From the
Laboratory of Infectious Disease
Immunology, Department of Microbiology, National Public Health Institute,
FIN-00300 Helsinki, Finland, ¶HELIOS Clinic/Franz
Volhard Clinic at the Max Delbrueck Center, Medical Faculty of the
Charité, 13122 Berlin, Germany, and
||Diabetes and Genetic Epidemiology Unit,
Department of Epidemiology and Health Promotion, National Public Health
Institute, FIN-00300 Helsinki, Finland
Received for publication, April 7, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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Six different human importin molecules have been identified:
importin
1 (Rch1, hSRP1
), importin
3 (Qip1), importin
4 (hSRP1
), importin
5 (hSRP1, NPI-1), importin
6,
and importin
7
(49).
Importin
proteins have remained structurally and functionally
conserved throughout evolution. The six human importin
proteins show
over 60% sequence similarity.
The crystal structures of two importin molecules, yeast importin
(10) and mouse
importin
2 (11), have
been determined. Importin
is composed of a large central domain that
consist of 10 tandemly repeated armadillo (arm) motifs, which are organized in
a superhelix flanked by small N- and C-terminal domains. The 10 arm repeats
generate an array of binding pockets that are situated within a long helical
surface groove. One binding pocket typically includes a tryptophan residue,
followed by an asparagine residue 4 residues downstream. The arm repeats are
variable within one protein, but they are remarkably conserved in sequence and
order when homologous proteins from yeast to humans are compared. The
N-terminal importin
binding domain of importin
mediates binding
to importin
(12,
13). In the absence of cargo
and importin
, the importin
binding domain blocks the NLS binding
site of importin
(14,
15). The C-terminal domain of
importin
mediates interactions with the export receptor CAS
(16,
17).
The NLS used by the classical nuclear import pathway is a short stretch of positively charged amino acids, arginines and lysines, that lack strict consensus sequence (18). A monopartite NLS, like that of simian virus 40 (SV40) large T antigen, is composed of a cluster of five to seven basic amino acids (19, 20). A typical bipartite NLS contains two clusters of basic amino acids separated by a linker of 1011 amino acids (21).
Monopartite NLSs have been shown to bind to the "major" binding
site of importin , which is formed by arm repeats 24
(10). The downstream cluster
of the bipartite NLS is also recognized by these arm repeats, whereas the
upstream cluster is recognized by arm repeats 7 and 8, also called the
"minor" binding site
(11).
Signal transducers and activators of transcription (STATs) are latent
cytoplasmic transcription factors that regulate the expression of a number of
genes involved in host defense and growth
(22). Binding of interferon
(IFN) to its specific cell-surface receptor leads to the activation of
receptor-associated Janus (Jak) tyrosine kinases, which phosphorylate STATs.
This leads to dimerization and translocation of STATs into the nucleus.
Dimerization is an essential and sufficient step for nuclear import
(23). Although bound by
importin 5, STATs do not contain a classical NLS
(24). Instead, it is supposed
that both STAT1 and STAT2 contain in the DNA binding domain a nonclassical NLS
that seems to become operative in STAT dimers
(2527).
In response to IFN-
stimulation, STAT1 homodimers are formed, whereas
IFN-
stimulation results in the formation of STAT1-STAT2 heterodimers.
Both STAT1 homodimers and STAT1-STAT2 heterodimers are specifically bound to
importin
5 (24,
26,
28). The STAT1-binding site of
importin
5 has been suggested to be located in the very C-terminal end
of STAT1 apart from the arm repeat domain
(24).
Also other regions or completely different mechanisms have been suspected
to affect the nuclear translocation of STATs. Strehlow and Schindler
(29) suggested that the most
N-terminal amino acids (the first 129 residues) regulate the nuclear import of
STAT molecules. Subramaniam et al.
(30) suggested that the
C-terminal nuclear localization sequence of IFN- regulates STAT1
nuclear import, and Bild et al.
(31) showed that
receptor-mediated endocytosis is necessary for STAT3 nuclear import.
