From the Terry Fox Molecular Oncology Group, Lady
Davis Institute, Jewish General Hospital, Montreal H3T 1E2, Canada, the
¶ Children's Hospital Research Foundation and Department of
Pediatrics, Ohio State University, Columbus, Ohio 43205, the
** Laboratory of Eukaryotic Gene Expression, NICHD, National Institutes
of Health, Bethesda, Maryland 20892, and the
Vienna Biocenter, Institute for
Microbiology and Genetics, Vienna 1030, Austria
Received for publication, December 13, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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We have previously reported a physical
association between STAT1 and the protein kinase double-stranded
RNA-activated protein kinase (PKR). PKR inhibited STAT1 function in a
manner independent of PKR kinase activity. In this report, we have
further characterized the properties of both molecules by mapping the
sites of their interaction. A STAT1 mutant unable to interact with PKR
displays enhanced interferon Cytokines and growth factors exert a diverse range of biological
activities, from host defense, growth regulation, to immunomodulation. Upon ligand binding to cell-surface receptors,
JAK1 kinases are activated
and proceed to phosphorylate the receptor on tyrosine residues, which
then function as docking sites for cytoplasmic transcription factors of
the STAT family (1, 2). STATs are subsequently activated by tyrosine
phosphorylation, dimerize by phosphotyrosyl·SH2 interactions, and
translocate to the nucleus to induce transcription of
cytokine-responsive genes (3). A single tyrosine phosphorylation site
in the carboxyl-terminal activation domain is absolutely essential for
STAT dimerization and DNA binding (3), whereas phosphorylation of a
serine residue in this region is important for transactivational
activity (4).
One of the major STATs intimately involved in both the innate and
acquired immune responses is STAT1. Upon virus infection or exposure to
interferons (IFNs), STAT1 is found in protein complexes that bind
specific DNA sequences upstream to genes responsible for host
resistance. For instance, IFN- We previously described an association between PKR and STAT1 (6). This
interaction takes place in unstimulated cells and diminishes upon
treatment with IFNs or dsRNA. Increased levels of PKR·STAT1
complex have a negative effect on STAT1 DNA-binding and
transactivation capacities. In this report we have mapped the
interaction sites between the two proteins and have identified a novel
function of STAT1. Specifically, we demonstrate that STAT1 functions as
an inhibitor of PKR activation and eIF-2 Cell Culture and Transfections--
HeLa S3, U3A,
STAT1+/+, STAT1 Plasmid Construction--
GST-PKRK296R, GST-PKR 1-262
(PKR N), and GST-PKR K296R 263-551 (PKR C) were generated as
previously described (25). GST-PKRLS4K296R was generated by
site-directed mutagenesis using the QuikChange site-directed
mutagenesis kit (Stratagene) with the following primer pairs
(Sheldon Biotechnology):
5'-d[CCAGAAGGTGAAGGTGGAGCATTGAAGGAAGCAAAAAATGCCGC]-3' and
5'-d[GCGGCATTTTTTGCTTCCTTCAATGCTCCACCTTCACCTTCTGG]-3'.
GST-PKRLS9K296R was generated by subcloning an EcoNI and
AflII fragment of PKRLS9. Truncated GST-PKR proteins were
generated with the following primers: pGEX forward primer (Amersham
Pharmacia Biotech); 5'-PKR 263, 5'-d[GGGGGATCCTAAAACCTCTTGTCCACAGTATAC]-3'; 5'-PKR 325, 5'-d[GGGGGGATCCGGCTGTTGGGATGGATTTGATTAT]-3'; 5'-PKR 367, 5'-d[GGGGGGATCCTTCTGTGATAAAGGGACCTTGGAA]-3'; 3'-PKR 262, 5'-d[GGGGGATCCTAAAACCTCTTGTCCACAGTATAC]-3'; 3'-PKR 324, 5'-d[CCCGGGCTAATTGTAGTGAACAATATTTACATGAT]-3'; 3'-PKR 366, 5'-d[CCCGGGCTATTCCATTTGGATGAAAAGGCACT]-3'; 3'-PKR 415, 5'-d[CCCGGGCTACTTAAGATCTCTATGAATTAATTTTTT]-3'; and pGEX reverse primer (Amersham Pharmacia Biotech). PCR fragments were cut with BamHI and/or SmaI and ligated to pGEX-2T.
