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INTRODUCTION |
Signal transducers and activators of transcription
(STATs)1 are latent
transcription factors, which function as signal transducers in the
cytoplasm and activators of transcription in the nucleus. Presently,
seven mammalian STAT proteins, STAT1, STAT2, STAT3, STAT4, STAT5a,
STAT5b, and STAT6 have been described. Binding of cytokines to their
specific cell surface receptors leads to the activation of Janus
tyrosine kinase (JAK)/STAT pathway (1). In response to type I IFNs
(IFN-
, -
, and -
), IFN-
/
receptor-associated JAK1 and
TYK2 are phosphorylated and activated (2, 3). JAKs in turn tyrosine
phosphorylate STAT1 and STAT2 at Tyr-701 and Tyr-690,
respectively. Phosphorylation triggers STAT1 and STAT2 to dimerize,
translocate into the nucleus, and, together with p48 protein, bind to
well-conserved DNA sequences in the promoter regions of
IFN-
/
-responsive target genes and activate gene transcription (4-8). Binding of type II IFN (IFN-
) to its receptor leads to activation of JAK1 and JAK2 and tyrosine phosphorylation of STAT1 (also
at Tyr-701). STAT1 homodimerizes, translocates into the nucleus, and
activates IFN-
-inducible gene expression (1, 9).
Both in cytokine-induced and uninduced cells the majority of STAT
protein seem to be associated with high molecular mass complexes, and
the amount of free STAT monomers is very small (10, 11). STAT1 has the
ability to form heterocomplexes with several other proteins and is
capable of being imported into the nucleus also in its unphosphorylated
form (12). Tyrosine phosphorylation triggered dimerization is crucial
in IFN-induced nuclear import of STATs (13, 14).
Active nuclear transport of large macromolecules occurs via
the nuclear pore complex (15). Proteins to be imported into the nucleus
contain a nuclear localization signal (NLS), which interacts with a
specific NLS receptor, usually importin-
(16). Importin
binds to
importin-
, which docks the NLS-containing cargo·importin-
/
complex at the cytoplasmic side of the nuclear pore (17, 18).
Importin
interacts with Ran GTPase and p10/NTF2, and the complex is
translocated into the nucleus in an energy-dependent manner
(15, 19, 20). Importin-
recognizes the classic mono- and bipartite
basic-type NLSs (16, 21). The classic monopartite NLS consists of one
and the bipartite NLS of two arginine/lysine-rich clusters of basic
amino acids separated by a spacer region ranging from the usual 10 amino acids up to 37 residues (22, 23). Although IFN-
-induced
nuclear import of STAT1 has been shown to be mediated by at least one
importin-
molecule, NPI-1 (24), and to be dependent on RanGTPase
(25), the molecular mechanisms or elements in STATs responsible for the
nuclear import have remained unresolved.
In the present work we show that STAT1 and STAT2 have a structural
arginine/lysine-rich element involved in IFN-induced nuclear import.
The structural element situates in the DNA-binding domain of the
molecule, and two of these elements, one in each monomer, are required
for nuclear import, because nuclear import-defective mutant STAT1
proteins inhibit the nuclear accumulation of STAT2 and vice
versa.
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MATERIALS AND METHODS |
Cells--
Human hepatocellular carcinoma HuH7 (26) cells were
maintained in minimal essential medium, supplemented with penicillin (0.6 mg/ml), streptomycin (60 mg/ml), glutamine (2 mM),
HEPES buffer, pH 7.4 (20 mM), and 10% fetal calf serum
(Integro, Zaandam, Netherlands). Before stimulation with IFNs, the
cells were cultured in the growth medium supplemented with 2% fetal
calf serum for 24 h. Monolayers and suspension cultures of
Spodoptera frugiperda Sf9 cells were maintained in
TNM-FH medium as described previously (27).
Interferons--
Human leukocyte IFN-
(6 × 106 IU/ml) was kindly provided by Dr. Kari Cantell at our
Institute (28). Human IFN-
(1 × 106 IU/ml) was
obtained from the Finnish Red Cross Blood Transfusion Service and was
prepared and purified as described (29).
Antibodies--
ANTI-FLAG M5 Abs (Sigma Chemical Co., St. Louis,
MO) were used in indirect immunofluorescence microscopy and in
immunoprecipitation (1:1000 dilution). Mouse anti-human STAT1 (1:50
dilution, Transduction Laboratories, Lexington, KY) and rabbit
anti-human STAT1 (STAT1 p91 c-24), STAT2 (STAT2 p113 c-20), and p48
(ISGF-3
p48 c-20) were obtained commercially (1:50-1:200 dilutions
in immunofluorescence microscopy, Santa Cruz Biotechnology, Santa Cruz,
CA). FITC- and TRITC-labeled goat anti-mouse and anti-rabbit
immunoglobulins were used as secondary Abs (1:200 dilution, Cappel,
Organon Teknika Co., West Chester, PA). In Western blotting secondary
horseradish peroxidase-conjugated goat anti-mouse immunoglobulins
(1:2000 dilution; Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA) were used. For the detection of tyrosine phosphorylation of STAT proteins, immunoprecipitated or gel-filtrated samples were stained
with rabbit or mouse anti-phosphotyrosine Abs (1:500 dilution, Transduction Laboratories), followed by biotin-SP-conjugated goat anti-rabbit or anti-mouse (1:10,000 dilution; Jackson ImmunoResearch Laboratories) and horseradish peroxidase-conjugated streptavidin (1:2000 dilution; Jackson ImmunoResearch Laboratories).