Influenza A virus nucleoprotein (NP) and SV40 large T antigen have
functioned as important model molecules for analyzing the nuclear import
machinery. Influenza A virus NP encapsidates the viral genome and is essential
for viral transcription, replication, and packaging. NP interacts with viral
RNA, NP, and other viral proteins as well as cellular proteins including
importins 1 and
5
(32,
33). Two NLSs have been
identified in this protein, an unusual NLS at the N terminus
(34) and a classical bipartite
NLS in the middle of the molecule
(35). Apparently, the
N-terminal NLS mediates NP interaction with importins
(34,
36). SV40 large T antigen has
a monopartite NLS that interacts with importin
(19,
20,
37).
Here we show by site-directed mutagenesis that importin 5 recognizes
the nonclassical STAT1, STAT2, and influenza A virus NP NLSs by its arm repeat
domain. The binding site for STAT NLS is composed of arm repeats 8 and 9 and
for influenza A virus NP of arm repeats 79. Both binding sites differ
from the previously described major and minor classical NLS binding sites of
importin
. We also show that influenza A virus NP binds to the C
terminus and SV40 large T antigen to the N-terminal NLS binding site of
importin
3, indicating that one importin
molecule is able to
use different binding sites for various NLSs.
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MATERIALS AND METHODS |
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Interferons and Other ReagentsHuman leukocyte IFN-
(6 x 106 IU/ml) was kindly provided by Dr. Kari Cantell at
our Institute (39).
35S-Labeled PRO-MIX (>1000 Ci/mmol) was obtained from Amersham
Biosciences.
AntibodiesIn Western blot analysis rabbit anti-STAT1 (c-24; 1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-STAT2 (c-20; 1:2000; Santa Cruz Biotechnology), and mouse monoclonal anti-SV40 large T antigen (sc-147; 1:1000; Santa Cruz Biotechnology) immunoglobulins were used as suggested by the manufacturer. Influenza A NP antibodies (40) were used at a 1:500 dilution. In Western blotting secondary horseradish peroxidase-conjugated goat anti-rabbit (1:2000; Dako, Glostrup, Denmark) or anti-mouse immunoglobulins (1:5000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used.
Plasmids and DNA ManipulationsThe importin 1 and
3 gene constructs encoding C-terminal His-tagged proteins have been
described previously (9). His
tags were replaced by GST tags from pFA6aGST-HIS3Mx6 via
BamHI/HindIII using PCR mutagenesis. Importin
and
5 cDNAs in GST expression vectors were kindly provided by Drs. D.
Görlich and M. Malim, respectively. To create N- and C-terminal point
mutations to GST-importin
5, we used QuikChangeTM Site-directed
mutagenesis kit (Stratagene, La Jolla, CA). The primers used are 5'-CAC
TGC AGT TTG AAT CAG CTG CGG TAC TGG CAG CTA TTG CTT CAG GAA ATT CTC (arm 2),
5'-GAT GTC CAG GAA CAG GCA GTC GCG GCT CTT GGC GCC ATT GCT GGA GAT AGT
ACC ATG (arm 3), 5'-CGG AAT GCA GTA GCG GCT TTG TCT AAT C (arm 4),
5'-GAA TCT ATC AAA AAG GAA GCA TGT GCG ACG ATA GCT GCT ATT ACA GCT GGA
AAT AGG GC (arm 7), 5'-CGG ACA AGA AAA GAA GCA GCT GCG GCC ATC ACG GCT
GCA ACT TCT GGA GGA TCA GCT G (arm 8), and 5'-GAT TGT ACA GGT TGC CCT
AGC CGG CTT GGA AGC TAT CCT GAG GCT TGG AGA AC (arm 9). To create arm repeat
mutations to GST-importin
3, we used QuikChangeTM Site-directed
mutagenesis kit (Stratagene). The primers used are 5'-GTC TGT GAG CAA
GCA GTG GCG GCA TTG GGA GCT ATC ATA GGT GAT GGG CCC CAG (arm 3) and
5'-GGC ACT CAA AAA GAA GCT GCT GCC GCC ATA AGT GCC TTA ACA ATT AGT GGA
AGG (arm 8). Human importin
7 gene
(9) (GenBankTM accession
number AF060543
[GenBank]
) was PCR-modified with oligonucleotides GAG CGG ATC CAC
CAT GGA GAC CAT GGC GAG CCC (5' oligonucleotide, initiation
codon underlined) and TTC TTA GGA TCC CTA TTA TAG CTG GAA GCC CTC
(3' oligonucleotide) to create BamHI cloning sites (in
boldface) on both sides of the gene coding region. After BamHI
digestion the insert was cloned into the pGEX-2T GST fusion vector (Amersham
Biosciences). Baculovirus expression constructs of SV40 large T antigen and
influenza A virus NP have been described previously
(26,
41). All DNA manipulations
were performed according to standard protocols, and the newly created gene
constructs were partially sequenced.
