Download -Independent Manner Nucleus in a Cytokine

Stat5B Shuttles Between Cytoplasm and
Nucleus in a Cytokine-Dependent and
-Independent Manner
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of June 15, 2017.
Rong Zeng, Yutaka Aoki, Minoru Yoshida, Ken-ichi Arai
and Sumiko Watanabe
J Immunol 2002; 168:4567-4575; ;
doi: 10.4049/jimmunol.168.9.4567
http://www.jimmunol.org/content/168/9/4567
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Copyright © 2002 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Stat5B Shuttles Between Cytoplasm and Nucleus in a
Cytokine-Dependent and -Independent Manner1
Rong Zeng,*‡ Yutaka Aoki,*‡ Minoru Yoshida,†‡ Ken-ichi Arai,*‡ and Sumiko Watanabe2*
R
oles of Janus kinases (Jaks)3 and Stats in cytokine signal
transduction were first established in IFN signaling pathways (1). Although the cytokine receptor superfamily is
not structurally related to the IFN receptor, the data indicated that
all members of the cytokine receptor superfamily use Jaks and
Stats (2). Furthermore, it is now well known that receptor tyrosine
kinases such as epidermal growth factor or insulin-like growth
factor activate the Jak and Stat pathways (3, 4). As observed initially in the IFN system, accumulating evidence suggests that Stats
are involved in the activation of cytokine-specific genes (3, 5–7),
and knock-out studies of various Stats strongly support this idea (8,
9). To date, Stats 1– 6 with similar structural features have been
identified (10). All of these members have a DNA-binding domain
located in the amino-terminal half, and linker and Src homology 2
(SH2) domains, followed by the transactivation domain, in their
C-terminal half (2). A conserved tyrosine residue is located in the
C-terminal region and phosphorylation of this residue plays an
essential role in dimerization and nuclear translocation of Stats.
Serine residues on the C-terminal side of this tyrosine are phosphorylated by extracellular signal-related kinase, p38 mitogen-activated protein kinase (MAPK), or c-Jun N-terminal kinase, and
*Department of Molecular and Developmental Biology, Institute of Medical Science,
and †Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan; and ‡Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan
Received for publication January 16, 2002. Accepted for publication February
21, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
R.Z. is recipient of the Okazaki International Scholarship.
2
Address correspondence and reprint requests to Dr. Sumiko Watanabe, Department
of Molecular and Development Biology, Institute of Medical Science, University of
Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address:
[email protected]
3
Abbreviations used in this paper: Jak, Janus kinase; SH2, Src homology 2; MAPK,
mitogen-activated protein kinase; NLS, nuclear localization signal; CRM1, chromosome region maintenance 1; NES, nuclear export signal; LMB, leptomycin B; ␤c, ␤
subunit; m, mouse; h, human; EGFP, enhanced green fluorescent protein; PI, propidium iodide.
Copyright © 2002 by The American Association of Immunologists
have been implicated in the transcriptional activity of Stat1 and
Stat3 (11).
Stats exist as monomers in the cytoplasm before receptor activation (1). Cytokine stimulation leads to Jak activation followed
by phosphorylation of tyrosine residues in the cytoplasmic part of
the cytokine receptor. Phosphotyrosine of the receptor recruits
Stats through their SH2 domain, making Jak2 accessible to phosphorylate Stats. Activated Jaks, which phosphorylates the C-terminal tyrosine residue of Stats, leads to Stat dimer formation by
the intermolecular interactions of the SH2 domain and the phosphorylated tyrosine (12). Once dimerized, Stats dissociate from the
receptors and translocate to the nucleus in the dimer form where
they bind to target DNA. Stats are thought to be translocated back
to the cytoplasm after dephosphorylation, which means the regulation of translocation of Stats is important for the regulation of
Stat activation (13). In contrast, the involvement of proteasomedependent or ubiquitin-associated degradation of Stats in the nucleus as a shut-off mechanism has also been proposed (14, 15).