Plasmids and DNA Manipulations--
Polymerase chain reaction
was used to modify STAT1, STAT2, and
p48 genes. The noncoding sequences were removed by
inserting unique BamHI or BclI restriction sites
immediately upstream of the first ATG codon and downstream of the STOP
codon of STAT1, STAT2, and p48 genes,
respectively. The primers used for STAT1 were GCA CAA (GGA
TCC) GCC ATG TCT CAG TGG TAC GAA CTT CAG-(sense) and AAA
AAT T(GG ATC C) CT ATA CTG TGT TCA TCA TAC TGT
C-(antisense); for STAT2 the primers were CTA ATC (TGA TCA)
GCC ATG GCG CAG TGG GAA ATG CTG CAG-(sense) and GAA ATG
(TGA TCA) CTA GAA GTC AGA AGG CAT CAA GGG-(antisense); and
for p48 the primers were GGA CAG GAT CCC GCC ATG
GCA TCA GGC AGG GCA CGC-(sense) and TGG GTC GGA TCC TCA TTA
CAC CAG GGA CAG AAT GGC TGC-(antisense) (BamHI sites for
STAT1 and p48 and BclI sites for
STAT2 in parentheses, initiation and STOP codons
underlined). The polymerase chain reaction products were
digested with BamHI or BclI, isolated from
agarose gel, and subcloned into the BamHI site of a modified
transient FLAG-tagged pCDNA 3.1(+) expression vector (Invitrogen,
Carlsbad, CA) or HA-tagged pBC12/CMV expression vector (30). To
construct a FLAG-tagged transient expression vector, oligonucleotides
with a new BamHI cloning site with
BclI-compatible ends (sense oligonucleotide, 5'-CTA GCA CCA
TGG ACT ACA AGG ACG ACG ATG ACA AGG GAT CCC; and antisense,
5'-TCG AGG GAT CCC TTG TCA TCG TCG TCC TTG TAG TCC ATG GTG; the
initiation codon is underlined) were synthesized. The
oligonucleotides were annealed, and the resulting double-stranded DNA
fragment was subcloned into the BamHI site of the pCDNA
3.1(+), to create a vector pCDNA 3.1(+)-FLAG-tag. All DNA
manipulations were performed according to standard protocols, and the
newly created gene constructs were partially sequenced.
Point mutations to the arginine/lysine-rich elements of STAT1 and STAT2
(FLAG-tagged STAT1 and STAT2) in the modified pCDNA 3.1(+)-FLAG-tag
expression vector were constructed using a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). Primers used were 5'-CAA
CTC AGT CCT GAT AGC TCC AGT TCC TTT AGG (Y701A), 5'-GCC CAA AAT GTT GAA
GGC CGC AAA TCC TTT TAC TG (R378A,K379A), 5'-GTG CCA GCA TTT GCC TGT
TCT GCC AAT TGC AGG TGC CG (K410A,K413A), and 5'-CCT CAT TCG TTG CGG
TGC CAG CAT TGG CCT GTT CTT TC (K413A,R418A) for STAT1 and
5'-GTC AGA ATG TTG AAC GCC GCG AAG CCT TGT AAT TG (R374A,K375A) and
5'-CTT ATT GCT GCC CGC TCC TGA ACC ACC TGA AGC TTG CTC CAC C
(R409A,K415A) for STAT2. The newly created mutations were
verified by sequence analysis. wtSTAT1, STAT1 Y701A, STAT1 K410A,K413A,
wtSTAT2, and wtp48 were also subcloned into the BamHI site
of the baculovirus expression vector pAcYM1 (27). Baculovirus expression vectors of wt murine JAK2 (31) and kinase-negative JAK2
L882G (knJAK2) (32) were kindly provided by Dr. Olli Silvennoinen (University of Tampere, Finland).
Transfections--
HuH7 cells were transfected with HA-STAT1-
and HA-STAT2-pBC12/CMV or FLAG-STAT1- and FLAG-STAT2-pCDNA 3.1(+)
gene constructs, using FuGENE6 transfection reagent (Roche Molecular
Biochemicals, Mannheim, Germany).
Immunoprecipitation, SDS-PAGE, and Western
Blotting--
Transfected HuH7 cells were left untreated or were
treated with IFN-
(1000 IU/ml, 30 min). The cells were collected,
washed with phosphate-buffered saline, and lysed in immunoprecipitation binding buffer (IP buffer) (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) on
ice for 30 min. The cell lysates were cleared by centrifugation. The
samples of cleared cell lysates were immunoprecipitated with monoclonal
anti-FLAG Abs bound to protein A-Sepharose CL-4B (Amersham Pharmacia
Biotech, Uppsala, Sweden) at 4 °C for 2 h. Immunoprecipitates
were washed three times, and Laemmli sample buffer was added (33).
