(Received for publication, November 21, 1996, and in revised form, March 26, 1997)
From the Transcription factors of the Stat gene family are
selectively activated by many hormones and cytokines. Stat5 originally
was cloned as a prolactin-stimulated DNA-binding protein, but is also activated by non-lactogenic cytokines in many cell types. The recent
identification of two distinct Stat5 genes, which encode a 94-kDa
Stat5a and a 92-kDa Stat5b as well as several lower molecular weight
isoforms, suggests additional complexity and combinatorial possibilities for transcriptional regulation. We now report a biochemical analysis of prolactin activation of Stat proteins in Nb2
lymphocytes, which was associated with: 1) rapid tyrosine phosphorylation of Stat5a, Stat5b, a COOH-terminally truncated 80-kDa
Stat5 form, Stat1 A variety of polypeptide hormones and cytokines use cytoplasmic
signal transducers and activators
of transcription (Stats)1 to
regulate expression of specific genes (1, 2). The ability of individual
cytokine receptors to activate overlapping, but distinct sets of homo-
and heterodimerizing Stat proteins contributes to their signal
specificity. For example, interferon- Two distinct Stat5 genes have recently been identified which encode the
highly homologous 94-kDa Stat5a and 92-kDa Stat5b proteins, as well as
shorter 78-80-kDa isoforms of each gene product (10-14).
Interestingly, the COOH-terminally truncated forms of Stat5a and Stat5b
have transdominant negative effects on transcription (15, 16).
Similarly, shorter forms of Stat1 and Stat3 have been identified, and
result from alternative splicing of mRNA (11, 12, 17, 18). This
multiplicity of Stat isoforms adds further combinatorial possibilities
to receptor-mediated transcriptional regulation. Thus, preferential
activation of Stat5a over Stat5b by granulocyte colony-stimulating
factor has suggested receptor selectivity in Stat5 recruitment (19).
Knowledge of how different combinations of Stat5 isoforms may be used
by distinct receptors therefore becomes critical to our understanding
of gene regulation by a large number of hormones and cytokines.
Thus far all Stat proteins have been shown to require phosphorylation
of a positionally conserved tyrosine residue corresponding to Tyr-701
of human Stat1, which in turn facilitates dimerization and binding to
DNA response elements (20, 21). Furthermore, inducible serine
phosphorylation of Stat1 The present study specifically set out to define and compare the
molecular activation of different Stat5 gene products by PRL in Nb2
lymphocytes, a well characterized model of PRL-induced cell
proliferation and signal transduction. We now show that multiple forms
of Stat5, including p94Stat5a, p92Stat5b, and a
COOH-terminally truncated 80-kDa Stat5 isoform, undergo marked
phosphorylation on tyrosine and serine residues in response to PRL.
Furthermore, PRL induced rapid and selective heterocomplex formation
without involving Stat1 Ovine PRL (NIDDK-oPRL-19, AFP-9221A) and human
PRL (NIDDK-hPRL-SIAFP-B2, AFP-2969A) were supplied by the National
Hormone and Pituitary Program, National Institutes of Health, Bethesda, MD. Polyclonal rabbit antisera specific to peptides corresponding to
the unique COOH termini of Stat1 The Nb2 cell line (8) was
originally developed by Dr. Peter Gout (Vancouver, Canada), the Nb2-SP
clone used in this work was provided by Dr. Henry Friesen (University
of Manitoba, Canada). Cells were grown in RPMI 1640 medium
(Mediatech, catalog no. 15-040-LM) containing 10% fetal calf serum
(Intergen, catalog no. 1020-90), 2 mM
L-glutamine, 5 mM HEPES, pH 7.3, and
penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively), at
37 °C with 5% CO2. Nb2 cells at a density of
1-1.5 × 106/ml were incubated for 20 h in
lactogen-free medium consisting of RPMI 1640, which instead of 10%
fetal calf serum contained 1% gelded horse serum (Sigma, catalog no.
