![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
RESEARCH PAPER
1 Abteilung Zelluläre Signalverarbeitung, Freie Universität Berlin, Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany , 2 Abteilung Molekulare Medizin, Freie Universität Berlin, Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany
 |
Abstract |
|---|
 Top Abstract ResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
[Keywords: Stat1; nuclear accumulation; DNA binding; dephosphorylation; transcription; cytokine signaling]
Received April 11, 2003; revised version accepted June 11, 2003.
). Canonical
signaling through the Janus tyrosine kinase (Jak)/Stat pathway begins with the
engagement of cytokines with their cognate receptor at the cell membrane
(Ihle et al. 1998). This
triggers the phosphorylation of signature tyrosine residues in the
intracellular receptor tails by noncovalently attached Jak kinases, which also
autophosphorylate on tyrosines (Briscoe et
al. 1996), and the phosphorylated receptors are thus turned into
binding partners for the Stat SH2 domain. Stat1 monomers form high-avidity
reciprocal homo- or heterodimers and detach from their receptor docking site
after phosphorylation of tyrosine residue 701 at their C terminus (Shuai et
al. 1993,
1994;
Greenlund et al. 1995). This
sequence of events results within minutes in the nuclear translocation of
activated Stats, where they can bind to palindromic DNA recognition sites
(GAS) and directly induce transcription
(Darnell et al. 1994). Stat1
consists of well-defined structural domains. The N-terminal domain engages in
protein binding such as cooperative dimer–dimer interactions
(Vinkemeier et al. 1996;
Xu et al. 1996). It is
connected to a large core domain containing the DNA-binding module
(Horvath et al. 1995), an SH2
domain responsible for dimer formation
(Shuai et al. 1994), and a
linker domain implicated in DNA binding
(Yang et al. 2002). A
transferable transactivation domain is located at the C terminus
(Müller et al. 1993).
The regulated nucleocytoplasmic translocation of Stat proteins is only
beginning to be understood, with Stat1 in interferon 
(IFN)
signaling as the best characterized example. Recently, we and others described
an unusual dimer-specific nuclear localization signal (dsNLS) comprising
residues 407–413 in the DNA-binding domain that mediates binding of
Stat1 dimers to importin 
/
and subsequent nuclear import
(Fagerlund et al. 2002;
McBride et al. 2002;
Meyer et al. 2002a). Prior to
stimulation with IFN, Stat1 is found in both the cytoplasm and the
nucleus because of signal-independent nucleocytoplasmic shuttling (Meyer et
al.
2002a,2002b).
The mechanism by which this occurs is not resolved, but differs from the
signal-induced nuclear import of activated Stat1
(Sekimoto et al. 1997;
Meyer et al. 2002a). Thus,
nuclear import does not necessarily result in nuclear accumulation, which is
only observed with tyrosine-phosphorylated Stat1 dimers containing a
functional SH2 domain (Mowen and David
1998). Loss of DNA binding was also described to cause defects in
nuclear accumulation, although conflicting data with various DNA-binding
mutants were reported (Herrington et al.
1999; McBride et al.
2000; Melén et al.
2001; Lillemeier et al.
2001). A leucine-rich export signal (NES) in the N-terminal region
contributes to nuclear export of Stat1, but pharmacological inactivation of
the NES-receptor CRM1 will not induce nuclear accumulation or suppress
nucleocytoplasmic shuttling of the unphosphorylated molecule
(Begitt et al. 2000;
Meyer et al. 2002a). Nuclear
accumulation of Stat1 during IFN signaling is transient, yet appears to
remain stable for several hours. Multiple mechanisms are involved in limiting
receptor signaling temporally. Receptor-associated protein tyrosine
phosphatases and the targeted degradation of the IFN receptor chains
play a prominent role (Kisseleva et al.
2002). Pulse-chase experiments demonstrated that Stat1 was not
phosphorylated on tyrosine when it reappeared in the cytoplasm after nuclear
accumulation (Haspel et al.
1996), and a phosphatase was identified recently that participates
in the dephosphorylation of activated Stat1 in the nucleus
(Haspel and Darnell 1999;
ten Hoeve et al. 2002).
Here, a coherent model is provided that describes Stat transcription factors as shuttling proteins that accumulate in the nucleus because of their nonspecific affinity for DNA. We identify and characterize nuclear retention mutants and show that tyrosine-phosphorylated Stat molecules are barred from nuclear exit. DNA-bound Stat1 is protected from dephosphorylation and is thus transiently retained in the nucleus. These data describe a mechanism that allows nuclear Stats to monitor receptor activity and that at the same time selectively promotes occupation of target regulatory elements.
|
Results |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
The analysis of nucleocytoplasmic transport of Stat1 has revealed its high,
energy-independent mobility in the cytoplasm, which is indicative of random
diffusion (Lillemeier et al.
2001). On the other hand, processes that require receptor
activation with vesicle formation or complexation with ligand have been
proposed to precede nuclear accumulation of Stat3 and Stat1, respectively
(Subramaniam et al. 2000;
Bild et al. 2002). To
investigate nuclear translocation, we purified the human Stat1 protein from
baculovirus-infected Sf9 cells (Fig.