SDS-PAGE was performed on 6-12% polyacrylamide gels. Proteins
separated on gels were transferred onto Immobilon-P membranes
(polyvinylidene difluoride, Millipore, Bedford, MA) and visualized with
the enhanced chemiluminescence system (ECL) (Amersham Pharmacia
Biotech, Buckinghamshire, UK) as recommended.
Electrophoretic Mobility Shift Assay (EMSA)--
Monolayers of
Sf9 cells were infected with STAT1, STAT1 Y701A, STAT1
K410A,K413A, STAT2, p48, wtJAK2, or knJAK2 recombinant baculoviruses.
28 or 38 h after infection, cells were disrupted in lysis buffer
(10 mM HEPES-KOH, pH 7.9, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol,
and 100 µM Na3VO4). Lysates were
cleared of insoluble material by centrifugation and used for
electrophoretic mobility shift assay using ISRE15 and GAS consensus
sequence oligonucleotides as described (34).
Gel Filtration--
Lysates (above) of baculovirus-infected
Sf9 cells were gel-filtrated in the above lysis buffer using a
24-ml Superose 12 fast protein liquid chromatography (Amersham
Pharmacia Biotech) gel filtration column. Molecular weight standards
were obtained from Sigma.
Indirect Immunofluorescence and Confocal Laser
Microscopy--
For indirect immunofluorescence and confocal laser
microscopy, transiently transfected HuH7 cells were grown on glass
coverslips, either treated with IFN-
(1000 IU/ml) for 45 min or left
untreated and performed as described previously (35). The cells
positive for FLAG tag were visualized and photographed on a Zeiss
Axiophot photomicroscope or a Leica TCS NT confocal microscope. In some experiments, to better visualize STAT and p48 proteins in
immunofluorescence, the cells were primed with IFN-
(10 IU/ml for
24 h), which is known to up-regulate their expression (34,
36).
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RESULTS |
Kinetics of IFN-induced Nuclear Import of STAT Proteins--
The
transport kinetics of endogenous STAT1 and STAT2 was analyzed by
treating HuH7 cells with 1000 IU/ml of IFN-
. As detected by confocal
laser microscopy (Fig. 1), nuclear import
of both STAT1 and STAT2 could be seen starting at 10 min after IFN-
treatment. The nuclear accumulation was at its maximum within 30-60
min, and the cytoplasmic recycling was completed within 3 h. The
kinetics of nuclear transport of STAT1 and STAT2 was dependent on IFN
dose, because low doses of IFN-
(1-10 IU/ml) resulted to reduced
transport kinetics (not shown). As visualized by confocal laser
microscopy (Fig. 2, a and
b), in untreated HuH7 cells STAT1 was located evenly in the
cytoplasm and nucleus, whereas STAT2 was predominantly cytoplasmic and
no colocalization with STAT1 could be seen in the cell nucleus. 30 min
after IFN-
treatment both STAT1 and STAT2 translocated effectively
to the cell nucleus and showed marked nuclear
colocalization (Fig. 2, c and d). To study the intracellular location of p48 protein, we carried out double-staining experiments in HuH7 cells (Fig. 3). p48
protein was predominantly nuclear already in untreated HuH7 cells, and
no detectable change in intracellular distribution was seen after
IFN-
treatment. STAT1, instead, was effectively imported into the
nucleus and showed marked colocalization with p48 protein 30 min after
IFN-
treatment (Fig. 3).

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Fig. 1.
Kinetics of nuclear transport of endogenous
STAT1 and STAT2 proteins in IFN- -treated HuH7
cells detected by confocal microscopy. For good visualizing
of STAT1 and STAT2, HuH7 cells were first primed with IFN- (10 IU/ml) for 24 h. The cells were left untreated or were treated
with 1000 IU/ml of IFN- for 10 to 180 min, fixed, and double stained
with anti-STAT1 and anti-STAT2 Abs. Stimulations and stainings were as
indicated in the figure. Colocalization of both proteins is shown in
yellow. Bar, 10 µm.
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Fig. 2.
Confocal images of indirect
immunofluorescence staining for STAT1 and STAT2 proteins in untreated
and IFN- -treated HuH7 cells.
a, HuH7 cells were left untreated or treated (c)
with 1000 IU/ml IFN- for 30 min and double stained with monoclonal
anti-STAT1 Abs, followed with TRITC-labeled anti-mouse Abs
(red) and polyclonal anti-STAT2 Abs, followed with
FITC-labeled anti-rabbit Abs (green). a and
c, focus was adjusted through the center of the nucleus.
b, staining profiles of STAT1 and STAT2 in untreated HuH7
cells (white line in a). d, staining
profiles of STAT1 and STAT2 in IFN- -treated HuH7 cells (white
line in c). Nuclear areas are indicated.
Bar, 10 µm.