H-1895). Cells were brought to a density of 5 × 107
cells/ml and preincubated at 37 °C for 10 min prior to stimulation with 100 nM of ovine PRL.
Frozen pellets from 1 × 108 Nb2
cells were thawed on ice and solubilized in 1 ml of lysis buffer
containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA,
50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate,
1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin. Cell
lysates were incubated at 4 °C for 60 min, and insoluble material
was pelleted at 12,000 × g for 30 min at 4 °C.
Depending on the experiment, clarified lysates were incubated with for
3 h at 4 °C with either monoclonal mouse anti-phosphotyrosine
antibodies (clone 4G10; Upstate Biotechnology, Inc. (UBI), catalog no.
05-321; 5 µg/ml), or polyclonal rabbit antisera (2 µl/ml) to
individual Stat proteins as specified. Antibodies were captured by
incubation for 60 min with protein A-Sepharose beads (Pharmacia Biotech
Inc., catalog no. 17-0780-01) and washed, and samples were subjected to
7.5% SDS-PAGE under reducing conditions according to Laemmli (27). The
proteins were then transferred to polyvinylidene difluoride membrane
(Millipore, catalog no. 1PVH 00010), using a semidry transfer unit
(Multiphor Novablot, Pharmacia) at constant current of 195 mA for 90 min. After transfer, the blots were incubated for at least 1 h at
room temperature in blocking buffer (0.02 M Tris-HCl, pH
7.6, 0.137 M NaCl, 1% bovine serum albumin, and 0.01%
sodium azide) before immunoblotting. Blots were exposed for 90 min to
primary antibodies diluted in blocking buffer at the following
concentrations: anti-phosphotyrosine mAb 4G10 (UBI, catalog no. 06-321, 1 µg/ml), anti-Stat1 mAb (1:2,500 dilution), anti-Stat3 mAb (1:2,500
dilution), and anti-pan-Stat5 mAb directed to a distinct internal
region common to Stat5a and Stat5b (1:250 dilution). The blots were
then incubated twice for 5 min in wash buffer (50 mM
Tris-HCl, pH 7.6, 200 mM NaCl, 0.25% Tween 20), followed
by incubation for 30 min with horseradish peroxidase-conjugated goat
antibodies to mouse or rabbit IgG (Kirkegaard and Perry Laboratories,
catalog nos. 074-1806 and 074-1506, respectively) at 500 ng/ml in
blocking buffer, followed by four 15-min incubations in wash buffer.
The blots were then incubated for 1 min with enhanced chemiluminescence
substrate (ECL) mixture according to the manufacturer's instructions
(Amersham, catalog no. RPN2106), and exposed to x-ray film for 1-5 min
(Eastman Kodak Co., catalog no. 165 1454).
[32P]Orthophosphate labeling of cell
proteins were carried out as described earlier (28). Labeled Stat5a and
Stat5b proteins were excised from polyvinylidene difluoride membranes
and hydrolyzed in 6 N HCl at 110 °C for 90 min.
Phosphoamino acid analysis was then performed as described earlier
(29).
Quiescent Nb2
cells were treated with or without PRL (100 nM) for 10 min,
pelleted by centrifugation, and immediately solubilized in EMSA lysis
buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.2% Nonidet P-40, 1 mM orthovanadate, 25 mM NaF, 200 µM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin). Lysates were incubated
on ice for 20 min, then clarified by centrifugation at 20,000 × g for 20 min at 4 °C. For the EMSA (30), 1 ng of
oligonucleotides corresponding to the PRL response elements of the rat
Cytokine-induced tyrosine phosphorylation plays
a key role in the activation of Stat transcription factors by
facilitating Stat dimerization, which is needed for nuclear
translocation and DNA binding (17). Thus, both homo- and
heterodimerization of Stats have been reported after interferon
receptor stimulation (31, 32). To assess whether the two recently
identified highly homologous Stat5 genes are differentially regulated
by PRL, we chose a biochemical approach and utilized the PRL-sensitive
rat Nb2 lymphoma cell line (8). The first step was to analyze
PRL-inducible tyrosine phosphorylation of major Stats and to compare
their dimerization patterns by immunopreciptation from lysates of
stimulated and unstimulated Nb2 cells with specific antisera.