Stat proteins have a mass over the generally considered largest
size for diffusion through the nuclear pore (16), and thus they are
assumed to be actively transported into and of the nucleus. However, the mechanism regulating the subcellular distribution of Stats
is not well understood. Stat1 dimers bind the nuclear import complex importin ␣, ␤ (17), thus indicating the involvement of nuclear
import machinery. Neither classical nuclear localization signals
(NLS) nor regions critical for this process have been found in
Stat1. Chromosome region maintenance 1 (CRM1)/exportin 1 was
identified as the nuclear export receptor (18, 19). CRM1 interacts
with Ras-like nuclear G protein GTPase, and this complex binds to
the nuclear pore to translocate nuclear export signal (NES)-containing proteins (20). NES is a short stretch of amino acids composed of leucine (or hydrophobic amino acid) spaced by two to
three lengths of amino acids. The functions of many signaling
molecules and transcription factors, including protein kinase inhibitor, MAPK kinase, I␬B, p53, and NFAT, were found to be
regulated by CRM1-dependent nuclear export (21). Leptomycin B
(LMB), an antifungal antibiotic blocking the yeast CRM1 function
(22), was recently shown to inhibit NES-dependent nuclear export
0022-1767/02/$02.00
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In response to cytokine stimuli, Stats are phosphorylated and translocated to the nucleus to activate target genes. Then, most are
dephosphorylated and returned to the cytoplasm. Using Ba/F3 cells, we found that the nuclear export of Stat5B by cytokine
depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor chromosome region maintenance
1. Interestingly, LMB treatment in the absence of cytokine led to the accumulation of Stat5B in the nucleus, suggesting that Stat5B
shuttles between the nucleus and the cytoplasm as a monomer without cytokine stimulation. This notion is supported by the
observation that LMB-induced accumulation of Stat5B in the nucleus was also observed with Stat5B having a mutated tyrosine
699, which is essential for dimer formation. Using a series of mutant Stat5Bs, we identified a part of the coiled coil domain to be
a critical region for monomer nuclear import and a more N-terminal region to be critical for the cytokine stimulation dependent
import of Stat5B. Taken together, we propose a model in which Stat5B shuttles between the nucleus and cytoplasm by two different
mechanisms, one being a factor-independent constitutive shuttling by monomeric form, and the other, a factor stimulationdependent one regulated by tyrosine phosphorylation and subsequent dimerization. The Journal of Immunology, 2002, 168:
4567– 4575.
4568
Materials and Methods
Chemicals and cytokines
FCS was purchased from Biocell Laboratories (Carson, CA), and RPMI
1640 and DMEM were from Nikken Biomedical Laboratories (Kyoto, Japan). Mouse (m) rIL-3 expressed in the silkworm Bombyx mori was purified as described (38). Human (h) GM-CSF and G418 were kind gifts from
Schering-Plough (Madison, NJ). Anti-Stat5 Ab (C-17) was obtained from
Santa Cruz Biotechnology (Santa Cruz, CA) and anti-Stat5 mAb was obtained from Transduction Laboratories (Lexington, KY). Anti-phosphoStat5A/B Ab was obtained from Upstate Biotechnology (Lake Placid, NY).
Anti-green fluorescent protein Ab (8362-1) came from Clontech Laboratories (Palo Alto, CA), and anti-FLAG Ab (F3165), from Sigma-Aldrich
(St. Louis, MO).
Cell lines and culture methods
The mIL-3-dependent pro-B cell line, Ba/F3 (39), was maintained in RPMI
1640 medium containing 5% FCS, 0.25 ng/ml mIL-3, 100 U/ml penicillin,
and 100 U/ml streptomycin. For cell washing or depletion, the same medium without mIL-3 was used. Various Ba/F3 cell clones expressing hGMCSFR␣ and hGM-CSFR␤ (Ba/F-GMR) and Stat5B or its mutants were
selected and maintained in the same type of medium with 500 ␮g/ml G418.
A monkey kidney epithelial cell line, COS7, was maintained in low glucose DMEM with 10% FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. Serum depletion was done by washing COS7 cells with DMEM two
times.
Construction and expression of Stat5B mutants
The mammalian expression vector pME18S used in this study contained
the SR␣ promoter (40). pME-Stat5B-enhanced green fluorescent protein
(EGFP) was generated by ligating together the EcoRI-SacI fragment of
Stat5B, which contained aa 1–771, the NotI-EcoRI fragment of the
pME18S vector, and the SacI-NotI fragment of pEGFP-1 (Clontech Laboratories, Palo Alto, CA).
Stat5B-FLAG was constructed by replacing the NarI-NotI fragment of
pME-Stat5B-EGFP with an NarI-NotI-digested PCR fragment amplified
with primers, designed to create FLAG tag DYKDDDDK (eight amino
acids) followed by a termination codon and NotI site. Sequences of the
primers used are as follows: 5⬘-CTGACGGATTCGTGAAGCCACAGA
TCA-3⬘ (sense) and 5⬘-CTAGTCTAGCGGCCGCTTGTCGTCGTCGTCC
TTGTAGTCCATGGTGGCGACCGGTG-3⬘ (antisense). For NES mutants, pME-F570A/F574A-EGFP, ⫺M578A/V580A-EGFP, and ⫺L656A/
L660A-EGFP, all of which contained two amino acid substitutions in the
conserved NES, were prepared. These point mutations were introduced by
PCR mutagenesis and the XhoI (1730)-AgeI (2149)-digested PCR product
containing these mutations at the NES site was used to replace the same
fragment in the wild-type Stat5B. pME-I324A-EGFP, which contained one
amino acid mutation at the NES1 site, was constructed by PCR mutagenesis, and the SapI-BstEII fragment was replaced with the corresponding
fragment of the wild-type Stat5B. NES-EGFP was constructed by ligating
fragments of the NES sequence, which had a start codon ATG at the N
terminus, into pME-EGFP. Construction of F mutants was done as described (41). For a series of N-terminal deletion mutants, the XhoI site
followed by ATG was placed at aa 85, 104, 138, 165 by PCR-based mutagenesis. XhoI and BstEII or XhoI and ApaLI fragments were then used to
replace the same fragment of the wild-type Stat5B. All the PCR products
and junctions of the ligated fragments were confirmed by sequencing, and
the size of proteins was examined by the expression in COS7 cells followed by Western blotting using anti-Stat, GFP, or FLAG Abs.
