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B, AP-1, NFAT, and STAT1 Nuclear Import in T Lymphocytes by Noninvasive Delivery of Peptide Carrying the Nuclear Localization Sequence of NF-B p501
Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, AP-1, NFAT, and
STAT from the cytoplasm into the nucleus. The first step in the nuclear
import process is recognition of a nuclear localization sequence (NLS)
within the karyophilic protein by a cytoplasmic receptor such as the
importin (karyopherin)-
subunit. The NLSs of NF-B, AP-1, and NFAT
differ and the NLS of STAT1 has not yet been identified. Herein we
demonstrate that the inducible nuclear import of NF-B, AP-1, NFAT,
and STAT1 in Jurkat T lymphocytes is significantly inhibited by a
cell-permeable peptide carrying the NLS of the NF-B p50 subunit. NLS
peptide-mediated disruption of the nuclear import of these
transcription factors results in inhibition of IB and IL-2 gene
expression, processes dependent on NF-B or the combination of
NF-B, AP-1, and NFAT. Further, we show that inhibitory NLS peptide
interacts in vitro with a cytoplasmic NLS receptor complex comprised of
the Rch1/importin (karyopherin)-ß heterodimer expressed in Jurkat T
cells. Taken together, these data indicate that the inducible nuclear
import of NF-B, AP-1, NFAT, and STAT1 in Jurkat T cells can be
regulated by NLS peptide delivered noninvasively to the cytoplasm of
Jurkat T cells to target members of the importin (karyopherin)-ß
NLS receptor complex. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B,3 AP-1,
NFAT, and STAT transcription factors play a key role in this process
(1, 2). Upon cellular activation, these factors translocate from the
cytoplasm to the nucleus where they bind DNA and regulate gene
transcription. NF-B, NFAT, and STAT are constitutively expressed and
sequestered as latent factors in the cytoplasm of resting T cells (3, 4). The NF-B p50/p65 heterodimer is complexed in the cytoplasm with
inhibitory proteins, the most extensively characterized being IB
(5, 6, 7). Cellular activation results in phosphorylation, ubiquitination,
and degradation of IB, thereby releasing the p50/p65 heterodimer
for import to the nucleus (8, 9, 10, 11, 12). The NFAT proteins exist as
phosphoproteins in the cytoplasm of resting T cells (13).
Activation-induced dephosphorylation by calcineurin, a
calcium/calmodulin-dependent serine-threonine phosphatase, results in
nuclear import of NFAT (14, 15), which binds DNA alone or in complex
with AP-1 proteins (16, 17, 18). Cytoplasmic STAT monomers are recruited to
activated growth factor or cytokine receptors where they are tyrosine
phosphorylated by Janus kinases, dimerized through their SH2 domains,
and then rapidly imported into the nucleus (19, 20). The AP-1 proteins
c-Fos and c-Jun are present at only minimal levels in resting T cells
in contrast to NF-B, NFAT and STAT (4). Cellular activation leads to
de novo synthesis and nuclear import of c-Fos and c-Jun, which bind DNA
alone or in complex with other transcription factor proteins (e.g.,
NFAT) (21).
For most karyophilic proteins, the first step in the nuclear import
process involves recognition of a nuclear localization sequence (NLS)
within the protein by a cytoplasmic NLS receptor complex (22). Most
well-characterized NLSs consist of clusters of basic amino acids
arranged in one of two motifs: a classical motif having a single
cluster of four or five basic residues, or a bipartite motif having two
clusters of basic residues separated by a spacer of approximately 10
amino acids (23, 24). Classical NLSs are found in the NF-B p50 and
p65 subunits (5, 6, 25, 26), while bipartite NLSs are found in the AP-1
subunits c-Fos and c-Jun (27, 28, 29). The NFAT proteins have recently been
shown to contain two sequences that can function as NLSs (30). However,
the primary nuclear targeting activity of NFAT appears to be
attributable to a unique sequence near the N terminus composed of three
basic amino acids flanked by a motif containing multiple phosphoserines
targeted by calcineurin (30, 31). Unlike the NF-B, AP-1, and NFAT
proteins, STAT1 appears to have a unique and as yet unidentified NLS
that is of neither classical nor bipartite type (32). In light of the
variations in NLSs present among these four distinct transcription
factors, it is unclear whether there are biochemical differences in the
pathway(s) responsible for their nuclear import.
