Serine Phosphorylation of GH-Activated Signal Transducer and Activator of Transcription 5a (STAT5a) and STAT5b: Impact on STAT5 Transcriptional Activity
Soo-Hee Park,
Hiroko Yamashita,
Hallgeir Rui and
David J. Waxman
Division of Cell and Molecular Biology, Department of Biology,
Boston University (S.-H.P., D.J.W.), Boston, Massachusetts 02215; and
Department of Pathology, Uniformed Services University of the Health
Sciences (H.Y., H.R.), Bethesda, Maryland 20814
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ABSTRACT
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Signal transducer and activator of transcription 5b (STAT5b),
the major liver-expressed STAT5 form, is phosphorylated on both
tyrosine and serine in GH-stimulated cells. Although tyrosine
phosphorylation is known to be critical for the dimerization, nuclear
translocation, and activation of STAT5b DNA-binding and transcriptional
activities, the effect of STAT5b serine phosphorylation is
uncertain. Presently, we identify Ser730 as the site of
STAT5b serine phosphorylation in GH-stimulated liver cells. We
additionally show that the serine kinase inhibitor H7 partially blocks
the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b
without inhibiting STAT5b nuclear translocation. Evaluation of the
functional consequences of STAT5b serine phosphorylation by mutational
analysis revealed an approximately 50% decrease in GH-stimulated
luciferase reporter gene activity regulated by an isolated
STAT5-binding site when STAT5b Ser730 was mutated to
alanine and under conditions where STAT5 DNA-binding activity was not
diminished. No decrease in GH-stimulated reporter activity was seen
with the corresponding STAT5a-Ser725Ala mutant; however, a
decrease in reporter activity occurred when the second established
STAT5a serine phosphorylation site, serine 779, was additionally
mutated to alanine. Unexpectedly, STAT5a-Ser725,779Ala and
STAT5b-Ser730Ala displayed approximately 2-fold higher GH-
or PRL-stimulated transcriptional activity compared with
wild-type STAT5b when assayed using an intact ß-casein
promoter-luciferase reporter. Finally, STAT5b-stimulated gene
transcription was abolished in cells treated with H7, but in a manner
unrelated to the inhibitory effects of H7 on STAT5b Ser730
phosphorylation. These findings suggest that the effects of STAT5b and
STAT5a serine phosphorylation on STAT-stimulated gene transcription can
be modulated by promoter context. Moreover, in the case of STAT5a,
phosphorylation of serine 779, but not serine 725, may serve to
regulate target gene transcriptional activity.
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INTRODUCTION
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SIGNAL TRANSDUCER AND activator of
transcription (STAT) factors are signal transducers that mediate the
effects of a broad range of hormones and cytokines on target gene
expression (1, 2). STAT protein activation is catalyzed by
a cell surface receptor-associated tyrosine kinase of the Janus kinase
(JAK) family, which phosphorylates the STAT protein on a single
C-terminal region tyrosine in response to hormone or cytokine
stimulation. Tyrosine-phosphorylated STATs undergo rapid homo- or
heterodimerization associated with STAT translocation to the nucleus,
where the dimeric, DNA-binding STAT activates target gene
transcription. Dephosphorylation catalyzed by a
phosphotyrosine-specific phosphatase (3) terminates STAT
signaling and returns the inactivated STAT protein to the cytosol.
In addition to this JAK-catalyzed tyrosine phosphorylation reaction,
STAT proteins may undergo serine phosphorylation in a manner that can
be cell type and stimulus dependent (4, 5, 6). In the case of
STAT1, -3, and -4, serine phosphorylation is directed at a conserved
PMSP motif located about 2030 amino acids C-terminal of the
conserved tyrosine phosphorylation site. This phosphorylation can be
stimulated by cytokine treatment and may be catalyzed by a downstream
kinase, e.g. a cytokine- or growth factor-activated MAPK.
Basal serine phosphorylation of STAT proteins has also been observed
(for a review, see Ref. 7). Mutation of the conserved PMSP
serine phosphorylation site (e.g.
STAT1-Ser727, STAT3-Ser727,
or STAT4-Ser721) decreases cytokine-induced
transcriptional activity, supporting the hypothesis that serine
phosphorylation is required for maximal transcriptional activity of
these three STATs and may modulate cytokine responses (4, 8, 9), at least on some promoters (10). Serine
phosphorylation can also negatively regulate cytokine-induced tyrosine
phosphorylation in the case of STAT3 (11). STAT nuclear
translocation and DNA-binding activity are generally not affected by
STAT serine phosphorylation, suggesting that serine phosphorylation
modulates STATs intrinsic transcriptional potential, which is
mediated by a C-terminal trans-activation domain just
downstream of the serine phosphorylation site.
Serine phosphorylation of STAT5a and the closely related (>90%
identical) STAT5b (12) has been observed in cells and
tissues stimulated with STAT5-activating ligands such as GH
(13, 14, 15), PRL (16, 17), and IL-2
(18). In PRL-stimulated cells, both STAT5 forms are
phosphorylated on a conserved serine residue
(STAT5a-Ser725 and
STAT5b-Ser730) located within a PSP
sequence, which corresponds in location to the PSMP serine
phosphorylation sequence of STAT1, -3, and -4 (19). STAT5a
is additionally phosphorylated at a second site, recently identified as
serine 779 (20, 21). STAT5 serine phosphorylation may in
part be mediated by the MAPK cascade, as suggested by binding
interactions between STAT5a and the MAPKs ERK1 and ERK2
(21) and by the inhibition of constitutive, but not
PRL-inducible, STAT5a serine 725 phosphorylation in Nb2 lymphocytes by
the MAPK pathway inhibitor PD98059 (19). Functional
studies of the effects of serine phosphorylation on STAT5s
transcriptional activity have not provided a consistent picture. In the
case of IL-2-activated STAT5 (STAT5a and/or STAT5b),
cytokine-stimulated reporter gene activity is abolished in cells
treated with the serine kinase inhibitor H7, which blocks STAT5 serine
phosphorylation (18). Similarly, GH-activated STAT5a
activity is blocked by inhibition of MAPK activity
(22), which may play a role in STAT5a serine
phosphorylation (21). In contrast, no difference in
PRL-stimulated STAT5 reporter gene activity was seen when cells were
transfected with serine to alanine mutant forms of STAT5b
(Ser730 to Ala) or STAT5a
(Ser725 mutated to Ala,
Ser779 to Ala, or the
Ser725,779Ala double mutant) compared with the
corresponding wild-type STAT5 forms (19, 20). Delayed
tyrosine dephosphorylation was, however, reported for STAT5a-S725A in
cells stimulated with PRL (20). These findings raise
the possibility that the consequences of STAT5 serine phosphorylation
may vary with the activating hormone or cytokine, or perhaps with the
target gene used to evaluate the impact of STAT5 serine
phosphorylation. These and related issues are investigated in the
present study, where we evaluate the functional consequences of STAT5
serine phosphorylation in GH-stimulated cells using site- specific
mutants of STAT5a and STAT5b. Our findings reveal that GH induces the
same pattern of STAT5 serine phosphorylation as that previously
reported for PRL. Moreover, we report that serine phosphorylation can
modulate the transcriptional activity of both STAT5a and STAT5b in a
promoter-dependent manner.