Subcellular localization of Stat5B
Subcellular localization was examined either by biochemical cell fractionation or histochemical analysis. Biochemical cell fractionation of Ba/F3
cells was done as described (41). Briefly, cells were incubated in buffer (10
mM HEPES (pH 7.9), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM
EDTA, 0.1 mM PMSF) for 15 min on ice, and Nonidet P-40 was added at
the final concentration of 0.6%. The cells were mixed vigorously for 15 s,
then centrifuged. Nuclear proteins were extracted as described (42).
For histochemical analysis, Ba/F3 cells were harvested on glass slides
(Matsunami, Kishiwada, Osaka) by use of Cytospin3 (Shandon, Pittsburgh,
PA) and COS7 cells were cultured in LabTekII chamber slides (Nunc,
Rochester, NY). These cells were fixed with 3.7% (volume/volume) formaldehyde in PBS for 15 min at room temperature. The fixed cells were then
rinsed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 15
min. In some cases, cells were incubated with the anti-FLAG M2 monoclonal Ab at 1 ␮g/ml in PBS and 1% BSA followed by FITC-rabbit antimouse IgG Ab (Zymed Laboratories, South San Francisco, CA) at 15
␮g/ml in PBS. Then, propidium iodide (PI; 5 ng/ml) was applied to stain
the nuclei of the cells. Slide glasses were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and analyzed with a laser scanning confocal imaging system (MRC-1024; Bio-Rad, Hercules, CA) or with a fluorescence microscope (BX50; Olympus, Tokyo, Japan). Images were
processed by using Adobe Photoshop (Adobe Systems, Mountain View,
CA). Each experiment was repeated at least twice, and images representing
over 80% of the total cell population are shown in the figures otherwise
indicated.
Transient transfection and Western blotting
Ba/F3 or COS7 cells were transfected with plasmid DNA by electroporation as described (30, 41). Briefly, cells in 200 ␮l of OPTI-MEM (Life
Technologies, Tokyo, Japan) were mixed with plasmid DNA and electroshocked with a gene pulser (Bio-Rad) at 960 ␮F, 200 V for Ba/F3 cells, or
at 100 V for COS7 cells. Two days after transfection COS7 cells were
lysed and subjected to SDS-PAGE followed by Western blotting. For the
luciferase assay, Ba/F-GMR cells were transfected with the ␤-casein promoter luciferase plasmid (7) by electroporation. After a 12 h of culture in
mIL-3 medium, the cells were depleted of mIL-3 for 5 h and restimulated
with hGM-CSF (10 ng/ml) for 5 h, and then the luciferase activity was
analyzed, as described (43).
Results
Nuclear export of Stat5B in Ba/F3 cells is LMB-sensitive
Many proteins are exported from the nucleus via the CRM1 (exportin 1) system (21). CRM1 recognizes its target protein through
a short stretch of amino acids composed of leucine (or hydrophobic amino acid) spaced by two to three lengths of amino acids
named NES (44, 45). When we examined the sequence of Stat5B,
we found at least three amino acid stretches that matched the consensus of NES; all of these sequences are highly conserved among
other members of the Stat family. LMB is a specific inhibitor of the
NES-dependent nuclear export receptor CRM1 (exportin 1) and
can be used to block the nuclear export pathway (18, 24, 25). We
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by specific binding to the CRM1 (23–25). Quite recently, the inhibition by LMB of Stat1 nuclear export was reported (26, 27).
The cytokines GM-CSF and IL-3 stimulate proliferation and
differentiation as well as promote the survival of various hemopoietic cells (28). GM-CSFR and IL-3R consist of two subunits, ␣
and ␤, both of which are members of the cytokine receptor superfamily (29). The ␣ subunit is specific to each cytokine, and the ␤
subunit (␤c) is shared by GM-CSF, IL-3, and IL-5 receptors (29).
