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INTRODUCTION |
Signal transducers and
activators of transcription
(STAT1 proteins) form a
family of seven latent cytoplasmic transcription factors, which
following activation in response to many different cytokines, dimerize
and translocate into the nucleus to activate gene transcription (1).
Stats can be activated by a broad spectrum of cytokines interacting
with specific cytokine receptors, leading to the activation of members
of the Janus tyrosine kinase (JAK) family. The activated JAKs
phosphorylate the recruited monomeric Stats on a specific tyrosine and
induce their dimerization. The activated dimers then translocate into
the nucleus, where they bind to specific DNA-response elements in the
promoters of target genes. This linear signal transduction pathway is
clearly an oversimplification and can be modulated by cross-talk with
other signal transduction pathways (e.g. by steroid
receptors, etc.) (2).
Most Stats bind to similar GAS (IFN-
-activated sequence) motifs
(TTCNnGAA) with slightly different affinities for optimal binding; n = 4 for Stat6 and n = 3 for
all other Stats (3-5). Non-receptor tyrosine kinases such as Src or
Abl can also directly phosphorylate Stat proteins without involving
JAKs (6-9). In addition to tyrosine phosphorylation, Stat proteins can
be phosphorylated on serine residues (10-14). While regulation of
Stats by serine phosphorylation is poorly understood, it has been shown
that this phosphorylation can modulate both their transcriptional
activity and DNA binding (13, 15).
The DNA binding region of Stat proteins is located between amino acids
350 and 500, and the activation domain is located in the C-terminal
region (16-18). C-terminal-truncated Stat proteins still retain DNA
binding activity, but may in some cases suppress transcriptional
activation of responsive genes in a dominant-negative manner (19-21).
There is some evidence that these naturally occurring dominant-negative
Stat5 isoforms may be functionally correlated with biological responses
(22), but the potential physiological significance of dominant-negative
Stat isoforms is still presently unknown. The N-terminal region of
Stats is required for cooperative binding of Stat dimers to form a
tetrameric structure (23, 24). For example, Stat5 forms tetramers on
the IL-2 response element in the human IL-2Ra promoter
(23-25), and the CIS gene (26), both of which contain two
adjacent Stat binding sites.
Stat5 plays a key role in mammary gland differentiation, as well as in
the prolactin (Prl)- induced expression of milk protein genes, such as
-casein (27, 28),
s1-casein (29),
-lactoglobulin (30), and whey acidic protein (31). However, the expression of Stat5 is
not restricted to the mammary gland. Both isoforms of Stat5 (Stat5a and
Stat5b, which share 93% identity at the amino acid level) can be
activated by many cytokines in addition to Prl, such as IL-2 (32, 33),
IL-3 (34, 35), IL-5 (35), IL-7, IL-15 (33), thrombopoietin (36),
erythropoietin, GM-CSF (35, 37), EGF (38), and platelet-derived growth
factor (39) in various cells and tissues, as well as by non-receptor
tyrosine kinases such as Src and Bcr-Abl (6, 7). Thus, Stat5 may play a
variety of regulatory roles that control different functions including
cell growth, survival, and differentiation. Recent studies have
demonstrated for example, that activated Stat5 may regulate cyclin D1
promoter activity resulting in cell cycle progression (40-42).
Targeted disruption of Stat5a or Stat5b genes in
mice results in a distinctive tissue-specific phenotype.
Stat5a knockout mice display, for example, defects in
mammary gland development and lactation during pregnancy (43), while
Stat5b knockout mice display sexually dimorphic liver gene
expression (44). There is also evidence suggesting that closely related
Stat5a and Stat5b may be differently activated (45) and exhibit
distinct biochemical differences (26) and distinct DNA binding
specificities (46). However, the latter studies are controversial,
because they were based on an incorrectly identified single amino acid
difference between murine Stat5a and 5b. Instead, recent studies on the
DNA binding properties of Stat5a and Stat5b suggest that
baculovirus-expressed Stat5a and Stat5b homodimers recognize a similar
optimal consensus sequence (47). Significant differences, however, have
been observed in the ability of Stat5a and Stat5b to form tetrameric
complexes on adjacent consensus and nonconsensus GAS sites, thus in
part explaining their ability to activate nonidentical sets of genes (48). Thus, the arrangement of weak affinity binding sites in pairs on
target genes may help provide selectivity in response to Stat5a
tetrameric complexes. Selectivity might also reflect the cooperation
with other transcription factors that interact with Stat5, and their
own adjacent weak affinity binding sites perhaps helping to stabilize
coactivator complexes (49).
Previous studies from our laboratory have suggested that Prl and Src
signaling pathways may differentially regulate Stat5a and Stat5b
nuclear translocation and retention (9). Thus, we postulated that the
regulation of specific gene targets involved in proliferation,
differentiation, or apoptosis by these two highly related isoforms of
Stat5 might also reflect their activation by different signal
transduction pathways. In this study, the DNA binding properties of
Stat5a and Stat5b induced by Prl and Src to a variety of GAS-like
sequences, such as
-casein, APRE (
2-macroglobulin), APRE-2
(double APRE site), p21 (SIE2), and p21 (SIE3) were analyzed. As
expected, Stat5a activated by Prl bound to the APRE-2 (double GAS site)
as a tetramer, while Stat5b activated identically bound as a dimer.
However, surprisingly, differences in the mobility of DNA binding
complexes of Stat5b induced by Src versus Prl were observed.
Based on immunoprecipitation studies and antibody supershift
experiments, we propose that Stat5b induced by Src versus
Prl may exhibit a different conformation at its C terminus, possibly
due to additional tyrosine phosphorylation. In contrast to Prl, which
induced only Tyr-699 phosphorylation of Stat5b, Src induced
phosphorylation on Tyr-699 as well as on additional tyrosines. To
identify the potential Src-induced tyrosine phosphorylation sites in
the C-terminal region of Stat5b in addition to Tyr-699, a series of
tyrosine to phenylalanine mutants were analyzed both individually and
sequentially by IP-Western blots. Based on this analysis, Tyr-724
(conserved in both Stat5a and Stat5b) and Tyr-679 (a unique site
present only in Stat5b, but absent in Stat5a) were shown to contribute
to the Src-induced tyrosine phosphorylation of Stat5b. Furthermore,
mutation of Tyr-679 to Phe in Stat5b altered its pattern of nuclear
localization upon activation by Src kinase, and decreased v-Src-induced
cyclin D1 promoter activity, suggesting that phosphorylation of this
tyrosine may influence protein-protein interactions most likely of the Stat5b C-terminal transactivation domain and possibly may influence cell cycle progression. Thus, depending on the signal transduction pathway responsible for activation, different structural conformations of activated Stat5b may result in selective biological responses.
