From the Department of Physiology, University of
Michigan Medical School, Ann Arbor, Michigan 48109-0622 and the
Departments of
Microbiology and Immunology,
Cell Biology, and
§§ Medicine, Baylor College of Medicine,
Houston, Texas 77030
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The mechanisms regulating the cellular
distribution of STAT family transcription factors remain poorly
understood. To identify regions of Stat5B required for ligand-induced
nuclear accumulation, we constructed a cDNA encoding green
fluorescent protein (GFP) fused to the N terminus of Stat5B and
performed site-directed mutagenesis. When co-expressed with growth
hormone (GH) receptor in COS-7 cells, GFP-Stat5B is
tyrosyl-phosphorylated, forms dimers, and binds DNA in response to GH
in a manner indistinguishable from untagged Stat5B. In multiple cell
types, laser scanning confocal imaging of GFP-Stat5B co-expressed with
GH receptor shows that GFP-Stat5B undergoes a rapid, dramatic
accumulation in the nucleus upon GH stimulation. We introduced alanine
substitutions in several regions of Stat5B and assayed for
GH-dependent nuclear localization. Only the mutation that
prevented binding to DNA (466VVVI469)
abrogated GH-stimulated nuclear localization. This mutant fusion protein is tyrosyl-phosphorylated and dimerizes in response to GH.
These results suggest that either high affinity binding to DNA
contributes to nuclear accumulation of Stat5B or that this region is
crucial for two functions, namely accumulation of Stat5B in the nucleus
and DNA binding. Thus, we have identified a mutant Stat5 defective in
nuclear localization despite its ability to be tyrosyl-phosphorylated
and to dimerize.
The Signal Transducers and
Activators of Transcription
(STAT)1 family of
transcription factors provide a crucial signaling link between
complexes of cytokine/hematopoietin receptors and Janus (JAK) family
tyrosine kinases at the plasma membrane and gene transcription in the
nucleus (1). Seven mammalian STAT genes have been identified and mouse
genetics have revealed functions for most that are well supported by
the in vitro experiments that led to their discovery (1, 2).
A general model for cytokine activation of STATs has been proposed,
based primarily on Stats 1-3 (2, 3). In this model, STATs exist as
monomers located in the cytoplasm prior to receptor activation,
although evidence for preassociation prior to activation exists (4).
Upon cytokine stimulation, phosphotyrosine residues in the receptor
recruit STATs through an SH2 domain interaction. Activated Janus
kinases phosphorylate STATs on a carboxyl tyrosine, promoting STAT
dimer formation by intermolecular SH2 domain interaction and
dissociation from the receptor complex. Once dimerized, STATs
translocate to the nucleus, bind DNA, and regulate gene transcription
(2, 3). Recent studies have shown that in some cells some STATs are
present in the nucleus prior to activation (5-9). Thus, it appears
that beyond the general mechanism outlined above, there may be cell
type-specific mechanisms that could further regulate some of the STATs.
Finding STATs in the nucleus of some cells prior to activation suggests
that constitutive nuclear import and export exist in these cells.
The mechanism by which activated STATs accumulate in the nucleus is
unknown. STATs have a mass above the upper limit for diffusion through
the nuclear pore (~45 kDa) (10-12) and thus are assumed to be
actively transported into the nucleus. The best characterized nuclear
import pathway involves binding of the transported protein to the
heterodimer protein complex importin Stat5 was first identified as a mammary gland factor required for
prolactin (PRL)-stimulated gene transcription (15, 16) but was found to
be activated by many hormones, growth factors, and cytokines (1, 17,
18). We now know that in humans and rodents Stat5 is actually two
distinct proteins arising from different genes (Stat5a and
Stat5b) (18-22). Stat5A and Stat5B are highly homologous,
differing mainly in their C terminus, and are expressed in most tissues
(18, 19). Genetic disruption of the Stat5a gene results in
mice deficient in mammopoiesis and lactogenesis (23). Knock-out of the
Stat5b gene removes the sexual dimorphism of body growth and
liver gene expression induced by growth hormone (GH) (24, 25). The
simultaneous deletion of both genes results in the most severe growth
and reproductive defects, revealing functional redundancy of the Stat5
proteins in physiological processes mediated by GH and PRL (25).
To probe the mechanisms regulating Stat5B localization within cells, we
constructed a green fluorescent protein (GFP)-Stat5B fusion protein. We
find that the fusion protein is tyrosyl-phosphorylated, dimerizes,
accumulates in the nucleus, and binds DNA upon cytokine receptor
stimulation in a manner indistinguishable from untagged Stat5B. We
identify by site-directed mutagenesis residues required for DNA binding
that are also needed for GH-dependent nuclear localization.
These studies identify for the first time a region of Stat5 required
for nuclear localization independent of those regions needed for dimerization.
Materials--
Human fibrosarcoma 2C4 and 2C4-GHR cells were
provided courtesy of G. Stark, Y. Han (Cleveland Clinic, Cleveland,
OH), and I. Kerr (Imperial Cancer Research Fund, London, UK). Rat
Stat5A cDNA (26) was kindly provided by J. Rosen (Baylor College of Medicine, Houston, TX); rat GH receptor (GHR) cDNA (27) was from G. Norstedt (Karolinska Institute, Stockholm, Sweden), and recombinant
human GH was the gift of Lilly. Prestained molecular weight standards
were from Life Technologies, Inc. All chemicals were reagent grade or better.