At least two known pathways, each utilizing distinct but related
proteins, have been identified for the nuclear import of karyophilic
proteins. The first pathway involves a heterodimeric NLS receptor
complex, known as importin-ß or karyopherin-ß, that
recognizes either classical or bipartite NLS sequences in many
karyophilic proteins and mediates their import into the nucleus (22).
Importin- (karyopherin-) binds to the NLS motif in the cytoplasm,
and importin-ß (karyopherin-ß1) docks the entire
complex at the cytoplasmic face of the nuclear pore. Once docking has
occurred, the complex is subsequently translocated through the pore and
into the nucleus by an energy-dependent process involving the GTPase
Ran/TC4 and the Ran interacting factor NTF2/p10 (22). Homologues of
importin- (60 kDa) have been described in several species including
Xenopus, yeast, Drosophila, mouse, and human. To
date, in addition to Rch1/hSRP1 (33, 34) and NPI-1/hSRP1 (35, 36),
four other human importin- homologues have been cloned including
Qip1 and hSRP1
(37, 38, 39, 40). Importin-ß (karyopherin-ß1)
(97 kDa) has also been characterized in several different species
(41, 42, 43). The second pathway of nuclear import involves an
importin-ß-related protein, termed transportin or
karyopherin-ß2, which binds the M9 sequence present in
the hnRNP A1 protein, but does not interact with classical or bipartite
NLSs (43, 44). Initial studies have shown that transportin or its yeast
homologue, Kap104p, can perform both the NLS-binding and nuclear pore
docking functions of the heterodimeric importin-ß complex (44, 45).
To investigate the mechanism by which the nuclear import machinery
recognizes NF-B, AP-1, NFAT, and STAT1, the new strategy of
noninvasive peptide delivery was used (46). Using this technique,
peptides bearing the NF-B p50 NLS were delivered into the cytoplasm
of intact cells and the activation-dependent nuclear import of each
transcription factor analyzed in the presence of the NLS peptides. Our
results demonstrate that the NF-B p50 NLS peptide significantly
inhibited the signal-dependent nuclear import of NF-B, AP-1, NFAT,
and STAT1 and the subsequent induction of genes activated by one or
more of these factors in human T lymphocytes. We also show that the
inhibitory p50 NLS peptide binds in vitro to the NLS receptor complex,
Rch1/importin-ß. These findings indicate that despite the presence of
diverse NLS motifs in these transcription factors, the nuclear import
of NF-B, AP-1, NFAT, and STAT1 can be regulated by a single NLS
peptide delivered noninvasively to the cytoplasm of human T cells to
target members of the importin (karyopherin) family of NLS receptors
(e.g., Rch1/importin-ß). This inhibition of transcription factor
nuclear import was subsequently coupled to attenuation of gene
transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The peptides listed in Table I
were synthesized manually by a stepwise solid-phase synthesis method
using Boc chemistry as previously described (46). Polyclonal antiserum
specific for IB (amino acids 1–29) and NF-B p50 (amino acids
1–21) were generated as described (12). Polyclonal c-Jun antiserum and
affinity-purified polyclonal c-Fos Ab were gifts from Dr. Stephen Hann
(Vanderbilt University, Nashville, TN). The mAb 7A6, specific for human
NFATc (NFAT2), was a gift from Dr. Gerald Crabtree (47). Polyclonal Abs
and mAbs to ISGF3/STAT1 (Transduction Laboratories, Lexington, KY) were
gifts from Dr. Mark Boothby (Vanderbilt University). Polyclonal Abs to
Rch1 and importin-ß were gifts from Dr. Dirk Görlich (41).
Affinity-purified horseradish peroxidase-conjugated goat
anti-rabbit and goat anti-mouse IgG were purchased from Pierce
(Rockford, IL). Expression plasmids encoding full-length human
Rch1 and importin-ß were generously provided by Dr. Dirk
Görlich (41). Recombinant Rch1 and importin-ß were expressed in
Escherichia coli and purified from cell lysates by standard
chromatographic procedures using Q Sepharose Fast Flow followed by
Superdex 200 (Pharmacia Biotech, Piscataway, NJ).