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EXPERIMENTAL PROCEDURES |
Plasmids and Mutagenesis--
The expression vectors for
rat-Stat5a, rat-Stat5b, a C-terminal-truncated isoform, Stat5b
40,
and for the long form of the PrlR have been described previously
(50-53). c-Src kinase-active construct (54) was kindly provided by Dr.
Sara Courtneidge at Sugen Corporation (South San Francisco, CA). v-Src
cDNA was kindly provided by Dr. Hiromitsu Hanafusa (The Rockefeller
University, New York, NY) and cloned into pEFIRES vector in our
laboratory by Dr. Alexander Kazansky. The
674CD1LUC plasmid
containing the cyclin D1 promoter-driven luciferase reporter, as well
as several other cyclin D1 constructs were kindly provided by Dr.
Richard G. Pestell (Lombardi Cancer Center, Georgetown University,
Washington, D. C.). Stat5aW37A and Stat5bW37A mutants were kindly
provided by Dr. Warren J. Leonard (National Institutes of
Health, Bethesda, MD). Stat5bS730A was provided by Dr. Robert Kirken
(University of Texas Medical School, Houston, TX). Stat5b mutants were
prepared using the QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA) as described by the manufacturer with
oligonucleotide primers designed to alter tyrosine residues (TAC or
TAT) to phenylalanines (TTC or TTT). The following mutants of rat
Stat5b were generated by this method: Y699F, Y699F/Y742F,
Y699F/Y739F, Y699F/Y724F, Y699F/Y683-82F, Y699F/Y679F, Y699F/Y668F,
Y699F/Y742F/Y739F, Y699F/Y742F/Y739F/Y724F, Y699F/Y742F/Y739F/Y724F/Y683-82F,
Y699F/Y742F/Y739F/Y724F/Y683-82F/Y679F, Y699F/Y742F/Y739F/Y724F/Y683-82F/Y679F/Y668F. For the
dominant-negative C-terminal-truncated isoform Stat5b
40, the Y699F
mutant was also generated. All mutations were confirmed by direct
sequence analysis.
Cell Culture, Transient Transfections, and Luciferase
Assays--
HeLa cells were maintained in Opti-MEM media (Invitrogen),
supplemented with 3% fetal bovine serum and gentamicin (50 µg/ml) in
a 37 °C and 5% CO2 incubator. DNA constructs (10 µg)
were transiently transfected into HeLa cells by using a calcium
phosphate precipitation technique (5Prime-3Prime, Boulder, CO). For the
co-transfection experiments, 4 µg of Stat5a or Stat5b expression
vector and 6 µg of PrlR or Src kinase construct were utilized. Before
Prl or Src induction, the cells were incubated overnight in medium
containing 10% charcoal-stripped horse serum, insulin (5 µg/ml),
gentamicin (50 µg/ml), and hydrocortisone (1 µg/ml) and then (for
the Prl experiments only) stimulated with ovine Prl for 30 min (1 µg/ml, lot AFP-10677C, kindly provided by the National Hormone and
Pituitary Program, NIDDK, National Institutes of Health, Bethesda, MD). Alternatively, for reporter assays, Stat5a/5b-deficient MEFs derived from Stat5a/5b knockout mice (66) kindly provided by Dr. James Ihle,
were transiently co-transfected with 0.5 µg of Stat5b or a
Stat5bY679F mutant, 1 µg of v-Src and 1 µg of a
674CD1-Luc reporter construct. Experiments were performed in 6-well plates, and
each transfection was done in triplicate. Lysates were prepared 24 h after transfection, and luciferase activity was measured using a
Promega luciferase kit (Madison, WI). Luciferase values were normalized
to protein amount by Western blot analysis for Stat 5b.
Antibodies--
The specific, affinity-purified rabbit
polyclonal antiserum generated against peptides from the unique amino
acid epitopes present in the C-terminal regions of Stat5a and Stat5b
have been characterized previously (50, 52). The N-terminal Stat5
(N-20) antibody was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Phosphotyrosine antibody (PY-20) was purchased from
Transduction Laboratories (Lexington, KY). v-Src antibody was
purchased from Calbiochem (La Jolla, CA). The site-specific antibody to
Ser-730-phosphorylated Stat5b was kindly provided by Dr. Robert Kirken
(University of Texas Medical School, Houston, TX).
Preparation of Cells Extracts--
Cells were rinsed twice with
ice-cold phosphate-buffered saline (9.1 mM dibasic sodium
phosphate, 1.7 mM monobasic sodium phosphate, 150 mM sodium chloride, pH 7.4) and scraped into 2 volumes of
Wu buffer (51) containing 10 mM HEPES, pH 7.4, 1.5 mM magnesium chloride, 0.1 mM EGTA, 10%
glycerol, 1 mM dithiothreitol, 10 µg/ml aprotinin, 10 µg/ml antipain, 10 µg/ml benzamidine, and 5 µg/ml leupeptin.
After lysis in Wu buffer, cells were centrifuged for 15 min at 4 °C
and 14,000 rpm. Supernatant was retained as the cytoplasmic extract.
After washing of remaining pellets twice with 5 volumes of Wu buffer,
they were resuspended in two volumes of the same buffer with 400 mM of sodium chloride. The samples were incubated on ice
for 10 min at 4 °C and then centrifuged for 15 min at 4 °C and
14,000 rpm. The supernatants were dialyzed in Invitrogen microdialysis
units for 2 h at 4 °C against 1 liter of Wu buffer with 100 mM sodium chloride and were retained as nuclear extracts.
Protein concentrations were determined using a Bio-Rad protein assay.
Oligonucleotide Labeling and Electrophoretic Mobility Shift
Assays (EMSA)--
Five oligonucleotides were designed, annealed, and
used for the EMSA experiments (Table I).
-Casein and APRE-2 were
end-labeled using the Klenow fragment of DNA polymerase with
[
-32P]CTP, while all others were end-labeled by T4
polynucleotide kinase with [
-32P]ATP. EMSA were
performed as described previously (55). Briefly, nuclear extracts (10 µg for each sample) were preincubated in 10 µl of Wu-buffer
containing 100 mM sodium chloride and 2 µg of poly(dI-dC)
(Amersham Biosciences) for 15 min on ice. If antibody was used for
supershift analysis, it was included in the 10 µl of Wu buffer. After
preincubation to block nonspecific binding and allow the antibody to
bind, 5 µl of binding mixture (50 µg/ml p(dN)6, 2.5 mg/ml bovine serum albumin, 4% Ficoll 400, and 10% glycerol) with
labeled probe (20,000 cpm) was added to each sample and incubated on
ice for 15 min. The reaction mixture was loaded onto a 4%
polyacrylamide gel in 0.25× Tris borate-EDTA buffer, electrophoresed,
dried, and subjected to autoradiography. Immunoprecipitation and
Western blot analysis were performed as described previously (9).