Immunoblotting with anti-phosphotyrosine antibodies revealed that both
Stat5a and Stat5b were markedly tyrosine-phosphorylated following PRL treatment for 15 min (Fig. 1A, panel
1, lanes g-j). The treatment also stimulated tyrosine
phosphorylation of Stat1
There is considerable evidence to suggest that the DNA binding activity
of Stat proteins is critically dependent upon selective dimerization
after tyrosine phosphorylation. To test for inducible heterodimerization among PRL-activated Stat transcription factors, we
performed sequential cross-blotting of parallel sets of samples to
those analyzed for tyrosine phosphorylation (Fig. 1A,
panels 2-5). These experiments showed that a significant
fraction of Stat5a and Stat5b molecules heterodimerized and could be
immunoprecipitated with antibody to the heterologous Stat5 from lysates
of PRL-stimulated cells (Fig. 1A, panels 4 and
5). In contrast, PRL induced no detectable heterodimerization of Stat1 Having demonstrated
that PRL selectively induced heterodimerization of Stat5a and Stat5b,
we investigated how the tyrosine phosphorylation kinetics of these two
Stats corresponded with their dimerization kinetics. Nb2 cells were
stimulated with PRL for varying times up to 60 min. Stat5a and Stat5b
were immunoprecipitated from cell lysates with specific antibodies and
blotted with anti-phosphotyrosine antibodies or specific antisera to
Stat5a or Stat5b. As shown in Fig. 1B (panels 1 and 2), marked tyrosine phosphorylation of both Stat5a and
Stat5b was detected within 1 min of PRL exposure, and maximal levels
were reached within 2 min of stimulation. Interestingly, these elevated
tyrosine phosphorylation levels were sustained for at least 60 min.
Cross-probing the stripped immunoblots with antibodies to the
heterologous Stat5 protein showed that heterodimerization of Stat5a and
Stat5b paralleled their tyrosine phosphorylation kinetics (Fig.
1B, panels 3 and 4). This supports the
concept of Stat dimerization via phosphotyrosyl-binding SH2-domains
(22, 31, 38). Furthermore, reprobing of the same blots with antibodies to the homologous Stat5 showed that the levels of immunoprecipitated Stat5 proteins remained constant over the 60-min time course (Fig. 1B, panels 5 and 6). Collectively,
experiments using reciprocal co-immunoprecipitation and immunoblotting
as shown in Fig. 1 suggest that PRL induces a significant degree of
heterodimerization of Stat5a and Stat5b, although further studies are
needed to elucidate the stoichiometry of homo- and heterodimers.
The results reported in Fig. 1 are based
on the use of specific antisera directed to the unique COOH termini of
Stat5a or Stat5b (10). Interestingly, when a polyclonal anti-Stat5
serum that recognizes shared internal epitopes of Stat5a and Stat5b (6)
was used to immunoprecipitate lysates of PRL-treated Nb2 cells, we
observed a second inducibly tyrosine-phosphorylated protein with an
apparent molecular mass of 80 kDa in addition to the
tyrosine-phosphorylated 92-94-kDa forms of Stat5a/b (Fig. 2A, lane b). We identified this
80-kDa protein as a truncated Stat5 molecule by immunoblotting with
either a monoclonal anti-pan-Stat5 antibody whose epitope is located in
the central portion of both Stat5a and Stat5b (Transduction
Laboratories catalog no. S21520; Fig. 2A, lanes c
and d), or the polyclonal anti-pan-Stat5 serum (Ref. 6; Fig.