1A). Additionally, the recombinant protein was phosphorylated in
vitro, and a pure preparation of correctly tyrosine-phosphorylated Stat1 was
isolated (Fig. 1A). These
proteins were microinjected into HeLa cells and the cell line U3A (data not
shown), which does not express Stat1
(Müller et al. 1993). We
initially microinjected unphosphorylated Stat1 into the cytoplasm of HeLa
cells and followed its intracellular redistribution. The Stat1 protein entered
the nucleus rapidly and showed a pancellular distribution after 
30 min
(Fig. 1B). This situation was
stable, and nuclear accumulation did not occur even after 3 h (data not
shown). Nuclear microinjection of unphosphorylated Stat1 similarly led to a
pancellular distribution 30 min after injection
(Fig. 1B).
|
Next, tyrosine-phosphorylated Stat1 was injected into the cytosol of untreated HeLa cells and U3A cells. Figure 1C shows the nuclear accumulation that resulted 15 min after injection. Serum starvation for 4 h prior to microinjection resulted in identical accumulation (data not shown). After nuclear microinjection of tyrosine-phosphorylated Stat1, a slow release of Stat1 into the cytosol was observed. We traced the microinjected material with antibodies directed against unphosphorylated as well as phosphorylated Stat1. Even 2 h after nuclear microinjection, we were unable to detect Stat1 immunoreactivity in the cytoplasm (data not shown). However, after 6 h, considerable amounts of Stat1 were found outside of the nucleus, because an almost pancellular distribution was observed with a Stat1-specific antibody (Fig. 1D,F). In contrast, the remaining tyrosine-phosphorylated Stat1 was confined to the nuclear compartment (Fig. 1E,F). These data confirm that unphosphorylated Stat1 is capable of nucleocytoplasmic shuttling, yet accumulation only occurred with the tyrosine-phosphorylated species. Conversely, nuclear export required dephosphorylation. Notably, nuclear translocation and the subsequent accumulation of tyrosine-phosphorylated Stat1 were entirely independent of IFN stimulation or receptor activation.
Stat1 nuclear accumulation is a dynamic process sustained by
continuous shuttling and kinase activity
Nuclear accumulation of Stat1 in response to stimulation of cells with
IFN can last for several hours. Given that Stat1 has a slow turnover
time (t1/2 > 8 h;
Lee et al. 1997) but
dephosphorylation is rapid (t1/2 < 15 min;
Haspel et al. 1996), we wanted
to determine how long Stat1 molecules remained nuclear. We first observed the
effects of aborted receptor signaling on the persistence of nuclear
accumulation. Nuclear accumulation was induced by treating HeLa cells with
IFN for 30 min. Subsequently, the medium was replaced with medium
containing staurosporine to block further tyrosine kinase activity
(Shuai et al. 1992). This
resulted in the complete dephosphorylation of Stat1 after 60 min and a
collapse of nuclear accumulation that closely mirrored the kinetics of
tyrosine dephosphorylation (Fig.
2A,B, top panels). When we additionally included leptomycin B, an
inhibitor of the export receptor CRM1
(Kudo et al. 1998), the rapid
termination of nuclear accumulation was not prevented, confirming
CRM1-independent nuclear export (Begitt et
al. 2000; data not shown). On the contrary, inactivating tyrosine
phosphatases by treatment of cells with pervanadate
(Gordon 1991) not only
suppressed Stat1 dephosphorylation but also extended the duration of nuclear
accumulation (Fig. 2A,B, bottom
panels).
|
We then used an antibody-microinjection assay, which we introduced earlier
(Meyer et al. 2002a), to
reveal ongoing nucleocytoplasmic shuttling of Stat1 during stable nuclear
accumulation. Cells treated with IFN for 30 min were injected with
Stat1 antibodies in the cytoplasm and then incubated in the continuous
presence of IFN for a further 30 min. As can be seen from
Figure 2C (first row), this
resulted in the significant loss of Stat1 from the nucleus because of
antibody-induced precipitation in the cytoplasm. However, treatment of cells
with the tyrosine phosphatase inhibitor vanadate precluded the
antibody-induced nuclear clearance of Stat1, because even after 90 min of
incubation there was no translocation of Stat1 to the cytoplasm
(Fig. 2C, second row). The bar
diagram in Figure 2C gives a
quantitative summary of the above injection data. Additionally, the export
specifically of unphosphorylated Stat1 from the nucleus of vanadate-treated
cells was investigated. We made use of a mutated Stat1, the tyrosine
phosphorylation site of which, Tyr 701, was defective
(Shuai et al. 1993). We
prepared purified protein of this mutant and injected it into the nucleus of
cells that had been stimulated with IFN and treated with
vanadate/H2O2 (Fig.
2C, third row). Contrary to tyrosine-phosphorylated wild-type
Stat1, which was retained in the nucleus by vanadate treatment (second row),
the unphosporylated Stat1Y701F was still capable of nuclear export during
phosphatase inhibition. In another control experiment, we performed nuclear
microinjections of a canonical Stat1-derived nuclear export signal linked to a
fusion of green-fluorescent protein with glutathione-S-transferase
(GFP-NES-GST). This reporter construct also was quickly exported into the
cytoplasm irrespective of vanadate/H2O2
(Fig. 2C, fourth row). These
observations demonstrated the highly dynamic nature of Stat1 nuclear
accumulation, with kinase and phosphatase activities being the antagonistic
determinants for its duration. Importantly, tyrosine-phosphorylated Stat1 was
blocked from nuclear exit. In addition, these data showed that repeated cycles
of nuclear import and export of Stat1 during the accumulation phase were
required to maintain a stable accumulation in the nucleus.
Stat1 DNA-binding mutants discriminate nuclear retention from nuclear
import
Nuclear accumulation was shown to depend on the integrity of the
DNA-binding domain, which can be attributed to the need for a functional dsNLS
located here (Fagerlund et al.
2002; Meyer et al.
2002a). In addition, vicinal residues unrelated to the dsNLS were
also demonstrated to influence nuclear buildup of activated Stat1
(McBride et al. 2000).