Indirect Immunofluorescence--
For indirect immunofluorescence
cells were cultured on glass coverslips coated with
poly-D-lysine, fixed and stained as described in (9).
Images were obtained using a DeltaVision deconvolution microscope and
softWoRx imaging workstation from Applied Precision, Inc. (Issaquah, WA).
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RESULTS |
DNA Binding Specificity of Stat5a and Stat5b Induced by
Prl--
To explore DNA binding specificities of rat Stat5a and Stat5b
to different DNA sequences that contain the consensus Stat5 binding
motif, e.g.
-casein GAS site, as compared with those promoters with minor differences, e.g. p21-SIE2 and APRE GAS
sites (Table I), HeLa cells were
transfected with expression vectors for Stat5a or Stat5b and PrlR.
After stimulation with Prl for 30 min, nuclear extracts were prepared,
and EMSA was performed (Fig. 1,
panel A). As expected, following Prl activation, both Stat
5a and Stat 5b formed complexes with the single GAS sites in the
-casein, p21-SIE2, and to APRE probes with different intensities most likely reflecting slightly different affinities to the consensus and nonconsensus Stat5 binding sites (Fig. 1, panel A,
lanes 4 and 6). A slight difference in the
mobility of Prl-inducible DNA binding complexes for Stat5a and Stat5b
was observed (compare lanes 4 and 6, Fig. 1,
panel A), which also was expected based upon previous
observations (9, 46). Two supershifted complexes were observed in
experiments using the anti-Stat5a C-terminal-specific antibody with all
of the indicated probes (Fig. 1, panel A, lane 7), while only one supershifted complex was seen with the
anti-Stat5b C-terminal-specific antibody (Fig. 1, panel A,
lane 8). To control for Stat5a and Stat5b activation in
these transiently transfected cells, immunoprecipitation (IP)
experiments were performed using an anti-Stat5 N-terminal (N-20)
antibody, which recognizes both Stat5 isoforms, followed by
immunoblotting with an anti-phosphotyrosine antibody (Fig. 1,
panel B). Similar levels of tyrosine phosphorylation were
observed for both Stat5a (Fig. 1, panel B, lane
4) and Stat5b (Fig. 1, panel B, lane 6).
Direct Western blot analysis also indicated a similar level of
expression of both proteins (Fig. 1, panel C). This
experiment established the basic parameters for investigating Stat5a
and Stat5b-DNA interactions using the nuclear extracts from Prl-treated
HeLa cells.

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Fig. 1.
DNA binding and tyrosine phosphorylation of
Stat5a and Stat5b induced by Prl. A, HeLa cells were
co-transfected with expression vectors for Stat5a or Stat5b and PrlR.
Nuclear extracts were prepared from non-stimulated cells (lanes
1, 3, and 5) and cells stimulated for 30 min
with Prl (lanes 2, 4, and 6). For
supershift assays, 1 µl of anti-Stat5a (lane 7) or
anti-Stat5b (lane 8) C-terminal specific antibodies were
added. EMSA were performed with the indicated probes. The positions of
the Stat5-DNA binding complexes are indicated by arrows at
the left and the supershifted complexes by arrows
at the right. B, IP-Western. IP by anti-Stat5a or
anti-Stat5b N-terminal-specific antibodies, separated by SDS-gel
electrophoresis, blotted, and probed with anti-phosphotyrosine PY-20
antibody. C, direct Western blot analysis of cell extracts
with anti-Stat5a and anti-Stat5b C-terminal antibodies.
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Stat5a Binds as a Tetramer to the Duplicated APRE Probe That
Contains Two Stat Sequences, whereas Stat5b Binds as a
Dimer--
Since both Stat5a and Stat5b dimers exhibited decreased
affinity for the APRE probe containing a single, nonconsensus Stat5 binding site, we next wanted to confirm whether the expected
differences in binding of the Stat5a tetramer to two copies of the APRE
element (APRE-2) (Table I) as compared with Stat5b dimer would be
observed following Prl activation. Both Stat5a and Stat5b induced by
Prl formed DNA complexes with the expected mobilities and intensities (Fig. 2, panel A). A slower
mobility and more intense Stat5a tetrameric complex was observed bound
to the APRE-2 probe (Fig. 2, panel A, lane 4), in
contrast to the faster mobility, weaker Stat5b dimer complex (Fig. 2,
panel A, lane 10). To verify whether Stat5a bound
as a tetramer to APRE-2, DNA binding analysis of N-terminal mutants,
Stat5aW37A and Stat5bW37A were performed. Since the Trp-37 residue is
required for efficient tetramerization of Stat5 (23), this mutation
should block tetramer formation and cooperative DNA binding of Stat5a
to APRE-2. As shown in Fig. 2, panel A, this N-terminal
mutation of Stat5a completely inhibited its DNA binding activity to
APRE-2 (lanes 7 and 8), while the comparable mutation of Stat5b did not exhibit any significant effect on DNA binding (Fig. 2, panel A, lanes 13 and
14). Thus, following Prl activation Stat5a binds as a
tetramer to the duplicated APRE sequence whereas Stat5b binds as a
dimer, similar to previous results obtained with the human
CIS promoter (26). Again, the supershift with the
anti-Stat5b-specific antibody showed only one complex (Fig. 2,
panel A, lanes 11 and 14), while
multiple complexes were detected with the anti-Stat5a-specific antibody
(Fig. 2, panel A, lane 5). Finally, we
investigated the DNA binding activity of Stat5b and its N-terminal
mutant activated by Src tyrosine kinase to the APRE-2 probe (Fig. 2,
panel B). Both Stat5b (lane 5) and the Stat5bW37A
mutant (lane 6) exhibited a similar mobility DNA binding complex when induced by Src suggesting that regardless of the mechanism
of activation Stat5b interacted with the APRE-2 probe as a dimer.

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Fig. 2.