2A, lanes c-f). The 80-kDa protein was not
recognized by antibodies specific to the COOH termini of either Stat5a
or Stat5b (Fig. 2A, lanes g-j). Furthermore, the
kinetics of tyrosine phosphorylation of the p80 Stat5-like protein
paralleled that of Stat5a and Stat5b (Fig. 2B, upper
panel). Based upon these observations and the reported existence
of alternatively spliced 80-kDa variants of Stat5 (11-14), we conclude
that PRL activates a COOH-terminally truncated form of Stat5 in Nb2
cells. Further studies are needed to determine whether p80Stat5
represents a short form of Stat5a or Stat5b. This is in contrast to an
80-kDa short form of Stat5a with a presumed internal deletion that was
inducibly tyrosine-phosphorylated by granulocyte colony-stimulating factor in myeloid cells and could be recognized by a COOH-terminal anti-Stat5a serum (19).
The COOH-terminally truncated forms of Stat molecules, including the
80-kDa splice form Stat3 In addition to phosphorylation of the positionally
conserved tyrosine residue corresponding to Tyr-701 of human Stat1, it has been reported that Stat1 requires phosphorylation of Ser-727 for
full transcriptional activity (22, 23). Specifically, the
serine-threonine kinase ERK2, one of the MAPKs, has been implicated in
IFN
Thus far, we have only examined PRL-inducible serine phosphorylation in
Nb2 cells. However, we have now extended these observations to include
IL2 induction of serine phosphorylation of both Stat5a and Stat5b in
several target cells (44). This supports the notion of serine
phosphorylation of Stat5 molecules as a general activation mechanism.
On the other hand, cell specific differences may exist. In a separate
study of HC11 mouse mammary epithelial cells, constitutive rather than
PRL-inducible serine phosphorylation of Stat5a or Stat5b was observed
(40).
We next evaluated the ability of PRL-activated Stat5a
and Stat5b to bind to oligonucleotide probes corresponding to the PRL response elements of the
The present study provides initial evidence to suggest a
differential involvement of Stat5a and Stat5b molecules in PRL
receptor-mediated signal transduction. Particularly intriguing is the
finding of a Stat5b complex that bound preferentially to an
oligonucleotide corresponding to the PRL response element of the
In conclusion, we demonstrate that PRL stimulates rapid tyrosine
phosphorylation of multiple forms of Stat5, including
p94Stat5a, p92Stat5b, and a COOH-terminally truncated
80-kDa Stat5 variant. This event is paralleled by heterodimerization of
Stat5a and Stat5b, and consistent with the model of
phosphotyrosyl-dependent intermolecular bridging via
SH2-domains of Stat proteins. Furthermore, we demonstrated inducible
phosphorylation of both Stat5a and Stat5b on serine and tyrosine, but
not threonine residues, thus providing direct evidence for the
existence of a PRL-activated Stat5 serine kinase. Finally, we report
the formation of a Stat5a/b complex that binds equally well to the
promoters of
Intramural Research Support Program,
Laboratory of
Biochemistry and Metabolism, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892, and the ** United States Food and Drug
Administration, Center for Biologic Evaluation and Research, Division
of Cytokine Biology, Bethesda, Maryland 20814
, and Stat3; 2) rapid and selective formation of
Stat5a/b heterodimers, without involvement of Stat1
or Stat3; 3)
marked serine, but not threonine phosphorylation of Stat5a and Stat5b;
and 4) the appearance of two qualitatively distinct Stat5 protein
complexes, which discriminated between oligonucleotides corresponding
to the prolactin response elements of the
-casein and interferon
regulatory factor-1 gene promoters. Collectively, our analyses showed
that Stat5a and Stat5b respond similarly to prolactin receptor
activation, but also suggested that the two genes have evolved unique
properties that may contribute to the specificity of receptors that
utilize Stat5 signaling proteins.
activates Stat1, Stat2, and
Stat3 and exerts antiviral and growth-inhibitory effects in target
cells (3). Prolactin (PRL), on the other hand, activates Stat1, Stat3,
and Stat5 (4-6), and stimulates
-casein synthesis in mammary
epithelial cells and proliferation of Nb2 lymphocytes (7-9).