Therefore, we analyzed the effects that DNA binding has on Stat1 nuclear
retention and also took nonspecific protein–DNA interactions into
account. Guided by the Stat1–DNA cocrystal structure
(Chen et al. 1998), we mutated
residues in the DNA-binding domain that potentially make backbone contacts
with phosphate groups in the DNA and are not part of the dsNLS
(Fig. 3A). We reasoned that
introducing positive charges in these positions (termed
Stat1dnaplus; Thr327Arg; Val426His; Thr427His) might increase
electrostatic attraction to the polyanionic DNA, whereas negative charges
(termed Stat1dnaminus; Val426Asp; Thr427Asp) might potentially
reduce electrostatic interactions with DNA. The Stat1 variants were expressed
well and phosphorylated on tyrosine 701 in response to IFN
(Fig. 3B). Additionally, we
included another DNA binding mutant, named Stat1dnaoff here, that
was previously characterized by Darnell and coworkers
(Yang et al. 2002) and
displays a markedly increased off-rate from GAS sites. First, we observed the
nucleocytoplasmic distribution of GFP-fusion proteins before and after
IFN stimulation and found accumulation of Stat1dnaplus and
dnaoff, whereas the Stat1dnaminus mutant failed to
accumulate in the nucleus in response to IFN
(Fig. 3C). Addition of a
negative charge in position 327 (Thr to Glu) of Stat1dnaminus also
resulted in defective nuclear accumulation, albeit less severely, because a
partial accumulation could still be induced by IFN (data not shown). We
therefore used the double mutant dnaminus for further
experimentation.
|
DNA-binding activity was investigated in mobility shift assays with an
optimal Stat1-binding site (M67), and both Stat1dnaplus and
dnaminus failed to bind (Fig.
3D). Because this experiment can only reveal sequence-specific DNA
interactions, we used a different approach to also determine nonspecific
binding. We used oligonucleotides harboring either tandem optimal
Stat1-binding sites or a TTC-polymer with a single site weakly resembling a
cognate GAS element (see Materials and Methods). These were coupled to agarose
beads and then mixed with extracts from IFN-treated U3A cells
expressing similar amounts of tyrosine-phosphorylated wild-type Stat1
(Stat1wt) or either of these mutants. As expected, Stat1wt showed a strong
preference for the optimal binding site
(Fig. 3E). However, the mutant
Stat1dnaplus was capable of binding to both the specific and the
nonspecific site with almost equal avidity
(Fig. 3E). In contrast,
Stat1dnaminus showed only a very weak interaction with either
sequence (Fig. 3E). Thus,
Stat1dnaplus bound to DNA, albeit nonspecifically, whereas
dnaminus did not. To exclude the possibility that the observed
defect in DNA binding was caused by an inability to dimerize, we cotransfected
U3A cells with Stat1dnaminus tagged with either GFPor Flag and
confirmed the formation of GFP/Flag-tagged heterodimers
(Fig. 3F). We also tested
nuclear extracts from IFN-stimulated U3A cells for the presence of
tyrosine-phosphorylated Stat1dnaminus and detected ample amounts
similar to wild type (Fig. 3G).
Additionally, we were able to induce the nuclear accumulation of
Stat1dnaminus– GFPafter nuclear microinjection of
GFPantibodies into IFN-stimulated HeLa cells
(Fig. 3H). Importantly, the
antibody-induced nuclear accumulation of Stat1wt and Stat1dnaminus
could not be discriminated, thus indicating unperturbed import of the mutant.
Moreover, blocking of tyrosine dephosphorylation with
vanadate/H2O2 during 30 min of IFN stimulation
rescued the accumulation defect of Stat1dnaminus
(Fig. 3C).
These results identified nonspecific DNA binding as a sufficient
requirement for nuclear accumulation and distinguished
STAT1dnaminus from a recently characterized import mutant
(Meyer et al. 2002a). Thus, we
determined defective retention as a cause preventing accumulation in the
nucleus.
DNA-bound Stat1 is protected from tyrosine dephosphorylation
The above experiments demonstrated the need for tyrosine dephosphorylation
prior to nuclear export of Stat1, and they identified diminished DNA-binding
activity as the cause of defective nuclear retention. At first we therefore
examined the influence of DNA binding on the dephosphorylation of Stat1
variants in vitro. Highly pure recombinant tyrosine-phosphorylated Stat1 was
incubated in the absence and presence of DNA with purified TC-PTPa (also
called TC45; Ibarra-Sanchez et al.
2000), a ubiquitously expressed Stat1-specific nuclear tyrosine
phosphatase (ten Hoeve et al.
2002). We used recombinant full-length TC45 as well as a truncated
version that lacks a noncatalytic segment of 35 amino acids at its C terminus.
This variant enzyme was demonstrated to exhibit increased phosphatase activity
(Hao et al. 1997). The two
preparations differed in their Stat1 dephosphorylation activity, but the
specificity of the reaction was unchanged by the truncation (data not shown).
The Stat concentration was set equal to the Kd value of 1 nM
(Vinkemeier et al. 1996). As
can be seen in Figure 4A, Stat1
was efficiently dephosphorylated by TC45. In the presence of DNA, however, the
tyrosine dephosphorylation of Stat1 was strongly reduced in a
concentration-dependent manner (Fig.
4A). Notably, the dephosphorylation reaction was sensitive also to
alterations of the DNA sequences used, because GAS sites protected Stat1
better from dephosphorylation than unrelated DNA sequences
(Fig. 4A). Control reactions
with an artificial substrate showed that TC45 was not generally inhibited in
the presence of DNA (see Materials and Methods). We then examined the in vitro
dephosphorylation of the two DNA-binding mutants Stat1dnaminus and
dnaplus, which display oppositional accumulation behavior. Similar
to Stat1wt, the mutants were efficiently dephosphorylated by TC45 in the
absence of DNA, and the addition of DNA to the reaction afforded protection
from dephosphorylation (Fig.
4B,C). However, Stat1dnaminus, the mutant that shows
deficient accumulation, was only very poorly protected from dephosphorylation
even at the highest DNA concentrations used (25-fold molar excess) and
regardless of its sequence (Fig.
4B). An entirely different result was found with the accumulating
mutant Stat1dnaplus, which was strongly protected from
dephosphorylation by DNA. Moreover, opposite to Stat1wt, the protective
influence of DNA had no sequence-specific component, as both the
GAS-containing oligonucleotide and a nonspecific DNA were equally efficient in
preventing Stat1 inactivation (Fig.