Truncated STAT1 proteins were generated by PCR from the template
pGEX-5X-3-HA-STAT1 (26) using the following primers: 5'-HA-STAT1,
5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTATGTCTCAGTGGTACGAACTTCAG]-3'; 5'-HA-STAT1 304, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTCGCACCTTCAGTCTTTTCCAG]-3'; 5'-HA-STAT1 343, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTGTGAAGTTGAGACTGTTGGTGAAA]-3'; 5'-HA-STAT1 365, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTGATAAAGATGTGAATGAGAGAAATAC]-3'; 5'-HA-STAT1 380, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTTTCAACATTTTGGGCACGCACAC]-3'; 5'-HA-STAT1 414, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTAATGCTGGCACCAGAACGAATG]-3'; 5'-HA-STAT1 519, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTCTGAACATGTTGGGAGAGAAGC]-3'; 5'-HA-STAT1 611, 5'-d[GGGGGGATCCACCATGGCATACCCATACGACGTCCCAGATTACGCTGCCATCACATTCACATGGGTG]-3'; 3'-STAT1 303, 5'-d[GGGGCGGCCGCCTAGTCCCATAACACTTGTTTGTTTTT]-3'; 3'-STAT1 342, 5'-d[GGGGCGGCCGCCTAAGTGAACTGGACCCCTGTCTTC]-3'; 3'-STAT1 348, 5'-d[GGGGCGGCCGCCTACAACAGTCTCAACTTCACAGTGAA]-3'; 3'-STAT1 357, 5'-d[GGGGCGGCCGCCTAATTATAATTCAGCTCTTGCAATTTCA]-3'; 3'-STAT1 364, 5'-d[GGGGCGGCCGCCTAAAATAAGACTTTGACTTTCAAATTATAAT]-3'; 3'-STAT1 379, 5'-d[GGGGCGGCCGCCTACTTCCTAAATCCTTTTACTGTATTTC]- 3';
3'-STAT1 413, 5'-d[GGGGCGGCCGCCTATTTCTGTTCTTTCAATTGCAGGTG]-3';
3'-STAT1 518, 5'-d[GGGGCGGCCGCCTACTGGTCCACATTGAGACCTCT]-3'; 3'-STAT1
610, 5'-d[GGGGCGGCCGCCTACCCTTCCCGGGAGCTCTCA]-3'; and the pGEX reverse primer. PCR products were BamHI-NotI-digested,
subcloned into the mammalian expression vector, pcDNA3.1/zeo, and
confirmed by sequencing. Site-directed mutagenesis was carried out
with the following primer pairs: R346A,
5'-d[CAGTTCACTGTGAAGTTGGCACTGTTGGTGAAATTGCAAG]-3' and
5'-[CTTGCAATTTCACCAACAGTGCCAACTTCACAGTGAACTG]-3'; R346A/L347D, 5'-[CAGTTCACTGTGAAGTTGGCAGACTTGGTGAAATTGCAAGAGCTG]-3' and
5'-[CAGCTCTTGCAATTTCACCAAGTCTGCCAACTTCACAGTGAACTG]-3'; and
R346A/L347D/L348D,
5'-[GTTCACTGTGAAGTTGGCAGACGACGTGAAATTGCAAGAGCTG]-3' and
5'-[CAGCTCTTGCAATTTCACGTCGTCTGCCAACTTCACAGTGAAC]-3'. PCR products were ligated to pcDNA3.1/zeo-HA-STAT1 by EcoNI
restriction digest. pBABE-HA- STAT1 Cell Extract Preparation, Immunoprecipitation, and Immunoblot
Analysis--
Cell extract preparation, immunoprecipitation, and
immunoblotting were performed as previously described (6). The
following antibodies were used: STAT1 DNA Binding and Transactivation
Assays--
Electrophoretic mobility shift analysis was performed
using the dsDNA c-Fos c-sis-inducible element (SIE,
5'-GATCGTGCATTTCCCGTAAATCTTGTCTACAATTC-3') according to protocols
previously described (6, 28). The Dual Luciferase system (Promega)
was used to assess the transactivation potential of STAT1. Briefly,
STAT1 Isoelectric Focusing and PKR in Vitro Kinase
Assays--
Isoelectric focusing and immunoblot analysis of yeast
eIF-2 GST Pull-down Assays--
Protein production and extraction were
performed according to previously described protocols (25, 30).
Normalized GST fusion proteins were co-incubated with HeLa whole cell
lysates or [35S]methionine in vitro translated
proteins, washed, subjected to SDS-PAGE, and visualized by fluorography
(25).
Yeast Plasmids, Transformations, Growth Protocols, and Protein
Extractions--
Wild-type and mutants of HA-STAT1 1-413 were
subcloned by restriction digest of BamHI-NotI
sites into a modified form of the yeast expression vector, pEMBL/yex4
(29), containing a NotI site in the multiple cloning site.
Transformation of yeast strains H2544 and J110 and growth analyses were
performed as previously described (31).
The catalytic domain of PKR specifically associates with the
DNA-binding domain of STAT1. To map the PKR·STAT1
interaction we performed a series of binding assays using full-length
GST-PKRK296R mutant (GST-PKR WT could not be overexpressed in bacteria
(32)) or truncations of PKR bearing either the dsRNA-binding (GST-PKR N, amino acids 1-262) or catalytic (GST-PKR C, amino acids 263-551) domain (Fig. 1A). HeLa S3
extracts expressing human HA-STAT1 (IFN-
)-induced transactivation
capacity compared with STAT1. This effect appears to be mediated by the higher capacity of STAT1 mutant to heterodimerize with STAT3. Furthermore, expression of STAT1 mutant in STAT1
/
cells enhances both the antiviral and antiproliferative effects of IFNs
as opposed to STAT1. We also provide evidence that STAT1 functions as
an inhibitor of PKR in vitro and in vivo. That
is, phosphorylation of eIF-2
is enhanced in STAT1
/
than STAT1+/+ cells in vivo, and this
correlates with higher activation capacity of PKR in
STAT1
/
cells. Genetic experiments in yeast demonstrate
the inhibition of PKR activation and eIF-2
phosphorylation by STAT1
but not by STAT1 mutant. These data substantiate our previous findings on the inhibitory effects of PKR on STAT1 and implicate STAT1 in
translational control through the modulation of PKR activation and
eIF-2
phosphorylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
induces formation of the
heterodimeric ISGF3, whereas IFN-
induces binding of homodimeric STAT1 (2, 3). Moreover, dsRNA, an intermediate produced during virus
replication, can also activate STAT1 DNA binding (5, 6). The
non-redundant role of STAT1 in the antiviral response is further
appreciated by findings that stat1 null mice (STAT1
/
) are highly susceptible to microbial infection
(7, 8). IFN signaling leads to the expression of a number of genes, one of which encodes for the dsRNA-dependent protein kinase,
PKR (9, 10). PKR is a serine/threonine protein kinase that displays two
distinct activities: (i) autophosphorylation upon dsRNA binding (9, 10)
and (ii) phosphorylation of the eukaryotic translation initiation
factor eIF-2
(9, 10), a modification resulting in inhibition of
protein synthesis (11). Several studies with cultured cells provide
evidence for antiviral (12, 13), antiproliferative, and tumor
suppressor functions of PKR (9, 10). However, pkr null
(PKR
/
) mice exhibit a modest susceptibility to viral
infection (14-17) and show no signs of tumor formation (14, 17),
suggesting that the lack of PKR can be compensated by other PKR-like
molecules (9, 10). This hypothesis is supported by the recent
identification of the PKR-related genes, PERK/PEK
(18) and the mouse homolog of the yeast eIF-2
kinase, GCN2 (19).