or Stat3 is needed for full transcriptional
activation (22, 23), and the mitogen-activated protein kinase (MAPK)
p42ERK2 has been implicated in the phosphorylation of
Stat1
(22, 24). However, Stat5 homologues lack the putative MAPK
phosphorylation site (X-Pro-X-Ser-Pro)
corresponding to Ser-727 of human Stat1
(22). This finding raises
the possibility that Stat5 activities are regulated different than
other Stats.
and Stat3, which also became tyrosine-phosphorylated. Finally, we demonstrate the formation of a
Stat5a/b-containing complex that bound equally well to oligonucleotide probes corresponding to the PRL-response elements of the
-casein and
interferon regulatory factor (IRF1) gene promoters. In contrast, a
slower migrating complex that contained Stat5b and not Stat5a bound
exclusively to the
-casein promoter and not to the IRF-1 probe.
Collectively, these observations demonstrate that the highly homologous
Stat5a and Stat5b proteins respond biochemically in a similar manner to
PRL receptor activation. However, the data also suggest that the two
gene products, which differ most in their COOH-terminal transactivation
domains, have evolved unique properties that may contribute specificity
to the large number of Stat5-activating receptors.
Materials
, Stat2, Stat3, Stat5a, and Stat5b
were generated as described previously (10, 25, 26). A polyclonal
antiserum that recognized both Stat5a and Stat5b was also developed as
described previously (27). Each of these antisera was useful for
detection of rat, mouse, and human Stat proteins by immunoprecipitation
and immunoblotting. In addition, a monoclonal anti-pan-Stat5 antibody,
directed to a distinct internal region shared by Stat5a and Stat5b, and
monoclonal antibodies for immunoblotting of Stat1 and Stat3, were
purchased from Transduction Laboratories, Inc. (catalog nos. S21520,
S21120, and S21320, respectively).
-casein (5
-agatttctaggaattcaaatc-3
) or human IRF-1
(5
-gatccatttccccgaaatga-3
) genes that had been end-labeled using
polynucleotide kinase and [
-32P]ATP, were incubated
with 10 µg of protein from cellular lysates in 30 µl of binding
mixture (50 mM Tris-Cl, pH 7.4, 25 mM
MgCl2, 5 mM dithiothreitol, 50% glycerol) at
room temperature for 20 min, with preincubation of samples with 1 µl
of either normal rabbit serum or antisera specific to Stat proteins as
indicated. Polyacrylamide gels (5%) containing 5% glycerol and
0.25 × TBE were prerun in 0.25 × TBE buffer at 4-10 °C
for 1.5 h at 270 V. After loading of samples, the gels were run at
room temperature for approximately 3 h at 250 V. Gels were
dried by heating under vacuum and exposed to x-ray film (X-Omat,
Kodak).
PRL Induces Tyrosine Phosphorylation and Heterodimerization of
Stat5a and Stat5b
and Stat3, but not of Stat2 (Fig.
1A, panel 1, lanes a-f), consistent
with previous results (5, 6). Thus, by the use of specific antibodies, these data extend the previous evidence of Stat5 activation by PRL to
define specific products of the two homologous Stat5a and Stat5b
genes.
Fig. 1.
PRL induces tyrosine phosphorylation and
heterodimerization of Stat5a and Stat5b. A, general
screening for Stat tyrosine phosphorylation and dimerization. Quiescent
Nb2 cells were incubated with medium () or 100 nM oPRL
(+) for 15 min at 37 °C, and lysates were immunoprecipitated
(IP) with anti (
)-Stat1
(lanes a and b),
-Stat2 (lanes c and d),
-Stat3 (lanes e and f),
-Stat5a (lanes g and h), or
-Stat5b (lanes
i and j). Parallel samples were blotted for either
phosphotyrosine (
PY; panel 1),
-Stat1
(panel 2),
-Stat2 (not shown),
-Stat3 (panel
3),
-Stat5a (panel 4), or
-Stat5b (panel
5). B, kinetic analysis of tyrosine phosphorylation and
heterodimerization of Stat5a and Stat5b. Quiescent Nb2 cells were
incubated with medium (
) or 100 nM oPRL (+) at 37 °C
for various times as indicated, and lysates were immunoprecipitated (IP) with either
-Stat5a (panels 1,
3, and 5), or
-Stat5b (panels 2,
4, and 6). Parallel samples were blotted for
either phosphotyrosine (
PY; panels 1 and
2),
-Stat5a (heterologous antiserum; panels 3 and 6),
-Stat5b (homologous antiserum; panels
4 and 5). Note that Stat5a/b heterodimerization occurs
immediately after PRL stimulation, and parallels the tyrosine
phosphorylation kinetics.