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Human Jurkat T cells were grown in RPMI 1640 (Cellgro, Fisher,
Atlanta, GA) supplemented; with 50 U/ml penicillin, 50 µg/ml
streptomycin, and 10% heat-inactivated FBS. For each assay, 5 x
106 Jurkat T cells were pelleted, suspended in 4.5 ml fresh
culture media, and incubated at 37°C (5% CO2) for 60
min. Peptides were dissolved in 0.5 ml HBSS without Ca2+ or
Mg2+ (Cellgro) and added to the cells for 30 min before the
addition of either a combination of PMA (5 nM; Sigma, St. Louis,
MO)/ionomycin (2 µM; Calbiochem, La Jolla, CA) or IFN- (1 U/ml;
Sigma). Cytoplasmic and nuclear extracts were prepared as described
(48) with minor modifications. Briefly, cells were washed with ice-cold
PBS and then lysed by incubation in 200 µl of buffer A (10 mM HEPES,
pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.4% Nonidet P-40, 1 mM
DTT, 0.5 mM PMSF, and 1 µg/ml each of leupeptin, aprotinin,
pepstatin, chymostatin, and antipain) on ice for 5 min. Nuclei were
pelleted by centrifugation, the supernatant saved as the cytoplasmic
extract, and the nuclear pellet washed in 1 ml buffer A. Nuclei were
resuspended in 25 µl (STAT1) or 100 µl (NF-B, AP-1, and NFAT) of
buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT, 1 mM PMSF, and 1 µg/ml each of leupeptin, aprotinin, pepstatin,
chymostatin, and antipain). Nuclear extracts prepared from
IFN--stimulated cells were made fourfold more concentrated than
extracts prepared from cells stimulated with PMA/ionomycin because of
the relatively lower concentration of STAT1 found in Jurkat T cells.
Samples were vortexed for 15 min at 4°C, centrifuged, and the
supernatant saved as the nuclear extract. The protein concentration of
the nuclear extracts was determined using the Pierce BCA protein assay
and equalized among samples using buffer B. For cells treated with
IFN-, buffers A and B also contained 1 mM
Na3VO4 and 10 mM NaF. Intracellular peptide
concentrations were determined by cell extract ELISA. Briefly,
cytoplasmic and nuclear extracts from peptide-treated cells were
combined and 25, 50, or 100 µl used to coat the wells of a 96-well
plate. For comparison, adjacent wells were coated with extracts from
untreated cells to which known amounts of peptide were added. Following
a 16-h incubation, all wells were washed with PBS plus 0.05% Tween 20,
blocked with BSA, and incubated with an Ab specific for the LMP
residues present in each peptide (see Table I
) (46). Peptide-Ab
complexes were detected with anti-rabbit IgG conjugated with
alkaline phosphatase and quantitated by ELISA.
Electrophoretic mobility shift assay (EMSA)
The following oligonucleotides, with the transcription factor
recognition sites underlined, were used: NF-B,
5'-AGCTTAGAGGGGACTTTCCGAGAGGA-3' (8); AP-1,
5'-GATCCATGACTCAGAGGAAAACA-3' (49); NFAT,
5'-GATCTTTACATTGGAAAATTTTAT-3' (50); NFAT/AP-1,
5'-GATCCAGAAAGGAGGAAAAACTGTTTCATACAG-3' (18);
STAT1, 5'-GATCGTCGACATTTCCCGTAAATC-3' (51); and NF-Y,
5'-GATCTGAGAATTTTCTGATTGGTTCTGGCGAGTTTGG-3' (52).
Double-stranded oligonucleotides (40 pmol) were labeled with 50 µCi
[32P]dATP (3000 Ci/mmol, DuPont-New England Nuclear,
Boston, MA) and 5 U of E. coli DNA polymerase I (Klenow
fragment) at room temperature for 30 min. Labeled oligonucleotides were
separated from unincorporated [32P]dATP by
chromatography on Sephadex G50 (Sigma). DNA-binding reactions were
performed in a final volume of 20 µl. NF-B- and AP-1-binding
reactions contained 5 µl nuclear extract, 1 µg poly dI-dC, 1 µg
salmon sperm DNA, EMSA buffer (20 mM HEPES, pH 7.9, 5% glycerol, and 1
mM EDTA), and 100,000 cpm 32P-labeled oligonucleotide.