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RESULTS
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Serine Phosphorylation of STAT5b in GH-Stimulated Cells
STAT5a and STAT5b carry out distinct functions in endocrine target
tissues. STAT5a is the principal mediator of mammopoietic and
lactogenic signaling stimulated by PRL, whereas STAT5b is an important
determinant of sexual dimorphic liver gene expression induced by GH
(23). To ascertain whether these distinctive biological
roles of STAT5a and STAT5b, might in part reflect their differential
serine phosphorylation in response to GH and PRL, we first investigated
whether GH induces phosphorylation of STAT5b on the same serine residue
(Ser730) as that reported previously for PRL
(19).
Initial experiments were carried out using COS-1 cells transfected with
GH receptor and either STAT5b or the site-specific mutant STAT5b-S730A
and then stimulated with GH. Western blotting with anti-STAT5b antibody
revealed multiple protein bands, which were previously identified as
differentially phosphorylated forms of STAT5b (Fig. 1A
). STAT5b band 0 (Fig. 1A
) migrates as
a doublet of bands, neither of which appears to be phosphorylated, as
shown previously by phosphatase treatment experiments, whereas STAT5b
band 1a corresponds to serine-phosphorylated STAT5b (14).
This latter conclusion is supported by the absence of STAT5b band 1a in
unstimulated cells transfected with STAT5b-S730A (Fig. 1A
, lane 3
vs. lane 1). STAT5b band 2, previously identified as STAT5b
phosphorylated on both tyrosine and serine, is the major GH-induced
phosphorylated form of wild-type STAT5b (lane 2). In contrast,
STAT5b-S730A was converted to a doublet of proteins after GH treatment
(Fig. 1A
, lane 4 vs. lane 3). Both bands of the doublet were
phosphorylated on tyrosine, as shown by immunoprecipitation with
anti-STAT5b antibody followed by antiphosphotyrosine 4G10 Western
blotting (data not shown; also see below). The lower band corresponds
in mobility to STAT5b phosphorylated on tyrosine alone, i.e.
STAT5b band 1, whereas the upper band of the doublet remains
unidentified (band X). The STAT5b doublet dominates in
STAT5b-S730A-transfected cells treated with GH and is not further
converted to the doubly phosphorylated STAT5b band 2, presumably
because of the block in the secondary serine phosphorylation on residue
730 (see below). The precise relationship between STAT5b-S730A bands 1
and band X is uncertain. The two proteins may correspond to the
tyrosine-phosphorylated counterparts of the two marginally resolved
STAT5b protein forms seen in unstimulated cells (both designated band
0; Fig. 1A
, lanes 1 and 3).

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Figure 1. GH-Stimulated Phosphorylation of STAT5b on
Ser730 and Tyr699
A, COS-1 cells were transiently transfected with wild-type (wt) STAT5b
or STAT5b-S730A as indicated. Thirty-six hours later, cells were either
treated with 200 ng/ml GH for 30 min or were untreated. Total cell
extracts were analyzed on Western blots probed directly with
anti-STAT5b antibody (lanes 14) or analyzed by immunoprecipitation
with anti-STAT5b antibody followed by sequential Western blotting using
anti-pS730-STAT5b and anti-pY699-STAT5b antibody (lanes 59). Band 0
represents nonphosphorylated STAT5b, bands 1 and 1a represent
monophosphorylated forms of STAT5b (tyrosine and serine phosphorylated,
respectively), and band 2 represents STAT5b phosphorylated on both
tyrosine and serine. Band X was shown to be a tyrosine-phosphorylated
form of STAT5b; its serine phosphorylation status could not be
determined. STAT5b (and STAT5a) were not detected in untransfected
COS-1 cells (lane 5). STAT5b bands 0, 1, and X were poorly resolved in
the gel shown in lanes 59. Protein recovery was low in the samples
shown in lanes 2 and 4. B, CWSV-1 cells were treated with 200
µM H7 beginning 1 h before GH treatment. Cells were
then stimulated with GH in the continued presence of H7 for the times
indicated (lanes 710). Control CWSV-1 cells were treated with GH in
the absence of H7 (lanes 16). Total cell extracts were analyzed by
immunoprecipitation with anti-STAT5b antibody, followed by sequential
Western blotting with anti-pS730 STAT5 and anti-STAT5b antibodies. C,
Control CWSV-1 cells (lanes 17 and 15), or cells pretreated with H7
for 1 h (lanes 814) were stimulated with GH for 20 min.
Incubation in the absence of GH (but in the continued presence of H7)
was continued up to 120 min. Shown is a Western blot of cell
extracts, probed sequentially with anti-pY699-STAT5b and anti-STAT5b
antibodies. In view of the low STAT5b protein recovery in several of
the samples in B and C, the specific pSer730 content and
the specific pTyr699 content of STAT5b (pS730-STAT5b or
pY699-STAT5b normalized to total STAT5b protein) were determined by
densitometric quantitation using ImageQuant software. These
normalized data are graphed as a function of time of GH stimulation at
the bottom of B and C, respectively. The basal
pSer730 level in the absence of GH was set at 1.0 in B, and
the maximal pY699-STAT5b signals at 20 min were set at 1.0 in C.