4C). Additionally, we explored the influence of tandem GAS sites
on the dephosphorylation of wild-type Stat1. Expectedly, effective protection
from dephosphorylation at subsaturating DNA concentrations was observed only
with tandem GAS sites (Fig.
4D). In conclusion, Stat1 that was not bound to DNA underwent
tyrosine dephosphorylation at maximum rate, whereas DNA binding protected this
molecule from inactivation in a sequence-dependent manner.
|
DNA binding determines the duration of nuclear accumulation
We then examined in vivo the rate of tyrosine dephosphorylation and its
influence on the intranuclear mobility of Stat1 as well as on the duration of
nuclear accumulation. Reconstituted U3A cells were treated with IFN for
30 min to induce Stat1 phosphorylation. Thereafter, the medium was replaced
with growth medium alone or medium containing staurosporine to expose tyrosine
dephosphorylation by blocking continuous kinase activity. As is shown in
Figure 5A, the initial
concentrations of activated Stats were similar for all variants after 30 min
of IFN treatment, indicating their comparable facility to become
tyrosine-phosphorylated. However, impeding tyrosine kinase activity with
staurosporine revealed the rapid dephosphorylation not only of Stat1wt but
also of dnaminus and dnaoff. In contrast,
Stat1dnaplus was much less sensitive to the cessation of kinase
activity, as the level of activated protein remained high, indicating strongly
reduced tyrosine dephosphorylation (Fig.
5A).
|
Next, the mobility of the Stat1 DNA-binding mutants before and during
nuclear accumulation was determined by FRAPanalysis (fluorescence recovery
after photobleaching). As is shown in Table
1, in unstimulated cells, the Stat1 variants did not differ from
one another because we determined almost identical recovery rates.
Interestingly, mobility was consistently lower in the cytoplasm. Yet, after
IFN stimulation, mobility differences between the Stat1 mutants became
apparent. Wild-type Stat1 was highly mobile in the nucleus, as were the
dnaoff and dnaminus mutants. These proteins had recovery
times of 20 sec (Fig. 5B). Remarkably, the mutant Stat1dnaplus showed a significantly reduced
intranuclear mobility, and the recovery time was extended to 200 sec.
Thus, DNA binding differences were reflected in an altered mobility in the
nuclear compartment, with enhanced binding correlating with reduced mobility.
Importantly, fluorescence recovery was complete for all Stat1 variants,
indicating that no immobile population was present within the nucleus during
accumulation.
|
Figures 1 and
2 indicated that
tyrosine-phosphorylated Stat1 was trapped in the nucleus. We therefore
examined how the inhibition of tyrosine phosphatases influences the mobility
of Stat1 inside the nucleus. This was achieved by treating
IFN-stimulated cells with vanadate/H2O2.
Strikingly, preventing tyrosine dephosphorylation had little impact on the
intranuclear mobility of all Stat1 proteins investigated
(Fig. 5B), because recovery
times increased only by a factor of 2.5 or less
(Table 1). Notably,
fluorescence recovery of all Stat1 variant proteins was complete even in the
absence of phosphatase activity, and the mobility continued to differ between
the DNA binding mutants (Fig.
5B). Compared with other studies, the mobility of Stat1 was much
faster than the core histones and similar to other inducible transcriptional
regulators (McNally et al.
2000; Kimura and Cook
2001; Lillemeier et al.
2001). In conjunction with the in vitro dephosphorylation data, we
conclude that the release of Stat1 from DNA did not require enzymatic
activity. Because progression of the enzyme reaction was proportionate to the
Stat–DNA exchange reaction, it follows that the dephosphorylation of
Stat1 was kinetically controlled.
The above data for the DNA-binding mutants indicated that the inactivation
of Stat1 is inversely correlated in vitro and in vivo with the avidity of the
interactions of Stat1 with DNA. Hence, the resulting differences in terms of
tyrosine dephosphorylation ought to be reflected in varied nuclear retention.
Because a complete loss of DNA binding was associated with a radical abortion
of nuclear accumulation (see Stat1dnaminus in
Fig. 3C), more subtle
modifications of the Stat1–DNA interactions might influence the duration
of nuclear accumulation less severely. As
Figure 5C shows, this is, in
fact, the case. Although the IFN stimulus was identical for all
mutants, the duration of Stat1 nuclear accumulation differed predictably
according to the avidity of Stat1 DNA binding. The mutant
Stat1dnaplus with improved binding to nonspecific DNA showed
transient, yet prolonged nuclear accumulation, whereas the diminished DNA
binding of Stat1dnaoff led to an abbreviated nuclear accumulation
phase (Fig. 5C). We infer the
following: Impaired DNA binding resulted in accelerated inactivation of Stat1
in the nucleus. Therefore, in the face of decreasing Stat1 rephosphorylation
because of terminated receptor signaling, the steady-state concentration of
activated Stat1 required for an observable nuclear accumulation was depleted
sooner. The opposite of this was true for improved DNA binding
interactions.
Interferon-induced nuclear accumulation is not indicative of
transcriptional activity
To our knowledge, there are only examples in which IFN-induced
nuclear accumulation of full-length Stat1 is simultaneously accompanied by the
induction of responsive target genes. Therefore, we tested the gene-activation
ability of the mutant Stat1dnaplus, which shows prolonged nuclear
accumulation, and its direct opposite Stat1dnaminus, which is
incapable of nuclear accumulation. As shown in
Figure 6, the two Stat1
variants did not differ in their inability to induce a GAS-driven reporter
gene in response to IFN. Therefore, nuclear accumulation does not
restore target gene recognition if the Stat1 dimer has lost the capacity to
discriminate GAS sites from unrelated sequences.
|
|
Discussion |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
). These circuits allow the nucleus to precisely detect and
respond to changes of the primary signal input at the cell membrane. A model
is shown in Figure 7 that
explains the molecular basis of the signal-dependent nuclear accumulation of
the transcription factor Stat1 and outlines the constitutive cycling of Stat1
both in resting cells and during stimulation with IFN.
|
Nuclear translocation and accumulation of activated Stat1 can occur without
receptor activation, as demonstrated by microinjection of in vitro
phosphorylated protein (Fig.