NFAT- and STAT1-binding reactions contained 5 µl nuclear extract, 2
µg poly dI-dC, and 100,000 cpm 32P-labeled
oligonucleotide. NF-Y-binding reactions contained 5 µl nuclear
extract, 2 µg poly dI-dC, EMSA buffer, and 50,000 cpm
32P-labeled oligonucleotide. All binding reactions were
incubated at room temperature for 20 min and terminated by the addition
of 2 µl of 10x EMSA gel-loading dye (0.25% bromphenol blue, 0.25%
xylene cyanol, and 50% glycerol). NF-B, AP-1, and NFAT samples were
run in 4% polyacrylamide/0.5x TBE (45 mM Tris-borate and 1 mM EDTA)
gels prerun in 0.5x TBE for 60 min at 100V. STAT1 samples were run in
4% acrylamide/Tris-glycine (50 mM Tris, 380 mM glycine, and 2 mM EDTA)
gels as described previously (8). Gels were dried onto chromatography
paper (Whatman 3Chr; VWR, Atlanta, GA) and exposed to Fuji BAS-IIIs
phosphorimager plates (Fuji, Tokyo, Japan) for quantitation and
then to Kodak Biomax MR autoradiography film (Eastman Kodak, Rochester,
NY). Ab supershift analyses of DNA-binding complexes were performed by
adding subunit-specific antiserum to the nuclear extract immediately
before addition of other binding-reaction components. EMSAs to detect
AP-1 in cytoplasmic extracts were performed as described above with the
exception that the nuclear extract was replaced with 10 µl of 10x
concentrated cytoplasmic extract prepared by centrifugation in
Microcon-30 concentrators (Amicon, Beverly, MA).
Immunoblotting
Nuclear proteins (50 µg) were precipitated with 10% TCA and
the protein pellet resuspended in SDS-PAGE sample buffer (50 mM
Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, 2% 2-ME, and
0.025% bromphenol blue). Samples were heated at 100°C for 5 min and
run in a 10% SDS polyacrylamide mini gel (53). Cytoplasmic proteins
(100 µg) were mixed with SDS-PAGE sample buffer, heated at 100°C
for 5 min and run in an 8% SDS polyacrylamide mini gel (53). Proteins
were transferred to nitrocellulose (Pharmacia Biotech) and blocked with
5% dry milk in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween
20) at room temperature for 1 h. Immunoreactive proteins were
detected using Abs specific for NF-B p50 (1:8000), c-Jun (1:6000),
NFATc (1:6000), IB (1:2500), Rch1 (1:5000), or importin-ß
(1:5000). After incubation with primary Abs, blots were incubated with
goat anti-rabbit or anti-mouse horseradish
peroxidase-conjugated Ab (1:20,000; Pierce), developed with supersignal
horseradish peroxidase enhanced chemiluminescence reagent (Pierce), and
exposed to Kodak Biomax MR film.
RNA isolation and ribonuclease protection assays
Total RNA was prepared from 107 Jurkat T cells using Trizol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s recommendations. Anti-sense RNA probes were generated using the Riboprobe Gemini system (Promega, Madison, WI) according to the manufacturer’s recommendations. The human IL-2 anti-sense RNA probe (280 nucleotides) was generated from pGEMIL-2 (a gift from Drs. Geraldine Miller and Marty Reich, Vanderbilt University). The human ß-actin anti-sense RNA probe (255 nucleotides) was generated from a plasmid provided by Boehringer-Mannheim (Mannheim, Germany). Ribonuclease protection assays were performed using the RPA II system (Ambion, Austin, TX) as described by the manufacturer. Briefly, 30 µg Jurkat total RNA and 160,000 cpm 32P-labeled IL-2 probe or 10 µg Jurkat total RNA and 80,000 cpm 32P-labeled ß-actin probe were coprecipitated with ethanol. Pellets were resuspended in hybridization buffer, heated to 90°C for 3 min, and annealed at 42°C for 16 h. Annealed samples were digested with RNase A and T1 and precipitated with ethanol. The resulting precipitate was resuspended in gel-loading solution, heated to 90°C for 3 min, and run in a 5% polyacrylamide/TBE gel containing 8 M urea. The gel was exposed to Kodak Biomax MR film and then to Fuji BAS-IIIs phosphorimager screens for quantitation.
Preparation of NLS peptide/biotinylated-BSA conjugates and in vitro binding assay
The cysteine-modified NF-B p50 wild-type (CYN50) and mutant
(CYN50 M) NLS peptides (Table I), were conjugated to BSA and the
peptide-BSA conjugates were biotinylated as described (54). The NLS
peptide/biotinylated-BSA conjugate (20 µg) and BSA (200 µg) were
added to 100 µl of Jurkat T cell whole-cell extract and incubated at
4°C for 60 min with gentle rocking. Aggregates were removed by
centrifugation in a microcentrifuge at 10,000 rpm for 10 min (4°C).