phosphorylation in
vitro and in vivo. A mutant of STAT1, which was unable
to interact with PKR, could not inhibit PKR function in yeast and was
better able to mediate the transcriptional, antiviral, and
antiproliferative responses of IFNs compared with STAT1. Taken
together, these findings not only support our previous observations,
but they also provide strong evidence for tight regulation of PKR and
STAT1 functions by virtue of their interaction. In addition, our data
suggest that STAT1 has a dual role in regulation of gene expression by functioning as a transcriptional factor and possibly as translational regulator through PKR activation and eIF-2
phosphorylation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
, PKR+/+, and
PKR
/
cells were maintained in Dulbecco's modification
of Eagle's medium supplemented with 10% calf serum, 2 mM
L-glutamine, and 100 units/ml penicillin/streptomycin (Life
Technologies). For IFN treatment, cells were incubated with 1000 IU/ml
of recombinant murine IFN-
/
(Lee Biomolecules) or 100 IU/ml of
IFN-
(PharMingen). Double-stranded RNA transfections were performed
as previously described (6). Transient transfections were performed
with LipofectAMINE Plus reagent (Life Technologies). T7 vaccinia virus
transient transfections were carried out using the recombinant vaccinia
virus, vTF7-3, encoding the bacteriophage T7 RNA polymerase (20).
In vivo [35S]methionine experiments were
performed as previously described (21). pBABE, pBABE-HA-STAT1
WT, or
TM STAT1
/
cells were generated as previously described
(22). Cell cycle analyses and CPE assays were performed as described in
previous studies (Refs. 23 and 24, respectively).
WT and TM were generated by
ligation into BamHI restriction sites.
(Santa Cruz Biotechnology); HA
(12CA5, Roche Molecular Biochemicals); STAT2 (Upstate Biotechnology
Inc.); STAT3 (Santa Cruz); PKR; GST (Amersham Pharmacia Biotech);
Myc (9E10, Roche Molecular Biochemicals); eIF-2
;
phosphoserine 51 of eIF-2
(Research Genetics Inc.); FLAG (M2,
Kodak); HA horseradish peroxidase antibody (3F10, Roche Molecular
Biochemicals); phosphotyrosine (4G10/PY20, UBI and Transduction
Laboratories); and phosphoserine 727 of STAT1
(27). Proteins were
visualized by ECL (Amersham Pharmacia Biotech).
/
cells or cells expressing STAT1 WT or TM were
transfected with Renilla luciferase (pRL-TK) and pGL-2XIFP53
GAS luciferase. Twenty-four hours after transfection, cells were
replated and treated with IFN-
for 18 h before harvesting. The
results presented represent quadruplicate experiments where GAS
luciferase activity was normalized to Renilla luciferase activity.
were performed as previously described (29). PKR in
vitro kinase assays were carried as previously described (6).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
were incubated with GST-PKR
fusion proteins, subjected to SDS-PAGE, and immunoblotted with an
anti-HA antibody. As shown in Fig. 1B (top
panel), both full-length GST-PKR (lane 5) and the
carboxyl terminus of PKR (lane 4) interacted with STAT1
whereas binding to the amino terminus was not detectable (lane
3). Furthermore, this interaction is specific for STAT1, because
we could not detect binding of GST-PKR proteins with other STATs
(Fig. 1B).
View larger version (40K):
[in a new window]
Fig. 1.
The kinase domain of PKR
specifically interacts with STAT1 in vitro.
A, GST alone, GST-PKR N, GST-PKR C, and GST-PKR FL were
subjected to SDS-PAGE and visualized by Coomassie Blue staining.
B, GST-PKR fusion proteins were co-incubated with protein
extracts of HeLa S3 cells transfected with HA-STAT1
(top), STAT2 (second panel), STAT3 (third
panel), or myc-STAT5B (bottom panel). Bound proteins
were revealed by immunoblotting with HA (top panel), STAT2
(second panel), STAT3 (third panel), or Myc (bottom
panel) antibodies. C, GST alone, GST-PKRK296R,
GST-PKRK296RLS4, and GST-PKRK296RLS9 were co-incubated with protein
extracts from HeLa S3 cells transfected with HA-STAT1
. After
GST-pull down, proteins were detected by immunoblotting with HA
(top panel) whereas the GST fusion proteins were visualized
by Coomassie Blue staining (bottom panel).