1C). This observation extends the previous finding that
dimerization is sufficient for nuclear import of activated Stat1
(Milocco et al. 1999). These
results do not rule out that there are events besides the routine import
machinery that contribute to the nuclear translocation of activated Stat1.
However, it seems unlikely that such events are necessary for anything other
than tyrosine phosphorylation. These data are in agreement with a proposed
"soft-wired" mechanism for cytoplasmic transport of Stat1
(Lillemeier et al. 2001).
Our results indicate that nucleocytoplasmic shuttling of Stat1 is crucial
even during nuclear accumulation. However, shuttling was observed only with
the unphosphorylated molecule. Blocking of tyrosine dephosphorylation by
vanadate induced nuclear accumulation of the retention mutant
Stat1dnaminus (Fig.
3C). The nuclear export of wild-type Stat1 was blocked after
stimulation with IFN in the presence of vanadate, whereas nuclear
export of both a Stat1 variant with a mutated tyrosine phosphorylation site
and an export reporter construct were not affected by this treatment
(Fig. 2C). In addition, we did
not observe nuclear export of phosphorylated Stat1 after its nuclear
microinjection (Fig. 1D,E).
Interestingly, the dephosphorylation of recombinant Stat1 in the nucleus was
slow in comparison to the endogenous protein. This could be caused by the high
concentration of the injected material or by the absence of Arg 31
methylation, which has been shown to hamper Stat1 dephosphorylation
(Zhu et al. 2002). We conclude
that tyrosinephosphorylated dimers were incapable of nucleocytoplasmic
shuttling and required inactivation before release into the cytoplasm.
Nevertheless, the cycling rate was high, as cytoplasmic trapping depleted
nuclear Stat1 in 30 min (Fig.
2C), and blocking of kinase activity led to a collapse of nuclear
accumulation in <60 min (Fig.
2B). This is in good accord with the progression of tyrosine
dephosphorylation (t1/2 < 15 min;
Haspel et al. 1996).
Therefore, Stat1 must continuously shuttle in and out of the nucleus with
constant reactivation at the cell membrane to maintain a steady level of
transcriptionally active dimers in the nucleus. These results explain why
protracted nuclear export of unphosphorylated Stat1 resulted in reduced
transcription (Begitt et al.
2000). Fast dephosphorylation with concomitant nuclear export and
reactivation in the cytoplasm are both essential for dynamic signaling. In
this way, the degree of receptor activity at the cell membrane is immediately
reflected by the level of activated Stat1 in the nucleus. Such shuttling was
also proposed for Stat6 in interleukin-4 signaling
(Andrews et al. 2002).
An essential conceptual addition to the standard model of Stat function is
the distinction between nuclear import and nuclear retention of activated
Stat1. Judged by several criteria, such as the abundance of phosphorylated
Stat1 in nuclear extracts, the antibody-induced nuclear precipitation after
IFN stimulation, and the rapid nuclear accumulation induced by an
export block through preventing tyrosine dephosphorylation
(Fig. 3), the
IFN-induced nuclear import of Stat1dnaminus proceeded
unaltered, but nevertheless the mutant was incapable of nuclear accumulation.
These results indicated that nuclear accumulation was not caused simply by
accelerated nuclear import after cytokine stimulation. Rather, there must
exist a retention mechanism affecting nuclear export. By modulating the
nonspecific affinity of dimeric Stat1 for DNA, we were able to engineer
mutants with increased or abolished nuclear retention. This was exemplified by
the opposed nuclear accumulation abilities of Stat1dnaminus and
dnaplus, neither of which could discriminate GAS elements from
nonspecific sites, although they differed from one another in their overall
DNA affinity. To date, nonspecific DNA binding of Stats has received little
attention. We show that nonspecific interactions with DNA are observable for
wild-type Stat1 (Fig. 3E), and
binding to DNA outside of consensus binding elements is common for
transcriptional regulators (Lin and Riggs
1975). We also point out the fact that GAS sites are generally
poorly conserved, making it difficult to predict actual chromatin binding
sites (Decker et al. 1997),
indicating that the distinction between "specific" and
"nonspecific" binding sites is a gradual one.
Figure 5 demonstrates that
nuclear accumulation of Stat1dnaoff is shortened, despite the fact
that this mutant was activated to a similar extent as wild-type Stat1 and
despite its normal ability to recognize GAS sites
(Fig. 3D). Moreover, we found
that microinjection of phosphorylated Stat1wt into the cytoplasm of
unstimulated cells (Fig. 1C) or
IFN-stimulated cells (data not shown) gave similar results. We
therefore consider it unlikely that the amount of nuclear Stat1 is regulated
by the availability of GAS binding sites. In addition, several DNA-binding
mutants that cannot recognize GAS sites have been reported, but not all of
them have lost the ability to accumulate in the nucleus
(McBride et al. 2000;
Lillemeier et al. 2001). There
are no data available concerning the nonspecific DNA binding of these mutants,
but it could well account for the observed differences.
Nonetheless, because tyrosine-phosphorylated Stat1 was not exported readily
but was retained in the nucleus, DNA binding per se cannot account for nuclear
retention. Rather, the nuclear export was controlled by tyrosine
dephosphorylation. This together with the observation that loss of DNA binding
resulted in loss of nuclear accumulation led to the conclusion that DNA
binding and dephosphorylation were interrelated. And, indeed, DNA-bound Stat1
was protected from phosphatase attack both in vitro and in vivo (Figs.