[View Larger Version of this Image (44K GIF file)]
, Stat2, or Stat3 (Fig. 1A,
panels 2-5). Based upon these results and the dimerization
model proposed by Darnell and colleagues (22, 31), we conclude that PRL
specifically induces heterodimerization of Stat5a and Stat5b, and
homodimerization of Stat1
and Stat3. Furthermore, we observed that
PRL induced a significant retardation of the electrophoretic mobility
of Stat5b, but not of Stat5a, resulting in a Stat5b protein doublet
(Fig. 1A, panels 4 and 5). PRL induced
similar shifts of Stat1
and Stat3 proteins. This type of mobility
shift on SDS-PAGE is often caused by protein phosphorylation (33-35),
and similar observations have been reported for Stat1
and Stat3
after stimulation with interferon and insulin (36, 37). Intriguingly,
Stat5a showed a significantly lesser mobility retardation than did
Stat5b in PRL-stimulated Nb2 cells (Fig. 1A, lanes
g-j, bottom two panels), possibly reflecting
functional differences between Stat5a and Stat5b.
Fig. 2.
PRL induces tyrosine phosphorylation of an
80-kDa form of Stat5. A, quiescent Nb2 cells were incubated
with medium () or 100 nM oPRL (+) for 15 min at 37 °C,
and lysates were immunoprecipitated with a polyclonal
-Stat5 serum
that does not discriminate between Stat5a and Stat5b. Five identical
sets of samples were separated by SDS-PAGE, and blotted in parallel
against either phosphotyrosine (
PY; lanes a
and b), monoclonal
-pan-Stat5 (lanes c and
d), polyclonal
-pan-Stat5 (lanes e and
f),
-COOH terminus of Stat5a (lanes g and
h), or
-COOH terminus of Stat5b (lanes i and
j). Note that an inducibly tyrosine-phosphorylated 80-kDa
protein that is immunoprecipitated with
-pan-Stat5 serum is
recognized by two
-pan-Stat5 antibodies, but not by antisera
directed to the unique COOH termini of either Stat5a or Stat5b.
B, kinetic analysis of tyrosine phosphorylation of
p80Stat5. Quiescent Nb2 cells were incubated with medium (
)
or 100 nM oPRL (+) at 37 °C for various times as
indicated, and lysates were immunoprecipitated with
-pan-Stat5 serum
and blotted with phosphotyrosine antibodies (upper panel) or
with monoclonal
-pan-Stat5 antibodies (lower panel). Note
that tyrosine phosphorylation of p80Stat5 is parallel to that
of p92-94Stat5 proteins, and also that p80Stat5
undergoes a gradual mobility shift with time.
[View Larger Version of this Image (53K GIF file)]
and 78-80-kDa forms of Stat5, act as
dominant negative regulatory partners that lack a transactivation domain (15, 16, 39). It is therefore reasonable to anticipate that the
PRL-sensitive p80Stat5 in Nb2 cells exerts a transdominant
negative activity. Interestingly, in parallel experiments conducted
with PRL-responsive human breast cancer cell lines, we have not
observed PRL-induced p80Stat5 activation, whereas the protein
response is present in 32D myeloid cells stably transfected with PRL
receptors (data not shown). Collectively, these observations suggest
that p80Stat5 is variably expressed in PRL target cells and may
constitute an important modulator of the transcriptional potency of
Stat5 complexes. Cellular variability in Stat isoform expression may markedly influence the signaling repertoire of individual
receptors.