We previously reported that binding of PKR to STAT1 is
independent of RNA but requires an intact RNA-binding domain, because the dsRNA-binding-defective mutant PKRLS4
(Arg58-Ser59-Lys60 to
Gly58-Ala59-Leu60) (33) does
not interact with mouse STAT1 in NIH3T3 cell extracts stably expressing
this PKR mutant (6). Based on this, we proposed that the integrity of
the dsRNA-binding domain of PKR plays a role in PKR·STAT1 interaction
in vivo (6). The observation, however, that the carboxyl
terminus of PKR is required for binding to STAT1 in vitro
(Fig. 1B) prompted us to examine the interaction of STAT1
with GST-PKR bearing either the LS4 or the LS9 mutation (Ala66-Ala68 to
Gly66-Pro68), which also abolishes RNA binding
(33) (Fig. 1C). The RNA-binding-defective mutants were
expressed and purified in the GST-PKRK296R background, because
expression of LS4 and LS9 in the GST-PKR WT background could not be
achieved (data not shown). HeLa S3 extracts containing HA-STAT1 were
incubated with GST-PKRK296R, GST-PKRK296RLS4, or GST-PKRK296RLS9, and
GST-PKR bound proteins were subjected to immunoblotting with anti-HA
antibody. All GST-PKR mutants interacted with STAT1 equally well (Fig.
1C, top panel), suggesting that mutations within
the dsRNA-binding domain do not interfere with PKR binding to STAT1
in vitro.
To map the region on STAT1 that facilitates its interaction with PKR,
we used truncated STAT1 proteins corresponding to its amino-terminal,
DNA-binding, linker, SH2, or transactivation domains (Fig.
2A). The pull-down assays were
performed with GST-PKR C, because this protein binds to STAT1 (Fig.
1A) and is more stable than GST-PKRK296R (data not shown).
We observed that GST-PKR C specifically interacted with the DNA-binding
domain of STAT1 (Fig. 2B, lane 14). Upon truncation of the
STAT1 DNA-binding domain from the carboxyl-terminal end, a critical
junction is reached when binding to PKR is retained (middle
panel, HA-STAT1 1-364, lane 13) and when binding is
abolished (HA-STAT1 1-342, lane 12). Further truncation of
STAT1 from the amino terminus also presented a similar junction between
amino acids 343-365 (Fig. 2C, lower panel,
compare lanes 25 and 26). Moreover, truncation of
the carboxyl terminus of the DNA-binding domain of STAT1 at positions
348 and 357 still retained binding to GST-PKR (Fig. 2D,
lanes 10 and 11), suggesting that the region of
interaction lies between amino acids 343 and 348 (-sheet 3) on the
DNA-binding domain of STAT1 (33, 34).
|
Mutations of STAT1 That Affect Binding to PKR-- To identify amino acids in STAT1 that form contacts with PKR, we next constructed mutations within amino acids 343-348 of HA-STAT1 1-413 that abolished binding to PKR. Alanine-scan mutagenesis of each of the six amino acids did not yield a point mutant of HA-STAT1 1-413 that disrupted interaction with PKR (data not shown). However, a three-amino acid substitution (TM; Arg346-Leu347-Leu348 to Ala346-Asp347-Asp348) within this region disrupted the ability of STAT1 to interact with GST-PKR C (Fig. 2E, lane 6). Interestingly, HA-STAT1 1-413 TM possessed faster mobility on SDS-PAGE gels compared with WT most likely through changes in the overall charge of the molecule.
To further characterize the interaction of full-length HA-STAT1 TM
with PKR, we utilized the human fibrosarcoma, U3A cell line, which
lacks endogenous STAT1 (35). HA-STAT1
WT or TM were co-transfected
with PKRK296R into U3A cells, after which, the protein extracts were
immunoprecipitated against PKR and immunoblotted with HA antibodies. As
seen in Fig. 2F, STAT1
WT associated with both
transfected and endogenous PKR (upper panel, lanes
2 and 5). Conversely, STAT1 TM binding with endogenous
PKR was completely abolished (lane 3) and displayed very
marginal binding to transfected PKR, which was detectable only after
long exposures (lane 6). In contrast to HA-STAT1 1-413 TM,
full-length STAT1
TM did not display any difference in its migration
pattern compared with STAT1
WT. These in vitro findings
were also verified in vivo by the yeast two-hybrid assay
(data not shown). Taken together, it appears that the DNA-binding
domain of STAT1 interacts with PKR in vitro and in
vivo.
DNA Binding and Transcriptional Properties of STAT1 TM--
To
test the ability of STAT1 TM to respond to IFN- treatment, we
performed transient transfection assays in STAT1
/
cells
using STAT1 WT or STAT1 TM and a luciferase reporter construct driven
by two copies of the GAS element from the IFP53 gene (28). As shown in Fig. 3A, we
observed that luciferase expression in cells transfected with STAT1 WT
was induced by IFN-
treatment. However, in cells transfected with
STAT1 TM, we observed, after normalization to Renilla
luciferase, a much higher basal luciferase activity (~5-fold) that
could be slightly induced by IFN-
stimulation.
|
To better characterize STAT1 TM, we infected STAT1/
fibroblasts with retroviruses harboring the puromycin-resistant gene
and HA-STAT1
WT or HA-STAT1
TM. As a control, retroviruses
containing only the puromycin-resistant gene were used. After puromycin
selection, polyclonal populations of STAT1 WT-expressing cells showed
~5-fold greater expression over STAT1 TM pools (Fig. 3B,
compare lanes 2 and 3). Transactivation assays
using the 2XIFP53-GAS luciferase reporter correlated with our findings
in transient transfection experiments that STAT1 TM confers higher
basal reporter activity, which can be induced by IFN-
treatment
(Fig. 3B). We next tested whether STAT1 TM could bind DNA
after IFN treatment. Although we could not detect ISGF3 formation in
STAT1 TM cells in response to IFN-
/
(data not shown), IFN-
stimulation resulted in the formation of DNA-binding complexes
consisting of either STAT1·STAT3 heterodimers or STAT3 homodimers,
but not that of STAT1 homodimers (Fig. 3C, compare
lanes 7-16). This finding is consistent with previous
reports that STAT3 is activated following IFN treatment (3). Moreover,
the intensity of the STAT3 homodimer appears to be higher compared with
control or STAT1 WT cells (compare lanes 2, 4,
and 6).