Construction of GFP-Stat5B cDNA--
pEGFP-C1
(), which encodes a red-shifted variant of
GFP optimized for fluorescence intensity and high expression in mammalian cells, was used to construct a cDNA encoding a GFP-Stat5B fusion protein. The BglII-EcoRI fragment of rat
Stat5B cDNA (28) was first subcloned into pEGFP-C1 (designated
pEGFP- Site-directed Mutagenesis--
GFP-Stat5B mutants were generated
using the QuickChangeTM site-directed mutagenesis kit
(Stratagene) as described by the manufacturer. Amino acids
Tyr568,
Lys582-Lys583-Lys586, or
466VVVI469 were replaced by alanine. The
oligonucleotide primers were as follows (lowercase represents
mutations): 5'-GCCAGGACGGAATgcCACTTTCTGGC-3' and
5'-GCCAGAAAGTGgcATTCCGTCCTGGC-3' (Tyr568);
5'-GGAGGTGTTAgcGgcACATCTCgcGCCTCACTGGAACG-3'and
5'-CGTTCCAGTGAGGCgcGAGATGTgcCgcTAACACCTCC-3' (Lys582-Lys583-Lys586); and
5'-CCTTGTCGCTCCCGGcGGcGGcGgcCGTGCATGGCAGCC-3' and
5'-GGCTGCCATGCACGgcCgCCgCCgCCGGGAGCGACAAGG-3' (466VVVI469). All mutations were verified by
DNA sequencing. For the 466VVVI469 mutant, two
clones were analyzed for tyrosyl phosphorylation, dimerization, and
cellular localization with similar results.
Cell Culture and Transfections--
2C4, 2C4-GHR, COS-7, and
NIH-3T3 cells were cultured at 37 °C in Dulbecco's modified
Eagle's medium supplemented with 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, 0.25 µg/ml amphotericin B, and either 10% fetal bovine
serum (2C4, 2C4-GHR, COS-7) or 9% calf serum (NIH-3T3). Medium for all
lines except COS-7 was further supplemented with 5 mM
sodium pyruvate. COS-7, 2C4, and 2C4-GHR cells were transiently
transfected by calcium phosphate precipitation (29) by incubating
subconfluent cultures with DNA precipitates for 6-16 h. For
transfection of 2C4 and 2C4-GHR cells, 20 µg/ml CalPhos maximizer
() was used. The cultures were then rinsed
twice with Dulbecco's modified Eagle's medium and fed with culture
medium. To equalize the amount of DNA transfected in non-imaging
experiments, empty pEGFP-C1 was used. NIH-3T3 cells were transfected
with Lipofectin (Life Technologies, Inc.) as described by the
manufacturer. Cells were harvested or used for imaging between 48 and
72 h post-transfection. 2C4 cells stably transfected with a
mammalian expression vector containing the cDNA for human GHR
(2C4-GHR) have been described previously (30).
Immunoprecipitation and Western Blotting--
10-cm plates of
cells were incubated in serum-free medium containing 1% bovine serum
albumin for 6-16 h and then treated with 500 ng/ml human GH at
37 °C. Cells were rinsed three times with ice-cold
phosphate-buffered saline containing 1 mM
Na3VO4, lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM Na3VO4), and
scraped from the plates on ice. Cellular proteins were precipitated
from the supernatant (2 h on ice) with Phosphatase Treatment--
Cell lysates were immunoprecipitated
with Electrophoretic Mobility Shift Assays (EMSAs)--
EMSAs were
performed using nuclear extracts of COS-7 cells transfected with
cDNAs encoding rat GHR and either Stat5B or GFP-Stat5B (wild-type
or VVVI mutant). Forty-eight hours after transfection and 16 h
after serum deprivation, cells were treated with 500 ng/ml GH for 5-60
min and nuclear extracts prepared (31). The extracts were incubated
with or without 1 µg of Confocal Fluorescence Microscopy--
Confocal imaging was
performed with a Noran OZ laser scanning confocal microscope equipped
with a 60× Nikon objective. GFP was excited at 488 nm by a
krypton-argon laser, and fluorescence above 500 nm was captured. Cells
were grown on glass coverslips attached to the bottom of a 60-mm
culture dish, transfected with cDNA, incubated in serum-free medium
for 6-16 h, and then imaged at room temperature in Krebs-Ringer
phosphate buffer (128 mM NaCl, 7 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4,
1 mM NaHPO4, 10 mM glucose, pH 7.4)
containing 0.1% bovine serum albumin. The contribution of cellular
autofluorescence was judged to be less than 1%. Preliminary experiments revealed that repeated laser exposure inhibited
GH-dependent migration of GFP-Stat5B into the nucleus,
presumably because of phototoxicity. Thus, the following protocol was
adopted. Once control images were obtained, cell location was recorded
by capturing a low-power image, and the cells were stimulated with GH
in a 37 °C incubator. Following the times indicated in the figures, the same cells were found and imaged a second time. The presented images are representative of at least three separate experiments during
which at least 20 cells were imaged. For quantitative analysis of
fluorescence distribution, cells were fixed (3.7% paraformaldehyde in
phosphate-buffered saline for 10 min) following stimulation. The mean
intensities of neighboring cytosolic and nuclear regions (approximately
5 µm2 each) were calculated using Adobe
PhotoshopTM, corrected for background, and expressed as
nuclear-to-cytosol fluorescence ratios.
Characterization of GFP-Stat5B--
As a tool for the study of the
cellular localization of Stat5B, we constructed a GFP-Stat5B fusion
protein. We first examined whether this protein, when expressed in
COS-7 cells, is regulated by GH in a manner similar to untagged Stat5B.
To examine whether GH stimulates tyrosyl phosphorylation of GFP-Stat5B,
cells transfected with cDNAs encoding rat GHR and either Stat5B or
GFP-Stat5B were treated with or without GH for 15 min. Proteins were
immunoprecipitated with
We next sought to determine whether GFP-Stat5B is capable of forming
dimers in response to GH-induced tyrosyl phosphorylation. To analyze
dimer formation, we utilized the ability of Stat5B to form dimers with
Stat5A in response to many cytokines, including GH (33). COS-7 cells
were transiently transfected with cDNAs encoding GHR, Stat5A, and
either GFP, Stat5B, or GFP-Stat5B. Stat5A was immunoprecipitated with
Stat5A-specific antibody, and precipitated proteins were probed with
Stat5B-specific antibody. GH induced co-immunoprecipitation of Stat5B
(Fig. 1B, lanes C and D) and GFP-Stat5B (Fig. 1B, lanes E and F)
with Stat5A. No proteins were detected when Stat5A was co-expressed
with GFP alone (Fig. 1B, lanes A and
B), showing that
Based on studies of Stat5B regulation by GH in liver (34), we predicted
that the multiple Stat5B and GFP-Stat5B bands seen in
immunoprecipitates from GH-treated cells reflect differential phosphorylation of Stat5B on serines/threonines and tyrosines. To test
for differential phosphorylation, we treated Stat5B immunoprecipitates with the general phosphatase alkaline phosphatase (AP). AP reduced the
three Stat5B and GFP-Stat5B bands seen in immunoprecipitates from
GH-treated cells to predominantly the fastest migrating band, with a
variable amount of a slower migrating band (Fig.