GM-CSF and IL-3 induce tyrosine phosphorylation of ␤c and various cellular proteins and activate expression of early response
genes and cell proliferation in hemopoietic cells and in fibroblasts
(30, 31). The ␤c contains conserved box 1 and box 2 regions and
eight tyrosine residues located in the cytoplasmic region (32). GMCSF activates Jak2 and Stat5 A and B in various hemopoietic cells
(33, 34). Stat5A and Stat5B genes encode proteins that are ⬃95%
identical in amino acid sequence (35). Although Stat5 is activated
by various cytokines such as erythropoietin, IL-3/IL-5/GM-CSF,
prolactin, growth hormone and thrombopoietin, Stat5A and/or
Stat5B knockout mice show a role for physiological responses associated with growth hormone and with prolactin (36). As other
Stats may play pivotal roles for cell differentiation and the function
of various cells, including hemopoietic cells, these results suggest
that Stat5A/B are obligate mediators of mammopoietic and lactogenic signaling rather than of cell proliferation (37).
We analyzed the nuclear import and export of Stat5B through
␤c signals in the mouse IL-3-dependent cell line Ba/F3 and the
monkey kidney epithelial cell line COS7. In addition to the nuclear
import in response to ␤c signals, interestingly, we found that
Stat5B shuttles between the nucleus and the cytoplasm as a nonphosphorylated form regardless of cytokine stimulation. This finding reveals a new and unique feature of Stat5B transport.
NUCLEAR TRANSLOCATION OF STAT5B
The Journal of Immunology
4569
Addition of LMB leads to the accumulation of Stat5B in the
nucleus in the absence of cytokine stimulation
FIGURE 1. LMB-sensitive nuclear export of Stat5 in Ba/F3 cells. A,
Effects of LMB on disappearance of Stat5 from the nucleus were analyzed.
Ba/F-GMR cells were depleted of cytokine and LMB (0, 2, or 11 ng/ml)
was added simultaneously with stimulation by hGM-CSF (10 ng/ml) as
indicated. After 30 min of stimulation, cells were depleted of GM-CSF and
cultured in the depletion medium containing the same concentration of
LMB. Nuclear proteins were extracted and run through SDS-PAGE followed by Western blotting by using anti-Stat5 Ab. B, Nucleocytoplasmic
translocation of Stat5B fused with EGFP or FLAG at the C terminus. Ba/F3
cells stably expressing Stat5B-EGFP or transiently expressing Stat5BFLAG were depleted of mIL-3 for 5 h and then stimulated with mIL-3 (1
ng/ml) for 1 h. Then cells were depleted of mIL-3 in the presence or
absence of LMB. Cells were harvested on glass slides and stained with PI
(for Ba/F3-Stat5B-EGFP), or PI and anti-FLAG Ab conjugated with FITC
(for Ba/F3-Stat5B-FLAG). Images of EGFP and FITC are shown.
asked if LMB affects the nuclear export of Stat5B. We have been
analyzing hGM-CSF receptor signaling in mIL-3 dependent Ba/F3
cells expressing the hGM-CSF receptor (Ba/F-GMR). Ba/F-GMR
can survive and proliferate in the presence of either mIL-3 or
hGM-CSF (30). Ba/F-GMR cells were depleted of mIL-3 for 5 h
and then stimulated with hGM-CSF (10 ng/ml) in the presence or
absence of LMB. Then, hGM-CSF was washed out, and cells were
harvested at indicated time points. The same concentration of the
LMB was present in the washing and depletion medium. Nuclear
proteins were subjected to Western blotting using an anti-Stat5 Ab.
In the absence of LMB, Stat5 accumulated in the nucleus by 30
min of GM-CSF stimulation (Fig. 1A, lanes 1 and 2) and then
disappeared from the nucleus 2– 4 h after the depletion of hGMCSF (lanes 3 and 6). When LMB was added, the disappearance of
Stat5 was inhibited in a dose-dependent manner (lanes 4 and 5) at
2 h after the depletion. Even at 4 h after depletion, the dose-dependent effects of LMB were still evident (lanes 7 and 8). These
results indicate that Stat5 was translocated from the nucleus by a
CRM1-dependent pathway.
In the course of our experiments, we found that the addition of
LMB in the absence of cytokine also resulted in accumulation of
Stat5B in the nucleus. As shown in Fig. 1A, Stat5B disappeared
from the nucleus in the cytoplasm 4 h after cytokine depletion in
Ba/F3 cells. At that time point, we added LMB and examined its
effect on the subcellular localization of Stat5B. As shown in Fig.
2A, Stat5B disappeared from the cytoplasm and accumulated in the
nucleus within 15 min after the addition of LMB. When we transfected COS7 cells with Stat5B-EGFP, Stat5B was exclusively localized in the cytosol (Fig. 2A) because there is no stimulus that
induces the nuclear translocation of Stat5B in COS7 cells. We then
examined the effects of LMB on Stat5B subcellular localization in
COS7 cells. Stat5B began to accumulate in the nucleus by LMB
addition and only a trace amount was detectable in the cytoplasm
after 60 min. These results indicate that Stat5B shuttles between
the nucleus and the cytoplasm, even in the absence of cytokines.
