SH2 domain function
resulted in the inability of STAT1 to translocate to the nucleus
despite its tyrosine phosphorylation.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
, IFN, and IFN were
generous gifts from Hoffman LaRoche, Chiron, and Genentech,
respectively. 50 mM sodium vanadate and 100 mM
hydrogen peroxide (both from Fisher) were incubated in Dulbecco's
modified Eagle's medium without fetal bovine serum for 15 min prior to
addition to cells.
-glycerophosphate, 1 mM sodium vanadate and 1 mM
phenylmethylsulfonyl fluoride. Lysates were centrifuged at 13,000 rpm
for 5 min, and protein concentration was determined by Lowry (Bio-Rad
Protein Assay).
RI (5'-AATTAGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3') as described previously (39).
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
Gln mutation in the SH2
domain to render it incapable of binding phosphotyrosine and preventing
dimerization was unsuccessful, because a STAT1 carrying such a mutation
did not become tyrosine phosphorylated in response to IFN or epidermal
growth factor (34, 37).
Gln mutation
in the SH2 domain. Indeed, exposure of either wild-type 2ftgh cells or
STAT1-deficient U3A cells stably expressing STAT1-SH2mut to H/V
resulted in the IFN-independent tyrosine phosphorylation of STAT1 or
STAT1-SH2mut, respectively (data not shown). However, this treatment
appeared to be extremely toxic to cells, because we found that it not
only failed to target tyrosine phosphorylated wild-type STAT1 into the
nucleus, but it also prevented the nuclear translocation of wild-type
STAT1 when IFN was added after H/V, despite the fact that this
costimulation resulted in a further increase in STAT1 tyrosine
phosphorylation (data not shown).
/. In contrast,
IFN was still unable to induce tyrosine phosphorylation of
STAT1-SH2mut (Fig. 1B, lane 8). Interestingly, we
were also able to detect a weak but reproducible tyrosine
phosphorylation of STAT1-SH2mut through stimulation with IFN/
alone but not with IFN. This is in contrast to previous reports
suggesting that STAT1 could not become phosphorylated in the absence of
a functional SH2 domain; however, these studies were predominantly focused on STAT1 activation by IFN or epidermal growth factor. Furthermore, we believe that the use of phosphoSTAT1-specific antiserum
results in increased sensitivity compared with phosphotyrosine blots,
allowing for the detection of low levels of STAT1 phosphorylation. It
thus appears that the priming of cells with a subthreshold concentration of h/v facilitates an increase in the
activation of STAT1-SH2mut after IFN/ while preserving the
IFN-dependent nature of the stimulation. Earlier
studies showed that in some cases the autophosphorylation sites of
receptors are dispensable for STAT activation, leaving the possibility
that the STAT-SH2 domain binds to a receptor-associated, tyrosine
phosphorylated protein (24, 45). In contrast, our findings suggest for
the first time the presence of an alternate mechanism for recruitment of a STAT protein to an activated cytokine receptor that does not
involve the SH2 domain of the STAT protein.
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Fig. 1.
Tyrosine phosphorylation of STAT1 in the
absence of STAT1-SH2 domain or STAT2. 2ftgh (A), U3A
reconstituted with STAT1-SH2mut (B), and U6A (C)
were stimulated for 30 min with either IFN (500 units/ml), IFN
(500 units/ml), or IFN (10 ng/ml) without (lanes 1-4) or
with (lanes 5-8) prior priming of cells for 30 min with h/v
(10 µM/5 µM). Whole cell extracts (15 µg)
were subjected to immunoprecipitation with antiserum directed against
the C terminus of STAT1, and isolated proteins were resolved on a 7.5%
SDS-polyacrylamide gel. After transfer to Immobilon polyvinylidene
difluoride membrane, activated STAT1 was detected with a polyclonal
anti- (Tyr701)PhosphoSTAT1 antibody. Loading of equal
amounts of STAT1 was confirmed by reprobing with STAT1 antiserum (data
not shown). CTL, control.
Because it had been reported that STAT2 is essential for activation of
STAT1 by IFN (27), we decided to test whether h/v priming could also
overcome this requirement. Indeed, whereas IFN/ alone activates
STAT1 only weakly in STAT2-deficient U6A cells (Fig. 1C,
lanes 2 and 3), the pretreatment with h/v before addition of IFN/ restored the ligand-induced tyrosine
phosphorylation to levels comparable with those seen in wild-type cells
(Fig. 1C, lanes 6 and 7). As was the
case with STAT1-SH2mut, h/v treatment alone did not lead to any
detectable tyrosine phosphorylation in the U6A cells (Fig.