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Western blotting using phospho-STAT5-specific antibodies
(anti-pS730-STAT5b and anti-pY699-STAT5b) further supported these STAT5
band identifications (Fig. 1A
, lanes 59). Thus, STAT5b was basally
phosphorylated on Ser730 (band 1a; Fig. 1
, middle panel, lane 6), and GH stimulated the
formation of STAT5b phosphorylated on both Ser730
and Tyr699 (band 2; Fig. 1
, lane 7). STAT5b
Ser730 phosphorylation was blocked in cells
transfected with STAT5b-S730A (Fig. 1
, lanes 8 and 9, middle
panel), whereas GH stimulated Tyr699
phosphorylation of STAT5b-S730A to form a band that migrated just below
band 2 (band 1/band X; Fig. 1
, upper panel, lane 9). [Of
note, STAT5b bands 0, 1 and X are poorly resolved in the right
panel of Fig. 1A
(c.f. lanes 1 and
4).]
We next investigated whether GH induces phosphorylation of
STAT5b on Ser730, e.g. via a
GH-stimulated serine kinase, and whether
phospho-Ser730-STAT5b serves as a substrate for
the GH-stimulated tyrosine phosphorylation reaction. These studies were
carried out using the GH-responsive liver cell line CWSV-1, where all
the components required for GH-induced STAT5b tyrosine and serine
phosphorylation are expressed endogenously (14, 24).
CWSV-1 cells were stimulated with GH for varying periods of time, and
cell extracts were prepared and analyzed by immunoprecipitation with
anti-STAT5b antibody, followed by sequential probing with the
antibodies shown in Fig. 1B
. As reported previously (14),
STAT5b is basally phosphorylated on serine in CWSV-1 cells
(c.f. presence of STAT5b band 1a in unstimulated cells; Fig. 1B
, lane 1, lower panel). At least a portion of this
phosphorylation is on Ser730, as indicated by the
reactivity of band 1a with
phospho-Ser730-specific anti-STAT5 antibody
(anti-pS730; upper panel). Moreover, GH-induced tyrosine
phosphorylation of Ser730-phosphorylated STAT5b
was readily detectable, as revealed by the appearance of the
pS730-reactive STAT5b, band 2 (lane 2). The GH-stimulated increase in
total pS730-STAT5b normalized to total STAT5b
immunoreactivity (lane 2 vs. lane 1, lower
portion of Fig. 1B
) suggests that STAT5b
Ser730 can be phosphorylated by a
GH-stimulated serine kinase in addition to the basal
Ser730 kinase activity. Both the basal and the
GH-stimulated STAT5b Ser730 kinase activity are
partially inhibited by the serine kinase inhibitor H7 (Fig. 1B
, lanes 7
and 8 vs. lanes 1 and 2). This conclusion is supported by
densitometric analysis of pS730-STAT5b immunoreactivity normalized to
total STAT5b protein (lower portion of Fig. 1B
). Moreover,
the specific phospho-Ser730 content of STAT5b is
seen to decline back to the basal level from its peak 30 min after GH
stimulation.
We cannot determine from the above data whether the
diphosphorylated STAT5b (band 2) is preferentially formed by
GH-stimulated tyrosine phosphorylation of preexisting
phospho-Ser730-STAT5b (band 1a) or by tyrosine
phosphorylation of STAT5b band 0, followed by a secondary,
GH-stimulated Ser730 phosphorylation of a STAT5b
band 1 intermediate. Support for the latter possibility is provided
by Western blot analysis of GH-stimulated CWSV-1 extracts using
antibody specific for STAT5b phosphotyrosine 699. Figure 1C
shows that
GH stimulates the transient formation of pY699-STAT5b at 5 min,
followed by conversion to band 2 in an apparent serine phosphorylation
reaction (lanes 3 and 4 vs. lane 2). In cells treated with
H7, this secondary conversion to STAT5b band 2 is partially blocked.
This finding supports the partial inhibition of GH-stimulated S730
phosphorylation shown in Fig. 1B
(note the persistence in Fig. 1C
of
the pY699-STAT5b-immunoreactive doublet, even at 4080 min; lanes
1113 vs. single band at 20 min in lane 15 in the absence
of H7). Densitometric analysis of the
phospho-Tyr699 signals normalized to total STAT5b
protein verified that H7 treatment slows down the decay in STAT5b
signaling, as shown previously (24).
Transcriptional Activity of Site-Specific STAT5 Serine Mutants
To ascertain the functional significance of GH-stimulated STAT5
serine phosphorylation, serine to alanine mutations were introduced at
the conserved PSP serine phosphorylation site of both STAT5 forms
(STAT5a Ser725 and STAT5b
Ser730). The effects of these site-specific
mutations on GH-stimulated, STAT5-dependent gene transcription were
evaluated in transfection experiments using a luciferase reporter gene
driven by four copies of a STAT5-binding site derived from the promoter
of the rat ntcp gene (25). These studies were
carried out in the liver cell line HepG2, which has low endogenous GH
receptor and STAT5, but serves as a useful model for STAT reporter gene
studies and for expression of GH-regulated liver promoters (25, 26). Wild-type STAT5a and wild-type STAT5b
trans-activated ntcp promoter activity in these
cells by 32- to 40-fold after GH stimulation (Fig. 2A
). STAT5a-Y694F and STAT5b-Y699F, which
are mutated at the established STAT5 tyrosine phosphorylation site,
were inactive, consistent with the absolute requirement of STAT5
tyrosine phosphorylation for activation of gene transcription. By
contrast, mutation of the PSP serine phosphorylation site had a more
subtle effect on STAT5-dependent transcriptional activity. No
significant change in ntcp promoter activity was seen with
STAT5a-S725A compared with wild-type STAT5a, whereas a substantial
(
50%) reduction in activity was seen with the corresponding
STAT5b-S730A mutant (Fig. 2A
). This effect was observed in each of four
independent HepG2 transfection experiments and was confirmed in a
second cell model, transfected COS-1 cells (Table 1
).

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Figure 2. Requirement of Ser730 Phosphorylation
of STAT5b for Maximal Transcriptional Activation of ntcp
Reporter Gene in HepG2 Cells
A, Expression plasmids (200 ng) encoding STAT5a, STAT5b, or the
indicated Tyr to Phe or Ser to Ala STAT5 mutants were transfected into
HepG2 cells together with the firefly luciferase reporter
4xNTCP-Luc (200 ng). pRL-tk-Luc plasmid
(Renilla luciferase) was used as an internal standard
for transfection efficiency. Twenty-four hours later, the cells were
either treated with GH for 1820 h or were untreated. Total cell
extracts were prepared, and relative luciferase activity was assayed as
described in Materials and Methods. Data
shown are the mean ± SD (n = 3), with wild-type
STAT5 expression plasmid activity set at 100. B, EMSA analysis showing
gel-shift complex formed by STAT5a or STAT5b or the indicated STAT5
mutants using a ß-casein probe. Samples were
stimulated with GH overnight, followed by the addition of a fresh
aliquot of GH 30 min before harvesting the cells for EMSA analysis.
