From the Department of Microbiology and Cell Science and the § Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida 32611
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ABSTRACT |
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Cytokines such as interferon-gamma
(IFN- The interferons (IFNs)1
are cytokines that use the well studied JAK/STAT pathway for signal
transduction to the nucleus (1). This pathway is initiated by the
binding of the ligand to the extracellular domain of the appropriate
receptor complex followed by the activation of select members of the
JAK family of tyrosine kinases at the intracellular cytoplasmic domain
of the receptor subunits. These tyrosine kinases in turn phosphorylate
appropriate members of the STAT family of transcription factors present
in the cytoplasm, thereby targeting these factors, through unknown mechanisms, for translocation to the nucleus to activate transcription. Transcription factors the size of STATs must be taken into the cell
nucleus by an active transport process through the nuclear pore complex.
Active nuclear import of a large number of nuclear proteins occurs
through the Ran/importin pathway (for review, see Ref. 2). In this
pathway, the nuclear protein initially binds to a heterodimeric nuclear
transport protein called importin which contains a As mentioned above, the nuclear import of STAT1 requires active
transport. Indeed, it has been shown recently that the
IFN- It has been well documented that human and murine IFN- Given the properties of this sequence, we tested in this study whether
the above polybasic sequence in the COOH-terminal domain of IFN- Cell Culture Peptide Synthesis--
Peptides used in this study (see Table I)
were synthesized on a PerSeptive Biosystems 9050 automated peptide
synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl)
chemistry as detailed previously (10).
Preparation of Import Substrates (APC
Conjugates)--
Allophycocyanin (APC) activated with the bifunctional
cross-linker succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate was purchased
from Prozyme (San Leandro, CA) and used according to the
manufacturer's suggestions. Briefly, peptides or human IFN- Nuclear Transport Assays--
Transport assays with mouse A31
cells were based on methods described previously (for review, see Ref.
20). Cells (both A31 and WISH cells) grown on coverslips were washed at
4 °C with transport buffer: 20 mM HEPES, pH 7.3; 110 mM potassium acetate; 5 mM sodium acetate; 2 mM magnesium acetate; 1 mM EGTA; 2 mM dithiothreitol; 10 µg/ml each leupeptin, pepstatin,
and aprotinin; and 2 mM dithiothreitol. Cells were then
permeabilized with digitonin (at 40 µg/ml) in transport buffer for 5 min at 4 °C. After washing with transport buffer, cells were
incubated with the transport reaction mixtures for 30 min at 30 °C.
Complete reaction mixtures (60-µl final volume) contained 20 mM HEPES, pH 7.3; 110 mM potassium acetate; 5 mM sodium acetate; 2 mM magnesium acetate; 1 mM EGTA; 2 mM dithiothreitol; 10 µg/ml each
leupeptin, pepstatin, and aprotinin; 0.5 mM GTP; 2.5 mM ATP; 5 mM phosphocreatine (Calbiochem); 50 units/ml creatine phosphokinase (Calbiochem); approximately 200 nM appropriate import substrate; and 20 µl of rabbit
reticulocyte lysate (untreated; Promega, Madison, WI). Coverslips were
washed in transport buffer containing 1% bovine serum albumin, mounted
on slides, and observed under a fluorescence microscope
(cooled-CCD deconvolution microscopy).
For ATP depletion experiments reticulocyte lysates were first treated
with a mixture of hexokinase (~300 units/ml), glucose (8 mM), and apyrase (0.2 units/ml) at 30 °C for 15 min
before the addition of the rest of the components. For GTP dependence, GTP was omitted from the reaction mixture, and the reticulocyte lysate
was incubated at room temperature with the analog GTP
For peptide competition experiments, peptides were added at 600-fold
molar excess, calculated with respect to APC, in the presence of all
other components except the import substrate. After incubation for 5 min at room temperature, the APC substrate was then added and the
mixture incubated with the cells.
The competence of the putative nuclear localization sequence
within the COOH-terminal domain of IFN- In this study, we used as substrate a peptide corresponding to amino
acids 95-132 of murine IFN-), which utilize the well studied JAK/STAT pathway for nuclear
signal transduction, are themselves translocated to the nucleus. The
exact mechanism for the nuclear import of IFN-
or the functional
role of the nuclear translocation of ligand in signal transduction is
unknown. We show in this study that nuclear localization of IFN-
is
driven by a simple polybasic nuclear localization sequence (NLS) in its COOH terminus, as verified by its ability to specify nuclear import of
a heterologous protein allophycocyanin (APC) in standard import assays
in digitonin-permeabilized cells. Similar to other nuclear import
signals, we show that a peptide representing amino acids 95-132 of
IFN-
(IFN-
(95-132)) containing the polybasic sequence 126RKRKRSR132 was capable of specifying
nuclear uptake of the autofluorescent protein, APC, in an
energy-dependent fashion that required both ATP and GTP.
