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(Received for publication, August 13, 1996, and in revised form, October 15, 1996)
From the Department of Anatomy and Cell Biology and the
§ Division of Molecular Oncology, Biomedical Research
Center, Osaka University Medical School, 2-2 Yamada-oka, Suita,
Osaka 565, Japan
ABSTRACT In response to interferon- INTRODUCTION Homeostasis in cells is maintained by the exchange of molecules,
such as proteins and RNAs, through the nuclear pore complex (NPC)1 in response to intracellular changes
and extracellular signals. Selective nuclear import of karyophilic
proteins has been shown to be directed by short amino acid sequences
termed nuclear localization signals (NLSs). This transport process into
the nucleus can essentially be divided into two steps: (a)
NLS-dependent binding to the cytoplasmic face of the
nuclear pore followed by (b) an energy-dependent
translocation through the NPC.
During the first step of nuclear import, an NLS-containing protein
forms a stable complex with ~60- and 97-kDa proteins to target the
nuclear pores (1, 2, 3, 4, 5, 6, 7, 8, 9). In subsequent steps, which require a small G
protein, Ran, the NLS-containing protein is translocated into the
nucleus (10, 11). Experiments using nonhydrolyzable GTP analogues in an
in vitro transport assay with digitonin-permeabilized cells
or overexpression of mutant yeast Ran, which is stabilized in GTP-bound
form, indicated that hydrolysis of GTP by Ran is requisite for nuclear
import (10, 11, 12). These factors have been shown to participate in the
nuclear import of single (for example, SV40 large T-antigen) or
bipartite (for the case of nucleoplasmin) basic type NLS containing
proteins, which are constitutively transported into the nucleus
immediately after their synthesis in the cytoplasm. The mechanism and
the factors that are required for extracellular
signal-dependent nuclear transport of proteins that
pre-exist or are anchored in the cytoplasm, however, remain
unknown.
Studies of cytokines have revealed essential components of the cytokine
signaling pathway. A family of latent cytoplasmic transcription factors
termed Stats (proteins acting as signal transducers and activators of
transcription) are activated by a variety of cytokines and growth
factors and are generally thought to play a pivotal role in the
function of these factors (13, 14, 15). For example, in response to
IFN- This study, which focuses on Stat1 as a model protein, was undertaken
in order to better understand the machinery required for extracellular
signal-induced protein import into the nucleus. To understand the
mechanism of IFN- MATERIALS AND METHODS Full-length cDNA of
human Stat1 was inserted into pGEX-5X-3 (Pharmacia Biotech Inc.) in
frame with a tag encoding an influenza virus hemagglutinin (HA)
epitope, YPYDVPDYA, at the N-terminal end. Recombinant Stat1 was
expressed by 0.1 mM IPTG for 20 h at 20 °C in
Escherichia coli strain BL21(DE3) as GST-fusion proteins and
purified with glutathione-Sepharose (Pharmacia) following the
manufacturer's recommendations. The GST portion was cleaved with
Factor Xa, and Stat1 was further purified by gel chromatography. Mutant
Stat1s were expressed and purified as wild type.
pET-3d expression constructs of wild type and mutant Ran proteins were
introduced into E. coli strain BL21(DE3). Recombinant Ran
proteins were expressed by 1 mM IPTG for 6 h at
37 °C (wild type) or for 12 h at 20 °C (mutants) and
purified essentially as described previously (19). 25 mM
EDTA and 2 mM GTP (for GTP-Ran and GTP-Ran-G19V) or GDP
(for GDP-Ran, GDP-Ran-G19V, and GDP-Ran-T24N) were added to Ran, and
after incubation for 1 h on ice, MgCl2 was added to a
final concentration of 50 mM. The samples were then
subjected to a Superose 12 column (Pharmacia), equilibrated with
transport buffer (20 mM HEPES, pH 7.3, 110 mM
potassium acetate, 5 mM sodium acetate, 2 mM
magnesium acetate, 0.5 mM EGTA, and 1.5 mM
2-mercaptoethanol). The nucleotides bound to Ran were determined by
HPLC (20).
Mutations were introduced by the method using polymerase chain reaction
(21) with appropriate oligonucleotides. All mutants were verified by
DNA sequencing.
To prepare SV40 T-antigen NLS conjugated BSA (SV40 T-NLS-BSA), the
synthetic peptides (CYGGPKKKRKVEDP) were chemically conjugated with BSA
as described previously (22).