Because FCS contains trace amount of cytokines, we next did similar experiments using COS7 cells in the absence of FCS. As in the
presence of FCS, Stat5B accumulated in nucleus after LMB treatment in the absence of FCS and cytokines, indicating that the
accumulation of Stat5B was not caused by FCS.
Tyrosine phosphorylation of Stat5B is not essential for factorindependent shuttling
Because Stats are believed to form dimers after tyrosine phosphorylation induced by cytokines (12), it is speculated that Stats exist
as monomers in the absence of cytokines. To determine whether
the cytokine independent shuttling of Stat5B was conducted with
Stat5B as monomers, we next constructed a tyrosine mutant of
Stat5B. Tyrosine 699 of Stat5B, which is phosphorylated in response to IL-3 or GM-CSF and plays an essential role in dimerization (46), was replaced with phenylalanine and the mutant was
fused with EGFP or FLAG at its C terminus (Fig. 2B). Ba/F3 or
COS7 cells were transiently transfected with the F mutant constructs, and the effects of LMB on subcellular localization of
Stat5B in the absence of cytokine were examined. As shown in
Fig. 2B (upper panel), Stat5B-F-EGFP or Stat5B-F-FLAG accumulated in the nucleus by adding LMB in the absence of cytokines.
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Because there is another possibility, that Stat5 is activated by
LMB and remains for a longer time in the nucleus, we examined
whether the Stat5 was activated by the addition of LMB. Tyrosine
phosphorylation, DNA binding, and ␤-casein luciferase activation
were not observed by the addition of LMB in the absence of cytokine (data not shown). These results thus indicate that LMB inhibits the nuclear export of Stat5 in Ba/F3 cells.
For more detailed analysis of the nuclear shuttling of Stat5, we
tagged mouse Stat5B with EGFP or FLAG at the C terminus (Fig.
1B). Subcellular localization of Stat5B-EGFP in stable clones of
Ba/F3 cells and of transiently expressed Stat5B-FLAG in Ba/F3
cells was examined under a fluorescence microscope. By cytokine
depletion, both fusion proteins accumulated in the cytoplasm, and
then were translocated to the nucleus after cytokine stimulation.
Although the C-terminal 15 amino acids of Stat5B were deleted at
the junction between Stat5B and EGFP or FLAG, Stat5B-EGFP
and Stat5B-FLAG both behaved in a similar manner as wild-type
Stat5B. Nuclear export of these proteins was inhibited by LMB,
and their tyrosine phosphorylation, DNA binding activity, and
transactivation potential were all equivalent to those observed with
wild-type Stat5B (data not shown). When we added cycloheximide
to block de novo protein synthesis, essentially the same pattern
was observed (data not shown).
4570
NUCLEAR TRANSLOCATION OF STAT5B
In COS7 cells, the addition of LMB also resulted in accumulation
of Stat5B in the nucleus. Taken together, these results suggest that
cytokine-independent shuttling of Stat5B occurs with Stat5B in the
monomer state.
Different region required for factor-dependent and -independent
imports of Stat5B
To determine the region of Stat5B required for subcellular localization, we constructed a series of truncation mutants from the N
terminus of Stat5B, as shown in Fig. 3A. These mutants were transiently expressed in COS7 cells, and the subcellular localization in
the presence or absence of LMB (20 ng/ml, 60 min) was analyzed
(Fig. 3A). All mutants accumulated in the cytoplasm in the absence
of LMB, suggesting that the nuclear export of mutants ⌬N104,
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FIGURE 2. Stat5B shuttles between cytoplasm and nucleus
in the absence of cytokines. A, Effects of LMB on factor-depleted Ba/F3 and COS7 cells. As schematically shown, Ba/FStat5B-EGFP were depleted of cytokine for 5 h. LMB (20 ng/
ml) was added and cells were harvested at the indicated time
points. COS7 cells were transfected with Stat5B-EGFP and cultured for 2 days, then LMB was added and the subcellular localization of Stat5B-EGFP was examined at the indicated time
points. In the FCS depletion experiments, cells were washed
with DMEM 16 h before LMB addition and no FCS was contained in following steps. B, Effect of mutation of tyrosine 699 of
Stat5B on factor-independent shuttling. Mutant Stat5B-EGFP or
FLAG, which contained substitution of tyrosine 699 (Stat5B-FEGFP, Stat5B-F-FLAG) with phenylalanine, was introduced to
Ba/F3 cells and COS7 cells, and the effects of LMB were examined as described in Fig. 1B.