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Fig. 3.
Colocalization of STAT1 and p48 proteins in
HuH7 cells detected by confocal microscopy using indirect
immunofluorescence staining. The cells were first primed with 10 IU/ml of IFN- for 24 h, then treated with 1000 IU/ml of IFN-
for 45 min, or left untreated and double-stained with anti-STAT1 and
anti-p48 Abs. Bar, 10 µm.
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STAT1 and STAT2 Contain an Arginine/Lysine-rich Structural Element
which Regulates Their Nuclear Import--
Because IFN-induced nuclear
import of STAT1 has been suggested to be mediated by NPI-1 (24), we
reasoned that STATs are likely to contain an arginine/lysine-rich
element that would be involved in their nuclear import. In most
DNA-binding proteins the NLS overlaps or is immediately adjacent to the
DNA-binding domain (37). Although STATs do not contain a classic NLS,
computer analysis of the STAT1 structure (38) revealed a cluster of
basic amino acids in the DNA-binding domain of the molecule within the DNA-binding domain (Fig. 4, a
and c). This element includes amino acids Arg-378, Lys-379,
Lys-410, Lys-413, and Arg-418. All of these amino acids are
conserved in STAT1, STAT3, and STAT4 proteins and are mostly conserved
in STAT2 (human and pig), which is lacking the lysine corresponding to
K410 of STAT1. STAT5a, STAT5b, and STAT6 are more distantly related to
the other STAT molecules (Fig. 4b), and they lack the basic
residues corresponding to Arg-378 and Lys-379 of STAT1. Instead, they
have two additional arginine or lysine residues further downstream
(Fig. 4a).

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Fig. 4.
Comparison of the DNA-binding domains of STAT
proteins and localization of arginine/lysine-rich element.
a, alignment of the DNA-binding domain of human STAT
proteins. Identical amino acids are shown by an asterisk.
Arginine and lysine residues involved in arginine/lysine-rich element
are marked by red dots and nuclear export signal (NES (47))
in yellow. b, dendrogram showing relationships
between the DNA-binding domain of human STAT proteins. The dendrogram
and the alignment in Fig. 4a and 4b were
constructed using the PILEUP program in the GCG software package
(Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI).
c, space-filling representation of human STAT1 monomer
(Rutgers Protein Data Bank accession number 1bf5). DNA is shown in
blue, STAT1 DNA-binding domain in white, tyrosine
phosphorylation site at 701 in black, and amino acids
involved in arginine/lysine-rich surface element in red.
Views are shown along the DNA axis and from the side. The enlargement
shows individual amino acids comprising the arginine/lysine-rich
element. Representations were done using the program RASMOL (41).
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To analyze whether this structural site rich in basic amino acids is
involved in nuclear import of human STAT1 and STAT2, we made a series
of double point mutations and studied the nuclear import of the
FLAG-tagged, mutated STAT proteins in transiently transfected HuH7
cells by indirect immunofluorescence microscopy (Fig.
5). HuH7 cells were selected, because
they were effectively transfected and possessed excellent cellular
morphology in microscopic studies. Although HuH7 cells had their own
functional STAT1 and STAT2 proteins, using FLAG-tagged gene constructs,
we were able to follow the transport of transfected mutant proteins. To
rule out the possibility that the FLAG-tag affected the transport, we
also carried out the experiments with influenza virus hemagglutinin (HA)-tagged STAT gene constructs with identical results (not shown). Weak basal nuclear accumulation of intrinsic (Fig. 1) as well as of
transiently expressed (Fig. 5) STAT1 and to a lesser extent of STAT2
was observed in HuH7 as well as in several other cell lines
studied.

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Fig. 5.
Subcellular localization of transiently
expressed wt and mutant STAT1 and STAT2 proteins in
IFN- - and
IFN- -stimulated HuH7 cells. The known
functional domains of STATs are schematically shown in the figure.
FLAG-tag was expressed in the N-terminal end of both STAT1 and STAT2.
Alignments of the DNA-binding domain of both STAT1 and STAT2 are shown.
Amino acids mutated to alanine are shown in black squares.
HuH7 cells were transiently transfected with FLAG-tagged wt or point
mutated STAT1 and STAT2 gene constructs as shown
in the figure. At 48 h post-transfection the cells were treated
with IFN- or IFN- (1000 IU/ml) for 30 min and fixed. Staining was
performed with monoclonal Abs against the FLAG-epitope, followed by
staining with FITC-labeled anti-mouse immunoglobulins. All experiments
were repeated three to four times, and typical subcellular localization
of STATs is presented in each picture. The relative effectiveness of
IFN-induced nuclear import is presented after the sequence of each gene
construct (+++, +, ). Bar, 10 µm.
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Although FLAG-tagged transiently expressed wild-type STAT1 protein was
readily transported into the nucleus after IFN-
or IFN-
stimulation, all arginine/lysine mutant STAT1 proteins showed impaired
IFN-induced nuclear import (Fig. 5). Fig.