Rabbit anti-Ran antibodies were prepared by
subcutaneous injection of purified recombinant Ran and affinity
purified by purified full-length Ran-conjugated Sepharose 4B. For
microinjection experiments, preimmune IgG and affinity purified
anti-Ran antibodies were concentrated by ultrafiltration using
Centricon 30 (Amicon). The affinity purified anti-Ran antibodies were
found to inhibit the nuclear import of SV40 T-NLS-BSA in
vivo.2 Anti-HA (12CA5) antibody was
purchased from Boehringer Mannheim, and monoclonal anti-Stat1 antibody
was purchased from Transduction Laboratory.
Cells were
grown on coverslips in Dulbecco's modified Eagle's minimal essential
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum. Purified recombinant Stat1 (3 mg/ml) was microinjected into the
cytoplasm, and cells were then treated with IFN- RESULTS AND DISCUSSION In order to investigate the mechanism of
IFN-
It is well known that certain WGA-reactive nuclear pore proteins appear
to play an important role in nuclear transport (23, 24, 25) and that WGA
inhibits NLS-mediated active nuclear import (26, 27, 28). When WGA was
co-injected with the recombinant Stat1, the
IFN- IFN-
It is well known that a small G protein,
Ran, is essential for SV40 T antigen-NLS containing substrates to
translocate through the nuclear pore in vitro (10, 11). In
order to determine if Ran also mediates IFN-
Ran has GTPase activity and its activity is known to be essential for
translocation of SV40 T-NLS-BSA (10, 11, 12). In order to determine if GTP
hydrolysis on Ran is required for IFN-
The loss of activity of chromatin-associated protein RCC1, which
mediates the GDP-GTP exchange of Ran (33), resulting in the
accumulation of GDP-bound Ran, leads to suppression of nuclear import
of NLS-BSA (34). To better understand the effect of GDP-Ran on nuclear
import of Stat1, we prepared another mutant Ran protein, Ran-T24N,
which has little or no binding affinity to GTP but has GTPase activity
and thus accumulates in the GDP-bound form and binds tightly to RCC1
(35). The guanine nucleotide binding to Ran-T24N loaded with GDP
(GDP-Ran-T24N) was determined to be nearly 100% GDP. When wild type
GDP-Ran was microinjected with Stat1, Stat1 was transported in a normal
fashion to the nucleus as described above. In contrast, injection of
the GDP-Ran-T24N blocked nuclear import of Stat1 (Fig. 4) as well as
SV40 T-NLS-BSA (data not shown) in vivo, although the
inhibition was less effective than that by GTP-Ran-G19V. This is
consistent with recent data that show that nuclear import of SV40 T
antigen-NLS conjugated substrate and snRNP was inhibited by Ran-T24N
in vitro (31). These results suggest that GDP-bound mutant
Ran reduces both constitutive and conditional nuclear import of
proteins, although further experiments are required to understand the
exact mechanism by which GDP-bound mutant Ran suppresses the nuclear
import.
In this study, we established an in vivo system that is
useful for understanding extracellular signal-dependent
nuclear import of proteins. The experimental evidences presented here
indicate that Stat1 is actively transported in response to IFN- FOOTNOTES REFERENCES
Volume 271, Number 49,
Issue of December 6, 1996
pp. 31017-31020
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
-dependent Nuclear Import of Stat1 Is
Mediated by the GTPase Activity of Ran/TC4*
,,
(IFN-), Stat1
enters the nucleus, where it activates transcription. In order to
better understand the mechanism of the extracellular signal-induced
protein import into the nucleus, we have established an in
vivo assay system that uses recombinant Stat1 protein as a model
transport substrate. Using this system, we found that Stat1 is actively
transported through the nuclear pores in an
IFN--dependent manner and tyrosine (Tyr701)
phosphorylation of Stat1 is actually required for its nuclear import.
When the antibody against Ran, which was identified as an essential
factor for active nuclear protein transport, was injected, the
IFN--dependent nuclear transport of Stat1 was completely inhibited. Furthermore, nuclear import of Stat1 was suppressed by
microinjection of two mutant Ran proteins, one defective in GTP
hydrolysis (G19V) and the other with little or no binding to GTP
(T24N), both of which are known to act as dominant negative inhibitors
of nuclear import. These results indicate that the conditional nuclear
import of Stat1 requires GTP hydrolysis by Ran.
, a specific tyrosine residue (Tyr701) of Stat1 is
phosphorylated and is then translocated into the nucleus where it
directly activates target genes by binding to specific promoter
sequences (16, 17, 18). In contrast to the detailed understanding of the
activation step and the binding of Stat1 to DNA, the NLS of Stat1 has
not yet been identified, and very little is known about the
translocation step from the cytoplasm to the nucleus in this signaling
pathway.