The supernatant was incubated at 4°C for 60 min with 25 µl of
packed ultralink immobilized streptavidin (Pierce). Following
incubation, bound proteins were washed six times with wash buffer (20
mM Tris-Cl, pH 7.5, 80 mM NaCl, and 2 mM MgCl2) and eluted
with 200 µl of 1 M MgCl2. Eluted proteins were
precipitated with 10% TCA, run on an 8% SDS-PAGE mini gel, and
Western blotted with Abs to Rch1 and importin-ß.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, AP-1,
NFAT, and STAT1
Jurkat T cells were stimulated with either a combination of PMA
and ionomycin or IFN-. The combination of PMA and ionomycin mimics
costimulation through the TCR and CD28 (55). The level of transcription
factor nuclear import was determined by measuring DNA-binding activity
using an EMSA performed on nuclear extracts. As shown in Fig. 1, the DNA-binding activities of NF-B,
AP-1, NFAT, and STAT1 were maximally induced by 30, 60, 10, and 10 min,
respectively. The relatively slower rate of induction of AP-1
DNA-binding activity in nuclear extracts is consistent with the
requirement for de novo AP-1 synthesis following agonist stimulation.
Because NFAT DNA binding to a human DNA probe requires AP-1, the
kinetics of NFAT induction are coincident with that of AP-1. However,
NFAT binding to a murine DNA probe (50), independent of AP-1
complexation, showed a rapid kinetics. During the same experimental
time period, the constitutively expressed nuclear CCAAT-binding factor,
NF-Y, remained unchanged with either PMA/ionomycin or IFN-
treatment. The identity of the proteins in mobility-shifted complexes
was confirmed by Ab supershift analysis using antiserum specific for
each transcription factor subunit (data not shown).
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We have previously shown that a cell-permeable peptide carrying
the NLS from NF-B p50 (SN50, Table I) blocks the nuclear import of
NF-B in endothelial and monocytic cell lines (46). Because AP-1 and
NFAT contain NLSs that differ from the p50 NLS, and the STAT1 NLS has
not yet been identified, we used the SN50 peptide to test whether the
signal-dependent nuclear import of these transcription factors can be
regulated by the same NLS peptide that blocked NF-B nuclear
translocation. As shown in Fig. 2, the
SN50 peptide inhibited not only NF-B nuclear import but also the
nuclear import of AP-1, NFAT, and STAT1. In respect to NFAT, inhibition
was observed toward both its forms, one requiring AP-1 for DNA binding
and detectable with human DNA probe (NFAT/AP-1 in Fig. 2), and a second
form that does not require AP-1 complexation for binding to murine DNA
probe (NFAT in Fig. 2). The inhibitory activity of the SN50 peptide was
concentration-dependent (Fig. 2) and was evident for up to 2 h of
stimulation (data not shown). Within the time frame of this experiment,
the constitutively expressed NF-Y was not affected by the SN50 peptide.
Nevertheless, a possibility of reduction of NF-Y content in the nuclear
extract by longer treatment of cells with SN50 peptide cannot be
excluded.
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used to stimulate the
cells. As shown in Fig. 3, the SN50
peptide (75 µM) efficiently inhibited STAT1 nuclear import in cells
stimulated with a low dose of IFN- (1 U/ml), but this inhibitory
effect was not observed in cells stimulated with a high dose of IFN-
(100 U/ml). In this experiment, it is apparent that the levels of STAT1
DNA-binding activity in nuclear extracts from cells stimulated with low
or high doses of IFN- were similar (Fig. 3). Also, we have found
that STAT1 DNA-binding activity in cytoplasmic extracts from cells
stimulated with low and high doses of IFN- were similar (data not
shown). Together, these data indicate that the loss in efficacy of the
SN50 peptide at high doses of IFN- is not caused by an increased
cytoplasmic pool of activated STAT1 saturating the peptide inhibition
of the import process.
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B, AP-1, NFAT, and STAT1 was not affected when SN50
peptide (20 µM final concentration) was added to EMSA reactions (data
not shown). Nuclear import inhibition by the SN50 peptide requires the NLS motif
To determine whether the observed inhibition of NF-B, AP-1,
NFAT, and STAT1 nuclear import by the SN50 peptide was dependent on the
p50 NLS motif, we assessed the inhibitory effect of cell-permeable
peptides containing mutations in the NLS sequence. Fig. 4A shows that the inhibitory
effect of the SN50 peptide on the nuclear import of all four
transcription factors was significantly reduced when only 2 of 10 NLS
residues were mutated (SN50M). The inhibitory effect was completely
lost when 7 of 10 NLS residues were mutated (SM). To confirm the EMSA
results, Western blots were performed on the same nuclear extracts. As
shown in Fig. 4B, SN50 peptide caused a significant
reduction in the intranuclear levels of NF-B p50, c-Jun, and NFATc
proteins. The mutant peptide, SN50M, was much less effective in
blocking transcription factor nuclear import. However, some reduction
in the intranuclear levels of these proteins, in particular NFATc, was
evident in cells treated with the SN50M peptide and this result was
consistent with the reduction in the DNA-binding activities observed by
EMSA (Fig. 4A). In these studies, the SM peptide did not
significantly affect the intranuclear levels of any of these
transcription factors. As expected, no inhibitory effect was seen with
the p50 NLS peptide lacking the cell-permeable hydrophobic sequence
(N50) necessary for cellular delivery of the peptides (46).