6 shows the nuclear import of the
corresponding STAT forms in a quantitative manner. In STAT1 R378A,K379A
and K413A,R418A mutant proteins nuclear import was partially inhibited,
whereas STAT1 K410A,K413A protein appeared to be fully nuclear
transport incompetent after stimulation with either IFN type (Fig. 5,
j, k, l). A revertant, where residues 413 and 418 where mutated back to those of the wild-type STAT1, showed normal IFN-induced nuclear translocation (Fig. 5,
p, q, r and Fig. 3). Mutation in
tyrosine 701 of STAT1 (STAT1 Y701A in Fig. 5), which has previously
been shown to result in lack of tyrosine phosphorylation and subsequent
dimerization (39), rendered the protein cytoplasmic. The corresponding
mutations in STAT2 molecule showed similar effects to those seen in
STAT1 (Figs. 5 and 6). IFN-
-induced nuclear import of STAT2
R374A,K375A and STAT2 R409A,K415A proteins were partially and
completely blocked, respectively (Fig. 5, u, v,
w, x). It is unlikely that p48 protein, which
together with STAT1 and STAT2 proteins forms the ISGF3 complex (4),
would be involved in STAT1·STAT2 heterodimer transport, because both
transfected as well as intrinsic p48 protein was always found in the
nucleus and its cellular distribution was not altered by IFN treatment
(Fig. 3).

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Fig. 6.
A graphical representation of subcellular
localization of transiently expressed wt and mutant STAT1 and STAT2
proteins in IFN- - and
IFN- -stimulated HuH7 cells as presented in the
Fig. 5. Subcellular localization of STAT1 and STAT2
proteins was divided in three categories; I, mainly cytoplasmic
(open bars); II, cytoplasmic and nuclear (shaded
bars); and III, mainly nuclear (solid bars). The
results represent the means of three individual experiments, in which
100 cells were counted.
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Tyrosine Phosphorylation of STAT Mutant Proteins--
Although the
tyrosine phosphorylation site is far from the arginine/lysine-rich
element of STAT proteins (Fig. 4c), we considered the
possibility that mutations in the arginine/lysine-rich element of STAT1
or STAT2 proteins would affect their tyrosine phosphorylation properties. Therefore, we transfected HuH7 cells with wt or mutant STAT genes for 48 h, followed by stimulation with
IFN-
for 1 h. Cells were collected, and transgene STAT protein
expression was analyzed by anti-FLAG Abs. All STAT1
constructs were equally well expressed, and in response to IFN-
stimulation there was a clear increase in the molecular weight of all
tested STAT1 proteins except Y701A mutant construct (Fig.
7a). Immunoprecipitation, followed by Western blotting with anti-P-Tyr Abs, revealed that all
STAT forms except STAT1 Y701A were readily tyrosine-phosphorylated (Fig. 7b). This result suggests that point mutations in the
arginine/lysine-rich element do not affect IFN-induced phosphorylation
status of STAT proteins.

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Fig. 7.
IFN- -induced
tyrosine phosphorylation of wild-type and mutant STAT1 and STAT2
proteins. HuH7 cells were transiently transfected with wt or
mutated STAT gene constructs for 48 h. a,
transfected cells were left untreated or stimulated with IFN- (1000 IU/ml for 30 min). The cells were collected and prepared for Western
blot analysis. Transfected FLAG-STAT1 was detected with monoclonal
anti-FLAG Abs, followed by ECL detection. Tyrosine-phosphorylated and
unphosphorylated forms of STAT1 s are shown by arrows.
b, transiently transfected HuH7 cells were either stimulated
with IFN- (1000 IU/ml for 30 min) or left unstimulated.
Immunoprecipitation was carried out with anti-FLAG Abs, and tyrosine
phosphorylation was detected on Western blots with anti-phosphotyrosine
Abs, followed by ECL detection. Tyrosine-phosphorylated STAT1 and STAT2
are shown by arrows (STAT1 P-Tyr and STAT2
P-Tyr).
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Nuclear Import Defective STAT Mutant Proteins Inhibit the Nuclear
Import of Heterologous STAT Proteins--
Because STAT1 and STAT2
heterodimerize after stimulation with IFN-
, we studied whether the
transport-defective STATs inhibit the nuclear import of heterologous
STAT molecules. wt and transport-defective STAT proteins were
transiently expressed in HuH7 cells, and colocalization of STAT1,
STAT2, and p48 proteins was analyzed. IFN-
-induced nuclear import of
STAT2 in wt STAT1 transfected cells was readily seen (Fig.
8a), and it was comparable to
that seen in Figs. 1 and 5. Instead, the cytoplasmic FLAG-STAT1
K410A,K413A mutant effectively blocked the nuclear import of endogenous
STAT2, functioning as a dominant negative molecule in IFN-
-treated
cells (Fig. 8a).

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Fig. 8.