1C, lane 5).
To analyze the DNA binding capabilities of tyrosine phosphorylated
STAT1-SH2mut or of STAT1 activated by IFN in the absence of STAT2,
we performed electrophoretic mobility shift assays using the GRR
sequence of the high affinity FcRI promoter as a probe. As shown in
Fig. 2C, tyrosine
phosphorylated STAT1 in U6A cells lysates was able to bind to the GRR
as expected and confirmed the results of the phosphoSTAT1 Western
blots. In contrast, tyrosine phosphorylated STAT1-SH2mut was unable to
form a complex capable of binding the GRR (Fig. 2B) or to
assemble with activated STAT2 into the interferon-stimulated response
element-binding ISGF3 complex (data not shown). These results confirm
that the SH2 domain of STAT1 is required for formation of a dimerizing
DNA binding complex but is expendable for achieving tyrosine
phosphorylation in response to IFN/.
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Our primary goal was to investigate whether the dimerization of STAT1
was required for its nuclear translocation or whether tyrosine
phosphorylated STAT1 is transported into the nucleus as a monomer. We
therefore resorted to immunohistochemistry to analyze the subcellular
localization of tyrosine phosphorylated STAT1-SH2mut or of
IFN-activated STAT1 in the absence of STAT2. As shown in Fig.
3A (upper panel),
IFN-activated, tyrosine phosphorylated STAT1 translocated
efficiently into the nucleus, with h/v priming apparently enhancing the
level of translocation (Fig. 3A, lower panel).
STAT2-deficient U6A cells displayed, in good correlation with the low
level of tyrosine phosphorylation of STAT1, only marginal translocation
of STAT1 to the nucleus in response to IFN/ alone (Fig.
3C, upper panel). However, h/v priming was able
to facilitate substantial nuclear presence of STAT1 in response to
IFN/ (Fig. 3C, lower panel).
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In contrast, no STAT1-SH2mut could be detected in the nucleus after any
of the treatments (Fig. 3B), despite proper phosphorylation of the protein on Tyr701 in response to IFN/ after
h/v priming. These results demonstrate that the tyrosine
phosphorylation per se, although required, is not sufficient to target
STAT1 to the nucleus. Rather, the dimerization of STAT proteins appears
to be the essential mediator of nuclear translocation. It is
theoretically possible that h/v priming is able to promote the tyrosine
phosphorylation of STAT1-SH2mut in response to IFN/ but is unable
to facilitate any additional phosphorylation on serine/threonine
residues that could be required for STAT1 translocation to the nucleus.
However, thus far the only reported ligand-induced serine
phosphorylation of STAT1 occurs on Ser727 (46), a residue
that is absent in the STAT1 splice variant. Nevertheless, STAT1
is still able to translocate to the nucleus in response to IFN stimulation.
In conclusion, our results show for the first time that STAT1 can be
rapidly activated in a ligand-dependent manner even in the
absence of a functional SH2 domain, demonstrating that the binding of
the STAT1-SH2 domain to receptor phosphorylation sites is not the only
mechanism that allows STAT1 to interact with an activated cytokine
receptor. In contrast, an intact SH2 domain is required in order for
STAT1 to be imported into the nucleus. These results lead to the
conclusion that STATs need to form homo- or heterodimers in the
cytoplasm to cross the nuclear membrane.
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ACKNOWLEDGEMENTS |
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We thank Dr. Andrew Larner for the generous gift of STAT1 and STAT2 antisera and Drs. George Stark and Xin-Yuan Fu for providing the mutant cells lines and the STAT1 plasmids, respectively. We are also grateful to Dr. Robert Rickert for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA80105.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the Markey Foundation.
§ Recipient of the Sidney Kimmel Scholar Award. To whom correspondence should be addressed: University of California, San Diego, Dept. of Biology, Bonner Hall 3138, 9500 Gilman Dr., La Jolla, CA 92093-0322. Tel.: 619-822-1108; Fax: 619-822-1106; E-mail: midavid{at}ucsd.edu.
The abbreviations used are: STATs, signal transducers and activators of transcription; IFN, interferon; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
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