Samples were normalized for STAT5 protein content (determined by
Western blotting), with the exception of the sample in lane 6, which
had a high STAT5a protein content. STAT5b EMSA complexes migrate faster
than STAT5a EMSA complexes, as shown previously (41 ).
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EMSA analysis verified that GH activated the DNA-binding activity of
both STAT5b and STAT5b-S730A, as determined using a
ß-casein promoter STAT5-binding site probe. Similarly, GH
activated STAT5a and STAT5a-S725A DNA-binding activity (Fig. 2B
). By
contrast, mutation of the STAT5 tyrosine phosphorylation site abolished
STAT5 DNA-binding activity, as anticipated (Fig. 2B
, lanes 4 and 10).
We conclude that phosphorylation of STAT5b at
Ser730 is not required for DNA-binding activity,
but is required to achieve full activation of ntcp promoter
activity, as demonstrated in both HepG2 and COS-1 cells. In the case of
STAT5a, phosphorylation of the corresponding
Ser725 is not required for maximal
transcriptional activity.
Role of STAT5a Ser779 Phosphorylation
In addition to Ser725 phosphorylation,
STAT5a can be constitutively phosphorylated at a second site,
identified as Ser779 (20, 21). To
investigate the significance of Ser779
phosphorylation, we prepared and then assayed the transcriptional
activity of STAT5a expression plasmids containing a serine to alanine
mutation at position 779 alone, or serine to alanine mutations at
positions 725 and 779 in combination. Figure 3A
shows that GH-stimulated
ntcp promoter activity was significantly lower in the case
of the double serine mutant STAT5a construct in both HepG2 and COS-1
cells. Moreover, the fold stimulation of promoter activity was
substantially decreased (32- to 38-fold for wild-type STAT5a
vs. 12- to 15-fold for STAT5a-S725,779A; Table 1
), despite
the expression of the STAT5a mutant protein at a level at least as
great as wild-type STAT5a (Fig. 3B
, lanes 710 vs. lanes 3
and 4). A less dramatic decrease in transcriptional activity was seen
in the case of STAT5a-S779A (Table 1
), indicating that phosphorylation
of STAT5a on Ser779 in the context of
phosphorylation on Ser725 is required for full
STAT5a transcriptional activity. Phospho-STAT5a analysis confirmed that
mutation of the STAT5a serine phosphorylation sites did not alter
tyrosine phosphorylation of STAT5a at Tyr699
(Fig. 3B
, middle panel, lanes 8, 10, and 12 vs.
lane 4). Moreover, mutation of Ser779 did not
decrease phosphorylation of Ser725 (Fig. 3B
, upper panel, lanes 9 and 10 vs. lanes 3 and
4).

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Figure 3. Requirement of STAT5a Serine Residues 725 and 779
for Full Activation of ntcp Promoter Activity in COS-1
and HepG2 Cells
A, Relative luciferase activity was determined in cells transfected
with the indicated STAT5a expression plasmids as described in Fig. 2 .
B, Shown is a STAT5a Western blot of COS-1 cell extracts transfected as
described in A and treated with GH, as indicated. Cell extracts were
immunoprecipitated with anti-STAT5a antibody followed by sequential
Western blotting with anti-pS725-STAT5a, anti-pY694-STAT5a, and
anti-STAT5a antibody. STAT5a was not detected in untransfected COS-1
cells (lanes 1 and 2). Anti-pS725-STAT5a cross-reacted with a minor
band migrating just below pS725-STAT5a (c.f.
upper blot of B, lanes 7, 8, 11, and 12).
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STAT5 serine phosphorylation can be stimulated by PRL and by a variety
of other activators of cytokine receptor signaling pathways. To
determine whether the consequences of STAT5 serine phosphorylation
differ between receptors, we examined the effects of the STAT5 serine
mutations on ntcp promoter activity in cells cotransfected
with PRL receptor and treated with PRL. Figure 4A
(left panel) shows that PRL
activation of STAT5a or STAT5b led to a substantial increase in
ntcp reporter activity. This gene activation was reduced in
cells transfected with the serine- mutated STAT5 forms,
STAT5a-S725,779A and STAT5b-S730A, just as it was in GH-stimulated
cells.

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Figure 4. Influence of STAT5 Serine Mutations on PRL- or
GH-Stimulated STAT5 Activity Assayed with ß-casein and
ntcp Reporters
STAT5 reporter plasmid, 4xNTCP-Luc or
pZZ1-Luc, was cotransfected in COS-1 cells with
wild-type STAT5a or STAT5b or the indicated Ser to Ala mutated STAT5
expression plasmid. Cells were transfected with PRL receptor for
PRL-activated reporter activity (A) or with GH receptor for
GH-activated reporter activity (B) and the internal control plasmid
pRL-tk-Luc. Twenty-four hours after transfection, cells were stimulated
with 10 nM PRL for 1820 h (A). For the time-course study
(B and C), the cells were stimulated with GH for times ranging from
322 h. For all three panels, relative luciferase activities were then
determined as described in Fig. 2 . Values shown are the mean ±
SD (n = 3).
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Impact of STAT5 Serine Mutations on ß-Casein Promoter
Activity
We next investigated whether target gene promoter context may
influence the effect of the STAT5 serine mutations on the STATs
transcriptional activity. For these experiments we used the reporter
construct pZZ1 (27), which is comprised of 344 nucleotides
of the rat ß-casein proximal promoter linked to a
luciferase reporter gene. This promoter contains a STAT5-binding site
with the same core sequence (TTC-TTG-GAA) as the ntcp
promoter STAT site used in all of the experiments presented above, but
is flanked by a nonconsensus STAT5 site; combined, the pZZ1 STAT
sequence constitutes a strong tetrameric STAT5-binding site (28, 29). Figure 4A
(right panel) shows that ß-
casein promoter activity is strongly activated in COS-1 cells
transfected with PRL receptor together with either STAT5a or STAT5b and
stimulated overnight with PRL. However, in sharp contrast to the
reduced transcriptional activity obtained in parallel experiments with
the ntcp promoter-based luciferase reporter (left
panel), ß-casein promoter transcriptional
activity was increased to an approximately 2-fold higher level in cells
transfected with either STAT5a-S725,779A or STAT5b-S730A compared with
the corresponding wild-type STAT5 proteins. A similar stimulatory
effect of the STAT5 serine mutations on ß-casein promoter
activity was observed in GH receptor-transfected COS-1 cells stimulated
with GH (data not shown; also see below).