DNA binding of Stat5a, Stat5b, and their
N-terminal mutants Stat5aW37A and Stat5bW37A induced by Prl and
Src. A, DNA binding induced by Prl. HeLa cells were
transfected either with Stat5a or Stat5b or Stat5aW37A or Stat5bW37A
constructs and the PrlR expression vector. Nuclear extracts were
prepared from unstimulated (lanes 1, 3,
6, 9, and 12) or Prl-stimulated cells
(lanes 2, 4, 5, 7,
8, 10, 11, 13, and
14) and assayed in EMSA with the APRE-2 probe. The positions
of Stat5 tetrameric and dimeric DNA binding complexes are indicated by
arrows at the left and supershifted complexes by
arrows at the right. B, DNA binding of
Stat5b and Stat5bW37A-mutant induced by Src. 1, EMSA was
performed with nuclear extracts of HeLa cells transfected with Stat5b
or Stat5bW37A and Src using the APRE-2 probe. The positions of Stat5
complexes are indicated by arrows at the right
and supershifted complexes by arrows at the left.
2, direct Western blot analysis of cell extracts performed
with anti-Stat5b C-terminal specific antibody.
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DNA Binding Complexes of Prl- and Src-inducible Stat5b Have
Different Electrophoretic Mobilities--
Previously it had been shown
that Src induction resulted in the selective translocation and
retention of Stat5b as compared with Stat5a in the nucleus, whereas no
differences in their nuclear localization and retention were found
following Prl activation (9). While the precise mechanisms responsible
for these differences were unknown, analysis of chimeric proteins
suggested that the critical determinants resided in the C-terminal half
of Stat5b. To examine possible differences in the DNA binding
specificities of Stat5b induced by Src versus Prl, a Stat5b
expression vector was co-transfected into HeLa cells along with either
Src or PrlR, as described under "Experimental Procedures."
Surprisingly, the DNA-bound complexes of Stat5b induced by Prl (Fig.
3, panel A, lane 5)
exhibited a consistently slower mobility compared with those induced by
Src with all five probes examined (Fig. 3, panel A,
lane 6). Interestingly, the supershift of Stat5b-DNA
complexes induced by Prl with the anti-Stat5b C-terminal specific
antibody was almost complete (Fig. 3, panel A, lane
7), while only a partial supershift of Stat5b-DNA complexes
induced by Src was observed in parallel (Fig. 3, panel A,
lane 8). This result suggested that the C terminus of Stat5b
bound to DNA following Src induction might be less accessible to the
anti-Stat5b C-terminal specific antibody than it was following Prl
induction.

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Fig. 3.
Different mobilities and antibody supershift
properties of Prl- and Src-induced Stat5b DNA binding complexes.
A, DNA binding of Stat5b induced by Prl (lane 5)
and by Src (lane 6) with indicated oligonucleotide probes.
The positions of Stat5b-DNA complexes (5b) are indicated by
arrows on the right and supershifted complexes
(ss) by arrows in the center.
B, Western blot analysis was performed with anti-Stat5b
C-terminal-specific antibody. The blot was stripped and reprobed with
an anti-Src antibody.
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Unexpectedly, when the IP-Western controls were performed, different
levels of tyrosine phosphorylation of Stat5b activated by Prl (Fig.
4, panel A, lane 5)
as compared with Src (Fig. 4, panel A, lane 6)
were detected when Stat5b was immunoprecipitated using the anti-Stat5b
C-terminal-specific antibody. Remarkably, the level of total Stat5b
protein, detected by IP, was also different for Stat5b activated by Prl
(Fig. 4, panel B, lane 5) versus Src (Fig. 4, panel B, lane 6), while direct Western
blotting indicated that roughly equivalent amounts of Stat5b protein
were present in the cell extracts (Fig. 4, panel C,
lanes 5 and 6). Because proteins detected by IP
are more likely to exist in their native conformation, as compared with
the denatured state present in SDS-PAGE and Western blotting, we
hypothesized that this difference in the total levels of Stat5b
detected following IP might be explained by the decreased accessibility
of the C-terminal epitope following Src as compared with Prl
activation. To test this hypothesis, we performed IP-Westerns using an
anti-Stat5b N-terminal antibody. Interestingly, these experiments
showed similar levels of Stat5b expression and tyrosine phosphorylation
induced by Prl (Fig. 4, panels D and E,
lanes 5 and 6) and by Src (Fig. 4, panels
D and E, lanes 5 and 6).

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Fig. 4.
Stat5b induced by Src contains a site(s) of
tyrosine phosphorylation in addition to Tyr-699. All experiments
were repeated three times and revealed similar results; representative
pictures are shown. A, IP-Western. HeLa cells were
co-transfected with Stat5b or Stat5bY699F mutant along with PrlR or Src
expression vectors. After stimulation with Prl for 30 min, cell
extracts were immunoprecipitated with an anti-Stat5b
C-terminal-specific antibody and subjected to Western blotting with
anti-phosphotyrosine antibody (PY-20). B, the Western blot
was stripped and reprobed with anti-Stat5b C-terminal-specific
antibody. C, direct Western blot analysis of cell extracts
performed with anti-Stat5b C-terminal specific antibody. D,
IP of cell extracts performed with the anti-Stat5b N-terminal antibody
followed by Western blotting with the anti-phosphotyrosine (PY-20)
antibody. E, the Western blot was stripped and reprobed with
anti-Stat5b N-terminal antibody.
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Remarkably, using an anti-Stat5b C-terminal antibody for
immunostaining, we observed routinely that the percentage of cells with
nuclear staining for Stat5b induced by Src was ~60-70% less than
that observed in cells induced by Prl (data not shown), confirming the
results of the immunoprecipitation and antibody supershift experiments
described above. Based on all these results taken together, we
postulate that the DNA-bound complexes of Stat5b, stimulated with Src
and Prl have different conformations of their C-terminal
transactivation domains, which might be a result of differential phosphorylation.