Baculovirus ExpressionHuman Tyk2 cDNA in the baculovirus expression plasmid pVL1392 and SV40 large T antigen cDNA in the baculovirus expression plasmid were kindly provided by Dr. Sandra Pellegrini (Institute Pasteur, Paris) and Dr. J. M. Pipas (University of Pittsburgh, Pittsburgh (41)), respectively. Wt STAT1 and STAT2 baculovirus constructs were as described (25). Influenza A (PR8) virus NP gene was inserted into the BamHI site of the pAcYM1 expression plasmid, and recombinant viruses were obtained by plaque purification as described (38). For protein production Sf 9 cells were coinfected with Tyk2 and STAT protein-expressing baculoviruses for 42 h. Influenza A NP and SV40 large T antigen were produced without coinfection with Tyk2. Virus-infected Sf 9 cells were collected, and the whole cell extracts were prepared by disrupting the cells in 50 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100 (immunoprecipitation (IP) buffer) on ice for 30 min. The cells were disrupted with a syringe. Cell extracts were clarified by Eppendorf centrifugation (13,000 rpm, 10 min).
Importin Binding Assay, SDS-PAGE, and Western BlottingHuman
importins ,
1,
3,
5, and
7 as well as
mutants in the arm repeats 2, 3, 2 + 3, 2 + 4, 3 + 4, 2 + 3 + 4, 2 + 3 + 7, 2
+ 3 + 8, 7, 8, and 9 of importin
5 and mutants in the arm repeats 3 and
8 of importin
3 were expressed in Escherichia coli BL21 cells
as GST fusion proteins under
isopropyl-1-thio-
-D-galactopyranoside induction. Bacteria
were lysed in IP buffer with 5 mg/ml lysozyme (Sigma) at room temperature for
30 min, briefly sonicated, and clarified by Eppendorf centrifugation (13,000
rpm, 5 min). From 0.1 to 1.0 ml of bacterial cell extracts containing
GST-importin proteins were allowed to bind to 25 µl of
glutathione-Sepharose 4 Fast Flow beads (Amersham Biosciences) in IP buffer
with agitation at +4 °C for 60 min, followed by washing twice with the
buffer. From 0.1 to 1.0 ml of baculovirus cell extracts containing STATs,
influenza A NP, or SV40 large T antigen as well as 50 µl of in
vitro translated influenza A virus NP protein (TNT Coupled
Reticulocyte Lysate Systems, Promega, Madison, WI) were allowed to bind in IP
buffer to Sepharose-immobilized GST-importins
3 and
5 or
GST-importin
at +4 °C for 2 h, followed by washing 3 times with the
buffer. Sepharose beads were dissolved in 50 µl of 2x Laemmli sample
buffer, and the proteins were separated on 8% SDS-PAGE
(42). The gels were stained
with Coomassie Brilliant Blue or transferred onto Immobilon-P membranes
(polyvinylidine difluoride; Millipore, Bedford, MA), followed by staining with
primary and secondary antibodies and visualization of the proteins with the
enhanced chemiluminescence system (Amersham Biosciences) as recommended by the
manufacturer. 35S-Labeled protein samples were separated on 8%
SDS-PAGE. The gels were stained and treated with Amplify reagent (Amersham
Biosciences) as recommended and autoradiographed.
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RESULTS |
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The central domain of importin is composed of 10 arm repeats
(Fig. 1B). The well
conserved tryptophan and asparagine residues in arm repeats 24 and
79 in the helical surface groove of the importin
molecule are
thought to be crucial for NLS binding
(10,
11)
(Fig. 1, B and
C). An alignment of the central domain of human importin
molecules is shown in Fig.