The ability of STAT1 to be phosphorylated upon IFN stimulation was also
examined (Fig. 3D). To compare STAT1 phosphorylation per
equal amounts of STAT1 protein, we used a 5-fold higher amount of STAT1
TM extracts versus STAT1 WT before and after IFN
stimulation. These reactions were also normalized to total protein
concentration by the addition of treated or untreated
STAT1/
control extracts. Although STAT1 WT was
tyrosine-phosphorylated following IFN-
/
or IFN-
treatment
(top panel, lanes 5 and 6), we failed
to detect STAT1 TM tyrosine phosphorylation (lanes 8 and
9). In contrast, phosphorylation of serine 727 did not
significantly differ between STAT1 WT and STAT1 TM after IFN treatment
(middle panel, lanes 5-6 and 8-9).
Reprobing of the membrane with antibodies to HA revealed the hypo- and
hyperphosphorylated forms of STAT1 usually observed after IFN treatment
(lower panel). Because STAT1 is also known to form
heterodimers with STAT3 following IFN stimulation (3), we tested
whether STAT1 TM could associate with STAT3. A much higher amount of
STAT3 co-precipitated with STAT1 TM before and after IFN treatment
(Fig. 3E, top panel, lanes 7-9),
although STAT1 protein levels were approximately equal (bottom
panel). We also analyzed expression and activation of STAT3 in the
same protein extracts used for the STAT1/STAT3 co-immunoprecipitation. STAT3 phosphorylation was slightly elevated (~50%) in cells
expressing STAT1 TM before or after treatment with either IFN-
/
or IFN-
(Fig. 3F, lanes 7-9). This increase
in STAT3 activity may account for increased STAT3 DNA binding in STAT1
TM cells.
STAT1 TM Enhances the Antiviral and Antiproliferative
Effects of IFNs--
The biological effects of STAT1 TM activation
were examined by cell cycle analysis after treatment with IFN-/
or IFN-
(Fig. 4A). A
greater proportion of STAT1 TM-expressing cells (IFN-
/
, 6-8%;
IFN-
, 10-11%) were arrested in G0/G1
phase after treatment with either type I or type II IFNs (right
panel) compared with control (left panel) or STAT1
WT-expressing (middle panel) cells. In addition, the ability
of STAT1 TM cells to resist virus infection was also investigated.
Control, STAT1 WT, and STAT1 TM cells were primed with IFNs and
subsequently infected with serially diluted VSV. The amount of virus
needed to induce CPE was qualitatively measured. As shown in Fig.
4B (upper panel), STAT1 TM cells that were
treated with IFN-
were ~50-fold more resistant to VSV infection compared with STAT1 WT cells, and ~104-fold more
resistant versus control STAT1
/
cells. In
contrast, IFN-
/
-treated STAT1 TM cells were 10-fold more
susceptible to VSV CPE compared with control, STAT1 WT, and STAT1+/+ cells. Interestingly, even untreated STAT1 TM
cells provided a greater degree of protection compared with STAT1 WT
and STAT1+/+ cells. This enhanced ability of STAT1 TM cells
to resist virus infection was also observed after
encephalomyelocarditis virus infection (data not shown). Western
blotting against STAT1
revealed that STAT1 TM is expressed at much
lower levels than STAT1 WT and endogenous STAT1 from
STAT1+/+ cells (bottom panel). Taken together,
these data suggest that STAT1 TM enhances the antiproliferative and
antiviral effects of IFNs on a per molecule basis.
|
STAT1 Functions as a PKR Inhibitor in Vitro--
To gain better
insight into the PKR·STAT1 interaction, we next mapped the
STAT1-binding site on PKR (Fig.
5A). In agreement with earlier
results, the full-length kinase domain (lane 4), but not the
dsRNA-binding domain (lane 3), bound PKR. Truncation of the
kinase domain from either the amino or carboxyl terminus (Fig.
5A) defined a region critical for STAT1 binding: amino acids 367-415 (lanes 5-9). Interestingly, this corresponds to
the same region in the large lobe of the PKR kinase domain, to which
the vaccinia virus K3L protein binds to block PKR activation (36, 37).
In view of this fact, we tested whether the two proteins could compete
for binding to PKR. Bacterially expressed FLAG-K3L was co-incubated
with GST-PKR C and extracts from HeLa cells expressing increasing
amounts of HA-STAT1. Immunoblotting was performed to detect either
binding of HA-STAT1
or FLAG-K3L to GST-PKR C. As seen in Fig.
5B, STAT1 displaced K3L from PKR in a
dose-dependent manner (bottom panel, lanes
10-13), indicating that K3L and STAT1 bind to the same region on
PKR.
|
The PKR pseudosubstrate, K3L, has been shown to bind PKR and block
access of eIF-2 to the catalytic pocket of PKR (29, 37). To address
the importance of STAT1 binding to PKR, we investigated the ability of
STAT1 to act as a cellular inhibitor of PKR. A series of in
vitro assays were performed to assess the effect of STAT1 on PKR
activation. Human PKR was activated by reovirus dsRNA in
vitro from HeLa S3 cells in the presence of increasing amounts of
recombinant full-length STAT1 (Fig. 5C, middle
panel). We observed that PKR autophosphorylation diminished in a
dose-dependent manner by the addition of STAT1 (top
panel), although PKR protein levels were equal (bottom
panel).