2, lanes B and C).
The changes in band pattern produced by AP treatment are the result of
dephosphorylation and not protein degradation since sodium
orthovanadate inhibited the mobility changes (Fig. 2, lane
D). The incomplete dephosphorylation of Stat5B seen in immunoprecipitates from GH-treated cells is thought to result from
limited access of AP to one of the phosphorylated sites in the Stat5B
dimer. In support of Stat5B being phosphorylated on serines/threonines
as well as tyrosines, we have found that the serine/threonine-specific
phosphatase PP2A condenses the three Stat5B bands from GH-treated cells
to two, tyrosyl-phosphorylated bands (data not shown). Further support
for the multiple GFP-Stat5B bands not being truncated forms of Stat5B
is the finding that all bands are recognized by both antibody to the
N-terminal GFP tag and antibody to the 10 amino acids at the C terminus
of Stat5B which are unique to the B isoform (28). Also, the bands do
not arise from adventitious proteolysis during the immunoprecipitation since the same bands are seen in blots of the cell lysates (Fig. 2,
lane A). Overall, these data indicate that GFP-Stat5B, like untagged Stat5B, is phosphorylated at multiple sites.
To assess the ability of GFP-Stat5B to bind DNA, we performed EMSAs
with a probe corresponding to the GAS-like element of the
GH-dependent Nuclear Accumulation of
GFP-Stat5B--
The EMSAs indicated that GFP-Stat5B was present in
COS-7 cell nuclei following GH treatment (Fig. 3). We next sought to
verify the presence of GFP-Stat5B in nuclei by directly visualizing
GFP-Stat5B in single, living cells. For these experiments we employed
human fibrosarcoma 2C4 cells because they possess a more uniform
morphology than COS-7 cells and are easily transfected. Cells
transiently expressing GHR and GFP, GFP-Stat5B alone, or GHR and
GFP-Stat5B were imaged by confocal microscopy prior to and following
stimulation with GH (Fig. 4). In cells
expressing both GFP and GHR (Fig. 4, A and B),
GFP was found throughout the cytoplasm and nucleus, and this
distribution did not change upon GH treatment. Likewise, in cells
expressing GFP-Stat5B but not GHR (Fig. 4, C and
D), GFP-Stat5B was present both in the cytoplasm and nucleus
prior to and following GH addition. A similar subcellular distribution of GFP-Stat5B was seen in cells expressing GFP-Stat5B and GHR in the
absence of GH (Fig. 4E). GFP-Stat5B was also present in both
the cytosol and nucleus prior to activation when expressed in COS-7,
NIH-3T3, and 2C4-GHR cells (Fig. 5,
A, C, and E). The presence of some GFP-Stat5B in
the nucleus as well as in the cytoplasm is unlikely to be the result of
the GFP tag or overexpression since a similar distribution was seen for
the following: 1) untagged Stat5B expressed in COS-7 cells and
endogenous Stat5B in CHO-GHR cells (both detected by
immunocytochemistry); and 2) GFP-tagged Stat5B expressed at levels
barely above the limits of detection of GFP fluorescence (data not
shown).
GH stimulation of cells co-expressing GHR and GFP-Stat5B resulted in a
dramatic nuclear accumulation of GFP-Stat5B in the majority of cells
(>90%; Fig. 4F). Nuclear accumulation was detectable by
10-15 min of GH treatment and persisted for several hours with continuous exposure to GH (data not shown). Interestingly, GH stimulated the formation of intense, punctate patterns of GFP-Stat5B fluorescence in the nucleus (Fig. 4F) and cytoplasm (Fig.
5B) of ~80% and ~15% of cells, respectively. GH also
stimulated nuclear accumulation of GFP-Stat5B in COS-7, NIH-3T3, and
2C4 cells stably expressing GHR (2C4-GHR) (Fig. 5, A-F),
indicating that GH regulation of GFP-Stat5B localization is not
restricted to a single cell type. Immunofluorescent detection of
untagged Stat5B expressed with GHR in COS-7 cells showed similar
GH-regulated nuclear accumulation (data not shown), confirming that the
GFP tag does not influence Stat5B nuclear accumulation.
Determinants of Stat5B Nuclear Localization--
We next sought to
identify regions of Stat5B required for nuclear accumulation. We
performed site-directed mutagenesis on GFP-Stat5B, transiently
expressed the mutant proteins with GHR in 2C4 cells, and assayed for
GH-dependent nuclear accumulation. Based on sequence
similarity, the region between the DNA-binding and SH2 domains was
thought to be an SH3 domain (35). Since SH3 domains are known to be
important in regulating the localization of signaling proteins within
cells (36), we reasoned that this region of Stat5B might be important
for nuclear localization. We mutated to alanine a single amino acid
(Tyr568) that, based on the structure of SH3 domains, would
be predicted to abrogate binding to proline-rich sequences. This mutant
protein (GFP-Stat5BY) was present in the cytosol and
nucleus prior to stimulation and localized to the nucleus in response
to GH (Fig. 6, A and
B), similar to wild-type GFP-Stat5B. The recent report of
the crystal structures of Stat1 and Stat3 homodimers bound to DNA have
revealed that this region does not have the architecture of an SH3
domain and is now referred to as a linker domain (37, 38).
Clusters of basic amino acids in STATs might constitute a functional
nuclear localization sequence when present in the context of dimers.
