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* 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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 proteasome-dependent 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 by specific binding to the CRM1 (23, 24, 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). GM-CSF 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-phospho-Stat5A/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 hGM-CSFR and hGM-CSFR (Ba/F-GMR) and
Stat5B or its mutants were selected and maintained in the same type of
medium with 500 µg/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'-CTGACGGATTCGTGAAGCCACAGATCA-3' (sense) and 5'-CTAGTCTAGCGGCCGCTTGTCGTCGTCGTCCTTGTAGTCCATGGTGGCGACCGGTG-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 µg/ml in PBS and 1% BSA followed by FITC-rabbit anti-mouse IgG Ab (Zymed Laboratories, South San Francisco, CA) at 15 µg/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 µl of OPTI-MEM (Life Technologies, Tokyo, Japan) were mixed with
plasmid DNA and electroshocked with a gene pulser (Bio-Rad) at 960
µF, 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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. 1
A, lanes 1 and
2) and then disappeared from the nucleus 2–4 h after the
depletion of hGM-CSF (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.
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-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. 1
B).
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).
Addition of LMB leads to the accumulation of Stat5B in the nucleus in the absence of cytokine stimulation
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.
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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.
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, 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. 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-terminal 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.
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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/L660A-EGFP 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 -casein-luciferase 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.
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B). 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 the nuclear export of this mutant
(578723-F-EGFP), but further deletion up to 611 (611723-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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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-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
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.
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 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
To confirm the essential role of CRM1, we examined the CRM1/Stat5B interaction by coimmunoprecipitation using anti CRM1 or Stat5B Abs in Ba/F3 or COS7 cells, but we have not yet succeeded in obtaining clear results. As deduced from the high specificity of LMB for CRM1, the involvement of CRM1 is feasible but identification of the role of NES, which is essential, remains to be clarified as an important issue. Studies on the three-dimensional structure of Stat1 and Stat3 (58, 59) revealed that Stats dimerize through their SH2 domain and amino acids surrounding the phosphorylated tyrosine residue. As the SH2 domain serve as a binding site of homodimerization, this region may be masked during dimer formation and unmasked after dephosphorylation of tyrosine 699 followed by monomerization in the nucleus. Although there is no information on the monomer structure of Stat5B, it is possible that some intra- or intermolecular masking mechanism is involved in the regulation of the subcellular localization of Stat5B. This notion is supported by reports that artificial dimerization of Stats without tyrosine phosphorylation triggered nuclear translocation of Stats (60, 61).
We propose a model for the nucleocytoplasmic transport of Stat5B as
shown in Fig. 5. In the absence of
cytokine stimulation, the nonphosphorylated, monomeric form of Stat5B
shuttles continuously between the nucleus and the cytoplasm. Because
nonphosphorylated Stat5B is mainly located in the cytoplasm in the
absence of cytokine stimulation, the nuclear export rate may be much
faster than the nuclear import rate. Upon cytokine stimulation, Stat5B
is phosphorylated and dimerized, thereby masking the region responsible
for the CRM1-dependent nuclear export, leading to nuclear
translocation. When the stimulation is terminated, Stat5B dimers are
tyrosine dephosphorylated and cannot sustain their dimer form, and the
NES-like region within the SH2 domain is again exposed. By means of
this process, Stat5B monomers are sent back to the cytoplasm for the
next activation-inactivation cycle.
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Acknowledgments |
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Footnotes |
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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: sumiko{at}ims.u-tokyo.ac.jp
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.
Received for publication January 16, 2002. Accepted for publication February 21, 2002.
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-activated STAT1 by the ubiquitin-proteasome pathway. Science 273:1717.-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5. EMBO J. 19:392.[Medline]
subunit regulating multiple signaling pathways of granulocyte-macrophage colony-stimulating factor receptor. Mol. Cell. Biol. 18:742.c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol. Cell. Biol. 14:4335. promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8:466.-induced apoptosis in Stat1null cells due to low constitutive levels of caspases. Science 278:1630.-mediated signaling. Cell 96:121.[Medline]
-dependent nuclear import of Stat1 is mediated by the GTPase activity of Ran/TC4. J. Biol. Chem. 271:31017. homodimer bound to DNA. Nature 394:145.[Medline]
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