Stat5b Induced by Src Displays Additional (to Tyr-699) Tyrosine
Phosphorylation as Well as Serine Phosphorylation at Ser-730--
To
determine if there was differential phosphorylation of Stat5b as a
consequence of Src as compared with Prl activation, HeLa cells were
co-transfected with expression vectors for Stat5b, or a Stat5bY699F
mutant, and PrlR or Src. After stimulation with Prl for 30 min, cell
extracts were prepared and subjected to immunofluorescence, EMSA and
immunoblotting analysis. Since Tyr-699 of rat Stat5 is the site of
phosphorylation by JAK-2 and the primary regulator of Stat5
dimerization and subsequent DNA binding (27), the mutation of this Tyr
to Phe should block these processes. As expected, nuclear localization
and DNA binding of Stat5b induced by either Src or Prl was completely
prevented by the Y699F mutation (data not shown). However, IP-Westerns
performed with the anti-Stat5b C- and N-terminal-specific antibodies
followed by blotting with anti-phosphotyrosine antibody revealed
additional sites of tyrosine phosphorylation of Stat5b induced by
the Src tyrosine kinase (Fig. 4, panels A and
D, lane 9). In contrast, no additional tyrosine phosphorylation of Stat5bY699F in response to Prl was detected (Fig. 4,
panels A and D, lane 8). Equivalent
amounts of Stat5b were expressed (Fig. 4, panel C) and
immunoprecipitated by N-terminal Stat5b antibody (Fig. 4, panel
E) from all cell lysates. Differences in the levels of Stat5b
following activation by Src and Prl were once again detected when the
IP was performed with the anti-Stat5b C-terminal-specific antibody
(Fig. 4, panel B) as discussed above.
Ser-730 has been characterized previously as a major site of
Prl-inducible serine phosphorylation of Stat5b (11). Accordingly, we
wanted to determine if differential effects of Src and Prl on the
phosphorylation of Ser-730 in Stat5b might also explain the apparent
conformational differences observed in the C terminus. For this
purpose, a Stat5bS730A mutant was co-transfected into HeLa cells along
with PrlR and Src expression vectors. After stimulation with Prl, cell
extracts were prepared and subjected to IP-Westerns using the
anti-Stat5b N-terminal-specific antibody followed by blotting with a
specific anti-phosphoserine antibody (
-PS730) that recognizes
phospho-Ser-730 (Fig. 5). Activation by
both Prl (Fig. 5, panel A, lane 5) and Src (Fig.
5, panel A, lane 6) resulted in increased Stat5b
phosphorylation at Ser-730 and, as expected, in both cases the S730A
mutation completely prevented this increase (Fig.
6, panel A, lanes 8 and 9). These differences (Fig. 5, panel A,
lanes 5 and 6 compare with lanes 8 and
9) cannot be attributed to slightly different levels of
Stat5b and Stat5bS730A expressed in these experiments (Fig. 5,
panel B).

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Fig. 5.
Src-induced Stat5b serine phosphorylation at
Ser-730. A, HeLa cells were co-transfected with Stat5b
or Stat5bS730A mutant along with PrlR or Src constructs. After
stimulation with Prl, cell extracts were subjected to IP with
anti-Stat5b N-terminal antibody followed by Western blotting with the
site-specific phosphoserine ( -PS730) antibody. B, the
Western blot was stripped and reprobed with anti-Stat5b N-terminal
antibody.
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Fig. 6.
The dominant-negative C-truncated
Stat5b 40 mutant contains a site(s) of
Src-induced tyrosine phosphorylation in addition to Tyr-699.
A, location of tyrosine residues at the C-terminal end of
rat Stat5b and its dominant-negative isoform Stat5b 40. B,
IP-Westerns. HeLa cells were co-transfected with the expression vectors
for Stat5b, Stat5bY699F, Stat5b 40, Stat5b 40Y699F, and Src. Cell
extracts were immunoprecipitated with anti-Stat5b N-terminal antibody
and subjected to Western blotting with anti-phosphotyrosine antibody
(PY-200). C, the Western blot was stripped and reprobed with
anti-Stat5b N-terminal antibody. D, the level of tyrosine
phosphorylation was normalized to the recovery of total Stat5b and
Stat5b 40 proteins by densitometric scanning of Western blots using a
Molecular Dynamics densitometer with an ImageQuant program. The results
shown are representative of three independent experiments.
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Tyr-679 and Tyr-724 Are Potential Sites of Src-induced Tyrosine
Phosphorylation in Stat5b in Addition to Tyr-699--
Since
Src-induced tyrosine phosphorylation appeared to influence the
conformation of the C-terminus of Stat5b, it was important to determine
if the naturally occurring C-terminal-truncated isoform Stat5b
40
exhibited any differences in tyrosine phosphorylation on tyrosine
residues others than Tyr-699 relative to wild-type Stat5b. Since the
truncated Stat5b
40 mutant does not contain any additional tyrosines
as compared with wild-type Stat5b (Fig. 6, panel A), it was
predicted that the 40-amino acid C-truncation of Stat5b might not
affect the Src-induced tyrosine phosphorylation at residues other than
Tyr-699. To test this prediction, HeLa cells were co-transfected with
expression vectors for Stat5b, Stat5bY699F, Stat5b
40,
Stat5b
40Y699F, and Src. Cell extracts were prepared and
analyzed by IP-Westerns. As expected, both mutants Stat5bY699F
and Stat5b
40Y699F exhibited equivalent levels of tyrosine
phosphorylation at one or more additional tyrosines (Fig. 6,
panels B-D).
To determine the potential involvement of specific tyrosine residues,
other than Tyr-699, in the Src-induced tyrosine phosphorylation of
Stat5b, mutational analysis of all the tyrosines present at its
C-terminal end (Fig. 6, panel A) was then performed.
Tyrosine mutations were generated on the Stat5bY699F mutant by
replacing all the tyrosines from Tyr-742 to Tyr-668 with phenylalanine
in a sequential manner. Specific Tyr to Phe mutants were transfected into HeLa cells activated with Src. IP-Westerns were performed using
the specific anti-Stat5b N-terminal antibody for immunoprecipitation, followed by blotting with an anti-phosphotyrosine antibody. The level
of tyrosine phosphorylation (Fig. 7,
panel A) was normalized to the recovery of total Stat5b. No
significant difference in the level of tyrosine phosphorylation of
Stat5bY699F in response to Src was observed following mutation of
Tyr-742 and Tyr-739 to Phe. However, the additional mutation Y724F
resulted in a 2-fold decrease in phosphotyrosine levels. Interestingly,
when the two neighboring tyrosines, Tyr-683 and Tyr-682, were changed
to phenylalanines, an increase in the level of phosphotyrosine was
observed. Additional mutation of Tyr-679, the only conserved tyrosine
residue present in Stat5b but not in Stat5a, to Phe markedly reduced
the total level of Src-induced tyrosine phosphorylation. These
sequential Tyr to Phe mutation experiments suggested the possibility
that two tyrosines, Tyr-724 and Tyr-679, in addition to the major site of tyrosine phosphorylation at Tyr-699, might contribute to the level
of Src-induced tyrosine phosphorylation of Stat5b.

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Fig. 7.