138, and 165 occurred as in the wild type. When LMB was added,
⌬N104 and 138 translocated to the nucleus. In contrast, ⌬N165
remained in the cytoplasm in the presence of LMB, suggesting that
the region between residues 138 and 165 is essential for monomer
import of Stat5B. We next prepared stable clones of Ba/F3 cells
expressing ⌬N104, 138, and 165, and examined the subcellular
localization of the mutant Stat5B in these cells. Ba/F3 cell clones
expressing one of these mutants were depleted of mIL-3 for 5 h,
and then LMB was added and incubation was continued for 60
min. As shown in Fig. 3B (left column), all mutants accumulated
in the cytosol after factor depletion, as expected. ⌬N104-EGFP
and ⌬N138-EGFP accumulated in nucleus in the presence of LMB
as did wild-type Stat5B (right column). Further deletion up to residue 165 (⌬N165-EGFP) abrogated the response to the LMB.
The Journal of Immunology
4571
minal mutants can be tyrosine phosphorylated by mIL-3 stimulation (data not shown), thereby suggesting that the region up to
residue 104 is essential for the step of dimer formation or subsequent nuclear translocation.
A region containing SH2 is sufficient for nuclear export
Taken together, as in COS7 cells, the region between residues 138
and 165 is essential for cytokine-independent import of Stat5B
from the cytoplasm to the nucleus in Ba/F3 cells. Interestingly,
when we examined the nuclear import of factor-induced dimers
(center column, stimulated), none of the mutants could translocate
to the nucleus in response to mIL-3 stimulation. All these N-ter-
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FIGURE 3. Role of the N-terminal region for subcellular localization of
Stat5B in Ba/F3 and COS7 cells. A, Schematic representation of a series of
N-terminal deletion mutants of Stat5B and subcellular localization of these
mutants in COS7 cells. A series of N-terminal deletion mutants of Stat5B
were transfected to COS7 cells and their subcellular localization, in the
presence or absence of LMB, was examined. B, Ba/F3 cells stably expressing N-terminal truncation mutants of Stat5B were depleted of mIL-3 for 5 h
then restimulated with mIL-3 or treated with LMB (20 ng/ml). Subcellular
localization of Stat5B was examined.
We next analyzed the region required for the nuclear export of
Stat5B. Various mutants of Stat5B-EGFP containing a single or
double amino acid substitution at putative NES sites were constructed (Fig. 4A). Stable lines of Ba/F3 cells expressing these
mutants were established, and the subcellular localization was microscopically examined. Mutants I324A-EGFP and M578A/
V580A-EGFP accumulated in the cytoplasm after cytokine depletion (Fig. 4A). Both mutants translocated to the nucleus after
stimulation, and the nuclear export was inhibited by LMB (data not
shown). In contrast, F570A/F574A-EGFP and S656A/L660AEGFP were distributed both in the cytoplasm and nucleus, in the
absence of cytokines. We then analyzed the transcriptional activation potential of these mutants. ␤-casein is a target gene of Stat5,
and the promoter region covering ⬃0.3 kb is sufficient for the
induction by various stimulators including IL-3, GM-CSF (7).
Each NES mutant was introduced into Ba/F-GMR with ␤-caseinluciferase plasmids and the luciferase activity, in the presence or
absence of hGM-CSF, was analyzed. The mutants I324A-EGFP
and M578A/V580A-EGFP activated the ␤-casein promoter as did
the wild type, but the mutants F570A/F574A-EGFP and S656A/
L660A-EGFP could not activate ␤-casein luciferase (data not
shown). We constructed the same set of Stat5B mutants, but without EGFP, and essentially the same results were obtained in Ba/F3
cells (data not shown). These data suggest the possible involvement of either NES2 or NES3 in the nuclear export of Stat5B.
To determine whether one of the isolated Stat5B NES1, 2, or 3
regions would promote EGFP to cytoplasm as a nuclear export
signal, we constructed NES-EGFP in which each NES sequence
was fused with the N terminus of EGFP and the constructs were
introduced into COS7 cells. All the mutant fusion proteins were
localized in the nucleus in the absence of cytokine, and the subcellular localization did not change in the presence of LMB (Fig.
4B). Therefore, it seems clear that none of the putative NES regions alone can support transport of EGFP to the cytoplasm. Based
on these results, we next made deletion mutants from both the N
and C termini of Stat5B. All the mutants were transiently expressed in COS7 cells, and their subcellular localization in the
absence or presence of LMB was analyzed. We mutated tyrosine
699 of these mutants to exclude unexpected phosphorylation and
possible following dimerization with the SH2 region of endogenous Stat5B. N-terminal deletion up to residue 578 (⌬N578) resulted in the loss of NES1 and NES2, and this mutant exclusively
localized in the cytoplasm without LMB and accumulated in the
nucleus with LMB treatment (Fig. 4C). The mutant ⌬N578 redistributed into the nucleus in the presence of LMB, and it may have
been caused by the fact that EGFP tended to accumulate in the
nucleus. To test this hypothesis, we made the same type of mutant
using a FLAG tag. Our hypothesis was supported by the finding
that ⌬N578-F-FLAG localized in the cytoplasm both in the absence and presence of LMB and prolonged LMB treatment resulted in the nuclear localization of this mutant (data not shown).