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) (46), this assay demonstrated that both cell-permeable
peptides reached an intracellular level corresponding to approximately
4% of the total peptide added to the cells, whereas the
non-cell-permeable N50 peptide was not detected (data not shown).
The appearance of AP-1 in the nucleus following cellular activation
requires de novo synthesis of c-Fos and c-Jun (57). Therefore, it is
possible that the inhibitory effect of the SN50 peptide on AP-1 nuclear
import might be due to inhibition of the nuclear import of other
factors necessary for expression of these genes. We tested this
possibility by measuring the level of AP-1 in cytoplasmic extracts of
peptide-treated and control cells after PMA/ionomycin stimulation. As
determined by EMSAs of concentrated cytoplasmic extracts, cells
stimulated for 60 min in the presence of the SN50 peptide contained
approximately 60% AP-1 present in the cytoplasm of control cells (Fig. 4C). In contrast, SN50 caused near complete (>97%)
inhibition of AP-1 nuclear import as compared with control cells (Fig. 4A). These results indicate that SN50 exerted a twofold
effect on AP-1: First, by decreasing the amount of c-Fos and c-Jun
synthesized and second, by inhibiting the nuclear import of subunits
that are synthesized.
Inhibition of transcription factor nuclear import blocks IB
and IL-2 gene expression
Because the induction of IB gene transcription is dependent
primarily on NF-B (58, 59, 60, 61), we determined whether the SN50 peptide
blocked IB expression. Fig. 5A shows that, in the absence
of the SN50 peptide, IB was rapidly degraded within 20 min after
PMA/ionomycin stimulation and that the steady-state levels of IB
were reestablished after 90 min due to its rapid de novo synthesis. To
evaluate the effect of the SN50 peptide on the normal process of
IB degradation and resynthesis, we prepared cytoplasmic extracts
from peptide-treated and control cells 20 and 90 min after stimulation
with PMA/ionomycin. As shown in Fig. 5B, IB
degradation proceeded normally in the presence of SN50 peptide and the
other peptides used in this study, indicating that they did not
interfere with extracellular signal-induced phosphorylation and
proteolysis of IB. However, 90 min after stimulation a
concentration-dependent inhibitory effect of the SN50 peptide on
IB resynthesis was observed, while neither the SM nor the N50
control peptide significantly inhibited IB resynthesis. Thus,
inhibition of nuclear import of NF-B by SN50 peptide resulted in
concomitant inhibition of IB gene expression.
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B, AP-1, and
NFAT (62). Therefore, we investigated whether blockade of the nuclear
import of these transcription factors by the SN50 peptide exerted an
inhibitory effect on IL-2 gene transcription. First, we analyzed the
kinetics of IL-2 mRNA induction in Jurkat T cells in the absence of
peptide. Using a quantitative ribonuclease protection assay, we found
that IL-2 mRNA reached easily detectable levels after 90 min of
stimulation with PMA/ionomycin (data not shown). Consequently, we
determined the effect of the SN50 peptide on IL-2 mRNA synthesis 90 min
after stimulation. As shown in Fig. 6,
the SN50 peptide caused a concentration-dependent inhibition of
inducible IL-2 mRNA synthesis, but did not inhibit the constitutive
synthesis of ß-actin mRNA. At the concentration of 75 µM used in
many experiments, the inhibition of IL-2 mRNA synthesis by SN50 peptide
was 52 ± 7.5% (mean ± SEM).