Subcellular localization and nuclear import
of transiently transfected, FLAG-tagged STAT1 and
STAT2 gene constructs in
IFN- -treated HuH7 cells detected by confocal
microscopy. The cells were first transfected with FLAG-tagged gene
constructs as indicated in the figure. After 12 h, the cells were
primed with 10 IU/ml of IFN- or IFN- for 24 h
(IFN- -treated cells were primed with IFN- and vice
versa), then treated with 1000 IU/ml of IFN- or IFN- for 45 min, or left untreated, fixed, and double-stained with anti-FLAG,
anti-STAT1 (b) or anti-STAT2 (a) Abs as indicated
in the figure. Bar, 10 µm.
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To analyze the possible effect of STAT2 expression on IFN-
induction
in HuH7 cells both wt and nuclear transport-defective STAT2 were
transiently expressed, and colocalization of STAT1 and STAT2 was
detected. Overexpression of both wt and nuclear transport-defective
FLAG-STAT2 blocked IFN-
induction in transiently transfected HuH7
cells (Fig. 8b).
Transiently transfected FLAG-STAT1 Y701A did not inhibit
IFN-
-induced nuclear import of endogenous STAT2, suggesting that mutant STAT1 Y701A is incapable of forming dimers with endogenous STAT2
and thus does not block its nuclear import. It is likely that the weak
nuclear accumulation of STAT2 was due to the endogenous STAT1 in HuH7
cells (Fig. 8a). Transiently transfected transport-defective FLAG-STAT2 R409A,K415A, instead, completely blocked IFN-
-induced nuclear import of endogenous STAT1. This suggests that FLAG-STAT2 R409A,K415A mutant protein and endogenous STAT1 formed complexes in the
cytoplasm but they were not translocated into the cell nucleus after
IFN-
induction (Fig. 8a). The results suggest that in a
heterodimer both arginine/lysine-rich elements, one in each STAT, have
to be intact before IFN-induced nuclear import of STAT dimers can take place.
Nuclear Import Defective STAT1 K410A,K413A Is
Tyrosine-phosphorylated and Dimerized but It Does Not Bind DNA--
To
study STAT dimerization and DNA binding in a system lacking intrinsic
human STAT proteins we reconstituted STAT activation and DNA binding
analysis using a baculovirus expression system. We infected Sf9
cells with STAT1, STAT2, p48, and JAK2 protein-expressing recombinant
baculoviruses. Sf9 cells and a baculovirus expression system is
selected, because JAK2 kinase has earlier been shown to
tyrosine-phosphorylate and activate STAT1 protein (32). As shown in
Fig. 9 (a and b),
wtSTAT1 was tyrosine-phosphorylated, dimerized, and exhibited DNA
binding to the GAS oligonucleotide probe in the presence of wtJAK2
kinase, but not with that of knJAK2, as analyzed by gel filtration,
anti-phosphotyrosine blotting, and EMSA, respectively. The nuclear
import-defective FLAG-STAT1 K410A,K413A mutant was also
tyrosine-phosphorylated, and dimerized, but it was unable to bind
to the GAS oligonucleotide probe. The active ISGF3 complex bound ISRE15
oligonucleotide probe when Sf9 cells were coinfected with STAT1,
STAT2, p48, and wtJAK2 protein-expressing gene constructs, as detected
by EMSA (Fig. 9c). Instead, when STAT1 was replaced with the
nuclear import-defective STAT1 K410A,K413A mutant, or when wt JAK2 was
replaced with knJAK2, DNA-binding activity of the ISGF3 complex was not
seen or it was dramatically reduced, respectively (Fig.
9c).

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Fig. 9.
Gel filtration and EMSA analysis of GAS and
ISGF3 complex formation and DNA binding in baculovirus-infected
Sf9 cells. a, Sf9 cells were infected
with STAT1, wild type (wt)JAK2, and kinase negative (kn)JAK2
protein-expressing baculovirus gene constructs as indicated in the
figure. After 38 h the cells were collected, and cell lysates were
subjected to gel filtration in a Superose 12 column. Samples from gel
filtration fractions were run on 12% SDS-PAGE followed by Western
blots with anti-STAT1 or anti-phosphotyrosine Abs. The positions of
STAT1 monomers (85 kDa) and dimers (170 kDa) are marked with
arrows. b, Sf9 cells were infected with wt
baculovirus (E2) or coinfected with STAT1 and JAK2
protein-expressing baculovirus gene constructs as indicated in the
figure. After 28 h the cells were collected, and the lysates were
analyzed by EMSA using GAS oligonucleotide probe. c,
Sf9 cells were infected with wt baculovirus (E2) or
coinfected with STAT1, STAT2, p48, wtJAK2, and knJAK2
protein-expressing baculovirus gene constructs as indicated in the
figure. After 38 h the cells were collected, and the lysates were
analyzed by EMSA using ISRE15 oligonucleotide probe. In b
and c, supershift analyses were conducted with anti-STAT1
and anti-STAT2 antisera. Normal rabbit serum (NRS) was used
as a control antiserum. GAS and ISGF3 complexes are marked with
arrows.