activation of Stat1 (24). However, unlike other known Stats,
Stat5a and Stat5b lack classical X-Pro-X-Ser-Pro
MAPK phosphorylation sites. To the best of our knowledge, no direct
experimental evidence for inducible serine or threonine phosphorylation
of either Stat5a or Stat5b has thus far been presented. On the other
hand, the inducible shift in apparent molecular size of Stat5b, but not of Stat5a, on SDS-PAGE is consistent with increased protein
phosphorylation (33-37), and could reflect a difference of the extent
of phosphorylation between Stat5a and Stat5b. We therefore carried out
phosphoamino acid analysis of Stat5a and Stat5b proteins in Nb2 cells
treated with or without PRL for 10 min. Stat5a and Stat5b were
individually immunoprecipitated from lysates of Nb2 cells that had been
metabolically labeled with [32P]orthophosphate, and
examined for PRL-inducible phosphorylation by SDS-PAGE (Fig.
3A), followed by phosphoamino acid analysis after acid hydrolysis of the Stat5a and Stat5b proteins (Fig. 3B). As seen from Fig. 3A, PRL stimulated
incorporation of phosphate into both Stat5a and Stat5b, and subsequent
phosphoamino acid analysis revealed that both Stat5a and Stat5b were
inducibly phosphorylated on serine and tyrosine, but not on threonine
residues (Fig. 3B). Longer exposure times were generally
required to visualize phosphotyrosine than phosphoserine, and based
upon repeated analyses we propose that there is a higher number of
phosphorylated serine residues than phosphorylated tyrosines in both
Stat5a and Stat5b. Future studies will attempt to define the exact
stoichiometry and localize the phosphorylation sites.
Fig. 3.
PRL induces serine phosphorylation of Stat5a
and Stat5b. A, autoradiography of immunoprecipitated
(IP) Stat5a and Stat5b from
[32P]orthophosphate-labeled cells incubated with (+)
or without () PRL (100 nM) for 15 min at 37 °C.
B, phosphoamino acid analysis of Stat5a and Stat5b with (+)
or without (
) PRL stimulation. P-Ser, phosphoserine;
P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. Radioactive bands corresponding to either Stat5a or Stat5b were excised
and subjected to acid hydrolysis and thin layer electrophoresis, and
phosphate incorporated into amino acids was visualized by autoradiography.
[View Larger Version of this Image (42K GIF file)]
-Casein and IRF1 Gene
Promoters
-casein and IRF1 genes. The abilities of
antisera to Stat1, Stat3, and Stat5a/b to supershift individual complexes were tested. Nb2 cells were stimulated with PRL for 10 min,
lysed and clarified by centrifugation, and incubated with either normal
rabbit serum or with specific antisera as indicated. Intriguingly,
cellular Stat complexes from PRL-stimulated cells incubated in the
presence of normal rabbit serum displayed marked differences in their
ability to bind to the two oligonucleotide probes (Fig.
