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J Biol Chem, Vol. 273, Issue 46, 30218-30224, November 13, 1998
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From the Transcription factors of the Stat family are
controlled by protein kinases. Phosphorylation of a positionally
conserved tyrosine residue is obligatory for Stat dimerization, nuclear
translocation, and specific DNA binding. Studies of Stat1 and Stat3
have suggested that serine phosphorylation may also regulate function.
We now identify serine residues located in a conserved PSP motif of
Stat5a (Ser725) and Stat5b (Ser730) as
major phosphorylation sites, using mutagenesis, phosphoamino acid
analysis, and site-specific anti-Stat5-phosphoserine antibodies. Unexpectedly, phosphorylation control of this PSP motif differed between the highly homologous Stat5a and Stat5b proteins. Whereas Ser725 of Stat5a was constitutively phosphorylated both in
COS-7 cells and Nb2 lymphocytes, phosphorylation of Ser730
of Stat5b was markedly stimulated by prolactin. The data also suggested
the existence of a second major serine phosphorylation site in Stat5a.
Interestingly, constitutive phosphorylation of the PSP motif was
suppressed by PD98059 but not by staurosporine under conditions in
which both agents inhibited mitogen-activated protein kinases.
Furthermore, pretreatment of cells with staurosporine, PD98059, H7, or
wortmannin did not prevent either Stat5a or Stat5b from becoming
maximally serine-phosphorylated after prolactin exposure. We propose
that two pathways regulate Stat5 serine phosphorylation, one that is
prolactin-activated and PD98059-resistant and one that is
constitutively active and PD98059-sensitive and preferentially targets
Stat5a. Finally, phosphorylation of the PSP motif of Stat5a or Stat5b
was not essential for DNA binding or transcriptional activation of a
A variety of polypeptide hormones and cytokines use transcription
factors of the Stat family to regulate expression of genes that control
cellular growth and differentiation (1, 2). Although information on the
involvement of Stat proteins in the etiology and progression of disease
is still limited, fundamental knowledge of the function and regulation
of Stat proteins is expected to have important clinical and
pharmaceutical significance. There are currently seven known Stat genes
(2). Among these, Stat1 and Stat2 are important for the
antiproliferative and antiviral effects of interferons, whereas Stat3
regulates acute phase response genes (2). Furthermore, the highly
homologous Stat5a and Stat5b proteins (3-6) are essential mediators of
prolactin and growth hormone effects (7, 8) and have been implicated in
cytokine control of apoptosis, growth, and differentiation (9-11). Of
particular clinical relevance, constitutively activated Stat5 is
associated with several hematopoietic malignancies (12, 13), and both Stat5a and Stat5b were selectively downregulated in T lymphocytes from
patients with human immunodeficiency virus infection (14) and in T and
B lymphocytes from immunocompromised tumor-bearing mice (15).
The activity of Stat factors is under strict regulation by protein
tyrosine kinases. Phosphorylation of a positionally conserved tyrosine
residue is obligatory for dimerization, nuclear translocation, and
subsequent binding of Stats to specific promotor sequences (1).
Recently, studies have suggested that serine kinases also regulate the
activity of some Stat proteins. Specifically, serine residue 727 constitutes a major phosphorylation site in Stat1 The serine kinases responsible for Stat1 and Stat3 phosphorylation have
not yet been identified. However, amino acid Ser727 is
located within a consensus PXSP phosphoacceptor site for
mitogen-activated protein kinase
(MAPK)1 (21, 22), and
experimental evidence supports a role of the MAPK p42ERK2 in Stat1 To identify serine phosphorylation sites of Stat5 transcription
factors, a series of serine mutants of Stat5a and Stat5b were analyzed
using a COS-7 cell reconstitution system. COS-7 cells do not express
detectable levels of prolactin receptors, Stat5a, or Stat5b and have
been used for studies of prolactin receptor-mediated Plasmids and Mutants--
Expression vectors for mouse Stat5a
(pXM-Stat5a) and mouse Stat5b (pXM-Stat5b) were kindly provided by
Xiuwen Liu and Lothar Hennighausen (National Institutes of Health,
Bethesda, MD) (31). The ( Cell Culture and Transfections--
COS-7 cells (ATCC) were
grown in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum, 2 mM L-glutamine, and penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively) at
37 °C with 5% CO2. Transfections were done by the
DEAE-dextran method using subconfluent COS-7 cells in 100-mm dishes.