STAT1 Inhibits the Antiproliferative Properties of PKR in
Yeast--
To date, the best approach to test for the translational
function of PKR is in Saccharomyces cerevisiae. It has been
shown that high levels of PKR expression in yeast are toxic due to
inhibition of general translation (38). However, at lower levels of
expression PKR can substitute the function of GCN2 (39), the only
eIF-2 kinase known to exist in S. cerevisiae (40), by
phosphorylating eIF-2
on serine 51 to inhibit protein synthesis.
Through this approach, a number of PKR inhibitors have been identified
and characterized (31).
Given that PKR expression in yeast results in inhibition of cell growth
(38, 39), we wanted to analyze whether STAT1 could block this effect
when co-expressed with PKR. Yeast strain H2544, which lacks the yeast
eIF-2 kinase GCN2, contains a stable integration of human PKR WT
cDNA downstream to a galactose-inducible promoter, whereas the
isogenic strain J110 is identical except that the PKR cDNA was not
inserted. Previous studies have shown that induction of PKR expression
in H2544 results in inhibition of cell growth through phosphorylation
of eIF-2
(39). Conversely, co-expression of a PKR inhibitor, such as
K3L, leads to rescue of PKR-mediated growth inhibition (29). To test
the inhibitory activity of STAT1 on PKR, strains J110 and H2544 were
transformed with vector alone, K3L, HA-STAT1 1-413 WT, or HA-STAT1
1-413 TM. Transformants were streaked onto minimal media plates
containing either glucose or galactose as a carbon source, and the
effect of each of these proteins on PKR-mediated growth inhibition was
monitored. All transformants of the isogenic J110 strain grew well in
either glucose or galactose indicating that expression of these
exogenous proteins did not perturb normal yeast growth characteristics
(Fig. 6A, upper
plate, and Fig. 6B, upper graph). H2544
transformants containing only empty plasmid DNA demonstrated a
slow-growth phenotype after PKR induction (Fig. 6A,
lower plate, labeled C). However, expression of
K3L reversed this growth inhibitory phenotype (lower plate,
labeled K3L). Likewise, expression of STAT1 1-413 WT also rescued yeast growth (lower plate, labeled WT),
although in contrast, the interaction mutant of STAT1 was unable to
counteract the growth inhibitory effects of PKR (lower
plate, labeled TM). Growth curves to assess the degree
of rescue showed that the ability of HA-STAT1 1-413 WT transformants
to rescue growth was half as potent relative to K3L (Fig.
6B, lower graph). Because it was not possible to quantify the relative levels of K3L and STAT1 1-413 WT expression, the
degree of rescue may be dependent on their different levels of
expression. Efforts to rescue growth by full-length HA-STAT1
were
unsuccessful, because we could not detect HA-STAT1
expression in
yeast (data not shown). However, the truncated HA-STAT1 1-413 proteins
were readily detectable in yeast protein extracts (Fig. 6C)
as was the expression of human PKR in H2544 transformants (Fig.
6D, lanes 5-8). In correlation with the growth
curves, the extent of eIF-2
phosphorylation, as assessed by
isoelectric focusing experiments (29), was diminished in H2544
transformants expressing either K3L or HA-STAT1 1-413 WT (Fig.
6E, lanes 3 and 4). The induction of
PKR levels in Fig. 6D, lane 6, was probably
translational in nature as a result of inhibition of eIF-2
phosphorylation and up-regulation of PKR protein synthesis by K3L (Fig.
6E, lane 3). This PKR up-regulation was not
evident in lane 7 most likely due to the weaker inhibitory
effect of STAT1 1-413 WT on eIF-2
phosphorylation compared with K3L
(Fig. 6E, compare lanes 3 and 4).
|
Increased PKR Activation and eIF-2 Phosphorylation in
STAT1
/
Cells--
Next we investigated
whether the loss of STAT1 would augment PKR activity. To do so, protein
extracts from untreated or IFN-treated STAT1+/+ and
STAT1
/
cells were used to assess PKR activity in
vitro. We noticed that the basal activity of PKR was ~5-fold
higher in STAT1
/
cells compared with
STAT1+/+ cells (Fig.
7A, upper panel,
compare lanes 1 and 3), although PKR protein
levels were equivalent, as assessed by in vivo
[35S]methionine labeling (lower panel, compare
lanes 1 and 2). The increase in PKR activity
after IFN treatment in STAT1+/+ cells (top
panel) reflects increased PKR expression, whereas no such increase
in PKR activity/protein was observed in STAT1
/
cells.
This is consistent with the notion that transcriptional up-regulation
of PKR after IFN treatment is dependent on the JAK/STAT pathway
(upper panel, compare lanes 2 and 4).
To further substantiate our findings that PKR activity is elevated in
STAT1
/
cells, we compared the levels of eIF-2
phosphorylation in STAT1+/+ and STAT1
/
cells in vivo. The effect of STAT1 on eIF-2
phosphorylation in STAT1+/+ and STAT1
/
cells was examined by treatment with dsRNA and immunoblotting with a
phosphospecific antibody to phosphoserine 51 of eIF-2
(Fig.