1C. Typical arm repeats contain a tryptophan-asparagine
pair at analogous positions. A tryptophan-asparagine pair was found in arm
repeats 24 and 78. Arm repeats 5 and 6, which do not contain
this pair, have not been shown to participate directly in NLS binding. Arm
repeat 9 contains the conserved asparagine residue, but the tryptophan residue
is replaced either by an aspartic acid (importins
1,
3, and
4) or an asparagine (importins
5,
6, and
7). Based
on published crystal structures
(10,
11) and the alignment in
Fig. 1C, series of
mutations were created to analyze the interactions between importin
and various NLSs. Arm repeat 9 was included in our analysis, because the two
asparagine residues appeared as part of a possible NLS binding pocket
(Fig. 1C).
IFN-stimulated STAT Dimers Bind Specifically to Importin
5At present, STAT1 homodimers and STAT1-STAT2
heterodimers are the only STAT dimers that have been shown to interact with
importin
molecules
(24,
26,
28). Recent mutational
analyses suppose that leucine 407 and lysines 410 and 413 within the DNA
binding domain of STAT1 function as an NLS and mediate binding to importin
5 (25,
26,
28).
To determine the possible interactions of STAT1-STAT2 dimers with other
importin isoforms than 5, we stimulated the human NK-92 cell line with
1000 IU/ml of IFN-
for 30 min. Cell extracts were prepared, and the
cellular proteins were allowed to bind to Sepharose-immobilized GST-importins
1,
3,
5, or
7 at +4 °C for 2 h, followed by an
analysis of the bound STAT proteins by Western blotting. As shown in
Fig. 2, STAT1-STAT2
heterodimers were readily bound to importin
5, whereas no binding to
importins
1,
3, or
7 in IFN-
-stimulated cells was
detected. Clearly, binding of STAT1-STAT2 dimers to importin
5 is
highly specific.
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Tyrosine-phosphorylated STAT Dimers, Influenza A NP, and SV40 Large T
Antigen Bind to Different Importin IsoformsTo
characterize further STAT binding to different importin
isoforms, we
used a baculovirus expression system to reconstitute STAT activation
(26). Coinfection of
Sf 9 cells with recombinant Tyk2 and STAT protein expressing
baculoviruses resulted in an efficient expression and tyrosine phosphorylation
of STAT1 and STAT2 proteins. Tyrosine-phosphorylated STAT1 and STAT2 formed
dimers, which enabled us to analyze the potential interactions of dimeric STAT
complexes with importins (25,
26). As shown in
Fig. 3, baculovirus-expressed
STAT1 homodimers or STAT1-STAT2 heterodimers bound specifically to
Sepharose-immobilized GST-importin
5 but not to importin
1,
3, or
7 isoforms. These results, together with those shown in
Fig. 2 using natural
IFN-
-stimulated cells, show that the reconstituted STAT activation in
the baculovirus expression system is a reliable method to study STAT-importin
interactions.
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We also analyzed interactions of Sepharose-immobilized GST-importins
1,
3,
5, or
7 with baculovirus-expressed influenza
A NP and SV40 large T antigen. Two NLSs have been identified in influenza A
virus NP, a nonclassical NLS at the N terminus
(34) and a classical bipartite
NLS in the middle of the molecule
(35). It has been shown that
the N-terminal NLS of NP is involved in the interaction with importins
(34,
36). Here we show that
influenza A virus NP bound strongly to importins
1 and
5 and to
a lesser extent to importin
3, whereas no marked binding to importin
7 isoform was detected (Fig.
3C).
Importins 1 and
5 have been shown to bind efficiently SV40
large T antigen NLS peptide or T antigen NLS conjugated to BSA
(24,
4344).
Instead, our binding experiments of SV40 large T antigen with
Sepharose-immobilized GST-importins revealed that full-length T antigen bound
strongly to importin
3 and to a lesser extent to importin
1 but
not to importin
5 or
7 isoforms
(Fig. 3D), indicating
that the natural context of an NLS can greatly affect its accessibility to
importins.
A C-terminal Unique NLS Binding Site, Including Arm Repeats 8 and 9 of
Importin 5, Binds STATsBecause STAT NLS is formed
by a cluster of basic amino acids within the DNA binding domain of STAT1 and
STAT2 (25,
27) and recognized by importin
5 (26,
28), we reasoned that importin
5 arm repeats might be involved in STAT NLS binding. To analyze the
role of arm repeats in STAT-importin
5 interaction, we created several
mutations in the binding pockets of arm repeats 24 and 79 of
importin
5 (Figs. 1 and
4).