Three basic regions in the N terminus of Stat1 have been mutated
without any effect on nuclear accumulation in response to
interferon-
High affinity binding to DNA has been suggested to contribute to
nuclear accumulation of some transcription factors (39, 40). Hence, we
reasoned that disruption of Stat5B DNA binding might inhibit its
nuclear accumulation. A stretch of four amino acids
(466VVVI469) in the DNA binding domain of
Stat5B is critical for binding to the IRF-1 promoter (28). In the
context of GFP-Stat5B, we mutated to alanine these four amino acids
(GFP-Stat5BVVVI). Unlike wild-type GFP-Stat5B and the other
mutant GFP-Stat5Bs, GFP-Stat5BVVVI localized predominantly
to the cytoplasm prior to GH stimulation (Fig.
7, A and C) in
>95% of 2C4 cells. Furthermore, GH did not cause nuclear accumulation
of this protein (Fig. 7, B and D) at any stimulus
duration tested (5-90 min; data not shown). Yet, GH stimulated the
formation of punctate GFP-Stat5BVVVI fluorescence in the
cytoplasm in approximately 40% of cells (Fig. 7D), similar to wild-type GFP-Stat5B. GFP-Stat5BVVVI failed to localize
to the nucleus in COS-7 (Fig. 7, E and F) and
2C4-GHR cells (data not shown), indicating that the failure of mutant
GFP-Stat5VVVI to localize to the nucleus in response to GH
is not specific to a single cell type. Like GFP-Stat5BVVVI,
untagged Stat5BVVVI detected by immunocytochemistry was
located primarily in the cytoplasm, and its distribution did not change
with GH (data not shown). Thus, mutation of residues
466VVVI469 creates a genuine nuclear
localization defective Stat5B, independent of cell type and the
presence of the GFP tag.
To quantify our observations with GFP-Stat5B and its mutants, we
co-expressed wild-type or mutant GFP-Stat5B with GHR in COS-7 cells,
stimulated with GH for 40 min where appropriate, fixed the cells, and
measured the fluorescence intensity of the cytosol and nucleus in 20 cells per condition. Fig. 8 shows the
nuclear-to-cytosol fluorescence ratios (N/C) from a representative
experiment. Wild-type GFP-Stat5B, the linker domain tyrosine mutant,
and the lysine series mutants show similar cellular distribution, with
close to equal fluorescence in the nucleus and cytosol prior to
stimulation (N/C
Lack of GH-dependent nuclear accumulation of mutant
GFP-Stat5BVVVI could arise from a defect in some crucial
upstream signaling event. To test this idea, we co-expressed GHR with
GFP-Stat5B or GFP-Stat5BVVVI, treated the cells with or
without GH, and prepared cytosol and nuclear extracts. GFP-Stat5B or
GFP-Stat5BVVVI were immunoprecipitated with
By using the same cell extracts, we next sought to determine if mutant
GFP-Stat5BVVVI can bind DNA. As expected,
GH-dependent DNA binding is present in both cytosol and
nuclear extracts from cells expressing wild-type GFP-Stat5B and GHR
(Fig. 9B, lanes A-C and G-I). Both cytosolic
and nuclear extracts from cells expressing GFP-Stat5BVVVI
and GHR lack DNA binding activity (Fig. 9B, lanes D-F and J-L). Thus, although
GFP-Stat5BVVVI in the cytosol is tyrosyl-phosphorylated in
response to GH, it cannot bind DNA, as predicted (28).
To test whether the failure of GFP-Stat5BVVVI to accumulate
in the nucleus and bind DNA in response to GH might stem from an
inability to form dimers, we examined the ability of
GFP-Stat5BVVVI to dimerize with Stat5A. COS-7 cells
expressing GHR, Stat5A, and either GFP, wild-type GFP-Stat5B, or
GFP-Stat5BVVVI were treated with or without GH (Fig.
10). Immunoprecipitation with STATs play important roles in immune system function and hormonal
signaling. Numerous elegant studies have provided much insight into how
the STATs fulfill these roles (reviewed in Refs. 1-3). Yet, one key
aspect of their regulation is poorly understood: we do not know the
details of how the cellular localization of STATs is regulated. To
begin to probe the mechanisms that control the cellular distribution of
STATs, we constructed and characterized a GFP-Stat5B fusion protein. We
find that GFP-Stat5B is regulated by GH in a manner indistinguishable
from untagged Stat5B.
GFP-Stat5B is distributed equally in the cytosol and nucleus of COS-7,
2C4, and NIH-3T3 cells prior to GH stimulation. This distribution is
unlikely to result from constitutive activation of Stat5B since it was
present even when GHR was not co-expressed (Fig. 4C), and
GFP-Stat5B in the nuclear fraction of unstimulated cells was not
detectably tyrosyl-phosphorylated (Fig. 9A, upper and
lower panels, lane G). Also, this distribution is unlikely to be
an artifact of overexpression since the same distribution was seen even
in weakly fluorescent cells. Furthermore, we find endogenous Stat5B in
CHO-GHR cells present in both the nucleus and cytoplasm prior to ligand
stimulation. A similar distribution has been reported for endogenous
Stat5B in INS-1 insulinoma cells (9). However, Stat5B is restricted to
the cytoplasm prior to activation in the liver-derived CWSV-1 cell line
(41) and BRL-GHR hepatoma
cells.2 Significant cell
type-specific variability in the localization of other STATs has also
been documented. For example, Stat1 prior to activation is mostly
cytoplasmic in some cell types (7, 14, 42, 43) but distributed
throughout the nucleus and cytoplasm in other cells (5, 6, 8). Thus,
individual STATs may possess localization properties that are regulated
in a cell type-dependent manner.
We selected three regions of Stat5B to mutate and assay for
GH-dependent nuclear localization. Only mutation of
residues 466VVVI469 in the DNA binding domain
disrupted GH-dependent nuclear accumulation of Stat5B. Luo
and Yu-Lee (28) have shown previously that mutating these four amino
acids abrogates binding to the GAS sequence of the IRF-1 promoter in
response to PRL. We report here that these same amino acids are
required for GFP-Stat5B to bind to a In addition to Stat5B, Stats 1, 3, and 5A possess residues VVVI in
their DNA binding domain. Evidence for important functional differences
in the DNA binding domain of individual STATs is emerging. Mutation of
VVVI abrogates Stat5B nuclear localization in response to ligand
stimulation (Fig. 7), yet mutation of VVV in STAT3 does not (44, 45).