Tyr-679 and Tyr-724 are potential sites of
Src-induced tyrosine phosphorylation in Stat5b in addition to
Tyr-699. A, IP-Western. HeLa cells were transiently
co-transfected with plasmids encoding wild-type Stat5b or tyrosine
mutants generated on the Stat5bY699F in a sequential manner and Src.
B, HeLa cells were transiently co-transfected with
expression plasmids encoding Src and either wild-type Stat5b or
Stat5bY699F mutant, or the double mutants generated by replacing
tyrosine with phenylalanine on Stat5bY699F. Cell extracts were
prepared, immunoprecipitated with anti-Stat5b N-terminal antibody and
separated by SDS-PAGE. 1, the Western blot was performed
with anti-phosphotyrosine monoclonal antibody (PY-20). 2,
the Western blot was stripped and reprobed with anti-Stat5b N-terminal
antibody. 3, the Western blots were quantitated by scanning
on a Molecular Dynamics densitometer and quantitated using ImageQuant.
The level of tyrosine phosphorylation of each Stat5b mutant was
compared with wild-type Stat5b that is depicted as 100% recovery. The
results shown are representative of three independent
experiments.
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To confirm this result, individual tyrosine to phenylalanine mutations
at the C-terminal end of Stat5bY699F were generated, and these double
mutants were tested for their levels of Src-induced tyrosine
phosphorylation as described above. The results of the double mutant
analysis (Fig. 7, panel B) correlated well with those
obtained following the sequential mutation of all the C-terminal tyrosine residues (Fig. 7, panel A). No significant changes
in phosphorylation were observed for Y699F/Y742F and Y699F/Y739F mutants. In contrast, a decreased level of tyrosine phosphorylation was
seen with the Y699F/Y724F mutant, and an increased level of tyrosine
phosphorylation was once again observed in the Y699F/Y683-82F mutant.
Finally, very low levels of tyrosine phosphorylation (<1%) were
detected for mutant Y699F/Y679F. Tyrosine phosphorylation also was not
detected for the Y699F/Y668F mutant, but Tyr-668 is located within the
SH2 domain of Stat5b (Fig. 6, panel A).
Mutation of Tyr-679 to Phe Resulted in an Altered Pattern of Stat5b
Nuclear Localization Induced by Src Kinase, and Decreased v-Src
Activation of the Cyclin D1 Promoter--
Because Tyr-679 is a unique
site of tyrosine phosphorylation present in Stat5b, but not Stat5a, and
the mutation of this residue resulted in a major decrease in
Src-induced Stat5b tyrosine phosphorylation, the potential functional
consequences of phosphorylation of this particular tyrosine were
investigated using both cell and molecular approaches. Because of the
previously observed differences in nuclear localization of Stat5b as
compared with Stat5a following Src activation (9), the effects of
mutation of Tyr-679 to Phe, on the pattern of nuclear localization of
Stat5b were examined. For this study, HeLa cells were co-transfected
with expression vectors for either the wild-type Stat5b or the
Stat5bY679F mutant together with c-Src or PrlR and analyzed using
deconvolution microscopy (Fig. 8). As
expected, a similar punctate pattern of nuclear staining resistant to
Triton-csk treatment was observed for Stat5b activated either by Src
(Fig. 8A) or by Prl (data not shown). While the Stat5bY679F
mutant was localized in the nucleus in response to both prolactin and
Src activation (Fig. 8, C and B, respectively), a
significantly different pattern of nuclear staining was
observed for this mutant, as compared with wild-type Stat
5b. Thus, a punctate pattern of nuclear staining with a few large
speckles was observed for the Stat5bY679F mutant following Prl
activation (Fig. 8C) similar to that seen for wild-type
Stat5b after stimulation with either Prl or Src (Fig. 8A).
However, in cells co-expressing Stat5bY679F and Src, a very different
nuclear pattern with numerous tiny speckles was observed (Fig.
8B).

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Fig. 8.
The Stat5bY679F mutant induced by Src
exhibits a different pattern of nuclear localization as compared with
the wild-type Stat5b. HeLa cells were co-transfected with
expression vectors for Stat5b or Stat5bY679F together with
constitutively active Src kinase (panels A and B)
or PrlR with subsequent Prl induction for 30 min (panel C).
Nuclei were permeabilized and extracted as described previously
(9) following by fixing and staining with a specific C-terminal Stat5b
antibody conjugated with Texas Red (red) and with DAPI
(blue). Images were obtained using a DeltaVision
deconvolution microscope as described under "Experimental
Procedures."
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The effect of the Stat5bY679F mutation on Src-stimulated gene
transcription was evaluated in transient transfection experiments using
a cyclin D1 promoter-luciferase reporter construct previously shown to
respond to both v-Src and Stat5 activation (40, 42, 68). This is a
complex promoter, which responds to a numerous signal transduction
pathways, and contains multiple transcription factor binding sites,
including
481Stat5 (40, 42) and
58CREB/ATF-2 (68) binding sites. To
eliminate effects of endogenous Stat5b present in HeLa cells and to
provide a more appropriate genetic background in which to test specific
Stat5b mutants, these transfection experiments were carried out in
Stat5a/5b-deficient MEFs. These studies were performed with v-Src
rather than c-Src, because the increased transcriptional activation of
the cyclin D1-luciferase reporter in the Stat5a/5b-deficient MEFs
(6-7-fold, as compared with 1.5-2.0-fold) provided a better model in
which to compare wild-type and mutant Stat5b constructs (Fig.
9, panel A, lane 2). In cells co-expressing v-Src and wild-type Stat5b a slight increase in cyclin D1 promoter activity was observed (Fig. 9, panel A, lane 3) as compared with v-Src alone
(Fig. 9, panel A, lane 2). This is not unexpected
because the majority of v-Src-induced cyclin D1 promoter activity is
dependent on the CREB/ATF-2 site (68). Surprisingly, however, a
significant reduction in cyclin D1 promoter activity (comparable to
that seen with v-Src alone) was detected following transfection of the
Stat5bY679F mutant (Fig. 9, panel A, lane 4).
Western blot analysis indicated that approximately equivalent levels of
the wild-type and mutant Stat5b proteins were expressed in these
transient transfection assays (Fig. 9, panel B, lanes
3 and 4).

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Fig. 9.
The Stat5bY679F mutation decreases the
activity of v-Src-induced cyclin D1 promoter activity in
Stat5a/5b-deficient MEFs. A, MEFs were transiently
co-transfected with 1 µg of the 674 cyclin D1 promoter-luciferase
construct (lanes 1-4), v-Src (lanes 2-4),
Stat5b (lane 3), or the Stat5bY679F mutant (lane
4). After 24 h cells were harvested and assayed for
luciferase activity. The transfections were performed in triplicate,
and the experiment repeated at least three times with similar results.