The differential effect of STAT5 serine mutation on ntcp vs.
ß-casein promoter activity shown in Fig. 4A
suggests that
the effects of a mutational block in STAT5 serine phosphorylation may
be influenced by promoter context. To further investigate this finding,
we examined the effects of the STAT5b S730A mutation on reporter gene
activity in cells stimulated with GH for times ranging from 322 h. As
shown in Fig. 4B
, mutation of Ser730 to alanine
led to a decrease in GH-stimulated ntcp reporter activity
assayed at each time point. By contrast, this same mutation increased
ß-casein promoter activity when assayed 22 h after GH
addition, as seen in Fig. 4A
in the case of PRL stimulation, but had no
significant impact when the cells were assayed 3, 5, or 8 h after
GH addition (Fig. 4C
). Analysis of extracts prepared from GH-stimulated
cells revealed a higher level of Tyr699
phosphorylation in the STAT5b-S730A-transfected cells (Fig. 5A
, upper panel, lanes 814
vs. lanes 17). Correspondingly, a higher DNA-binding
activity was obtained at all time points examined (Fig. 5B
). This
increased activity of STAT5b-S730A largely reflects a higher level of
STAT5b-S730A protein expression (Fig. 5A
, lower panel, lanes
812 vs. lanes 15). Consequently, total cellular STAT5
DNA-binding activity is higher in the case of the STAT5b mutant (Fig. 5B
), and as a result there is a differential in the DNA-binding
activity found in STAT5b-transfected cells vs.
STAT5b-S730A-transfected cells that is maximal 22 h after GH
stimulation (Fig. 5C
). This finding provides an explanation for the
increased ß-casein promoter activity of STAT5b-S730A at
this time point (c.f. Fig. 4C
).

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Figure 5. Mutation of STAT5b Ser730 Results in
Higher Cellular STAT5b Protein Levels and DNA-Binding Activity
COS-1 cells were transiently transfected with plasmids encoding GH
receptor and either wild-type or S730A-mutated STAT5b. Thirty hours
later the cells were treated with GH as indicated. Cell extracts were
prepared and analyzed on Western blots probed with antibody to
pY699-STAT5b and STAT5b protein (A) or on EMSA gels
(ß-casein probe; B). C, PhosphorImager quantitation of
EMSA band intensities, with the data normalized to the EMSA signal
observed 30 min after GH stimulation. Graphed are the mean ±
SD for data points from 30 min to 22 h of GH
stimulation based on two independent experiments, one of which is shown
in B. The 30-min data point for wild-type STAT5b is set at 100.
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Inhibitory Effect of H7 on STAT5b Activity
The serine kinase inhibitor H7 has previously been used to
support the proposed importance of STAT serine phosphorylation for STAT
transcriptional activity (8, 18). We therefore
investigated whether the reporter-dependent changes in STAT5b activity
seen with STAT5b-S730A could also be observed in cells treated with H7,
which partially inhibits this serine phosphorylation reaction
(c.f. Fig. 1
). Table 2
shows,
however, that H7 treatment of COS-1 cells transfected with wild-type
STAT5b leads to a complete inhibition of GH-stimulated,
STAT5b-dependent transcription of the ntcp-luciferase
reporter gene, in contrast to the 50% activity decrease seen with the
STAT5b-S730A mutant. Moreover, H7 treatment led to a similar inhibition
of the transcriptional activity of STAT5b-S730A, demonstrating that the
inhibitory action of H7 is unrelated to its effects on STAT5b serine
phosphorylation. Complete inhibition of STAT5b and
STAT5b-S730A-stimulated transcription was also observed using the pZZ1
reporter (Table 2
). Finally, a similar inhibitory effect of H7 on
STAT5b-S730A transcriptional activity was obtained in cells stimulated
with GH for a shorter time period (3 h; c.f. 6 h GH
stimulation in Fig. 5A
; data not shown).
Mechanistic studies revealed that H7 did not block STAT5b tyrosine
phosphorylation (Fig. 1C
) or DNA-binding activity (data not shown).
Moreover, H7 treatment did not impair nuclear translocation of STAT5b
protein in its tyrosine-phosphorylated form, as revealed by
immunofluorescence microscopy using anti-STAT5b and anti-pY699-STAT5b
antibodies (Fig. 6
). We conclude that H7
inhibits STAT5b transcriptional activity at a stage that is downstream
of the STAT5b nuclear translocation and DNA-binding steps.

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Figure 6. Immunofluorescence Analysis of GH-Stimulated
STAT5b: Effect of H7 on Nuclear Localization of STAT5b
Shown are confocal microscope images of STAT5b (upper two sets
of panels) and anti-pY699-STAT5b indirect immunofluorescence
(lower two sets of panels) indicating no effect of H7
treatment (B and D vs. A and C) on STAT5b nuclear
translocation after GH stimulation. Control CWSV-1 cells or CWSV-1
cells pretreated with H7 for 1 h were untreated or were treated
with GH for 30 min in the absence or presence of H7 as indicated.