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Mutations in arm repeat 2 (W149A, T152A, and N153A), arm repeat 3 (W191A
and N195A), arm repeat 4 (W234A), arm repeat 7 (W360A, S363A, and N364A), or
different combinations of these had no effect on STAT1 homodimer binding to
importin 5 (Fig.
5A). Two amino acid substitutions in arm repeats 8 (W402A
and N406A) or 9 (N445A and N449A) instead completely prevented STAT1 binding
to importin
5 (Fig.
5A). These results show that in importin
5 the
very C-terminal arm repeats 8 and 9 form the binding site for STAT1. Also
STAT1-STAT2 dimers were recognized by arm repeats 8 and 9
(Fig. 5B). This NLS
binding site differs from the previously described minor NLS binding site that
is composed of arm repeats 7 and 8.
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The C-terminal Arm Repeats of Importin 5 Bind Influenza
A Virus NPBecause STAT1 homodimers and STAT1-STAT2 heterodimers
bound to the C-terminal arm repeats 8 and 9 of importin
5, it was of
interest to study the binding site for influenza A NP that also has a
nonclassical NLS. Our results show that the binding site for influenza A NP,
comprising arm repeats 79, is also located in the C-terminal half of
importin
5 (Fig.
5C). To confirm the results found with
baculovirus-expressed influenza A NP, we also used in vitro
translated NP protein in GST-importin binding experiments
(Fig. 5D). Both
methods gave quite similar results. A weak binding was detected with arm
repeat 9. Arm repeat 7 was not involved in STAT-NLS binding, but it was
absolutely necessary for influenza A NP binding. The importin
5-binding
site for influenza A NP thus seems to be a larger one compared with that of
STATs.
Influenza A Virus NP and SV40 Large T Antigen Bind to Different NLS
Binding Sites of Importin 3To identify influenza A
virus NP and SV40 large T antigen-binding sites of importin
3, we
mutated arm repeats 3 or 8 of the GST-importin
3 gene construct. Point
mutations in the arm repeat 8 of importin
3 clearly reduced but did not
completely prevent influenza A virus NP binding. Mutations in the arm repeat 3
instead had no effect on NP-importin
3 interaction
(Fig. 6A). The arm
repeat 3 mutant of importin
3 (W191A and N195A) had completely lost its
ability to bind SV40 large T antigen. Unlike the case of influenza A NP,
mutations in the arm repeat 8 (W390A and N394A) had no effect on T antigen
binding (Fig. 6B).
These results clearly show that one importin
molecule can use either
its N or C terminus for binding various NLS containing proteins.
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DISCUSSION |
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Crystallographic analyses of importins bound to NLS peptides show that also
the C-terminal minor binding site of importin , comprising arm repeats
7 and 8, is able to bind NLS peptides. The major NLS binding site of yeast
importin
molecule recognizes the classical monopartite NLS peptide
(SV40 large T antigen NLS, PKKKRKV) and the larger downstream basic cluster of
the classical bipartite NLS peptide (nucleoplasmin NLS, KRPAATKKAGQAKKKKLD).
The minor NLS binding site has a supportive role in binding the smaller
upstream basic cluster of the bipartite NLS
(10,
37). Mouse importin
showed a similar binding mode for the bipartite nucleoplasmin NLS as yeast
importin
, but SV40 large T antigen NLS peptide was bound almost
equally by the major and minor binding sites
(11). Also c-myc NLS
peptide (PAAKRVKLD) binds both to major and minor binding sites of yeast
importin
(37).
The C-terminal half of importin has been suggested to be involved
in NLS binding of several proteins. By using deletion analyses it has been
shown that the NLSs of a dimeric DNA-binding protein nuclear antigen 1 of
Epstein-Barr virus (KRPRSPSS) and the matrix protein of human immunodeficiency
virus (KKKYKL) are bound by the C-terminal half of importin
1
(45,
46). The NLS of a human
transcription factor LEF-1 (NLSKKKKRKREK) is recognized by importins
1
and
5 and was mapped to the C terminus of importin
1
(16). All these NLSs contain a
classical monopartite NLS consensus sequence K(K/R)X(K/R) (where
X is any residue). Herpesvirus saimiri open reading frame 57 is a
multifunctional trans-regulatory protein that interacts via its nonclassical
arginine-rich NLS with importins
1 and
5. By a series of
deletion mutants of importins
1 and
5, it was shown that both
importins bind herpesvirus saimiri open reading frame 57 NLS by their
C-terminal arm repeats (47).