-dependent nuclear translocation of
Stat1, we established and used an in vivo system in which
recombinant Stat1 translocates from the cytoplasm to the nucleus, after
cytoplasmic injection, in response to IFN-. The data collected
herein clearly show that tyrosine phosphorylation of Stat1 is essential
for its active nuclear import, and Ran/TC4 and its GTPase activity are required for the regulated nuclear import of Stat1.
(50 ng/ml, Genzyme)
for 30 min at 37 °C. Cells were fixed with 3.7% formaldehyde for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 5 min. After blocking with 3% skimmed milk in phosphate buffered saline,
subcellular localization of Stat1 was detected by indirect
immunofluorescence using anti-HA and fluorescein isothiocyanate-labeled
goat anti-mouse IgG. WGA (0.5 mg/ml), antibodies (18 mg/ml) or purified
Ran proteins (3 mg/ml) were co-injected with Stat1 and then cells were
treated with IFN-. For quantitation, photographic negatives were
analyzed by Adobe Photoshop, and fluorescence intensities of cytoplasm and nuclei were calculated by integrating the pixel intensities of the
fluorescence image using MacScope.
-dependent Nuclear Translocation of Recombinant
Stat1
-dependent nuclear translocation of Stat1, HA-tagged
human Stat1 was expressed in E. coli and then purified to
homogeneity (Fig. 1A). The tyrosine residue
(Tyr701) of recombinant Stat1 was phosphorylated by the
cytosol from human 293T cells expressing human Jak1, and the
phosphorylated form showed DNA binding activity in vitro
(data not shown). When the recombinant Stat1 was microinjected into the
cytoplasm of human FL cells, it remained predominantly in the cytoplasm
in the absence of IFN- stimulation (Fig. 1B,
b). However, after 30 min of treatment with IFN-, Stat1
localized in the nucleus in an extracellular
signal-dependent manner (Fig. 1B, c).
Endogenous Stat1 translocates into the nucleus in response to IFN-
within same time frame, as evidenced by indirect immunofluorescence
with anti-Stat1 antibody (data not shown). These findings indicate that
recombinant Stat1 is functionally active and, thus, the transport system developed herein is suitable for examining the intracellular behavior of Stat1, as well as for the assay of transport activity of a
variety of mutant Stat1s, which are exogenously injected.
Fig. 1.
Purification of recombinant Stat1 from
E. coli and IFN--dependent nuclear
translocation of purified Stat1. A, HA-tagged human Stat1
was expressed in E. coli as a GST fusion protein and purified to homogeneity. Left lane, crude extract;
center lane, GST-Stat1 fusion protein purified on
glutathione-Sepharose; right lane, purified recombinant
Stat1. Proteins were subjected to 10% SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Blue. B, purified
recombinant Stat1 (3 mg/ml) was microinjected into the cytoplasm of
human FL cells, and the cells were then treated with (c and
d) or without (b) recombinant human IFN- at 50 ng/ml for 30 min at 37 °C. Cells were fixed with 3.7% formaldehyde, and localization of Stat1 was visualized by indirect immunofluorescence with anti-HA tag. WGA (0.5 mg/ml) was co-injected with Stat1
(d). a, uninjected cells were stained with
anti-HA antibodies, as a control.
[View Larger Version of this Image (37K GIF file)]
-dependent nuclear import of Stat1 was inhibited (Fig. 1B, d), and endogenous native Stat1 was
also inhibited by WGA injecton (data not shown), indicating that Stat1
is actively but not passively transported into the nucleus through the
NPC.
stimulation causes phosphorylation of proteins (such as Jak
kinases), and staurosporine, a kinase inhibitor, which is known to
prevent the activation of Stat1 (16). Moreover, using cell lines that
express wild type and mutant Stat1, it has been demonstrated that
phosphorylation of Tyr701 is required for both nuclear
accumulation of Stat1 as well as the activation of specific genes (18).
In this study, we found that the IFN--dependent nuclear
translocation of Stat1 was inhibited in cells treated with
staurosporine (Fig. 2b). In additional
experiments, tyrosine 701 was substituted with phenylalanine or
glutamic acid by site-directed mutagenesis, and these mutant Stat1s
were purified to homogeneity. Neither of the Stat1 mutants translocated
into the nucleus, even in the presence of IFN- (Fig. 2, c
and d), indicating that phosphorylation, but not negative
charge at this position, is essential for the
signal-dependent nuclear import of Stat1. It has been
proposed that tyrosine phosphorylated Stat1 forms a homodimer through
an interaction between phosphorylated tyrosine and the SH2 domain of
another Stat1 (29). The data presented here are consistent with this
proposal and suggest that tyrosine unphosphorylated Stat1 localizes in
the cytoplasm as a monomer prior to cytokine stimulation, and when the
tyrosine residue is phosphorylated after stimulation, Stat1 forms a
homodimer that then actively translocates into the nucleus.