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DISCUSSION |
In the present work, we have characterized the kinetics of nuclear
import of IFN-stimulated STAT proteins and identified an arginine/lysine-rich element in the DNA-binding domains of STAT1 and
STAT2 that regulates their IFN-induced nuclear import. By indirect
immunofluorescence analysis, we observed that the kinetics of nuclear
import of STAT proteins was fast and transient, because already within
10 min after IFN-
stimulation, STAT proteins were found in the
nucleus and within 2-3 h they were recycled back to the cytoplasm
(Fig. 1). The kinetics of nuclear import of STATs correlates well with
the IFN-stimulated DNA-binding activity of ISGF3 and GAS complexes (34,
40). p48 protein was apparently not associated with STAT import,
because it was constitutively expressed in the cell nucleus and its
intracellular distribution was not changed during IFN-
stimulation.
In addition, colocalization of STAT1, STAT2, and p48 (Figs. 1, 2, and
3) was clearly visualized by confocal laser microscopy preferentially
in the nucleus, suggesting that heterodimers of STAT1 and STAT2 are
first transported into the nucleus where they then associate with the
p48 protein to form the ISGF3 complex.
In unstimulated cells STAT1 was evenly distributed in the cytoplasm and
the nucleus, whereas STAT2 was predominantly cytoplasmic (Figs. 1 and
2). IFN stimulation resulted in rapid nuclear accumulation and
colocalization of STATs (Figs. 1 and 5). This suggests that the
mechanism of constitutive nuclear accumulation of intrinsic or
transiently expressed STAT1 (and to lesser extent STAT2) may differ
from that of IFN-induced nuclear import. It is possible that in the
absence of IFN stimulation STATs recycle between the cytoplasm and the
nucleus with other transport proteins. Recently, it was shown that
STAT3 is present as high molecular mass complexes in the size range of
200-400 kDa and 1-2 MDa both in unstimulated and IL-6-stimulated
cells (11). One of the proteins that was found to interact with
STAT3 in an IL-6-dependent manner was chaperone GRP58·ER-60·ERp57. The results suggest that STATs interact with a
number of other cellular proteins some of which could be involved in
intracellular protein transport. In uninduced cells, STAT1 and STAT2
have been shown to exist as unphosphorylated heterodimers. This weak
association is accomplished by regions apart from the SH2 domain, which
is crucial in IFN-induced nuclear import competent dimer formation
(42).
IFN-
/
receptor (IFNAR) and IFN-
receptor (IFNGR) are each
composed of two integral membrane glycoproteins (IFNAR1 and IFNAR2; IFNGR1 and IFNG2). Cross-communication between these two receptors seems to be important at least in IFN-
signaling, where a
constitutive subthreshold of IFN-
/
signaling and an association
between IFNAR1 and IFNGR2 is needed for efficient signal transduction
(43). The only constitutive STAT binding site in uninduced cells is located in IFNAR2, the ligand binding component of IFNAR. IFNAR2 binds
constitutively the weakly associated STAT1·STAT2 heterodimer via the
STAT2 component. During IFN-
induction, a specific binding site for
STAT1 is formed on IFNGR1. It seems possible that IFNAR serves as an
efficient source for STAT1 during IFN-
induction. In cells lacking
IFNAR1 IFN-
-induced antiviral response is seriously impaired (43).
Interestingly, also STAT4 dimer formation by the IFN-
/
receptor
in human T-cells occurs via intermediates involving STAT2 (44).
The above results (43, 44) may give an explanation for our results
showing that STAT2 overexpression (wt or nuclear import-defective STAT2) inhibits IFN-
-induced STAT1 nuclear import. Apparently, there
is a very delicate balance between the amount of STAT1 and STAT2 in the
cell, because overexpression of STAT2 completely blocked STAT1 nuclear
import after IFN-
stimulation (Fig. 8b). It may be that
in STAT2 excess IFNAR2 is occupied by monomeric STAT2 molecules and
hence IFNAR cannot serve as the source for STAT1 molecules required by
IFNGR. In a situation of STAT2 overexpression also the low basal level
transport of STAT1 is blocked in uninduced cells (Fig. 8b).
It is thus apparent that STAT1 molecules in the cytoplasm interact, at
least weakly, with the unphosphorylated forms of STAT2 (42).
Members of the importin-
family recognize proteins with classic
mono- and bipartite arginine/lysine-rich NLSs (16, 21). Although STAT1
does not have a classic NLS, it has been suggested that STAT1 is
imported into the nucleus by the aid of one importin-
family member,
NPI-1 (24). The 3-D structure of NPI-1 has not been resolved, but
crystallographic analysis of yeast karyopherin
, another member of
this highly conserved importin-
family, has been resolved (45).
Karyopherin-
consists of a tandem array of ten armadillo repeats
forming a long helical surface groove that harbors the binding sites
for the classic mono- and bipartite NLSs (45). The binding site of
STAT1 was suggested to be located in the C-terminal region of NPI-1,
distinct from the binding site for classic NLS (24). The STAT NLS might
thus differ from the classic mono- or bipartite NLSs. We reasoned that
STAT1 could, however, contain an NLS consisting of basic amino acids,
but its structure would be different from classic NLSs. In other
transcription factors and DNA-binding proteins ~80% of the NLSs
overlap or are immediately adjacent to the nucleic acid-binding domains
(37). Analysis of the crystal structure of STAT1 (38) and STAT3 (46) revealed a structural arginine/lysine-rich element in the DNA-binding domain of the proteins. This element seemed like a good candidate for a
STAT NLS, because it was very well conserved in STAT1 to STAT4.