GH-treated cells were fixed and analyzed by immunofluorescence
microscopy with anti-STAT5b antibody (A and B) or anti-pY699-STAT5b
antibody (C and D). Propidium iodide was used to stain nuclei
(lower set of images in each panel). STAT5b or pY699-STAT5b
green fluorescence and propidium iodide red fluorescence images are
presented as grayscale images prepared using Adobe Photoshop.
|
|
 |
DISCUSSION
|
---|
STAT5a can be constitutively phosphorylated on serine at two
sites, Ser725 and Ser779,
whereas STAT5b, which lacks the COOH-terminal peptide sequence
corresponding to Ser779, can be phosphorylated at
a single serine, Ser730, in a manner that is
inducible by either PRL (19) or GH (this report). Whereas
mutation of STAT5a Ser725 had no significant
effect on STAT5 transcriptional activity, mutation of the corresponding
STAT5b Ser730 is presently shown to modulate
STAT5bs transcriptional activity. This modulation is manifest as a
decrease in transcription driven by a promoter sequence containing four
copies of a STAT5-binding site derived from the ntcp gene,
but leads to a time-dependent increase in transcription from a proximal
promoter fragment of the ß- casein gene. This latter
increase is associated with a higher cellular level of STAT5
DNA-binding activity in the case of the
Ser730-mutated STAT5b. These findings indicate
that the functional consequences of STAT5b serine phosphorylation can
vary from one promoter to the next, suggesting that STAT5b serine
phosphorylation may serve as a mechanism to differentially modulate the
expression of STAT5 target genes. This promoter-dependent effect of
STAT5b Ser730 phosphorylation appears to reflect
a change in STAT5bs intrinsic transcriptional activity, insofar as
the decrease in ntcp-luciferase reporter activity observed
with Ser730-mutated STAT5b occurred in cells
where there was an increase in STAT5 DNA-binding activity. Conceivably,
serine phosphorylation may modulate interactions between STAT5b and
other transcription factors bound to the same promoter, or perhaps may
influence the recruitment of STAT-interacting coactivator and
corepressor proteins (30, 31), which is likely to occur in
a promoter-dependent manner. Indeed, interferon-
-activated STAT1
interacts with the nuclear factor minichromosome maintenance-5 in a
phospho-Ser727-dependent manner (9).
Finally, changes in the trans-activation potential of STAT5
can also be achieved by mutations elsewhere in the C-terminal region
(e.g. increased activity of STAT5a-T757V)
(32).
The increased DNA-binding activity of STAT5b-S730A compared with
wild-type STAT5b appears to be due at least in part to increased
expression and/or stability of the Ser730-mutated
STAT5 protein. This raises the possibility that phosphorylation of
Ser730 in the wild-type protein may enhance the
turnover of STAT5b. Although the apparent increase in signaling of
STAT5b-S730A at longer times of GH stimulation could additionally
involve a decrease in the rate of STAT5b tyrosine dephosphorylation, as
was suggested earlier for STAT5a mutated at serine 725 and/or 779
(20), this has not been established. Independent of the
mechanism underlying this effect, the increased cellular DNA-binding
activity of STAT5b-S730A compared with wild-type STAT5b appears to
account for the enhanced ß-casein reporter activity seen
at longer times after GH stimulation. Nevertheless, ntcp
reporter activity was decreased under these same conditions,
highlighting the intrinsic differences in the effects of
Ser730 mutation on transcription of the two
STAT5-responsive reporter genes, discussed above.
Of the two STAT5a serine phosphorylation sites, residues 725 and 779,
Ser779 appears to be more important for maximal
ntcp promoter activity. This is supported by the decrease in
ntcp reporter activity in cells transfected with
STAT5a-S779A, but not in cells transfected with STAT5a-S725A. This
decrease cannot be explained by an effect of the
Ser779 mutation on the phosphorylation of STAT5a
on Tyr694 or Ser725 (Fig. 3B
). Both STAT5a serine residues are likely to be important, however,
as suggested by the more substantial decrease in ntcp
reporter activity displayed by the double mutant, STAT5a-S725,779A.
Similarly, mutation of Ser779, alone or in
combination with Ser725, led to an increase in
ß-casein promoter activity, a response that was also seen
with STAT5b-S730A. In contrast to STAT5b Ser730,
whose phosphorylation is inducible, STAT5a Ser725
and Ser779 can both be constitutively
phosphorylated in a variety of cells and tissues, including developing
mammary gland in the case of Ser779 (19, 20). It is unclear, however, whether both serine residues can be
simultaneously phosphorylated in a given STAT5a molecule, leaving open
the possibility that phosphorylation of Ser725
may inhibit, and thereby help regulate, Ser779
phosphorylation and the resultant
phospho-Ser779-dependent transcriptional
responses.
The stimulatory effects of the STAT5a-Ser779 and
STAT5b-Ser730 mutations on GH-induced
ß-casein promoter activity seen in the present study
contrast with the absence of a clear effect of these mutations in
previous studies in PRL-stimulated cells (19, 20). This discrepancy does not reflect differences in the
stimulating hormone, as we were able to duplicate our findings in
experiments in which STAT5a and STAT5b were activated via the PRL
receptor. Rather, it may relate to differences in the time-dependent
effects of the Ser730 mutation on
ß-casein promoter activity documented in the present
study. Also of note, the two previous studies were carried out in COS-7
cells under conditions where only a 2- to 3-fold stimulation of
ß-casein promoter activity was achieved, which may limit
or mask the modulatory effects of mutating the STAT5 serine
phosphorylation sites. In contrast, the present studies were carried
out in COS-1 cells under conditions where a 20- to 40-fold activation
of the ß-casein promoter was routinely achieved. Further
study will be required to clarify this point.
The serine kinase inhibitor H7, which inhibits GH-stimulated STAT5b
serine phosphorylation, strongly inhibited STAT5b-dependent reporter
gene activity. A strong inhibitory action of H7 was also seen with
STAT5b-S730A, independent of whether activity was assayed with the
ntcp or ß-casein promoter,
indicating that the transcriptional inhibition effected by H7 is
unrelated to the resultant changes in STAT5b
Ser730 phosphorylation. Mechanistic studies
revealed that H7 does not interfere with GH-stimulated STAT5b tyrosine
phosphorylation, nuclear translocation, or DNA-binding activity,
strongly suggesting that H7 exerts a specific inhibitory action at the
level of STAT5 trans-activation. This effect may thus be
distinct from the prolonged signaling by the GH receptor-JAK2 complex
after H7 treatment that we have previously described in GH-stimulated
liver cells (24). Conceivably, H7 may inhibit
STAT5-dependent transcription by altering the phosphorylation of a
STAT5b-interacting coactivator that is required for the STAT
transcriptional response. Alternatively, the inhibition by H7 of STAT5
transcriptional activity may be mechanistically linked to the prolonged
signaling by GH receptor-JAK2 to STAT5b by way of a block in
STAT5-stimulated transcription of feedback inhibitory regulators of GH
receptor-JAK2 signaling, such as SOCS/CIS proteins (33, 34). Further investigation is needed to address this issue.