4A,
5A). Thus, although the
enzymatic reaction was required for nuclear export, it could not be the
causative step for the release of Stat1 from its nuclear DNA tether. In
support of this idea, we found that inactivation of tyrosine phosphatases
reduced intranuclear mobility of Stat1 only marginally
(Fig. 5B;
Table 1). For these reasons,
DNA binding is sufficient but not necessary for nuclear accumulation. Any
process that interferes with the dephosphorylation of Stat1 in the nucleus,
for example, destruction by mutation of the Stat1 interface with the
phosphatase, should cause retention in the nucleus (see also vanadate
treatment in Fig. 3C). A
negative influence of DNA binding on Stat dephosphorylation was noted earlier
also for Stat5. Binding to the glucocorticoid receptor enhanced DNA binding of
Stat5, which correlated with its prolonged tyrosine phosphorylation in vivo
(Wyszomierski et al.
1999).
Central to the understanding of nuclear accumulation was the observation of
sequence-specific inhibitory effects of DNA on Stat1 dephosphorylation. It was
found that activated Stat1 is more efficiently protected by optimal GAS sites
than by nonconsensus oligonucleotides (Fig.
4A). Quantitative analysis of the Stat1–DNA exchange
reaction had revealed identical affinities for various sites, but despite
having similar apparent Kd values, the binding with DNA may differ
significantly in rates of association with and dissociation from the Stat1
protein (Vinkemeier et al.
1996). The Stat1 protein achieves equilibrium in DNA binding very
rapidly (<<30 sec); however, although the protein–DNA complex had a
half-life of no more than 3 min for any of the GAS sites investigated, the
off-times varied at least sixfold. Cooperative DNA binding further stabilized
the Stat1–DNA complex (see also Fig.
4D). Importantly, the data we present here show that conservation
of the active transcription factor was determined by the DNA site to which it
was bound. Therefore, Stat1 dimers bound to favorable sites, for example, the
promoter region of cytokine-regulated genes, are likely to be preserved
comparatively better than dimers occupying nonfunctional sites. As a result,
the intrinsic preference of Stat1 for its target genes would be transformed
into longevity of the transcriptionally active species. This way, the process
of nuclear accumulation might confer a selection advantage on Stat1 dimers
bound to GAS consensus promoter sites. Thus, in contrast to other gene
regulators (Tansey 2001),
control of transcription by Stat1 appears to be governed primarily by chance,
simply by falling off the DNA with concomitant inactivation at a frequency
dictated by the particular binding site.
|
Materials and methods |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Plasmids expressing Stat1 fused to GFP (pStat1–GFP) or
GST-NES–GFP(pGFP-h4C-GST) have been described
(Begitt et al. 2000).
Site-directed mutagenesis of pStat1–GFP(QuikChange, Stratagene)
generated (1) pStat1dnaplus, (2) pStat1dnaminus, (3)
Stat1dnaoff, and (4) pStat1-Y701F, in which (1) the codon for T327
was replaced by R, codons V426 and T427 by HH, (2) V426 and T427 by DD, (3)
K544 and E545 by AA, and (4) codon Y701 by F. Mutations were verified by DNA
sequencing. The coding sequence of full-length human TC45 (residues
2–387) was amplified by PCR from IMAGE clone IMAGp998B124644Q3 (RZPD
Deutsches Ressourcenzentrum) and cloned into the EcoRI and
BamHI sites of expression vector pASK-IBA3 (IBA). Cell culture and
transient transfections with lipofectamine were as described
(Begitt et al. 2000). Cells
were treated with IFN (5 ng/mL; Biomol), staurosporine (500 nM; Sigma),
leptomycin B (10 ng/mL; Sigma), Na-vanadate (0.8 mM), and
H2O2 (0.2 mM) as indicated in the figures.
Pull-down assay with biotinylated oligos
Duplex oligonucleotides (0.5 mL, 28 pmole/µL, with a 5' biotin on
one strand) containing either two optimal GAS sites
(5'-GAGACTCAGTTTCCCGTAAATCGTCCAGTTTCCCGTAA
AGACTATGC and its antisense), or a single GAS-like site
[5'-(TTC)4TAC(TTC)15 and its antisense] were
conjugated to streptavidin agarose (0.5 mL packed vol; Pierce) at 4°C for
1 h. Confluent U3A cells (one 10-cm dish) transiently coexpressing c-Eyk
kinase (Besser et al. 1999) and
a Stat1–GFPvariant were stimulated for 35 min with IFN and 15 min
in the additional presence of vanadate/H2O2 before lysis
in 400 µL of lysis buffer A (Meyer et
al. 2002a). Stat1 tyrosine phosphorylation was assessed by Western
blotting, and the extracts were diluted accordingly with buffer A. After
preclearing with streptavidin agarose, 250 µL of the normalized extracts
were rotated with 50 µL (packed vol) of DNA-conjugated beads at 4°C for
1.5 h. The beads were washed with lysis buffer A (three times with 500 µL),
and bound proteins were eluted by boiling in SDS sample buffer and analyzed by
Western blotting.
Electrophoretic mobility shift assay (EMSA) and analysis of
transcription
EMSA was performed as described with 5 µL of cytoplasmic extracts.
Luciferase reporter gene assays have been described
(Begitt et al. 2000).
Antibody microinjections, Western blotting, and
immunoprecipitation
Western blotting, immunoprecipitation, and antibody injections were done as
described (Meyer et al.
2002a). HeLa-S3 cells and U3A cells were injected with anti-Stat1
antibody C-136 (Santa Cruz) or the monoclonal anti-GFPantibody 2A3 (2.5 mg/mL;
a kind gift of M. Vigneron and C. Kedinger). After the indicated incubation
period, Stat1 was detected immunocytochemically with antibody C-24 (Santa
Cruz).
Purification and microinjection of recombinant Stat1
Baculoviruses expressing Stat1wt, Stat1dnaplus,
Stat1dnaminus, or Stat1Y701F, respectively, were produced with the
Bac-to-Bac system as specified by the manufacturer (GIBCO). Protein expression
in baculovirus-infected Sf9 insect cells, protein purification, and in vitro
tyrosine phosphorylation were done essentially as described
(Vinkemeier et al. 1996) with
the following modifications. Incubation of epidermal growth factor (EGF)
receptor with EGF and N-ethylmaleimide treatment of unphosphorylated Stat1
were omitted. Homogeneous tyrosine phosphorylation of the final preparation
was verified by MALDI analysis, as was the absence of Arg 31 methylation.