It will be important to determine if the isoleucine within VVVI of
Stat3 is needed for nuclear localization. Also, while conserved
residues EE and VVV in the DNA binding domain of Stat3 are required for
binding to DNA (44), residues EE in Stat5B are dispensable for DNA
binding (28). Hence, individual residues within the DNA binding domain
may have discrete functions that are specific for a given STAT.
How does mutation of 466VVVI469 prevent Stat5B
nuclear localization? Several possibilities exist. One model consistent
with our results is that in addition to being a DNA-binding
determinant, VVVI may also function as a protein-protein interaction
domain. In this model, VVVI would mediate the association of Stat5B
with nuclear import proteins or other proteins that would serve to shuttle Stat5B into the nucleus. In the case of Stats 1 and 3, the VVVI
region does not make direct DNA contact but contributes to a buttress
that supports the DNA binding loop (37, 38). Alternatively, mutation of
VVVI of Stat5B might disrupt the structure of the protein at a more
distant location, preventing the interaction of Stat5B with proteins
necessary for nuclear import. Several proteins known to associate with
Stat5B move between the cytosol and nucleus, including glucocorticoid
receptor (46), GHR (47, 48), and SH2 domain-containing protein tyrosine
phosphatase-1 (41). Interaction with these or other signaling proteins
may be instrumental in regulating the cellular distribution of Stat5B.
Alternatively, mutation of these residues may abrogate nuclear
localization by preventing binding to nuclear components, including DNA
and possibly the nuclear matrix. In this model, in cells in which
Stat5B is distributed throughout the cell prior to activation, non-activated Stat5B moves into and out of the nucleus by yet unidentified transport pathways, with export being faster than import.
This constitutive transport combined with the interaction of
non-activated Stat5B with nuclear components yields a near equal
distribution between cytosol and nucleus. Once Stat5B is tyrosyl-phosphorylated and dimerized, the significantly higher affinity
binding to nuclear components, including DNA, shifts the distribution
of Stat5B to being mostly nuclear. Mutation of residues VVVI prevents
both non-activated and activated Stat5B from binding to nuclear
components. Now the faster export dominates, shifting the equilibrium
distribution of Stat5BVVVI to being mostly cytoplasmic.
Finally, it is possible that mutation of residues VVVI prevents a
phosphorylation event required for nuclear accumulation. Based on
studies of rat liver Stat5B (34), we suspect that the differential
phosphorylation of GFP-Stat5BVVVI compared with wild-type GFP-Stat5B arises from GH-dependent serine/threonine
phosphorylation. Modulation of nucleocytoplasmic transport of
transcription factors by serine/threonine phosphorylation has
considerable precedence (11). Furthermore, phosphorylation of
retinoblastoma protein regulates its interaction with the nuclear
matrix (49). Identification of the serine/threonine phosphorylation
sites of Stat5B may provide further insight into the mechanisms
regulating nuclear localization.
At present, any of the above models seem plausible. Our challenge now
is to define the role of the DNA binding domain in Stat5B nuclear
localization. Doing so will shed light on how the STATs transduce
cell-surface signals into transcriptional regulation of diverse
physiological processes.
INTRODUCTION
Top
Abstract
Introduction
References
/
followed by
energy-dependent transport through the nuclear pore complex
requiring the GTPase activity of Ran/TC4 (10-12). STATs appear to lack
a conventional nuclear localization sequence, the single or dual
stretch of basic amino acids that bind to importin
. Yet, Stat1
dimers have recently been shown to associate with the importin
-homologue NPI-1 (13). Furthermore, IFN-
-stimulated Stat1 nuclear
accumulation requires NPI-1 (13) and the GTPase activity of Ran/TC4
(14). Perhaps STAT dimers possess the structure required for binding to
nuclear import proteins. In cells where STATs are found in the nucleus prior to activation, cytokine-induced nuclear accumulation might also
arise from down-regulation of nuclear export. Insight into how STAT
proteins interact with proteins needed for nuclear transport awaits
identification of a mutant STAT that can dimerize but is unable to
localize to the nucleus.
EXPERIMENTAL PROCEDURES
Stat5B). Polymerase chain reaction with Pfu DNA
polymerase (Stratagene) and oligonucleotides
5'-GAAGATCTATGGCAATGTGGATACAG-3' and 5'-GGAGCTGCGTGGCATAG-3' as primers
was used to engineer a BglII site and remove an in-frame stop codon upstream of the start codon of Stat5B. The polymerase chain
reaction product was purified, digested with BglII, and inserted into pEGFP-
Stat5B, yielding cDNA encoding GFP fused to
the N terminus of full-length Stat5B by a five amino acid linker (SGLRS). DNA sequencing (Sequenase 2.0; U. S. Biochemical Corp.) was
performed to verify the region created by polymerase chain reaction and
all junctions.
Stat5B (C-17, Santa Cruz
Biotechnology; 1:100) or
Stat5A (L-20, Santa Cruz Biotechnology;
1:100), immobilized on protein A-coated agarose beads (Repligen;
1.5 h at 8 °C), eluted by boiling, and separated by
SDS-polyacrylamide gel electrophoresis (5-12% gradient or 7.5%
gels). Proteins were transferred to nitrocellulose membrane (Amersham
Pharmacia Biotech) and detected by Western blotting using enhanced
chemiluminescence (ECL) (Amersham Pharmacia Biotech) with
Stat5B-specific, affinity purified polyclonal rabbit anti-rat
Stat5B
(1:5000) (28), Stat5A-specific
Stat5A (Santa Cruz Biotechnology;
1:2000),
-phosphotyrosine (4G10, Upstate Biotechnology; 1:7500), or
-GFP (8362-1, ; 1:500). For the
experiment shown in Fig. 9, cytosol and nuclear extracts were prepared
(31) and divided for immunoprecipitation or electrophoretic mobility
shift assay (see below). Extracts were diluted and supplemented where
appropriate to make final volumes (500 µl) and concentrations of NaCl
(150 mM), glycerol (7%), and Triton X-100 (0.2%) equal for immunoprecipitation from the cytosolic and nuclear fractions. The
Western blot data presented in the figures are representative of at
least three separate experiments except for Fig. 9, which was performed
twice with similar results.