Fig. 2.
Tyrosine phosphorylation is required for the
nuclear import of Stat1. a, recombinant Stat1 was injected
to the cytoplasm of FL cells, and then the cells were treated with
IFN-. b, FL cells were treated with 0.5 µM
staurosporine for 10 min prior to microinjection of wild type Stat1.
After injection, the cells were then treated with IFN- for 30 min at
37 °C in the presence of staurosporine. c and
d, Y701F or Y701E mutant Stat1 (3 mg/ml) was microinjected
to FL cells and stimulated with IFN-.
[View Larger Version of this Image (42K GIF file)]
-dependent
nuclear import of Stat1, we prepared polyclonal anti-Ran antibodies
that specifically recognized endogenous human Ran of FL cells (Fig.
3A). When the anti-Ran antibodies were
injected with Stat1, IFN--induced nuclear import of Stat1 was
strongly inhibited, whereas preimmune IgG had no effect on the process
(Fig. 3B). Nuclear import of native Stat1 was similarly
inhibited by injection of the antibodies (data not shown). These
results indicate that Ran participates in both constitutive and
extracellular signal-dependent, conditional nuclear import of proteins.
Fig. 3.
Effects of anti-Ran antibodies on nuclear
import of Stat1. A, whole cell extracts of FL cells were
resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted
with preimmune IgG or affinity purified anti-Ran antibodies.
B, preimmune IgG or affinity purified anti-Ran antibodies
(18 mg/ml) were microinjected with Stat1, and the cells were treated
with IFN-.
[View Larger Version of this Image (41K GIF file)]
-dependent nuclear
import of Stat1, we prepared a mutant Ran protein, Ran-G19V, that was
deficient in GTP hydrolysis. The guanine nucleotides binding to wild
type and Ran-G19V loaded with GTP (GTP-Ran and GTP-Ran-G19V,
respectively) were estimated by HPLC to be 70% GTP and 30% GDP and
with GDP (GDP-Ran and GDP-Ran-G19V, respectively) were determined to be
nearly 100% GDP. When buffer, wild type GTP- or GDP-Ran was
microinjected with Stat1, Stat1 was transported in a normal fashion to
the nucleus. In contrast, injection of GDP or GTP bound form of
Ran-G19V strongly inhibited IFN--dependent nuclear
import of Stat1 (Fig. 4). These results indicate that
Ran GTPase is required for IFN--dependent nuclear import
of Stat1. The mutant Ran protein also inhibited nuclear import of SV40
T-NLS-BSA (data not shown), which is consistent with the recent report
that injection of another GTPase-deficient mutant Ran-Q69L blocked the
nuclear import of SV40 T antigen in vivo (30) and that of
SV40 T-NLS conjugated BSA in vitro (31). More recently,
similar results were reported on the nuclear import of snRNP and
glucocorticoid receptor using Ran-Q69L or Ran-G19V (31, 32). These
findings with the Ran-G19V and Ran-Q69L mutant proteins, which are
insensitive to GAP activity, implicate that Ran acts as a common key
factor for constitutive and extracellular signal-dependent
nuclear import of proteins and snRNP import.
Fig. 4.
Effects of microinjected recombinant Ran
proteins on nuclear accumulation of Stat1. Stat1 was microinjected
with buffer (control) or recombinant Ran proteins (3 mg/ml)
in transport buffer, and the cells were treated with IFN-. Relative
nucleus to cytoplasm (N/C) ratio (average values
for 20 cells) was determined as described under "Materials and
Methods."
[View Larger Version of this Image (53K GIF file)]
through the nuclear pore depending on the phosphorylation of a single tyrosine residue (Tyr701). Although the NLS of Stat1 has
not yet been determined, IFN--dependent nuclear import
of Stat1 is mediated by Ran and requires its GTPase activity,
indicating that extracellular signal-dependent active nuclear import of Stat1 shares at least one common key factor, Ran,
which was first identified to support the translocation step of
constitutive nuclear import of SV40 T-NLS-bearing substrate.
Research Fellow of the Japanese Society for the Promotion of
Science.
¶
To whom correspondence should be addressed. Tel:
81-6-879-3211; Fax: 81-6-879-3219.
1
The abbreviations used are: NPC, nuclear pore
complex; IFN-, interferon-; NLS, nuclear localization signal; HA,
hemagglutinin; IPTG, isopropyl-
-D-thiogalactopyranoside;
GST, glutathione S-transferase; HPLC, high performance
liquid chromatography; BSA, bovine serum albumin; WGA, wheat germ
agglutinin.
2
T. Tachibana and Y. Yoneda, unpublished
data.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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