Purified protein (1 mg/mL in injection buffer with 2 mM DTT) was microinjected
with identical outcome into U3A cells and HeLa cells as described
(Begitt et al. 2000).
Afterward, the cells were left at 37°C in a humidified incubator for the
indicated times. Fixation with 3.7% formaldehyde (10 min) and permeabilization
with 0.2% Triton X-100 (5 min) were at room temperature, before
immunocytochemistry with anti-Stat1 antibody C-136 (1:1000) or C-24 (1:10000).
Tyrosine-phosphorylated Stat1 was detected with a specific antibody (1:1000;
Upstate) after 10 min of fixation in methanol at –20°C. Endogenous
Stat1 protein in HeLa cells was not detectable immunocytochemically at the
high antibody dilutions used.
Dephosphorylation assays
In vitro dephosphorylation reactions were performed at 30°C (or at
37°C with the full-length TC45) in 20 µL of reaction buffer (25 mM
Tris-HCl at pH 7.5, 0.5 mg/mL BSA, 10 mM DTT, 50 mM KCl, 5 mM EDTA, Complete
protease inhibitors; Roche) with 0.5 nM Stat1 dimers, and 1.5 U of the
truncated version of human TC45 (residues 1–352; Sigma) or 15 U of the
full-length phosphatase. Full-length TC45 was expressed in bacteria
(BL21pLysS) and purified by virtue of a C-terminal Strep-tag as recommended by
the manufacturer (IBA). Duplex oligonucleotides (GAS:
5'-AAGTCGTTTCCCGGAAATAGAA GATTATTATCATTAT-3' and its
antisense; Mut: 5'-AAGTC GAGGTACAGGTAAAGAAGAACCTCGTTGTCAC-3' and
its antisense; 2xGAS: 5'-GTTTCCCCGAAATTGACGGATTTC
CCCGAA AC-3' and its antisense) were added where indicated. The
DNA concentrations are given in the figures. Reactions were stopped after 60
min by boiling in SDS sample buffer and analyzed by Western blotting.
Dephosphorylation of the control substrate p-nitrophenyl phosphate (pNPP) was
not influenced by DNA (25 nM GAS; data not shown) For experimental details,
see Sigma product T 1196 information. The method described there was used to
determine the catalytic activity of full-length TC45. As expected, full-length
and truncated TC45 dephosphorylated pNPP with identical rates
(Hao et al. 1997). In vivo
dephosphorylation assays were performed with U3A cells transiently expressing
the indicated Stat1 variant proteins. Cells growing on 6-cm dishes were
transferred to six-well plates 24 h posttransfection and cultivated for
another 24 h, before stimulation with IFN for 30 min. Subsequently, the
medium was replaced by medium without or with staurosporine, and the
incubation was continued for the indicated times. Washed cells were lysed in
lysis buffer A (without glycerol phosphate) and extracts representing similar
cell numbers were analyzed by Western blotting.
Fluorescence microscopy and FRAP
Conventional fluorescence microscopy was as described
(Begitt et al. 2000).
FRAP(Axelrod et al. 1976) was
done with a LSM510 inverted confocal laser scanning microscope (Carl Zeiss
Jena) with a 100x/1.3 objective and an argon laser (
= 488 nm)
as excitation source. Fluorescence images were scanned as a time series with
250-msec intervals, and the relative fluorescence intensity was measured in a
bleached region of interest (ROI, 10 µm2). Fluorescence
intensity differences (Fu – Fb) between unbleached
ROI (Fu) and adjoining bleached ROI (Fb) were calculated
and used for an exponential curve fitting (prism program) to determine
recovery times. Recovery was considered complete if Fu –
Fb 
0.01 Fu. Cells were treated with IFN for
30 min, at which point incubation was continued without or with
vanadate/H2O2. FRAPwas performed before or 60 min after
IFN addition.
Quantification of immunofluorescence intensities
Images were acquired by confocal microscopy as described
(Meyer et al. 2002b). Stat1
immunofluorescence densities were determined both in the nucleus and in the
cytoplasm of the median slice (x/y image). Correction for
background fluorescence was done. For a number of cells (n), the
ratio of (cytoplasmic fluorescence density)/(nuclear fluorescence density) was
calculated and depicted in a bar diagram. The values are the mean and the
standard deviation. The data were analyzed by Student's t-test
(Fig. 1) or by ANOVA followed
by the Tukey multiple comparison test (Fig.
2). Differences were considered statistically significant at
p < 0.05.
|
Acknowledgments |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
Footnotes |
|---|
3 Corresponding author. E-MAIL
vinkemeier{at}fmp-berlin.de;
FAX 49-30-94793-179. 
|
References |
|---|
|
TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Axelrod, D., Koppel, D.E., Schlessinger, J., Elson, E., and Webb,
W.W. 1976. Mobility measurement by analysis of fluorescence
photobleaching recovery kinetics. Biophys. J.
16:
1055–1069.
Begitt, A., Meyer, T., van Rossum, M., and Vinkemeier, U.
2000. Nucleocytoplasmic translocation of Stat1 is regulated by a
leucine-rich export signal in the coiled-coil domain. Proc. Natl.
Acad. Sci. 97:
10418–10423.
Besser, D., Bromberg, J.F, Darnell Jr., J.E., and Hanafusa, H.
1999. A single amino acid substitution in the v-Eyk intracellular
domain results in activation of Stat3 and enhances cellular transformation.
Mol. Cell. Biol. 19:
1401–1409.