7B, top panel). These experiments showed that a
higher amount of eIF-2
was phosphorylated in STAT1
/
cells compared with STAT1+/+ cells (top panel,
compare lanes 1 and 3) and that this
phosphorylation was more highly induced after dsRNA treatment (compare
lanes 2 and 4). Taken together, the inhibition of
PKR activity by STAT1 in mammalian and yeast cells supports our
findings that STAT1 can inhibit PKR activity in vitro
and in vivo.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have mapped the sites of interaction between PKR and STAT1
in vitro using GST pull-down assays and also in
vivo. We found that STAT1 interacts with PKR on amino acids
367-415, an area located within the large lobe of the kinase domain of
PKR that is also bound by the vaccinia virus encoded PKR inhibitor, K3L (36, 37). Sequence comparison with other eIF-2 family members, GCN2,
HRI, and the newly discovered PERK/PEK revealed
that this part of the kinase domain is highly conserved and suggests
that these members might be capable of interacting with STAT1. Our previous observations that the dsRNA-binding mutant PKRLS4 was unable
to associate with STAT1 in vivo (6), at first, appear to be
challenged by our in vitro studies herein, which show that the carboxyl terminus of PKR is required for binding to STAT1 and that
GST-PKRK296RLS4 can interact with STAT1 (Fig. 1). One possible
explanation for this difference may be inferred, based on structural
data of the amino terminus of PKR (41) and also on previous mutational
studies (33), that the LS4 and LS9 mutations could introduce
conformational changes that would affect its interaction with STAT1
in vivo. Such conformational changes may not take place when
GST-PKRK296RLS4 and GST-PKRK296RLS9 are purified from bacteria. Another
conceivable explanation is that the association of PKR with STAT1
in vivo is facilitated by the presence of other protein(s) whose action is modulated by the amino terminus of PKR and may become
limited when the interaction is tested in vitro.
In the case of STAT1, amino acids 343-348 provide a major site of
interaction with PKR. This region of STAT1 corresponds to -sheet 3, a structure located on the back side of the DNA-binding domain of STAT1
(33, 34). Mutation of three amino acids within this region (STAT1 TM;
Arg346-Leu347-Leu348 to
Ala346-Asp347-Asp348) abolished
binding to PKR in vitro and in vivo.
Paradoxically, sequence alignment of this three-residue stretch of
wild-type STAT1 with other STAT members showed almost complete
conservation. Similarly, residues in
-sheet 3 of the DNA-binding
domain of STAT3 responsible for specific interaction with c-Jun are
also nearly identical between STAT members; however, c-Jun specifically interacts with STAT3 (42). Thus, our observation that PKR specifically interacts with STAT1 can probably only be explained in the context of
the presentation of
-sheet 3 in STAT1 compared with other STAT
molecules. In the end, structural analysis might be necessary to
determine the exact points of contact between PKR and STAT1.
In light of our previous observations that the ability of PKR to
interact with STAT1 can block STAT1 DNA binding (6), we examined
whether release of STAT1 from this inhibitory mechanism might augment
its transcriptional activity. In contrast to STAT1 WT, we were unable
to observe tyrosine phosphorylation of STAT1 TM following IFN
stimulation, and as a result, ISGF3 and STAT1 homodimer formation could
not be detected in DNA binding experiments. We believe that the
inability of STAT1 TM to be phosphorylated cannot be the result of a
misfolded protein, because this mutant is phosphorylated on serine 727 after IFN treatment. Rather, it appears that some undefined negative
regulatory mechanism is responsible for this phenomenon. The protein
kinase that directly phosphorylates serine 727 of STAT1 in
vivo is not as yet known (4). It is unlikely to be PKR because
STAT1 is not phosphorylated by PKR in vitro (6).
Interestingly, a recent report shows a defective serine 727 phosphorylation of STAT1 in PKR/
fibroblasts after IFN
stimulation providing evidence for an indirect role of the kinase in
this process (43). Although unable to bind DNA as a homodimer, STAT1 TM
was competent and more capable in forming DNA-binding complexes with
STAT3 relative to STAT1 WT cells. Interestingly, tyrosine
phosphorylation of STAT3 was elevated by 50% before and after IFN
treatment in polyclonal cell populations expressing STAT1 TM.
The mechanism behind this finding is not clear at this time,
but it might be possible that recruitment of STAT3 to the
JAK·receptor complex is more efficient in STAT1 TM cells compared
with STAT1 WT cells. Nevertheless, the net effect of increased
STAT1·STAT3 heterodimer and STAT3 homodimer formation probably
contributes to up-regulation of STAT1·STAT3 DNA-binding and
GAS-dependent transactivation. Because STAT3 homodimers
appear to play a minimal role in the transactivation of
IFN-dependent genes by IFN-
(44), it is likely that the
transcriptional activity observed in our reporter assays is
contributed by the STAT1·STAT3 heterodimer. This is the second
instance where a transcriptional role of STAT1 has been shown to
require serine 727 but not tyrosine 701 phosphorylation. An earlier
study demonstrated that mutation of serine 727 to alanine, but not
tyrosine 701 to phenylalanine, on STAT1 significantly ablated the
TNF-
-dependent induction of caspase genes (45). It was
speculated that STAT1 could potentially participate in signaling
pathways independent of its tyrosine phosphorylation state (46); such a
hypothesis is supported by our findings in here.
The antiproliferative and antiviral effects of IFNs were also
investigated, because a number of cell cycle regulatory proteins are
regulated at the transcriptional level through the JAK/STAT signaling
pathway (44). IFN-dependent cell cycle arrest, as measured
by FACS analysis, was significantly higher in cells expressing STAT1
TM versus control cells or cells expressing STAT1
WT. In
addition, the ability of STAT1 TM cells to resist VSV infection was
~10-fold greater than control or STAT1 WT cells following IFN-
stimulation. These differences become more significant when the
variation in the expression levels between STAT1 WT and TM is
considered (i.e. 5-fold higher expression of WT than STAT1 TM; Fig. 3B, lanes 2 and 3; Fig.
4B, bottom panel). Interestingly, even untreated
STAT1 TM cells provided greater protection relative to control and
STAT1 WT cells, suggesting that the anti-viral responses available
within STAT1 TM cells are already enhanced prior to IFN pretreatment.