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Our next objective was to determine whether inhibitory NLS peptide
interacts with the known cytoplasmic NLS receptor complexes expressed
in Jurkat T cells. As shown in Fig. 7A, Rch1 and importin-ß were
detected in Jurkat T cell extracts. In these experiments, purified
recombinant Rch1 and importin-ß were included as positive controls
for Ab reactivity. To determine whether the Rch1/importin-ß complex
in Jurkat T cells was capable of binding to the NLS sequence of NF-B
p50 NLS peptide, in vitro binding experiments were performed using
either the p50 wild-type (CYN50) or mutant (CYN50 M) NLS peptides (see
Table I) conjugated to biotinylated BSA. To capture NLS receptor
complexes, the NLS peptide/biotinylated-BSA conjugates were incubated
with whole-cell extracts from Jurkat T cells. The bound proteins were
recovered using streptavidin beads and analyzed by Western blotting. As
shown in Fig. 7B, Rch1 and importin-ß interacted with the
wild-type NLS peptide/biotinylated-BSA conjugate. The reduction of Rch1
and importin-ß binding to the mutant peptide/biotinylated-BSA
conjugate indicates the specificity of this interaction. However, there
was residual binding of Rch1 and importin-ß to the mutant peptide.
This residual binding provides an explanation for the observation that
the nuclear import of transcription factors was still partially
inhibited by the mutant peptide in vivo (see Fig. 4). As shown in Fig. 7C the specificity of Rch1 and importin-ß binding to the
wild-type NLS peptide/biotinylated-BSA conjugate was further supported
by the demonstration that this interaction was inhibited by a 50-fold
molar excess of the SN50 peptide used in experiments performed in
intact Jurkat T cells. These results indicate that the inhibitory SN50
peptide targets the Rch 1/importin-ß NLS receptor complex present in
Jurkat T cell extracts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, NFAT,
and STAT, which are sequestered in the cytoplasm, or AP-1 following its
de novo synthesis; 4) import of transcription factors into the nucleus
where they bind to DNA and activate transcription of a large subset of
genes; and 5) nucleocytoplasmic export of some transcription factors,
e.g., NFAT. Because distinct signal transduction pathways are involved
in the mobilization of NF-B, AP-1, NFAT, and STAT1 to the nucleus
following agonist stimulation, it was important to determine whether a
common nuclear import pathway(s) involved in signaling by each of these
transcription factors can be regulated by a single NLS peptide derived
from the NF-B p50 and delivered noninvasively to the cytoplasm of
Jurkat T cells. The results presented here indicate that following
stimulation of Jurkat T cells with PMA/ionomycin or low doses of
IFN- the nuclear import of NF-B, AP-1, NFAT, and STAT1 is
inhibited by SN50 peptide. The same SN50 peptide inhibited the
stimulus-dependent nuclear import of NF-B in endothelial and
monocytic cell lines (46). The SN50 peptide containing NLS made of
basic amino acids is unlikely to interfere with the nuclear export of
NFAT mediated by Ran and CRM1 (63) and enhanced by glycogen synthase
kinase-3 (64).
These data expand the range of inhibition of nuclear import by SN50
peptide to three other transcription factors important for T cell
function in the immune system. Unlike the NF-B and AP-1 proteins
that require classical or bipartite NLSs for nuclear import, the
NFATp/c proteins can be efficiently targeted to the nucleus by a short
sequence having only three basic residues (KRR or KRK) located in the
amino-terminal segment of the protein (30, 31). Calcineurin-mediated
dephosphorylation of nearby serine residues is postulated to expose
this region for binding by the nuclear import machinery (30). The
sequences required for activation-dependent nuclear import of STAT1
have not yet been identified but inspection of the STAT1 amino acid
sequence (65) reveals three clusters of basic amino acid residues that
have been postulated to function as NLSs. However, recent deletion and
mutagenesis experiments have shown that none of these sequences serve
as the NLS of STAT1, and it has been suggested that a unique type of
NLS is present within this protein (32).