A representative experiment is shown with error bars
denoting S.E. The differences observed with both wild-type Stat5b and
the Stat5bY679F mutant were found statistically significant with
p < 0.05. B, cell lysates were normalized
for Stat5b protein content by Western blotting using -Stat5b
antibodies. Western blots were quantitated by scanning on Molecular
Dynamics densitometer using an ImageQuant program.
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DISCUSSION |
Previous studies from our laboratory demonstrated that
ligand-dependent and -independent signal transduction pathways
may differentially regulate Stat5a and Stat5b nuclear translocation and, therefore, have the potential to differentially regulate Stat5-dependent gene expression (9).
Prl-dependent activation of Stat5a and Stat5b was dependent
on JAK2 activation and resulted in punctate detergent-resistant nuclear
complexes in COS and HeLa cells. In contrast, the selective
translocation of Stat5b to the nucleus following Src and Bcr/Abl
activation did not appear to be dependent on JAK2 activation. Analysis
of Stat5a and Stat5b chimeric proteins suggested that the determinant
responsible for these differences resides in the C-terminal half of the protein.
The present studies were initiated to determine if there might be
differences in the DNA binding specificity of Stat5b activated by Prl
versus Src. It had been suggested that a single amino acid in the DNA binding region of Stat5a and Stat5B could confer distinct DNA binding specificities (46). However, it is now apparent that this
amino acid change (E433G) does not even normally occur in murine
Stat5b, and that the DNA binding domain, and especially the amino acid
residues that contact the DNA helix, are highly conserved in Stat5a and
Stat5b from all mammalian species studied to date (56). Not
surprisingly, therefore, careful binding site selection studies
performed with baculovirus-expressed Stat5a and Stat5b have generated
identical consensus high affinity binding sites (47).
One principal difference between Stat5a and Stat5b appears to be in
their abilities to form tetramers and interact in a cooperative manner
with tandem nonconsensus Stat5 binding sites (23, 48). Tetramer
formation is regulated through interactions between
-helices present
in the N-terminal domain of Stats (18) and is critically dependent on a
single tryptophan residue (Trp-37) conserved in all Stats. In light of
these recent studies, it is not surprising that no major differences
were observed in Prl- versus Src-activated Stat5b binding to
single consensus or nonconsensus Stat5 binding sites or to tandem
nonconsensus Stat5 binding sites (Figs. 1 and 2). However, differences
in the mobility of Stat5b DNA complexes were observed, which were
dependent on the signaling pathway used for activation (Fig. 3). These
differences appear to reflect differences in antibody accessibility to
the C-terminal epitope in Stat5b. This was established by the direct
comparison in IP-Westerns of anti-Stat5 C-terminal and N-terminal
antibodies (Fig. 4).
Differences in the accessibility of the C terminus of Stat5b may exert
profound effects on Stat5b function. The C-terminal region of Stat5 has
the potential to form an amphipathic
-helix, and this region has
been shown to be required for both transcriptional activity and
proteasome-dependent turnover of tyrosine-phosphorylated Stat5 (16, 57). Naturally occurring isoforms of Stat5b containing a
40-amino acid C-terminal deletion result in the generation of a
dominant-negative protein with sustained DNA binding (16, 51). Thus, it
is likely that Src-induced changes in Stat5b C-terminal accessibility
will influence protein-protein interactions with co-activators and
other transcription factors leading to the differential regulation of a
subset of Stat5b-regulated target genes. In contrast, Prl-activation of
Stat5a, which is capable of tetramer formation will selectively
activate a subset of target genes containing tandem nonconsensus Stat5
binding sites.
The most likely explanation for these differences in antibody
recognition is that differential phosphorylation of Stat5b by Src and
Prl might result in different conformations of the C-terminal region of
the protein and altered protein-protein interactions. One likely
candidate was the phosphorylation of Ser-730 in Stat5b, since this has
been shown to be prolactin-regulated, and, although not essential for
DNA binding, may influence the duration of DNA binding following Prl
stimulation (10, 11). Since the inducible phosphorylation of Ser-730 in
Stat5b was observed following both Prl and Src activation, this does
not appear to be the basis for any conformational differences observed
(Fig. 5). However, a number of other serines in Stat5 are known to be
constitutively phosphorylated in mammary epithelial cells and may
provide other potential targets for Src-induced modifications (10).
Although no differences in the phosphorylation of Ser-730 were observed
following Prl- and Src-activation of Stat5b, additional tyrosine
phosphorylation of Stat5b was detected following Src activation of
Stat5b using a Stat5bY699F mutant, in which the principal site of
JAK2-dependent tyrosine phosphorylation was blocked (Fig.
4). Interestingly, EGF has been reported to induce Stat5a tyrosine
phosphorylation at a site or sites in addition to Tyr-694, the major
prolactin-regulated site of tyrosine phosphorylation in Stat5a (8).
This effect also appears to be mediated through the activation of c-Src
by the erbB receptor, since tyrosine phosphorylation was blocked by
pharmacological Src kinase inhibitors and a dominant negative c-Src.
Phosphorylation of Stat5a on tyrosines other than Tyr-694 has also been
observed in response to interleukin-3 and thrombopoietin stimulation
(58).
In the present study, all of the tyrosine residues C-terminal to the
SH2 domain of Stat5b were analyzed following mutagenesis to
phenylalanine, both sequentially and individually, in order to
determine potential sites of Src-induced tyrosine phosphorylation. These studies demonstrated that there are several tyrosines in addition
to Tyr-699 that are potential targets for Src-induced phosphorylation.
Specifically, Tyr-724 (conserved in both Stat5a and Stat5b) and Tyr-679
(unique in Stat5b and not present in Stat5a) were both potential
residues that contributed to the tyrosine phosphorylation of Stat5b in
response to Src kinase. Interestingly, these experimental results
coincided with predicted sites of tyrosine phosphorylation. Among 21 tyrosines present in Stat5b, 5 tyrosines (Tyr-548, Tyr-668, Tyr-679,
Tyr-699, and Tyr-724) were identified by the prediction network to be
potential sites of phosphorylation (59). Tyr-724 and Tyr-679 were
identified to be the most likely potential sites with predicted scores
of 0.860 and 0.832 respectively, followed by Tyr-699, with a score of
0.972. These three tyrosines (Tyr-699, Tyr-724, Tyr-679) are all
located in the C-terminal portion of Stat5, in a region that was
suggested to be highly disordered and extremely flexible by molecular
modeling based on the x-ray crystal structures determined for both
Stat1 and Stat3 (60, 61). While Tyr-699 has been shown to be critical for Stat5 dimerization and interacts with the SH2 domain, the functions
of Tyr-724 and Tyr-679 are less well defined. Conceivably, phosphorylation at these sites can influence the conformation of the C
terminus of Stat5b and protein-protein interactions, as illustrated by
the IP-Westerns performed with the antibodies to the unique C-terminal
epitope present in Stat5b as contrasted to those performed with the
anti-Stat5 N-terminal antibody (Fig. 4).