Site-directed mutagenesis of the conserved basic amino acids in the
arginine/lysine-rich structural element (Fig. 4) revealed that this
site was involved in the nuclear import of both STAT1 and STAT2. A
novel feature was that two intact elements, one in each monomer, were
required for the IFN-induced nuclear import of STAT dimers. Nuclear
import-defective STAT1 K410A,K413A or STAT2 R409A,K415A mutant proteins
were able to block the IFN-
-induced import of heterologous
endogenous STAT molecules (Fig. 8b), thus functioning as
dominant negative STAT forms. Therefore, we think that in STAT dimers
both arginine/lysine-rich structural elements are involved in binding
to transport proteins, which act as the first step in the cascade
leading to nuclear import. The question rises, whether this site binds
to an unknown "adapter" protein or is itself an NLS binding
directly to importin-
. A defect in STAT1 nuclear import is not due
to lack of dimerization, because in a reconstituted baculovirus
STAT activation system STAT1 K410A,K413A mutant was
tyrosine-phosphorylated and dimerized. Mutant STAT1 complexes were,
however, not able to bind GAS or ISGF3 oligonucleotide probes (Fig. 9).
This could be expected, because the site regulating nuclear import of
STAT1 is in the immediate vicinity of the STAT1·DNA interaction site
(Fig. 4). A recent study by McBride and coworkers (47) showed that
STAT1 nuclear export is leptomycin B-sensitive and regulated by CMR1
export protein. They also demonstrated that the nuclear export signal
(NES) of STAT1 is situated immediately adjacent (amino acids 400-409)
to the putative NLS of STAT1 described in the present paper. The close
proximity of NLS and NES in STAT1 would enable either import or export
function to take place. Leptomycin B treatment of IFN-stimulated
STAT1-transfected cells failed to show any nuclear accumulation of
STAT1 K410A,K413A (results not shown) further supporting the view that
the protein is fully incapable of entering the nucleus.
Karyopherin-
, the yeast homolog of NPI-1, is known to exist as
dimers, because it has been crystallized in this form (45). Amino acids
mediating the dimerization of karyopherin-
are highly conserved and
they are also found in other importin-
molecules, including NPI-1.
It has been hypothesized that importin-
dimers are inactive, because
the groove that harbors the binding sites for the mono- and bipartite
NLSs is not accessible in dimeric forms of importin-
. However, it is
possible that the C-terminal binding sites for STATs are accessible
also or only in NPI-1 dimers. The NPI-1 dimer would have the binding
sites for both of the two arginine/lysine-rich elements in STAT dimers.
This would lead to stabilization of the STAT·importin-
heterotetramer complex, importin-
binding, and nuclear translocation.
Different members of the STAT family are known to form homo- and
heterodimers (9, 40), and presumably the mechanism of nuclear import is
identical for most, if not all STAT proteins. Both the evolutionary
conservation in the arginine/lysine-rich elements in STAT1, STAT2,
STAT3, and STAT4 (Fig. 4, a and b) and the
similar three-dimensional structure of STAT1 and STAT3 favors the idea
that the nuclear import of these STAT molecules is regulated by the
structural element described by us in the present work. STAT5a, STAT5b,
and STAT6 seem to be different from STAT1-4, because they lack the
N-terminal basic cluster and instead have six basic amino acids within
nine residues at a site corresponding to residues Lys-410 to Arg-418 of
STAT1 protein (Fig. 4a). Presently, the three-dimensional
structure of STAT5 or STAT6 is not available, and therefore, it is not
known whether this site is situated on the surface of the molecule.
Experimental analysis of the possible involvement of this
arginine/lysine-rich element in STAT5 or STAT6 nuclear import is yet
lacking. It has also been suggested that the most N-terminal amino
acids (first 129 residues) may regulate STAT nuclear translocation,
because chimeric STAT1·STAT2 and STAT1·STAT5 proteins showed marked
defects in their activation and nuclear translocation (48). These
chimeric STAT proteins were, however, structurally altered, because
they showed e.g. changed DNA-binding properties (48).
Recently, it has also been suggested that the C-terminal nuclear
localization sequence of IFN-
regulates STAT1 alpha nuclear import
at an intracellular site (49).
In the present report we have shown that the IFN-
-induced STAT
nuclear import involves tyrosine phosphorylation and dimerization of
STAT1 and STAT2, followed by rapid nuclear translocation and association with the p48 protein. We present evidence that a structural arginine/lysine-rich element in the DNA-binding domain of STATs regulates their nuclear import, a mechanism that could be common to all
STAT proteins. We also demonstrate that the site regulating nuclear
import of STATs has to be intact in both molecules of STAT dimers for
the nuclear translocation to take place.