The kinase(s) that catalyze the constitutive phosphorylation of STAT5a
on Ser725 and Ser779 and
the signaling pathways that lead to the inducible phosphorylation of
STAT5b on Ser730 in response to GH or PRL
stimulation remain to be identified. Inhibitor studies suggest a role
for a MAPK-like activity in the constitutive phosphorylation of STAT5a
on Ser725, but not for the PRL-inducible
phosphorylation of STAT5b on the corresponding
Ser730 (19). Interestingly, in cells
in which the constitutive phosphorylation of STAT5a
Ser725 is blocked by the MAPK kinase inhibitor
PD98059, PRL can induce phosphorylation at that site (19),
demonstrating that Ser725 is intrinsically
responsive to PRL stimulation, in a manner that is analogous to the
inducible phosphorylation of Ser730 in the case
of STAT5b. The additional site of STAT5a phosphorylation, at residue
Ser779, is within the COOH-terminal 20 amino
acids of STAT5a, where the two STAT5 proteins are highly divergent in
sequence. This residue is thus absent from STAT5b. In vitro
phosphorylation of STAT5a by MAPK is strongly inhibited by mutation of
Ser779, as is the interaction of STAT5a with the
MAPK ERK1 and ERK2 (21), suggesting a role for MAPK in
this phosphorylation reaction as well. In other studies, carried out in
a different cell model, phosphorylation of STAT5a at
Ser779 was not blocked by inhibitors of MAPK or
PI3K (20). Interestingly, Ser779
occurs within a sequence (RLSPPA) that corresponds to a
consensus motif for phosphorylation by PKA, but not by nine other
serine protein kinases, as revealed by computer analysis using the
web-based PhosphoBase program (35). Accordingly, further
investigation of the role of PKA/cAMP-dependent signaling pathways in
the phosphorylation of STAT5a at this COOH-terminal site may be
warranted.
STAT5a and STAT5b play distinct physiological roles in mediating
hormonal responses to PRL (STAT5a) and GH (STAT5b) in the mammary gland
and liver, respectively (36, 37). Although this
differential endocrine function may largely reflect the distinct tissue
distributions of the two STAT5 forms, there is increasing evidence that
the biological properties of STAT5a and STAT5b, although very similar,
are distinguishable in several important ways. STAT5a and STAT5b not
only display biochemical differences in apparent DNA binding
specificity (38, 39) and propensity to bind to DNA as
tetramers (STAT5a > STAT5b) (29, 40), but they
exhibit potentially important differences in their regulation by serine
phosphorylation. Thus, the phosphorylation of STAT5a vs.
STAT5b on Ser725/730 is not only subject to
differential regulation (constitutive phosphorylation of STAT5a
vs. inducible phosphorylation of STAT5b), but leads to
modulatory effects on gene transcription only in the case of STAT5b. In
the case of STAT5a, such a modulatory effect requires phosphorylation
on Ser779, a residue unique to this STAT5
form.
 |
MATERIALS AND METHODS
|
---|
Plasmids and Preparation of STAT5 Mutant Constructs
STAT5 plasmids containing site-specific mutations of serine to
alanine (S725A and/or S779A for STAT5a, S730A for STAT5b) or tyrosine
to phenylalanine (Y694F for STAT5a, Y699F for STAT5b) were prepared
from double-stranded plasmid DNA using the QuickChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) and
oligonucleotide primers designed to introduce each mutation, as
described previously (19). Site-specific mutations were
verified by DNA sequence analysis. Expression plasmids for mouse STAT5a
and STAT5b (Dr. L. Hennighausen, NIH, Bethesda, MD), rat GH receptor
(Dr. N. Billestrup, Hagedon Research Institute, Gentofe, Denmark), and
human PRL receptor (Dr. P. Kelly, INSERM, Paris, France) were obtained
from the indicated sources. Luciferase reporter constructs containing
either four copies of a STAT5 binding site derived from the promoter of
the rat ntcp gene (4xNTCP-Luc) or the ß-casein
gene promoter (nucleotides -344 to -1; pZZ1-Luc) were respectively
provided by Drs. M. Vore (University of Kentucky, Lexington, KY) and B.
Groner (Institute for Experimental Cancer Research, Freiburg,
Germany).
Cell Culture and Transfections
COS-1 and HepG2 cells were maintained in DMEM containing 10%
FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin. For transient
transfections, cells were seeded in 24-well plates at a density of
1.3 x 105 HepG2 cells/well or 5 x
104 COS-1 cells/well. Fugene 6 transfection
reagent (Roche Molecular Biochemicals, Indianapolis, IN)
was used as described in the manufacturers protocol, at a ratio of
1.3:1 of Fugene 6/DNA (vol/wt). Each well received a total of 600 ng
DNA, including 150200 ng luciferase reporter plasmid, 50 ng GH
receptor, and 100200 ng STAT5 expression plasmid. pRL-tk-Luc plasmid
(Renilla luciferase; 50 ng DNA) was included as an internal
control for transfection efficiency. Twenty-four hours after
transfection, the cells were treated with rat GH (200 ng/ml) or rat PRL
(10 nM) for an additional 1824 h unless
specified otherwise. H7 (200 µM) was included
as indicated. Total cell extracts were prepared using 1x lysis buffer
(Promega Corp., Madison, WI) for measuring luciferase
activities. For Western blot and EMSA analysis, total cell lysates were
centrifuged for 30 min at 15,000 x g. Firefly and
Renilla luciferase activities were measured using a Dual
Reporter Assay System (Promega Corp.) and a Monolight 2010
luminometer (Analytical Luminescence Laboratory, San Diego, CA). Data
shown in the individual figures are relative values based on normalized
luciferase activity (i.e. firefly/Renilla
luciferase activities; mean ± SD for three
replicates).
Growth and passage of CWSV-1 cells was carried out as described
previously (14). For serine phosphorylation studies,
CWSV-1 cells were stimulated with GH at 200 ng/ml in the presence or
absence of H7 (200 µM) for varying periods of time. Total
cell extracts were prepared in lysis buffer containing 20
mM HEPES (pH 7.9); 1% Triton X-100; 1 mM each
of EDTA, EGTA, Na3VO4,
Na2P2O7,
and dithiothreitol; 0.5 mM phenylmethylsulfonylfluoride;
and 1 µg/ml each of pepstatin, antipain, and leupeptin. Total cell
extracts were passed through a 27-gauge needle seven times, adjusted to
150 mM NaCl, and centrifuged at 15,000 x g
for 30 min at 4 C. Protein concentrations were determined using the Dc
detergent protein assay kit (Bio-Rad Laboratories, Inc.,
Hercules, CA).