The present mutagenesis studies also suggested that Tyr-668 may be a
site for Src-dependent tyrosine phosphorylation of Stat5b, because no tyrosine phosphorylation was observed for the
Stat5bY699F/Y668F double mutant (Fig. 7B). However, this
site is located within the SH2 homology region (11, 48, 61-62) and,
therefore, mutagenesis of this tyrosine in addition to Tyr-699 may
influence the structure of the SH2 domain and dimerization of Stat5b,
thus preventing Src-induced tyrosine phosphorylation. Similar results
have been observed for IFN-induced Stat3 tyrosine phosphorylation when
analyzing a Y656F mutant (63).
Tryptic peptide mapping of Stat5b following activation by EGF has been
utilized in order to identify additional sites of tyrosine phosphorylation other than Tyr-699 (64). These studies identified two
series of five and three peptides other than the peptide containing the
major site of tyrosine phosphorylation, Tyr-699, suggesting that one or
more tyrosines may be sites of additional phosphorylation. However, due
to the low yields of the tyrosine phosphorylated peptides it was not
possible to perform amino acid analysis to identify the specific sites
of tyrosine phosphorylation induced by EGF. Instead, the authors also
employed site-directed mutagenesis to further characterize the putative
sites of tyrosine phosphorylation, similar to the approach employed in
our study. Mutation of Tyr-725 led to a slight, but consistent,
decrease in the EGF-induced tyrosine phosphorylation of Y699F Stat5b
(64), in agreement with our results obtained for the Src-induced
tyrosine-phosphorylated mutant, Y724F (Fig. 7, panels A and
B) (The different numbering reflects the use of rat
versus human Stat5b in that two studies.) However, two other
tyrosines Tyr-740 and Tyr-743 (designated as Tyr-739 and Tyr-742 in our
study) were also shown to be critical for EGF-induced tyrosine
phosphorylation (64), but interestingly these sites were not found to
play a significant role in Src-induced tyrosine phosphorylation of
Stat5b (Fig. 7, panels A and B). These
EGF-induced sites of tyrosine phosphorylation are present both in
Stat5a and Stat5b, and Stat5a is known to be activated by the erbB
family of receptor tyrosine kinases (8), suggesting that these
additional tyrosines cannot be responsible for the differential
activation of target genes by Stat5b. In fact, the reporter gene used
in these studies was a multimerized LHRR GAS site reporter construct, and mutation of specific tyrosines resulted in an increased basal activity of this artificial reporter construct. While this construct was induced by EGF, the parental
-casein gene promoter containing this site in its appropriate context is actually repressed by EGF
induction (65). On the other hand, our results indicated that Tyr-679,
the unique site of tyrosine phosphorylation in Stat5b, appears to
contribute to the of Src-induced tyrosine phosphorylation of Stat5b
(Fig. 7, panels A and B).
Using deconvolution microscopy to obtain high-resolution images, an
altered pattern of nuclear staining for the Src-induced Stat5bY679F
mutant as compared with wild-type Stat5b was observed (Fig. 8, compare
panels A and B). The observation that a mutation of a single tyrosine to a phenylalanine in Stat5b resulted in a
different pattern of nuclear localization helps support the biological
importance of Src-induced phosphorylation of Tyr-679. This altered
pattern of nuclear localization may reflect different conformations of
the C-terminal transactivation domain of Stat5b as indicated by the
IP-Western experiments shown in Fig. 4 and possibly its interactions
with co-activators such as p300/CBP and p160 (69). More detailed
co-localization experiments with different markers for splicing
factors, co-activators, PML bodies, etc. will be required to determine
the significance of these different patterns of nuclear localization
with respect to gene regulation.
The decrease in v-Src-induced cyclin D1 promoter activity observed in
the presence of the Stat5bY679F mutant (Fig. 9) also suggests that
phosphorylation of Tyr-679 may play a functional role in v-Src-induced
proliferation. While Stat5 has been shown to mediate the
transcriptional regulation of cyclin D1, thereby contributing to
cytokine- and prolactin-dependent growth of hematopoietic and breast cancer cells, respectively (40, 42), its role in v-Src
induction of cyclin D1 and proliferation is less clear. The cyclin D1
promoter contains binding sites for a number of transcription factors
other than Stat5, whose activity may be modulated either directly or
indirectly by Src kinase (68). Preliminary studies performed using
constructs containing mutations in the Stat 5 DNA binding site
(
674CD1LUC/SIE1-mut), as well as truncation mutants containing
only
163 bp of the cyclin D1 promoter (
163CD1LUC), suggest that
only part of the inhibitory effect of the Stat5bY679F mutant on v-Src
induced cyclin D1 promoter activity requires Stat5 DNA binding (data
not shown). Thus, it is likely that the altered conformation of the
Stat5b C-terminal region induced by phosphorylation of Tyr-679
influences cyclin D1 promoter activity in part through interaction with
other transcription factors, such as LEF1 and CREB/ATF2 and/or
co-activators as discussed previously.
In conclusion, these experiments demonstrate that
ligand-dependent signal transduction pathways exemplified
by Prl activation, and ligand-independent pathways as illustrated by
Src activation, may exert differential effects on the tyrosine
phosphorylation of Stat5b, and influence antibody recognition of its
C-terminal epitope. As demonstrated previously, Src activation may also
result in the preferential nuclear translocation and retention of
Stat5b. Activated Stat5b may then interact preferentially as a dimer
with single consensus Stat5 DNA binding sites. These differences may, therefore, help to explain the observation from gene knockouts in mice
that Stat5a and Stat5b play both "essential and nonredundant roles"
not only in cytokine signaling (66), but presumably also in mediating
the effects of other signaling pathways. One consequence of these
effects may be the potentiation of oncogenic signals leading to
malignancy. The definition of specific gene targets in different cell
types that are selectively regulated by these pathways is an important
area for future investigation.