Nuclear import was abolished when the above polybasic sequence was
deleted. Moreover, deletions immediately NH2-terminal of
this sequence did not affect the nuclear import. Thus, the sequence
126RKRKRSR132 is necessary and sufficient for
nuclear localization. Furthermore, nuclear import was strongly blocked
by competition with the cognate peptide IFN-
(95-132) but not the
peptide IFN-
(95-125), which is deleted in the polybasic sequence,
further confirming that the NLS properties were contained in this
sequence. A peptide containing the prototypical polybasic NLS sequence
of the SV40 large T-antigen was also able to inhibit the nuclear import
mediated by IFN-
(95-132). This observation suggests that the NLS in
IFN-
may function through the components of the Ran/importin pathway utilized by the SV40 T-NLS. Finally, we show that intact IFN-
, when
coupled to APC, was also able to mediate its nuclear import. Again,
nuclear import was blocked by the peptide IFN-
(95-132) and the SV40
T-NLS peptide, suggesting that intact IFN-
was also transported into
the nucleus through the Ran/importin pathway. Previous studies have
suggested a direct intracellular role for IFN-
in the induction of
its biological activities. Based on our data in this study, we suggest
that a key intracellular site of interaction of IFN-
is the one with
the nuclear transport mechanism that occurs via the NLS in the COOH
terminus of IFN-
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
and
subunit.
Nuclear proteins interact with the
subunit, importin
, of the
transporter via a specific nuclear localization sequence (NLS). These
NLSs generally consist of a cluster of basic amino acids or two short
clusters separated by a spacer of variable length (a bipartite NLS; 2).
The canonical simple polybasic NLS is represented by the sequence of
the NLS of the SV40 T-antigen, KKKRK. The
subunit, importin
, in
turn mediates the binding of the NLS-importin
complex to the GTPase Ran present at the nuclear pore complex. Nuclear import of this nuclear
protein-bearing complex through the nuclear pore is initiated by Ran
with the subsequent hydrolysis of GTP and ATP and hence is a strictly
energy-dependent process (2).
-activated STAT1 is transported into the nucleus via the
Ran/importin pathway by binding the importin
homolog NPI-1 (3, 4).
NPI-1 is known to interact with nuclear proteins via the above
polycationic NLSs. However, based on current knowledge, no polybasic
NLS has been identified on STAT1 (3, 4). Mutational analysis of STAT1
failed to reveal a conventional NLS on STAT1 which was responsible for
mediating the binding to NPI-1 or nuclear translocation of STAT1 (4).
These studies are consistent with the concept that the necessary basic
NLS required for STAT1 interaction with NPI-1 is provided for by
another molecule associated with STAT1. Based on the results of studies
from our laboratory on the COOH-terminal domain of IFN-
,
IFN-
(95-133), and its interaction with the
chain of the
receptor, we propose that this candidate chaperone function may be
served by the ligand IFN-
(5). Crucial to this hypothesis, as a
first step, is demonstrating that IFN-
contains a functional NLS. In
this report, we have specifically addressed this issue.
are
themselves translocated to the nucleus (6-8). The sequence of murine
IFN-
contains a putative polybasic NLS
126RKRKRSR132 within the above mentioned
COOH-terminal domain. It has been known for some time that this
conserved sequence in murine and human IFN-
is absolutely required
for the biological activities of the corresponding IFN-
(9-16). We
have also shown that both human and murine IFN-
interact with the
cytoplasmic domain of the
chain of the corresponding IFN-
receptor complexes via this COOH-terminal domain containing this
putative NLS sequence (17-19). STAT1 binds to the cytoplasmic domain
of the
chain of the receptor at a downstream site. The interaction
of the COOH terminus of IFN-
with the
chain is dependent on the
presence of the putative NLS sequence and in turn increases the
affinity of the receptor-activated tyrosine kinase JAK2 to an
immediately proximal site on the
chain of the receptor (18). JAK2,
along with JAK1, is involved in the receptor recruitment and
phosphorylation of STAT1, events that precede the subsequent nuclear
translocation of STAT1. Thus, this COOH-terminal domain of IFN-
has
many characteristics that it make a good candidate to be involved in
the chaperoning of STAT1
to the nucleus, probably as part of a
ligand-receptor-STAT1
complex.
can
function as an NLS and facilitate the nuclear translocation of a
heterologous protein. We demonstrate that this sequence is, indeed, an
NLS and can function to import an associated non-NLS heterologous
protein. The NLS functions in the strictly energy-dependent fashion typical of active nuclear import. Competition assays suggest that nuclear import driven by the IFN-
NLS may functionally overlap with the Ran/importin pathway utilized by the SV40 T-NLS.