Bild, A.H., Turkson, J., and Jove, R. 2002. Cytoplasmic transport of Stat3 by receptor-mediated endocytosis. EMBO J. 21: 3255–3263.[CrossRef][Medline]
Briscoe, J., Guschin, D., Rogers, N.C., Watling, D., Müller, M., Horn, F., Heinrich, P., Stark, G.R., and Kerr, I.M. 1996. JAKs, STATs and signal transduction in response to the interferons and other cytokines. Philos. Trans. R. Soc. Lond. B Biol. Sci. 351: 167–171.[Medline]
Brivanlou, A.H. and Darnell Jr., J.E. 2002. Signal
transduction and the control of gene expression.
Science 295:
813–818.
Chen, X., Vinkemeier, U., Zhao, Y., Jeruzalmi, D., Darnell Jr., J.E., and Kuriyan, J. 1998. Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell 93: 827–839.[CrossRef][Medline]
Darnell Jr., J.E. 1997. STATs and gene regulation.
Science 277:
1630–1635.
Darnell Jr., J.E., Kerr, I.M., and Stark, G.R. 1994.
Jak–STAT pathways and transcriptional activation in response to IFNs and
other extracellular signaling proteins. Science
264:
1415–1421.
Decker, T., Kovarik, P., and Meinke, A. 1997. GAS elements: A few nucleotides with a major impact on cytokine-induced gene expression. J. Interferon Cytokine Res. 17: 121–134.[Medline]
Fagerlund, R., Melén, K., Kinnunen, L., and Julkunen, I.
2002. Arginine/lysine-rich nuclear localization signals mediate
interactions between dimeric STATs and importin 5. J. Biol.
Chem. 277:
30072–30078.
Gordon, J.A. 1991. Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol. 201: 477–482.[Medline]
Greenlund, A.C., Morales, M.O., Viviano, B.L., Yan, H., Krolewski, J., and Schreiber, R.D. 1995. Stat recruitment by tyrosine-phosphorylated cytokine receptors: An ordered reversible affinity-driven process. Immunity 2: 677–687.[CrossRef][Medline]
Hao, L., Tiganis, T., Tonks, N.K., and Charbonneau, H.
1997. The noncatalytic C-terminal segment of the T cell protein
tyrosine phosphatase regulates activity via an intramolecular mechanism.
J. Biol. Chem. 272:
29322–29329.
Haspel, R.L. and Darnell Jr., J.E. 1999. A nuclear
protein tyrosine phosphatase is required for the inactivation of Stat1.
Proc. Natl. Acad. Sci.
96:
10188–10193.
Haspel, R.L., Salditt-Georgieff, M., and Darnell Jr., J.E. 1996. The rapid inactivation of nuclear tyrosine phosphorylated Stat1 depends upon a protein tyrosine phosphatase. EMBO J. 15: 6262–6268.[Medline]
Herrington, J., Rui, L., Luo, G., Yu-Lee, L.Y., and Carter-Su, C.
1999. A functional DNA binding domain is required for growth
hormone-induced nuclear accumulation of Stat5B. J. Biol.
Chem. 274:
5138–5145.
Horvath, C.M., Wen, Z., and Darnell Jr., J.E. 1995. A
STAT protein domain that determines DNA sequence recognition suggests a novel
DNA-binding domain. Genes & Dev.
9:
984–994.
Ibarra-Sanchez, M.J., Simoncic, P.D., Nestel, F.R., Duplay, P., Lapp, W.S., and Tremblay, M.L. 2000. The T-cell protein tyrosine phosphatase. Semin. Immunol. 12: 379–386.[CrossRef][Medline]
Ihle, J.N., Thierfelder, W., Teglund, S., Stravapodis, D., Wang,
D., Feng, J., and Parganas, E. 1998. Signaling by the cytokine
receptor superfamily. Ann. NY Acad. Sci.
865:
1–9.
Kimura, H. and Cook, P.R. 2001. Kinetics of core
histones in living human cells: Little exchange of H3 and H4 and some rapid
exchange of H2B. J. Cell Biol.
153:
1341–1353.
Kisseleva, T., Bhattacharya, S., Braunstein, J., and Schindler, C.W. 2002. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 285: 1–24.[CrossRef][Medline]
Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E.P., Yoneda, Y., Yanagida, M., Horinouchi, S., and Yoshida, M. 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242: 540–547.[CrossRef][Medline]
Lee, C.K., Bluyssen, H.A., and Levy, D.E. 1997.
Regulation of interferon- responsiveness by the duration of Janus
kinase activity. J. Biol. Chem.
272:
21872–21877.
Lillemeier, B.F., Köster, M., and Kerr, I.M. 2001. STAT1 from the cell membrane to the DNA. EMBO J. 20: 2508–2517.[CrossRef][Medline]
Lin, S. and Riggs, A.D. 1975. The general affinity of lac repressor for E. coli DNA: Implications for gene regulation in procaryotes and eucaryotes. Cell 4: 107–111.[CrossRef][Medline]
McBride, K.M., McDonald, C., and Reich, N.C. 2000. Nuclear export signal located within the DNA-binding domain of the STAT1 transcription factor. EMBO J. 19: 6196–6206.[CrossRef][Medline]
McBride, K.M., Banninger, G., McDonald, C., and Reich, N.C.
2002. Regulated nuclear import of the STAT1 transcription factor
by direct binding of importin-. EMBO J.
21:
1754–1763.[CrossRef][Medline]
McNally, J.G., Müller, W.G., Walker, D., Wolford, R., and
Hager, G.L. 2000. The glucocorticoid receptor: Rapid exchange
with regulatory sites in living cells. Science
287:
1262–1265.
Melén, K., Kinnunen, L., and Julkunen, I. 2001.
Arginine/lysine-rich structural element is involved in interferon-induced
nuclear import of STATs. J. Biol. Chem.
276:
16447–16455.
Meyer, T., Begitt, A., Lödige, I., van Rossum, M., and
Vinkemeier, U. 2002a. Constitutive and IFN--induced
nuclear import of STAT1 proceed through independent pathways. EMBO
J. 21:
344–354.[CrossRef][Medline]