Given the fact that STAT1 and K3L, a pseudosubstrate inhibitor of PKR,
can compete for binding with PKR, we next analyzed whether STAT1 could
inhibit PKR activation. We observed that recombinant STAT1 inhibited
PKR activation in vitro in a dose-dependent
manner. However, it is unlikely that STAT1 is a substrate or
pseudosubstrate for PKR, because sequence alignment of STAT1 with K3L
and eIF-2 did not reveal any significant similarities, findings that
coincide with our earlier observations that PKR does not phosphorylate STAT1 in vitro or in vivo (6). Instead, the
interaction of the two proteins might prevent opening of the cleft that
separates the two lobes of PKR's kinase domain, thus inhibiting both
PKR activation and eIF-2
phosphorylation.
The capacity of STAT1 to neutralize PKR activity is further appreciated
from in vivo studies. PKR exhibits the same substrate specificity as the yeast GCN2 kinase that regulates protein synthesis by phosphorylating eIF-2 to inhibit cell growth and replacement of
GCN2 with PKR leads to inhibition of translation and yeast growth (31).
As such, this system has been consistently used as a means to probe for
PKR activation, as well as for characterizing inhibitors of PKR, like
K3L (29). The observation that HA-STAT1 1-413 WT, but not the
interaction mutant, TM, was able to block PKR activity and subsequently
relieve the growth inhibitory effects of PKR, supports our model
whereby PKR activity is tightly regulated by its interaction with
STAT1. These results from yeast were further substantiated in
STAT1
/
cells where we observed augmentation of both PKR
activity and in vivo eIF-2
phosphorylation in comparison
to wild-type cells.
We rationalize that the interaction of PKR and STAT1 modulates cellular
proliferation in normally growing cells and in cells challenged by
viruses. In unstimulated cells, the balance of PKR·STAT1 heterodimer
formation versus their free monomers may dictate cellular proliferative capacity. For example, perturbing this equilibrium by the
expression of catalytic mutants of PKR would sequester a larger
fraction of STAT1 (6) and could contribute to increased virus
susceptibility and to the transformed phenotype of cells expressing
such mutants of PKR (9, 10). Conversely, loss of PKR's repressive
activity in PKR/
mice would favor the antiviral and
antiproliferative functions of STAT1. As such, PKR
/
mice are still capable of providing host resistance to many viruses (14, 17). It would thus appear that STAT1 provides a first line of
defense against virus infection, whereas the role of PKR in viral
resistance appears to be secondary. This is illustrated by the
observation that STAT1
/
mice are exquisitely sensitive
to a variety of pathogens (7, 8), whereas PKR
/
mice exhibit a modest sensitivity (14, 17). Increased eIF-2
phosphorylation in STAT1
/
cells could provide a
compensatory mechanism for the loss of STAT1 and may play a role in
differences in the susceptibility of STAT1
/
mice to
virus infection (8, 24). Therefore, eIF-2
phosphorylation may play a
role in the differential sensitivity of various viruses to interferon
treatment, an effect that could be mediated through the interaction of
STAT1 with PKR and other eIF-2
kinases, because they share high
sequence homology in the STAT1 binding region. Generation of mice
lacking multiple members of this family of kinases2 may be useful in
determining such virus-specific responses.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank T. Pannunzio and N. Tam for
assistance in some of the experiments; C. Schindler for STAT1 and
STAT2 cDNAs; M. Mathews for PKRLS4 and PKRLS9 cDNAs; A. Darveau, G. N. Barber, and J. C. Bell for PKR antibodies; M. Clemens for eIF-2
antibodies; C. Weissmann for PKR+/+
and PKR
/
cells; G. Stark for U3A cells; Y. Yoneda for
the pGEX-5X-3-HA-STAT1
; R. Schreiber for purified STAT1
protein;
N. Sonenberg for pGEX2TK-FLAG-K3L; J. E. Darnell, Jr. for pRC/CMV
STAT3; A. Veillette for pBABE/puro; N. Beauchemin for the
2
retroviral packaging cell line; and K. McDonald for assistance on flow
cytometry. We thank J. J. M. Bergeron and members of our
laboratory and of the Molecular Oncology Group for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by research grants from the Canadian Institutes of Health Research and the Human Frontier Science Program (to A. E. K.).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.
§ Recipient of a National Cancer Institute of Canada, Terry Fox studentship. Present address: The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016.
Recipient of a post-doctoral award from the Cancer Research
Society of Canada.
§§ A member of the Terry Fox Group in Molecular Oncology and recipient of a Canadian Institutes of Health Research Scientist Award. To whom correspondence should be addressed: Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Cote-Ste-Catherine Rd., Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260 (ext. 3697); Fax: 514-340-7576; E-mail: akoromil@ ldi.jgh.mcgill.ca.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011240200
2 J. C. Bell and D. Ron, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
JAK, Janus kinases;
STAT, signal transducer and activator of transcription;
SH2, Src
homology 2;
IFN, interferon;
dsRNA, double-stranded RNA;
PKR, interferon-induced dsRNA-activated protein kinase;
HRI, heme-regulated
inhibitor kinase;
PERK, PKR-like endoplasmic reticulum
kinase;
PEK, pancreatic eIF-2 kinase;
GCN2, general
control non-derepressible-2;
ISGF3, interferon-stimulated gene factor
3;
HA, hemagglutinin;
GST, glutathione S-transferase;
GAS,
-interferon-activating sequence;
VSV, vesicular stomatitis
virus;
CPE, cytopathic effect;
PAGE, polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
TM, triple-mutant.
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