Two micrograms of the prolactin receptor construct (p3PRLR), 2 µg of
one of the plasmids pXM-Stat5a or pXM-Stat5b, or mutants of pXM-Stat5a
or pXM-Stat5b, were used in each transfection. For luciferase and
The Nb2 cell line was originally established by Dr. Peter Gout
(University of British Columbia, Vancouver, Canada), and the Nb2-SP
clone used in this work was kindly provided by Dr. Henry Friesen
(University of Manitoba, Manitoba, Canada). Cells were grown in RPMI
1640 medium (Mediatech, catalog number 15-040-LM) containing 10% fetal
calf serum, 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 20-24
h in lactogen-free medium consisting of RPMI 1640 medium that instead
of 10% fetal calf serum contained 1% gelded horse serum (Sigma,
catalog number H-1895). Cells were preincubated at 37 °C for 30 min
with various inhibitors before stimulation with 100 nM
ovine prolactin (NIDDK-oPRL-19, AFP-9221A) for 15 min. Cell pellets
were frozen at Solubilization of Proteins, Immunoprecipitation, and
Immunoblotting--
Cells were solubilized in 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 phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin as
described (26). For immunoprecipitation from clarified cell lysates,
polyclonal rabbit antisera (2 µl/ml) specific to peptides
corresponding to the unique COOH termini of Stat5a or Stat5b were used
(27), and captured after incubation with protein A-Sepharose beads
(Amersham Pharmacia Biotech, catalog number 17-0780-01). Immunoblotting
was performed as described previously (26) using polyvinylidene
difluoride membranes (Millipore, catalog number 1PVH 00010), and
anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology,
catalog number 05-321; 1 µg/ml) or polyclonal rabbit antisera of
Stat5a and Stat5b (1:3000 dilution) as primary antibodies and
horseradish peroxidase-conjugated goat antibodies to mouse or rabbit
IgG as secondary antibodies in conjunction with enhanced
chemiluminescence substrate mixture (Amersham Pharmacia Biotech,
catalog number RPN2106).
[32P]Orthophosphate Labeling and Phosphoamino Acid
Analysis--
Transfected COS-7 cells were metabolically labeled with
0.75 mCi/ml [32P]orthophosphate for 2 h at 37 °C,
stimulated with 10 nM human prolactin for 30 min, and then
lysed and immunoprecipitated as described above. Proteins were eluted
from protein A-Sepharose beads, separated on SDS-PAGE (7.5%
polyacrylamide), and transferred to polyvinylidene difluoride
membranes. Labeled proteins were visualized by autoradiography and
analyzed by phosphoamino acid analysis as described earlier (33). Bands
corresponding to Stat5a or Stat5b proteins were excised and exposed to
limited hydrolysis in 6 N HCl at 110 °C for 60 min.
Samples were then dried, resuspended in water with phosphoamino acid
standards, and spotted onto a thin layer cellulose acetate gel.
One-dimensional thin layer electrophoresis was performed at 1500 V for
40 min in buffer containing pyridine:acetic acid:water at a 10:100:1890
ratio. Standards were visualized with ninhydrin, and samples were
analyzed by autoradiography.
Generation of Site-specific Anti-Stat5-Phosphoserine
Antibodies--
The phosphopeptide DQAP[pS]PAVC corresponding to
amino acid residues 721-729 of human Stat5a or 726-734 of human
Stat5b was synthesized, conjugated to keyhole limpet hemocyanin, and
used as an immunogen in rabbits (Genosys Inc., Woodlands, TX). The antiserum was first precleared by passing over a resin of immobilized, unphosphorylated peptide, followed by affinity purification against immobilized, phosphorylated peptide. For immunoblotting, blots were
incubated for 16 h with antibodies at a concentration
corresponding to 1:3000 dilution of original serum.
Electrophoretic Mobility Shift Assay--
Cells were treated
with or without 10 nM human prolactin as indicated,
pelleted by centrifugation, and immediately solubilized in
electrophoretic mobility shift assay lysis buffer and analyzed as
described earlier (26). 32P-Labeled oligonucleotide
corresponding to the prolactin response element
(5'-agatttctaggaattcaaatc-3') of the rat Luciferase and Targeting of Putative Phosphoacceptor Sites in Stat5a and
Stat5b--
Sequence alignments of various Stat proteins in the region
corresponding to the phosphorylated serine Ser727 of Stat1
and Stat3 showed that whereas Stat1, Stat3, and Stat4 have the
classical MAPK phosphorylation site PXSP (21, 22), Stat5a
and Stat5b contain instead a conserved PSP motif (Fig. 1A). A previous alignment
based solely on the sheep Stat5 sequence did not recognize this
difference (17). Peptide phosphorylation studies have suggested that
PSP motifs are less efficient phosphoacceptors for ERKs than
PXSP motifs (21). It was therefore of particular relevance
to determine whether Stat5 PSP motifs were phosphorylation sites. To
specifically analyze the involvement of the PSP serine residues of
Stat5a and Stat5b, mutants Stat5a-S725A and Stat5b-S730A were generated
(Fig. 1B). In addition, mutants Stat5a-S710A and Stat5b-S715A were also made based on the conserved nature and proximity
of these serine residues to the dimerization and trans-activation domains (Fig. 1B).