We found that the SN50 peptide efficiently inhibited the nuclear import
of STAT1 in Jurkat T cells stimulated with low doses of IFN-, but
with high doses this inhibitory effect was significantly decreased. In
contrast, we have determined that SN50 peptide inhibition of NF-B,
AP-1, and NFAT nuclear import does not change even when a 20-fold
higher concentration of agonist PMA, in combination with ionomycin, is
used to stimulate the cells (data not shown). Because the levels of
activated STAT1 in both nuclear and cytoplasmic extracts of Jurkat T
cells are not significantly different at low and high doses of IFN-,
the insensitivity of STAT1 nuclear import to inhibition by the p50 NLS
peptide at high doses of this cytokine does not appear to be simply a
consequence of increased cytoplasmic pools of activated STAT1
overcoming peptide inhibition of the import process. We have also
observed in THP-1 monocytic cells expressing much higher concentration
of STAT1 that at both low and high doses of IFN- tyrosine
phosphorylation of STAT1 occurs normally and is unaffected by the
presence of the SN50 peptide, thereby demonstrating that SN50’s effect
on STAT1 is not at the level of signal transduction (T.R.T. and J.H.,
unpublished observations). These findings suggest the intriguing
possibility that there are biochemical differences in the mechanism of
STAT1 nuclear import that depend on the concentration of IFN- and
that an alternative import pathway, resistant to inhibition by a
classical NLS peptide, is used at higher concentrations. In other cell
types (HeLa and 293T), high concentrations of IFN- (
2000 U/ml)
induced nuclear import of STAT1 mediated by NPI-1 (hSRP1). Moreover,
NLS peptides derived from wild-type SV40 T Ag did not inhibit
interaction between NPI-1 and STAT1 (32). These results support our
findings that high doses of IFN- induce nuclear import of STAT1 that
is insensitive to inhibition by NLS peptide. The involvement of
transportin (karyopherin-ß2) in this alternative pathway
seems unlikely because there are no regions within STAT1 with homology
to the M9 sequence of hnRNP A1 and transportin pathway is not sensitive
to inhibition by peptides representing classical NLS sequences (43, 44, 65). Nevertheless, nuclear import of STAT1 requires IFN--induced
phosphorylation of tyrosine 701, dimerization of the subunits, and the
GTPase activity of Ran/TC4 (66). The latter is needed for translocation
of karyophilic proteins across the nuclear pore following their
recognition and docking by either importin-ß
(karyopherin-ß1) or transportin
(karyopherin-ß2) NLS receptors (43).
We show that an inhibitory SN50 peptide containing the NLS of NF-B
p50 (SN50) binds to the Rch1 (importin-)/importin-ß heterodimer.
This interaction is specific for the NLS because mutation of two basic
amino acid residues within the sequence significantly reduces binding
of the peptide to Rch1 and importin-ß. These data suggest that the
mechanism by which the p50 NLS peptide inhibits nuclear import of
NF-B, AP-1, NFAT, and STAT1 involves direct disruption or blockade
of the importin-ß-transcription factor interaction. Because Rch1
contains the NLS-binding domain (67), it is likely that the p50 NLS
peptide binds directly to this subunit of the importin-ß complex,
an interpretation supported by the observation that the SN50 peptide
(see Table I) binds recombinant Rch1 but not importin-ß in vitro
(data not shown). The interaction of NLS peptides derived from
different karyophilic proteins in human T lymphocytes varies in respect
to different importin- (karyopherin-) proteins (68). It is
plausible that more than one importin- (karyopherin-) protein
interacts with transcription factors analyzed in these experiments.
The inhibition of the nuclear import of more than one transcription
factors by SN50 peptide as reported herein has practical implications
in view of growing use of this reagent as an inhibitor of the NF-B
nuclear translocation (69, 70). Interpretation of these data should
take into account the expanded range of SN50 peptide inhibitory
activity toward nuclear import of transcription factors other than
NF-B. The SN50 peptide also inhibited inducible expression of the
IB and IL-2 genes, processes dependent on NF-B alone or the
combination of NF-B, AP-1, and NFAT (58, 59, 60, 61, 62). In contrast to the
IB (MAD3) gene regulated primarily by NF-B, expression of IL-2
requires complex synergy between NF-B, AP-1, and NFAT and can
proceed when relatively low concentrations of these factors along with
constitutively nuclear factors are attained in the nucleus. Thus,
partial inhibition of nuclear import of NF-B, AP-1, and NFAT by the
SN50 peptide at 50 µM was apparently insufficient to cause
significant inhibition of IL-2 mRNA expression. Higher concentrations
of the SN50 peptide (75–100 µM) are required to achieve 50%
inhibition of IL-2 mRNA expression at 90 min following stimulation of
Jurkat T cells. This is, to our knowledge, the first evidence that gene
expression can be regulated in intact cells by the inhibition of
transcription factor nuclear import at the level of NLS recognition.
These findings are significant because they offer a new strategy to
exert an immunosuppressive effect on intact, activated T cells through
blockading nuclear import of key transcription factors involved in T
cell immune function.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jacek J. Hawiger, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232; E-mail address:
3 Abbreviations used in this paper: NF-B, nuclear factor- binding; AP-1, activator protein-1; NFAT, nuclear factor of activated T cells; NLS, nuclear localization sequence; EMSA, electrophoretic mobility shift assay.
Received for publication June 2, 1998. Accepted for publication July 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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