EXPERIMENTAL PROCEDURES
Mouse 3T3 cells (Balb/c, clone A31;
from ATCC) were grown in ATCC-modified Dulbecco's modified Eagle's
medium containing 10% bovine serum. WISH cells were grown in Eagle's minimal essential medium containing 10% fetal bovine serum. Cells were
plated onto coverslips for 24 h before use.
(Biosource International, Camarillo, CA) reduced with dithiothreitol (50 mM) was coupled at a 1:1 or 1:2 molar ratio
(APC:peptide) in 5 mM MES, pH 6.0, containing 5 mM EDTA. After the initial separation of uncoupled peptides
by gel filtration through an Econo 10DG column (Bio-Rad) in 20 mM HEPES, pH 7.3, any residual peptide was removed by
repeated concentration in the same buffer through a Centricon 50 ultrafiltration unit (MWCO 50,000; Amicon, Inc., Beverly, MA) and the
conjugate stored at 4 °C. Coupling efficiency (two or three
peptides/APC) and peptide removal were established by
SDS-polyacrylamide gel electrophoresis.
S (Calbiochem)
at 0.5 mM before the addition of other components.
RESULTS
to function in nuclear import was evaluated by testing its ability to mediate the nuclear import of a heterologous protein. This was performed using the standard
in vitro nuclear transport assay (20) in
digitonin-permeabilized mouse A31 cells.
(IFN-
(95-132); see Table I) coupled to the heterologous
autofluorescent protein APC to provide a substrate for the assay. As
shown in Fig. 1, essentially a 100% of
the A31 cells accumulated the IFN-
(95-132)-APC substrate in the
nucleus and nucleoli (compare Fig. 1, A and B).
This accumulation was inhibited at 4 °C (Fig. 1C) and was
dependent on the addition of reticulocyte-derived cytosolic factors
(Fig. 1D). Addition of a simple mixture of IFN-
(95-132)
with carrier protein without covalent coupling did not lead to any
nuclear staining (data not shown). These data show that the
IFN-
(95-132) was capable of mediating the nuclear import of a
heterologous protein when coupled to it.
Sequences of peptides used in this study
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Fig. 1.
The peptide IFN- (95-132) mediates the
nuclear import of the heterologous protein APC.
Digitonin-permeabilized mouse A31 cells were incubated for the duration
of the assay (30 min) with the complete import reaction mix containing
IFN-
(95-132)-APC as substrate at 30 °C (panel A) or
at 4 °C (panel C). Panel B is the
phase-contrast image of the field corresponding to panel A.
In panel D the cells were incubated with an import mixture
from which the reticulocyte lysate was omitted.
The transport of karyophilic proteins across the nuclear membrane via
the nuclear pore complex is an active, energy-dependent event (2). It depends strictly on the availability of both ATP and GTP.
This energy-independent binding to the nuclear pore complex leads to a
typical "rimming" pattern at the nuclear periphery (21). As shown
in Fig. 2, the nuclear import of
IFN-(95-132)-APC was strictly energy-dependent.
Cytosolic extracts depleted of ATP did not support the nuclear import
of IFN-
(95-132)-APC into the nucleus of A31 cells (compare Fig. 2,
A and B). In the absence of ATP the
IFN-
(95-132)-APC substrate accumulates as a rim at the nuclear
envelope. In a similar fashion, the absence of exogenous GTP coupled
with the addition of the nonhydrolyzable analog GTP
S also resulted
in failure of transport into the nucleus (Fig. 2C) and
resulted in the accumulation of the substrate at the nuclear periphery.
Thus, these data further demonstrate that the domain represented by
IFN-
(95-132) contains an NLS that functions in a strictly
energy-dependent fashion characteristic of nuclear import
signals (2).
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To establish further the specificity of this NLS-bearing domain, we
performed competition experiments using peptides to block the transport
of the IFN-(95-132)-APC substrate. The peptides used were the
following (see Table I): a peptide containing the NLS of the SV40
T-antigen (SV40 T-NLS); the IFN-
(95-132) peptide; and the peptide
IFN-
(95-125), which is derived from IFN-
(95-132) by deletion of
the polybasic putative NLS, 126RKRKRSR132. The
SV40 T-NLS is one of the best characterized NLSs that utilizes the well
studied Ran/importin pathway for nuclear import (2). It contains an NLS
that is a simple polybasic cluster similar to that in IFN-
(95-132).