Stat5B and incubated at 37 °C for 60 min in 100 µl of
dephosphorylation buffer (50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA, 0.2 mM MgCl2, 0.02 mM ZnCl2) containing 40 units of calf
intestinal alkaline phosphatase (AP; Boehringer Mannheim). As controls,
10 mM Na3VO4 was added to the dephosphorylation buffer or AP was omitted. The reaction was
terminated, and proteins were eluted by boiling in a 4:1 mixture of
lysis buffer and SDS-polyacrylamide gel electrophoresis sample buffer. The resultant dephosphorylated proteins were separated by
SDS-polyacrylamide gel electrophoresis and immunoblotted with
Stat5B
as described above.
Stat5B (C-17 10x, Santa Cruz
Biotechnology) and then with a probe corresponding to the PRL response
element of the
-casein promoter
(5'-AGATTTCTAGGAATTCAA-3'; 40,000 cpm, 5 × 10
15
mol) (32). Samples were analyzed on a non-denaturing polyacrylamide gel
and subjected to autoradiography. The EMSA experiments were performed
twice with similar results.
RESULTS
Stat5B and analyzed by Western blotting
using
Stat5B, anti-phosphotyrosine antibody (
PY), or
GFP (Fig.
1A). Antibodies directed
against the C terminus of Stat5B (
Stat5B) and the N-terminal GFP tag (
GFP) both recognized a protein with an apparent molecular mass appropriate for GFP-Stat5B (~120 kDa) in immunoprecipitates from cells transfected with GFP-Stat5B cDNA (Fig. 1A,
lanes E-H). Neither antibody detected a protein of smaller
size (except for endogenous Stat5B in the case of
Stat5B; Fig.
1A, lanes E-H, and data not shown) indicating
that the vast majority of expressed protein was full-length fusion
protein. In immunoprecipitates from cells transfected with Stat5B
cDNA,
Stat5B recognized an approximately 90-kDa protein (Fig.
1A, lanes A-D), appropriate for untagged Stat5B.
GH stimulated to similar extents tyrosyl phosphorylation of GFP-Stat5B
(Fig. 1A, lane H) and untagged Stat5B (Fig.
1A, lane D). Hence, GFP-Stat5B expressed in COS-7
cells is full-length (not truncated at either the N or C terminus), is
recognized by both
Stat5B and
GFP, and like untagged Stat5B is
tyrosyl-phosphorylated in response to GH.
View larger version (27K):
[in a new window]
Fig. 1.
GH promotes tyrosyl phosphorylation and
dimerization of GFP-Stat5B. A, COS-7 cells were
transfected without ( ) or with (+) cDNA expression vectors for
Stat5B, GFP-Stat5B, or GHR (all 5 µg). Forty-eight hours after
transfection and 16 h after serum deprivation, cells were treated
without (
) or with (+) 500 ng/ml GH for 15 min. Whole cell lysates
were immunoprecipitated (IP) with
Stat5B, Western blotted
with
PY (upper panel), and then reprobed with
Stat5B
and
GFP (lower panels). B, COS-7 cells were
transfected with 5 µg of cDNA expression vectors for GHR, Stat5A,
and either GFP (lanes A and B), Stat5B
(lanes C and D), or GFP-Stat5B (lanes
E and F). Cells were treated without (
) or with (+)
500 ng/ml GH for 30 min. Cellular proteins were immunoprecipitated with
Stat5A, blotted with
Stat5B (upper panel), and
reprobed with
PY (lower panel). The molecular weight of
prestained standards (×10
3) and the migration of
GFP-Stat5B, Stat5B, and Stat5A are indicated.
Stat5B did not cross-react with Stat5A.
The Stat5B which co-immunoprecipitated with Stat5A in response to GH
migrates as three distinct bands (Fig. 1B, lane D). Similarly, three GFP-Stat5B bands also co-immunoprecipitate with Stat5A in response to GH (Fig. 1B, lane F),
although the relative amounts differ when compared with Stat5B. The
lower level of GFP-Stat5B compared with Stat5B bound to Stat5A in Fig.
1B is assumed to be due to a lower level of expression of
GFP-Stat5B since in all experiments where it was examined, GFP-Stat5B
was expressed at lower levels than untagged Stat5B. Interestingly, co-expression of Stat5A with either Stat5B or GFP-Stat5B but not with
GFP leads to a low level of constitutive tyrosyl phosphorylation of
Stat5A (Fig. 1B, lanes A, C, and E).
In sum, these experiments show that activated GFP-Stat5B, like untagged
Stat5B, forms heterodimers with Stat5A.
View larger version (32K):
[in a new window]
Fig. 2.
Stat5B and GFP-Stat5B are phosphorylated at
multiple sites. COS-7 cells were transfected with 5 µg of
cDNA expression vectors for GHR and either Stat5B (upper
panels) or GFP-Stat5B (lower panels). Cells were
treated with (+) 500 ng/ml GH for 30 min. Stat5B immunoprecipitates
(lanes B-D) were incubated at 37 °C for 60 min in
dephosphorylation buffer with (lanes C and D) or
without (lane B) alkaline phosphatase and with (lane
D) or without Na3VO4 (lanes B
and C). Cell lysates (lane A) and
Stat5B
immunoprecipitates were immunoblotted with
Stat5B. The molecular
weight of prestained standards (×10
3) and the migration
Stat5B and GFP-Stat5B are indicated.
-casein promoter and nuclear extracts from COS-7 cells
overexpressing GHR with or without either Stat5B or GFP-Stat5B (Fig.
3). GH produced a similar
time-dependent increase in the formation of a DNA-binding complex in nuclear extracts from cells expressing Stat5B (Fig. 3,
lanes A-E) or GFP-Stat5B (Fig. 3, lanes G-K)
but not in extracts from cells expressing GHR alone (Fig. 3,
lanes M and N). This DNA-binding complex contains
Stat5B or GFP-Stat5B since pretreatment of the nuclear extracts with
Stat5B results in a supershifted complex (Fig. 3, lanes F
and L). We find that the DNA complex containing GFP-Stat5B
migrates slightly slower than the complex containing untagged Stat5B,
consistent with the addition of the GFP tag. Overall, Figs. 1 and 3
illustrate that the critical cytokine-regulated events of tyrosyl
phosphorylation, dimerization, and DNA binding are functionally intact
for GFP-Stat5B.