4). PRL induced the formation of three discernible complexes that bound to the
-casein probe: a slowly migrating complex indicated with an asterisk (Fig. 4, lanes
a and b) that did not bind to the IRF1 probe
(lane i), an intermediate complex that bound comparably well
to either probe, and a fast migrating complex that bound weakly to the
-casein probe and strongly to the IRF1 probe. Coincubation with
anti-Stat5a or anti-Stat5b sera alone supershifted significant portions
of the intermediate complex bound to the
-casein probe (lanes
c and d), and also the corresponding IRF1 complex
(lanes j and k). In both instances, the
anti-Stat5a serum was particularly efficient, although the two antisera
combined depleted the complex entirely (lanes e and
l). Intriguingly, the slow-migrating complex unique to the
-casein probe was almost completely supershifted by anti-Stat5b
serum. The effect of anti-Stat5a serum on the slow-migrating complex
was obscured by the appearance of a supershifted complex containing
Stat5a from the middle complex, but careful analysis of several
experiments indicates that the slow-migrating Stat5b complex did not
contain Stat5a. Although further work is needed to analyze the binding
of PRL-inducible Stat-DNA complexes and the accompanying physiological
impact on transcription rates of particular genes, these data clearly
indicate qualitative differences in PRL-induced Stat5a and Stat5b
complex formation within regulatory DNA sites. Finally, the
fast-migrating complex was shown to contain Stat1
, because it was
completely supershifted by anti-Stat1
serum (lanes f and
m). In contrast, an antiserum capable of supershifting
DNA-complexed Stat3 had no detectable effect on any of the
PRL-inducible complexes bound to either probe, indicating that Stat3
does not constitute a significant part of these particular complexes
(Fig. 4, lanes g and n). This is also consistent
with the observation that PRL-activated Stat3 did not heterodimerize
with Stat1
, Stat5a, or Stat5b (Fig. 1, panel 3), and that
each of the PRL-induced protein complexes binding to the
-casein and
IRF1 probes have been accounted for by supershift analysis. However,
PRL-activated Stat3 probably can interact with these or other response
elements under different conditions, but this remains to be
established. Most importantly, the data clearly indicate marked
differences in the ability of PRL-inducible Stat5 complexes to bind to
-casein and IRF1 probes, and that Stat5a and Statb appear to exist
in several distinct complex combinations.
Fig. 4.
Electrophoretic mobility shift assay of
PRL-inducible protein binding to PRL response elements of the
-casein and IRF1 gene promoters. Quiescent Nb2 cells were
incubated with medium (
) or 100 nM oPRL (+) for 10 min at
37 °C, and lysates corresponding to 10 µg of protein were
incubated either with normal rabbit serum (lanes a,
b, h, and j),
-Stat5a (lanes
c and j),
-Stat5b (lanes d and
k),
-Stat5a plus
-Stat5b (lanes e and
l),
-Stat1
(lanes f and m), or
-Stat3 (lanes g and n) in combination with
oligonucleotide probes corresponding to the PRL response elements of
either
-casein (lanes a-g) or IRF1 (lanes
h-n). Note qualitative differences in PRL-inducible complexes
bound to the two different response elements. An asterisk
(lane b) denotes a complex that preferentially binds to the
-casein promoter and is supershifted by
-Stat5b serum. A faster
migrating complex that is supershifted most efficiently by a
combination of Stat5a and Stat5b sera is indicated as
Stat5a/b, and the lower complex (Stat1
) is efficiently
supershifted with
-Stat1
serum.
-Stat3 serum does not have any
detectable effect on any of these complexes.
[View Larger Version of this Image (97K GIF file)]
-casein gene, but not to the PRL response element of IRF1. If Stat5a
or Stat5b were to have certain unique functions, or Stat5a/b
heterodimers constitute critical mediators, one would predict that mice
deficient in either transcription factor will have a phenotype.
Consistent with this notion, Stat5a-deficient mice are unable to
lactate (41). More specifically, in mammary glands of pregnant
Stat5a-deficient mice, the expression of
-casein is markedly less
affected than expression of the whey acidic protein, another mouse milk
protein under PRL control (42, 43). Studies are in progress to further define the specific functions of Stat5a and Stat5b.
-casein and IRF1, and a slower migrating complex that
contains Stat5b and not Stat5a, which binds exclusively to the
-casein promoter. Collectively, these data suggest that although
Stat5a and Stat5b respond biochemically very similarly to PRL and
probably have a significant functional overlap, the two proteins may
also have evolved unique properties that contribute to the specificity
of signals via receptors for PRL and possibly other cytokines.
*
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article were defrayed in part by the
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must therefore be hereby marked
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accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
USUHS School of Medicine, Bethesda, MD 20814. Tel.: 301-295-3801; Fax:
301-295-1640; E-mail: rui{at}usuhsb.usuhs.mil.
1
The abbreviations used are: Stat,
signal transducers and activators
of transcription; PRL, prolactin; oPRL, ovine prolactin; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.