As can be seen in Fig. 3, transport of
IFN-
(95-132)-APC was strongly inhibited in the presence of IFN-
(95-132) (compare Fig. 3, A and B) and
the SV40 NLS peptide (Fig. 3C) but not by the shortened
peptide IFN-
(95-125) lacking the putative NLS (Fig. 3D).
We repeatedly saw staining in the cytoplasm whenever the NLS-containing
peptides were added as competitors to block transport compared with
nuclear translocation in their absence.
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Several important conclusions can be drawn from these results. First,
the ability of IFN-(95-132) to inhibit transport of IFN-
(95-132)-APC clearly demonstrates that the nuclear import of
the substrate is specifically driven by the attached peptide IFN-
(95-132), further establishing the specific ability of
IFN-
(95-132) to act as an NLS. Second, the ability of the SV40
T-NLS to inhibit transport of the substrate suggests that the nuclear
import of IFN-
(95-132)-APC occurs through the Ran/importin pathway
that is utilized by the prototypical polybasic SV40 T-NLS. Third, the fact that IFN-
(95-125) that is deleted in the polybasic putative NLS is unable to block the import of IFN-
(95-132)-APC strongly suggests that the NLS is contained in the deleted sequence
126RKRKRSR132. This sequence is very similar to
that of the classical polybasic NLS contained in the SV40 T-NLS. Thus,
it is consistent that the SV40 T-NLS peptide can block the import of
IFN-
(95-132)-APC. We conclude that IFN-
(95-132) contains a
classical polybasic NLS that clearly functions in nuclear import in a
fashion similar to that of the prototypical SV40 T-NLS. The IFN-
NLS
probably utilizes components of the Ran/importin pathway common to the nuclear import pathway of the SV40 T-NLS.
To confirm further that the polybasic sequence
126RKRKRSR132 is responsible for the
nuclear localization properties of IFN-(95-132) we tested directly
the ability of the IFN-
(95-125)-APC conjugate to function as a
substrate in the nuclear import assays compared with
IFN-
(95-132)-APC. As shown in Fig. 4,
in contrast to IFN-
(95-132)-APC (Fig. 4A),
IFN-
(95-125)-APC (Fig. 4B and its phase-contrast image in 4D), which is deleted in the polybasic NLS region, was
not able to function as a nuclear import substrate. We also tested the
ability of a peptide derived from IFN-
(95-132) where the sequence
immediately NH2-terminal to the NLS has been deleted, namely IFN-
(121-132) (see Table I). IFN-
(121-132)-APC retained its ability to function as a substrate for nuclear import and appeared
as effective as IFN-
(95-132)-APC in nuclear import (Fig. 4C). These data clearly show that this polybasic sequence is
necessary and sufficient for the nuclear targeting properties of the
COOH-terminal domain IFN-
(95-132).
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Finally, we demonstrated in these assays that intact IFN- itself has
the ability to mediate nuclear import of a heterologous protein. As can
be seen in Fig. 5, IFN-
-APC was also
transported into the nucleus (Fig. 5A) in a fashion that was
strictly dependent on the presence of cytosolic factors (Fig.
5B). A lower signal was observed in these assays compared
with the transport of peptide-APC conjugates because of the lower
coupling efficiency to APC. Nuclear import of IFN-
-APC was strongly
inhibited by competition with the IFN-
(95-132) peptide and the SV40
T-NLS peptide. These data show that the nuclear import of IFN-
itself is mediated in a fashion similar to IFN-
(95-132) and the
SV40 T-NLS and probably occurs via the classical Ran/importin pathway
for nuclear import.
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DISCUSSION |
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In this report, we have demonstrated that the nuclear
translocation of IFN- occurs via a classical polybasic NLS sequence, 126RKRKRSR132, in the COOH terminus of the
molecule. This sequence functions in the nuclear import of IFN-
via
an active energy-dependent process. Competition studies
suggest that the NLS most likely utilizes components of the
Ran/importin pathway that is common to the nuclear import mechanism of
the prototype of this class of NLSs, namely the SV40 T-NLS. We have
shown previously that this sequence is a critical requirement for the
biological activity of the IFN-
molecule (9, 10). Several similar
studies using human IFN-
have shown that a highly homologous
polybasic region within the COOH terminus is absolutely required for
biological activity (11-16). Thus, in conjunction with these studies
our data show that the nuclear localization of IFN-
via this NLS
serves a crucial role in the biological activity of IFN-
.