We further deleted a part of Stat5B from the N terminus, and the
mutant ⌬N675-F-EGFP localized in the nucleus in the presence
and absence of LMB, suggesting that the region between 578 and
675 is essential for the export of Stat5B from the nucleus to the
cytoplasm. To identify the minimum region for the export of
Stat5B, we then deleted further from the C terminus of this mutant.
Deletion from the C terminus up to residue 723 did not abrogate
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NUCLEAR TRANSLOCATION OF STAT5B
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FIGURE 4. Region containing SH2 and NES3 of Stat5B is sufficient to direct EGFP from nucleus to cytoplasm. A, Schematic representation of putative
NES of Stat5B and other Stat family members and subcellular localization of mutants Stat5B-EGFP. Consensus sequences of NES and various Stat5BEGFP with mutation of putative NES are also shown. Ba/F3 cells stably expressing one of the Stat5B (1⬃771-EGFP) mutants containing mutations with
consensus amino acids of putative NES sequence (I324A-EGFP, F570A/F574A-EGFP, M578A/V580A-EGFP, S656A/L660A-EGFP) were depleted of
mIL-3 for 5 h. Subcellular localization was microscopically analyzed using EGFP fluorescence. B, Subcellular localization of EGFP fused with the putative
NES sequence derived from STAT5B. NES-EGFP, schematically represented, was transfected to COS7 cells and subcellular localization, in the presence
or absence of LMB, was examined. C, Deletion mutants Stat5B of N and/or C terminus fused with EGFP were transfected to COS7 cells and subcellular
distribution in the presence or absence of LMB was examined.
the nuclear export of this mutant (578⬃723-F-EGFP), but further
deletion up to 611 (611⬃723-EGFP) disrupted the nuclear export.
Taken together, the data indicate that the region between residues
578 and 723 is sufficient for the export of Stat5B from the nucleus
to the cytoplasm. Because this region contains a putative NES
motif (NES3) and this transport is LMB-sensitive, it seems likely
that the region mediates the CRM1-dependent nuclear export of
Stat5B.
Discussion
Cytokine stimulation-independent nuclear translocation of
Stat5B
Using LMB and various Stat5B mutants, we demonstrated evidence for new features of both the nuclear export and import of
Stat5B. We found that two different types of nuclear import systems and the LMB-sensitive nuclear export are coupled to regulate
The Journal of Immunology
4573
Statc required DIF signaling via sequences located in the N-terminal half of the Dd-Statc, suggesting that some factor-inducible
cue, which cannot be explained by the previous tyrosine-SH2
dimerization model, is required. Because Stat5B translocates to the
nucleus in response to LMB treatment even in the absence of FCS,
such a factor-inducible mechanism of monomer translocation is
less feasible.
Dimerization-dependent nuclear localization
Stat5B subcellular distribution. We found that Stat5B accumulated
in the nucleus in the presence of LMB, even in the absence of
cytokine stimulation. Because Stats are not tyrosine phosphorylated in the absence of cytokine stimulation, this nuclear translocation may have occurred with Stat5B in its monomer form. This
notion was supported by the finding that the Stat5B mutant with
tyrosine 699 replaced with phenylalanine could also translocate to
the nucleus with LMB treatment. Because some mutants that could
translocate to the nucleus cytokine independently could not translocate there by cytokine stimulation, it is likely that the region
required for cytokine-independent and -dependent nuclear translocation differs. These findings led to the idea that two different
mechanisms by which Stat5B is imported to the nucleus are
present. Monomer and dimer translocation of a single molecule
was noted in the case of MAP kinase (47). The size of MAPK (42
kDa) is small enough to pass through the nuclear pore and this
monomer translocation is assumed to occur by passive diffusion. In
contrast, the molecular mass of Stat5B is ⬃94 kDa, which is much
larger than the maximal size for passive diffusion. Therefore, the
cytokine-independent transport of Stat5B must be conducted by
active transport.