View larger version (30K):
[in a new window]
Fig. 3.
GH induces DNA binding activity of
GFP-Stat5B. COS-7 cells transfected with cDNA expression
vectors (5 µg) for GHR and Stat5B (lanes A-F), GHR and
GFP-Stat5B (lanes G-L), or GHR alone (lanes M
and N) were treated without (lanes A, G, and
M) or with 500 ng/ml GH for 5 (lanes B and
H), 10 (lanes C and I), 30 (lanes D, F, J, L, and N), or 60 min (lanes
E and K). EMSAs were performed using nuclear extracts
of these cells and a GAS-like PRL response element from the
-casein promoter. Where indicated, nuclear extracts were
preincubated without (
) or with (+)
Stat5B for 20 min.
View larger version (72K):
[in a new window]
Fig. 4.
GH promotes nuclear localization of
GFP-Stat5B. A-F, 2C4 cells transfected with cDNA
expression vectors (3 µg) for either GHR and GFP (A and
B), GFP-Stat5B (C and D), or GHR and
GFP-Stat5B (panels E and F) were imaged by laser
scanning confocal microscopy prior to (A, C, and
E) and following (panels B, D, and F)
40-60 min treatment with 500 ng/ml GH. Images in panels
A, C, and E are of the same cell as
B, D, and F, respectively. Scale
bar (F) represents 15 µm.
View larger version (63K):
[in a new window]
Fig. 5.
GH induces nuclear localization of GFP-Stat5B
in several cell types. A and B, COS-7 cells
transfected with cDNA expression vectors for GHR (3 µg) and
GFP-Stat5B (6 µg) were imaged by confocal microscopy prior to
(A) or 45 min following (B) treatment with 500 ng/ml GH. C and D, NIH-3T3 cells transfected with
GHR (3 µg) and GFP-Stat5B (6 µg) were treated without
(C) or with (D) 500 ng/ml GH for 40 min and
imaged. E-F, 2C4-GHR cells transfected with GFP-Stat5B (3 µg) were imaged prior to (E) or 40 min following
(F) treatment with 500 ng/ml GH. Images in all panels are of
different cells. Scale bar represents 15 µm.
View larger version (68K):
[in a new window]
Fig. 6.
GH induces nuclear localization of mutants
GFP-Stat5BY and GFP-Stat5BKKK. 2C4 cells
transfected with cDNA expression vectors for GHR (3 µg) and
either GFP-Stat5BY (tyrosine 568 to alanine; 6 µg)
(A and B) or GFP-Stat5BKKK (lysines
562, 563, and 568 to alanine; 6 µg) (C and D)
were imaged prior to (A and C) and following
(B and D) 45 min treatment with 500 ng/ml GH.
Images in A and B and images in C and
D are from the same cell. Scale bar represents 15 µm.
(13). We identified a different basic region consisting
of a cluster of three lysines between the linker and SH2 domains
(Lys582, Lys583, and Lys586) of
Stat5B and mutated the residues to alanine. This protein (GFP-Stat5BKKK) accumulated in the nucleus in response to
GH in a manner indistinguishable from wild-type GFP-Stat5B (Fig. 6, C and D), ruling out this region as a nuclear
localization determinant.
View larger version (80K):
[in a new window]
Fig. 7.
DNA binding domain mutant Stat5B
(GFP-Stat5BVVVI) does not localize to the nucleus in
response to GH. A-D, 2C4 cells transfected with
cDNA expression vectors for GHR (3 µg) and DNA binding domain
mutant GFP-Stat5BVVVI (amino acids
466VVVI469 to alanine; 6 µg) were imaged
prior to (A and C) and following (B
and D) 50 min treatment with 500 ng/ml GH. Images in
A and B and images in C and
D are from the same cell. Scale bar represents 10 µm. E-F, COS-7 cells transfected with GHR (3 µg) and
GFP-Stat5BVVVI (6 µg) were imaged prior to (E)
or following (F) treatment with 500 ng/ml GH for 30 min.
Images in E and F are from different cells.
Scale bar represents 15 µm.
1.2) and prominent GH-dependent nuclear
accumulation with GH (N/C
2.5-3.0). DNA-binding mutant
GFP-Stat5BVVVI is mostly cytoplasmic (N/C
0.4), and this
distribution does not change following treatment with GH.
View larger version (13K):
[in a new window]
Fig. 8.
Cellular distribution of GFP-Stat5B and its
mutants. COS-7 cells were transfected with cDNA expression
vectors for GHR (1 µg) and either wild-type GFP-Stat5B
(WT), linker domain mutant GFP-Stat5BY
(Tyr568), lysine series mutant GFP-Stat5BKKK
(Lys582-Lys583-Lys586), or DNA
binding domain mutant GFP-Stat5BVVVI
(466VVVI469) (2 µg). Cells were treated
without or with 500 ng/ml GH for 40 min, fixed, and imaged.
Bars represent the mean ± S.E. nuclear-to-cytosol
fluorescence intensity ratios of 20 cells for each condition. The
experiment was performed twice with similar results.
Stat5B and
probed with
PY. Like wild-type GFP-Stat5B,
GFP-Stat5BVVVI extracted from the cytosol is
tyrosyl-phosphorylated in response to GH (Fig.
9A, lanes A-F).
The lower level of tyrosyl-phosphorylated GFP-Stat5BVVVI in
the cytosol compared with wild-type GFP-Stat5B can be attributed to
lower expression of GFP-Stat5BVVVI compared with wild-type,
as seen by reprobing the membrane with
Stat5B (Fig. 9A,
lower panel, lanes A-F). Unlike wild-type GFP-Stat5B, tyrosyl-phosphorylated GFP-Stat5BVVVI is undetectable in
the nucleus (Fig. 9A, upper panel, lanes G-L). A
small amount of GFP-Stat5BVVVI is detected in the nuclear
fraction when the membrane is reprobed with
Stat5B (Fig.