Previous studies have suggested a biological role for intracellular
IFN- and IFN-
. These include the observations that (i) human
IFN-
delivered by a liposomal vector was able to activate murine
macrophages (22); (ii) secretion-defective human IFN-
expressed in
murine fibroblasts induced an antiviral state in these cells (23);
(iii) microinjected IFN-
can induce Ia expression on murine
macrophages (24); and (iv) intracellularly expressed IFN-
is also
active and can activate the DNA binding activity of the ISGF3
transcription complex containing STAT1
and STAT2 (25). Thus, the
IFN-
most likely interacts with intracellular element(s) to induce a
biological response. Based on our data in this study we suggest that a
key intracellular interaction is the one with the nuclear transport
mechanism which occurs via the NLS in the COOH terminus of IFN-
.
Our studies in this report have clearly identified for the first time
the NLS in IFN-. Although it appears critical for biological activity of IFN-
, the exact contribution of this NLS to biological function is less clear. Based on previous studies from our laboratory on the interaction of this domain of IFN-
with the receptor and its
effect on signaling components, we have speculated that a role for the
NLS could be as a chaperone for the nuclear localization of activated
STAT1
(5). Recent studies have shown that STAT1
is transported to
the nucleus via the Ran/importin pathway (3, 4). STAT1
binds to the
importin
homolog NPI-1 that mediates the nuclear import of
substrates, like the SV40 T-NLS, in a conventional NLS-dependent fashion (4). However, mutation analysis of
STAT1
has failed to reveal any sequence responsible for nuclear
import of STAT1
(4). These studies are consistent with a role for a
STAT1
-associated molecule that binds importin
and provides the
required NLS, i.e. the interaction of STAT1
with the
Ran/importin system could be indirect. We suggest that the NLS required
for nuclear import of STAT1
may be provided by the ligand IFN-
, which interacts intracellularly at the level of the nuclear transport mechanism. Others have noted the presence of putative NLSs in other
ligands and their receptors (26, 27) and have suggested a broad role
for the nuclear targeting of ligands and/or their receptors in
signaling to the nucleus (26). In fact, recent studies have
demonstrated that interleukin-1 and interleukin-5 possess functional
NLSs (28-30). We have found that when a comparison is made between
such cytokines and their signaling pathways, a remarkable correlation
exists between the ability of a cytokine to activate the JAK/STAT
pathway and the presence of a putative NLS in either ligand or receptor
(5). This raises the intriguing possibility that this model of
ligand/receptor-assisted nuclear translocation of STATs may extend to
other cytokine/receptor systems.
In this regard, as outlined in the Introduction, we have already
demonstrated that the COOH-terminal domain represented by IFN-(95-132) interacts specifically with the cytoplasmic domain of
the
chain of the IFN-
receptor (9, 17-19). This interaction is
also dependent on the presence of the NLS (9, 17). The
chain of the
IFN-
receptor is the subunit that binds activated STAT1
. We have
recently shown using immunofluorescence and immunoprecipitation techniques that after IFN-
treatment of intact WISH cells, the cytoplasmic domain of the
chain of the IFN-
receptor is also translocated rapidly to the
nucleus.2 This is specific to
the
chain of the receptor because the
chain of the receptor
complex, which does not bind STAT1
, did not undergo endocytosis or
nuclear translocation. Furthermore, the pattern of receptor and
STAT1
distribution throughout this process and the kinetics of
receptor
chain and STAT1
translocation to the nucleus are very
similar, consistent with the proposition that these events may be
coupled. Because a putative NLS sequence cannot be identified in the
receptor
chain, a likely scenario is that translocation of the
receptor complex bearing STAT1
after ligand-dependent
activation events is ultimately directed by the NLS in the ligand via
the interaction of the NLS-containing domain in the ligand with the
cytoplasmic domain of the receptor
chain. This scenario can be
tested and is being examined in our ongoing studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA69956 and CA38587 (to H. M. J.). This manuscript is Florida Agriculture Experiment Station Journal Series R-06627.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Microbiology
and Cell Science, Rm. 1052, Bldg. 981, P. O. Box 110700, University of
Florida, Gainesville, FL 32611. Fax: 352-392-5922; E-mail: prem{at}micro.ifas.ufl.edu.
2 J. Larkin III, M. R. Paddy, H. M. Johnson, and P. S. Subramaniam, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
IFN(s), interferon(s);
NLS, nuclear localization sequence;
APC, allophycocyanin;
MES, 4-morpholineethanesulfonic acid;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
JAK, Janus kinase;
STAT, signal transducer and activator of transcription;
NPI-1, nucleoprotein interactor-1..
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