The sequence of the NLS is RRPSR-PFRKP, and the arginine and lysine residues
were shown to be essential for importin binding. The C-terminal arm repeats of
importins
1 and
5 thus seem to be able to bind both classical
and nonclassical NLSs.
Importin molecules bind classical NLSs mainly through their basic
residues, although the surrounding amino acids also contribute to the binding
(10,
11,
37). It appears that the
nonclassical NLSs that are known to bind to importin
molecules contain
at least a few basic residues. The potential NLS of STATs is no exception. It
contains basic residues but also the adjacent residues are of importance
(25,
28). Our results, revealing
the STAT-binding sites in importin
5, further support the idea that a
cluster of basic residues in the DNA binding domain of STAT1 and STAT2 forms
an essential part of the STAT NLS.
STAT dimers contain two basic elements, one in each STAT monomer, and that
both of these elements are required for nuclear import is an extraordinary
feature (25,
27). One STAT dimer has been
shown to bind to two importin molecules
(26). Our hypothesis is that
one importin
5 molecule is bound to each of the STAT molecules in the
dimer, and additional interactions between the two importins or importins and
STATs are needed to stabilize the import complex. It has also been shown that
large cargoes need more than one importin molecule for a rapid nuclear import
to take place (48). A similar
dimeric basic type NLS has also been found in immediate early protein (IE1) of
baculovirus Autographa californica
(49).
It has been shown previously that importin 5 is necessary for
nuclear translocation of phosphorylated STAT, whereas importin
1 is not
(24). Our data provide further
evidence that importin
5 is the only isoform mediating nuclear import
of STAT1 homodimers, because isoforms
1,
3, and
7 did not
bind STAT1. Influenza A NP, on the other hand, is bound by importins
1,
3, and
5. Importins
1 and
5 are known to bind to
the N-terminal nonclassical NLS of influenza A NP
(34,
36). In the present study we
show that importin
5 uses its C-terminal arm repeats 79 for the
binding of NP NLS. Although importin
5 binds by its C-terminal arm
repeats both STATs and influenza A NP, the binding sites are not identical.
STATs need only arm repeats 8 and 9 for binding, whereas influenza A
NP-importin
5 interaction requires arm repeats 79.
In the present study, we show that importin 3 is able to use both N-
and C-terminal-binding sites for binding different nuclearly targeted
proteins. Previous experiments showed that SV40 large T antigen NLS peptides
and T antigen NLS coupled to BSA bound to the N-terminal major binding site of
importins
1 and
5
(24). Our studies show that
the full-length SV40 large T antigen is bound to the N-terminal major binding
site and influenza A virus NP, on the other hand, to the C-terminal binding
site of importin
3.
Our results suggest that the location and surroundings play an important
role in determining the accessibility of an NLS to importins. We show here
that the full-length SV40 large T antigen was recognized by importin 3
and weakly by importin
1. However, as a peptide or as a peptide
conjugated to a protein not naturally transported into the nucleus
(e.g. BSA), T antigen NLS has been shown to bind to importins
1,
5, and weakly to
3
(24,
43,
44). The accessibility of an
NLS can apparently be regulated by phosphorylation or structural masking
(50). SV40 large T antigen is
known to have a phosphorylation site affecting nuclear transport
(51).
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FOOTNOTES |
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To whom correspondence should be addressed: National Public Health Institute,
Mannerheimintie 166, FIN-00300 Helsinki, Finland. Tel.: 358-9-47448377; Fax:
358-9-47448355; E-mail:
krister.melen{at}ktl.fi.
1 The abbreviations used are: NLSs, nuclear localization signals; NP,
nucleoprotein; STAT, signal transducers and activators of transcription; IFN,
interferon; GST, glutathione S-transferase; BSA, bovine serum
albumin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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