9A, lower panel, lanes J-L); it may represent contamination from the cytosolic fraction or a small amount of GFP-Stat5BVVVI in the nucleus. In support of it being a
contaminant, GFP-Stat5BVVVI was undetectable in the nuclear
fraction of a second experiment (data not shown).
View larger version (28K):
[in a new window]
Fig. 9.
GH induces tyrosyl phosphorylation but not
DNA binding activity of mutant GFP-Stat5BVVVI.
A, COS-7 cells were transfected with cDNA expression
vectors (5 µg) for GHR, Stat5A, and either wild-type GFP-Stat5B
(GFP-5B, lanes A-F) or DNA-binding mutant
GFP-Stat5BVVVI (GFP-5BVVVI, lanes
G-L). Cells were treated without (lanes A, D, G, and
J) or with 500 ng/ml GH for 10 (lanes B, E, H,
and K) or 60 min (lanes C, F, I, and
L). Cytosol and nuclear extracts were immunoprecipitated
(IP) with Stat5B, blotted with
PY, and reprobed with
Stat5B. The molecular weight of prestained standards
(×10
3) are indicated. B, EMSAs were performed
using the cytosol and nuclear extracts from the experiment in
A and a GAS-like PRL response element from the
-casein promoter.
Stat5A and probing with
Stat5B or
PY reveals that tyrosyl-phosphorylated mutant GFP-Stat5BVVVI co-immunoprecipitates with Stat5A in
response to GH in a manner similar to wild-type GFP-Stat5B (Fig. 10,
lanes C-F). In this experiment, mutant
GFP-Stat5BVVVI was present in lysates at about 30% of the
level of wild-type GFP-Stat5B. When this difference in expression is
considered, the DNA-binding mutant GFP-Stat5BVVVI forms
dimers with Stat5A better than wild-type GFP-Stat5B. Interestingly, we
find that the slowest migrating band of wild-type GFP-Stat5B is absent
in GFP-Stat5BVVVI co-immunoprecipitates, suggesting
differential phosphorylation of the wild-type and mutant fusion
proteins. These data indicate that dimer formation is not sufficient
for tyrosyl-phosphorylated mutant GFP-Stat5BVVVI to localize to the nucleus and define the DNA binding domain as a novel
determinant of Stat5B nuclear accumulation.
View larger version (31K):
[in a new window]
Fig. 10.
GH promotes dimerization of DNA binding
domain mutant GFP-Stat5BVVVI. COS-7 cells were
transfected with cDNA expression vectors for GHR (5 µg), Stat5A
(5 µg), and either GFP (5 µg) (lanes A and
B), wild-type GFP-Stat5B (GFP-5B, 5 µg) (lanes
C and D), or DNA-binding mutant
GFP-Stat5BVVVI (GFP-5BVVVI, 15 µg)
(lanes E and F). Cells were treated without ( )
or with (+) 500 ng/ml GH for 30 min. Cellular proteins were
immunoprecipitated (IP) with
Stat5A, blotted with
Stat5B, and reprobed with
PY and
Stat5A. The molecular weight
of prestained standards (×10
3) and the migration
wild-type GFP-Stat5B, mutant GFP-Stat5BVVVI, and Stat5A are
indicated.
DISCUSSION
-casein promoter
probe. We find that the mutant protein, GFP-Stat5BVVVI, retains early signaling events; namely it is tyrosyl-phosphorylated and
forms dimers in response to GH. The failure of mutant
GFP-Stat5BVVVI to localize to the nucleus in response to GH
despite its ability to form dimers suggests that dimerization is not
sufficient for nuclear accumulation of Stat5B. To our knowledge, this
is the first identification of a region required for STAT nuclear
accumulation distinct from those regions needed for tyrosyl
phosphorylation and dimerization. Interestingly, Stat5BVVVI
is active as an inhibitor of PRL-induced IRF-1 transcription (28). The
inability of Stat5BVVVI to localize to the nucleus suggests
that this inhibition occurs in the cytoplasm.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. L. S. Argetsinger for critically reading the manuscript. We thank T. Komorowski for excellent technical assistance with confocal microscopy and P. Du and X. Wang for assistance with experiments and cell culture. We also thank Dr. G. Stark and Y. Han for kindly providing the 2C4 and 2C4-GHR cells; Dr. J. Rosen for providing the Stat5A cDNA; and Dr. G. Norstedt for the GHR cDNA. Confocal imaging facilities were provided by the Morphology and Image Analysis Core, Michigan Diabetes Research and Training Center (supported by MDRTC Grant DK-20572). Oligonucleotides were synthesized by the Biomedical Research Core Facilities, University of Michigan, and supported in part by the Cancer Center UM-MAC Grant 3 P30-CA46592 and by the Michigan Diabetes Research and Training Center.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants RO1-DK-34171 (to C. C.-S.) and RO1-DK-44625 (to L.-y. Y.-L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Research Service Award F32-DK-09756.
¶ Supported by a predoctoral fellowship from Rackham Graduate School, University of Michigan.
** Supported by National Institutes of Health Training Grant T32-DK-07696.
¶¶ To whom correspondence should be addressed: Dept. of Physiology, University of Michigan Medical School, 6804 Medical Science II, 1301 Catherine St., Ann Arbor, MI 48109-0622. Tel.: 734-647-2126; Fax: 734-647-9523; E-mail: cartersu{at}umich.edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
STAT, signal
transducers and activators of transcription;
SH2 domain, Src homology-2
domain;
GAS, interferon- activation sequence;
GH, growth hormone;
GHR, growth hormone receptor;
PRL, prolactin;
GFP, green fluorescent
protein;
EMSA, electrophoretic mobility shift assay;
IRF-1, interferon
regulatory factor-1;
GFP, green fluorescent protein;
AP, alkaline
phosphatase;
PY, anti-phosphotyrosine antibody;
N/C, nuclear-to-cytosol.
2 J. Herrington and C. Carter-Su, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|