Physiological relevance of monomer shuttling of Stat5B
Although there is no suggestion of monomer transport of Stat1,
there are several findings indicating the role of monomer or nonphosphorylated Stat1 in the nucleus. Unphosphorylated Stat1 has
been detected in the nuclei of rat liver cells (48). Stat1 mediates the
constitutive transcription of many genes such as cpp32 or ich-1,
and a mutant Stat1 in which the site of tyrosine phosphorylation
was mutated supports expression of these genes and TNF-induced
apoptosis (49). In the case of low molecular mass polypeptide 2
(LMP2), the same type of mutant Stat1 (Y701F), which does not
form a SH2-phophotyrosine-dependent dimer, binds to the IFN-␥activated sequence element and supports low molecular mass
polypeptide 2 expression (50). In this case, unphosphorylated Stat1
forms dimer through its N-terminal domain. These findings suggest the possibility that shuttling Stat5 mediates constitutive expression of target genes. Recently tyrosine phosphorylation independent nuclear translocation of a Dictyostelium Stat, Dd-Statc,
was reported (51). Interestingly, nuclear translocation of the Dd-
Involvement of NES and CRM1 for Stat5B nuclear exclusion
LMB is a specific inhibitor of the nuclear export receptor CRM1
(24) and we found that the nuclear export of Stat5 was blocked by
LMB. This effect of LMB on Stat1 nuclear export was reported
earlier, based on two independent studies (26, 27). Both of these
investigations and our results indicate multiple NES sequences
within Stat family members and conservation of all these motifs
among members of the Stat family. Nevertheless, all results suggest different regions as important motifs for nuclear export. We
found that two different mutations disrupted the correct subcellular
distribution of Stat5B. But in both cases, the mutant Stat5B was
also distributed in some amount in the cytoplasm rather than exclusively accumulating in the nucleus in the absence of cytokines,
hence, we could not conclude that the phenomenon means inhibition of nuclear export. In addition, these mutants could not be
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FIGURE 5. Schematic representation of Stat5B cytoplasm/nucleus
shuttling
Tyrosine phosphorylation of Stats by Jak2 followed by nuclear
translocation is a generally accepted model for all members of Stat
family and thus much attention has been paid to the mechanism of
the cytokine-dependent import. As is the case for other Stat members, only the tyrosine phosphorylated form of Stat5 was observed
to translocate to the nucleus in response to IL-3 or GM-CSF stimulation in Ba/F or COS7 cells (data not shown). Because the mutant ⌬N104, which cannot translocate to the nucleus upon IL-3
stimulation, was tyrosine phosphorylated by IL-3 or GM-CSF
stimulation (data not shown), we speculate that the mutant ⌬104 is
defective in some steps such as dimer formation and nuclear translocation. Thus, it is probable that the N-terminal region plays an
essential role in nuclear translocation of Stat5B. Proteins that
translocate to the nucleus usually contain the NLS. The best known
NLS consists of a single stretch of basic amino acids or a bipartite
sequence of basic amino acids (21). There is no documentation of
classical NLS within any member of the Stat family, and we also
could not find any consensus NLS within Stat5A and B. The involvement of the N-terminal region or DNA-binding domain in the
nuclear import of Stat1 and Stat5 has been reported (47, 52, 53).
Interestingly, the Stat5B N-terminal coiled coil domain possesses
leucine zipper-like motifs which have the potential to interact with
other proteins. Therefore, we speculate that this region may mediate the interaction of Stat5B with the nuclear import machinery,
probably through some other NLS-possessing adapter molecule.
Indeed, the N-terminal region of Stat5B was reported to interact
with other molecules such as Nmi or protein inhibitors of activated
Stats (54, 55). It remains to be examined if they act as adapters for
the nuclear import of Stats. Nuclear import in the dimer form has
been analyzed in the case of Stat1 induced by IFN-␥ and the involvement of Ran in the nuclear import was evident (56). In this
case, the interaction of IFN ␥ with NPI-1, but not with Rch1, has
been reported (17), but the involvement of any basic cluster of
amino acid stretch was not indicated, and the region responsible
for the interaction was not defined.
There is another report of identification of an essential region
within the DNA-binding domain of Stat5B for nuclear translocation (57). A mutant of this region can be tyrosine phosphorylated
by growth hormone stimulation followed by dimer formation.
4574
tyrosine phosphorylated. There are two different explanations for
the lack of tyrosine phosphorylation of these mutants, one is that
the mutants locate exclusively in the nucleus regardless of cytokine stimulation, and another is that a disrupted conformation of
these mutants resulted in a lack of response to IL-3 stimulation.
We identified that a portion of Stat5B, residues 578 –723, was
sufficient for the CRM1-dependent nuclear export. Interestingly,
this region contains SH2 as well as a consensus NES motif.
Model of Stat5B nucleocytoplasmic translocation
Acknowledgments
We thank Drs. Eisuke Nishida (Kyoto University) and Tohru Itoh (Tokyo
University) for discussion, and Saori Sato for technical assistance.
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sent back to the cytoplasm for the next activation-inactivation
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