EMSA Analysis
Total cell extracts (5 µg) were assayed for STAT5 DNA-binding
activity using a ß-casein STAT5 response element probe
(14). Gels were exposed to PhosphorImager plates
overnight, followed by quantitation of radioactivity intensity using a
Molecular Dynamics, Inc. PhosphorImager and ImageQuant
software (Sunnyvale, CA).
Western Blotting and Immunoprecipitation
Total cell extracts (2030 µg) were electrophoresed on 7.5%
Laemmli SDS gels, electrotransferred to nitrocellulose membranes, and
then probed with anti-STAT5b antibodies (catalogue no. sc-835,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Blocking
and probing conditions were described previously (41).
Probing with anti-pY699-STAT5b antibody (Cell Signaling Technology,
Beverly, MA) was performed with a 1-h incubation of the blot at room
temperature in TST buffer [10 mM Tris-HCl (pH 7.6), 0.1%
Tween 20, and 100 mM NaCl] containing 5% nonfat dry milk,
followed by incubation with anti-pY699 antibody (1:1000 dilution) in
5% BSA-TST buffer overnight at 4 C. Washings were carried out as
specified by the manufacturer. For STAT5 immunoprecipitation, CWSV-1
cells grown on 100-mm dishes were solubilized in 1 ml lysis buffer [10
mM Tris-HCl (pH 7.6), 5 mM EDTA, 50
mM NaCl, 30 mM
Na2P2O7
50 mM NaF, 1 mM
Na3VO4, 1% Triton X-100,
and 1 mM phenylmethylsulfonylfluoride] in the presence of
phosphatase inhibitors (14). Clarified total cell extracts
were incubated for 2 h on ice with 2 µl polyclonal rabbit
anti-STAT5b antiserum (antibody raised against a peptide corresponding
to amino acid residues 776786 of mouse STAT5b was obtained from Dr.
L. Hennighausen, NIH) (42). Immune complexes were captured
with protein A-Sepharose beads (Pharmacia Biotech,
Piscataway, NJ), electrophoresed on 7.5% Laemmli-SDS gels, and then
transferred onto nitrocellulose membranes (Millipore Corp., Bedford, MA). Membranes were blocked for 1 h at 37 C
with 5% nonfat dry milk in TST buffer. Incubations with site-specific
antiphosphoserine STAT5 antibody (anti-pS730) (19) were
carried out for 16 h at a dilution of 1:5000 in the cold-room.
Anti-pS730-STAT5 antibody was raised against the phosphopeptide
DQAP[pS]PAVC, corresponding to amino acid residues 726734 of human
STAT5b (19). Antibody binding was visualized on x-ray film
by enhanced chemiluminescence using the ECL kit from Amersham Pharmacia Biotech (Arlington Heights, IL; anti-STAT5b and
anti-pS730-STAT5b) or the SuperSignal ECL kit from Pierce Chemical Co. (Rockford, IL; anti- pY699-STAT5b).
Immunofluorescence Studies
CWSV-1 cells were seeded at about 60% confluence onto four-well
chamber slides (catalog no. 62409-294, VWR Scientific Products, Boston,
MA) in RPCD medium (14) containing 3% FBS and
allowed to adhere overnight. The medium was then replaced with
serum-free RPCD medium. The following day, the cells were pretreated
with H7 (200 µM) for 1 h as indicated, then treated
with GH (200 ng/ml) and H7 for 30 min. Cells were rinsed with ice-cold
PBS and fixed with 100% MeOH for 20 min at -20 C. Fixed cells were
blocked with 3% charcoal-stripped calf serum in PBS for 1 h at
room temperature and then incubated with anti-STAT5b antibody (1:500
dilution; Santa Cruz Biotechnology, Inc.) in blocking
solution overnight at room temperature. For anti-pY699-STAT5b
immunostaining, fixed cells were blocked with 5.5% charcoal-stripped
calf serum in TBST buffer [50 mM Tris-HCl (pH 7.4), 150
mM NaCl, and 0.1% Triton X-100] for 1 h at room
temperature and then incubated for 24 h at 4 C with
anti-pY699-STAT5b antibody (1:500 dilution; Cell Signaling Technology,
Beverly, MA) in TBS buffer [50 mM Tris-HCl (pH 7.4) and
150 mM NaCl] containing 3% BSA. The samples were then
washed (three times, 5 min/wash) with PBS containing 3% calf serum for
anti-STAT5b and with TBST for anti-pY699-STAT5b antibody. Cells were
then incubated for 1 h at 37 C with fluorescein
isothiocyanate-conjugated goat antirabbit IgG antibody (1 µg/ml;
Molecular Probes, Inc., Eugene, OR). Cells were
counterstained with 50 ng/ml propidium iodide (Sigma) to
localize nuclei. For confocal analysis, immunofluorescent cells were
scanned with an BX-50 confocal laser scanning microscope (Olympus Corp., New Hyde Park, NY) equipped with a x60 objective
(Carl Zeiss, New York, NY).
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Drs. L. Hennighausen, N. Billestrup, P. Kelly,
M. Vore, B. Groner, and F. Lemaigre for providing plasmid DNAs and
antibodies.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. David J. Waxman, Department of Biology, Boston University, Boston, Massachusetts 02215. E-mail:
djw{at}bio.bu.edu
This work was supported in part by NIH Grant DK-33765 (to D.J.W.).
Abbreviations: anti-pS730 antibody, Antibody specific for
phosphoserine 730 of STAT5b and phosphoserine 725 of STAT5a; anti-pY699
antibody, antibody specific for tyrosine 699 or 694 of STAT5b and
STAT5a, respectively; JAK, Janus kinase; PMSP motif, Pro-Met-Ser-Pro;
PSP, Pro-Ser-Pro; STAT, signal transducer and activator of
transcription; TST buffer, 10 mM Tris-HCl (pH 7.6), 5
mM EDTA, 50 mM NaCl, 30 mM
Na2P2O7 50 mM NaF, 1
mM Na3VO4, 1% Triton X-100, and 1
mM phenylmethylsulfonylfluoride.
Site-specific STAT5 mutants are designated using the single letter
amino acid code; thus, STAT5b-S730A corresponds to STAT5b with
Ser730 mutated to alanine.
Received for publication May 9, 2001.
Accepted for publication August 28, 2001.
 |
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