Effect of Alanine Substitution of Ser725 of Stat5a and
Ser730 of Stat5b on Prolactin-inducible Stat5 Serine
Phosphorylation as Judged by Phosphoamino Acid Analysis--
COS-7
cells were transiently transfected with expression plasmids for the
prolactin receptor and wild-type or mutant forms of Stat5a or Stat5b
proteins. Cells were metabolically labeled with
[32P]orthophosphate and stimulated with or without 10 nM prolactin for 30 min. Stat5a or Stat5b proteins were
immunoprecipitated from corresponding cell lysates, separated by
SDS-PAGE, and visualized by autoradiography (Fig.
2, A and B, upper
panels). The isolated Stat5a or Stat5b proteins were then excised
and subjected to phosphoamino acid analysis after limited acid
hydrolysis (Fig. 2, A and B, lower panels).
Wild-type Stat5a showed significantly higher levels of constitutive
phosphorylation than wild-type Stat5b when expressed in COS-7 cells
(Fig. 2, A and B, upper panels). This
constitutive phosphorylation of Stat5a was selective for serine
residues (Fig. 2A, lower panel). The extent of
inducible serine phosphorylation was therefore much less pronounced in
Stat5a than in Stat5b, whereas inducible tyrosine phosphorylation
levels were comparable.
The effect of mutation of Ser730 on Stat5b serine
phosphorylation was dramatic, because inducible phosphoserine levels
were reduced almost to background levels in mutant Stat5b-S730A (Fig.
2B). This analysis provided the first evidence that
Ser730 is the predominant serine phosphorylation site of
Stat5b. Furthermore, substitution of S715 of Stat5b with alanine led to
constitutively elevated phosphoserine levels of mutant Stat5b-S715A
(Fig. 2B). However, this hyperphosphorylated state did not
prevent mutant Stat5b-S715A from becoming inducibly
tyrosine-phosphorylated in response to prolactin. To address
whether Ser730 was the hyperphosphorylated residue in the
Stat5b-S715A mutant, we converted Ser730 of this mutant to
alanine and generated the double mutant Stat5b-S715A,S730A. This
co-mutation completely abolished the constitutive phosphorylation and
reduced inducible serine phosphorylation to the low levels observed in
the Stat5b-S730A mutant (Fig. 2B). This result was consistent with Ser730 as the hyperphosphorylated site of
Stat5b-S715A and supported the notion that Ser730 is a
predominant serine phosphorylation site of Stat5b. Furthermore, although of unknown significance, stoichiometrically low levels of
phosphothreonine were also induced in Stat5b but not in Stat5a.
The corresponding phosphoamino acid analysis of the Stat5a-S725A mutant
showed high constitutive phosphoserine levels, which suggested the
existence of an additional site distinct from Ser725 in
Stat5a (Fig. 2A, lower panel). However, a
significant and consistent reduction in phosphoserine content of
Stat5a-S725A compared with wild-type Stat5a indicated that
Ser725 of Stat5a was also a phosphorylation site. No
changes in phosphorylation were observed in mutant Stat5a-S710A, in
contrast to the increased levels of constitutive serine phosphorylation
in the matching Stat5b-S715A mutant. Finally, no constitutive or
inducible phosphotyrosine could be detected in mutants Stat5a-Y694F and
Stat5b-Y699F (Fig. 2, A and B), consistent with
the key regulatory role of these positionally conserved tyrosine
residues (30). On the other hand, both tyrosine mutants were
constitutively phosphorylated on serine, showing that serine
phosphorylation of both Stat5a and Stat5b can occur independently of
tyrosine phosphorylation. Thus, we conclude from these phosphoamino
acid analyses that Ser730 of Stat5b is a predominant and
inducible phosphorylation site, and that the corresponding
Ser725 of Stat5a was constitutively phosphorylated.
Finally, phosphoamino acid analysis suggested that a second
phosphoserine site existed in Stat5a.
Analysis of Phosphotyrosine and Phosphoserine Content of Stat5
Mutants by Immunoblotting--
To directly establish whether the PSP
motif is phosphorylated in both Stat5a and Stat5b, we generated
site-specific anti-Stat5-phosphoserine antibodies using a
phosphorylated peptide corresponding to this shared motif. Consistent
with marked constitutive phosphorylation of Ser725 of
wild-type Stat5a, these antibodies showed strong reaction to wild-type
Stat5a but not mutant Stat5a-S725A or the double mutant
Stat5a-S710A,S725A (Fig. 3A,
middle panel). Constitutive serine phosphorylation of
Ser725 was also verified in mutants Stat5a-S710A and
Stat5a-Y694F.
Examination of Stat5b mutants with the same antiphosphoserine
antibodies gave a similar pattern, except that basal phosphorylation of
Ser730 was much lower and was inducible by prolactin,
consistent with phosphoamino acid data. No immunoreaction toward
proteins carrying the Stat5a-S725A or Stat5b-S730A mutations was
observed, attesting to the specificity of the anti-Stat5-phosphoserine
antibodies. Parallel examination of tyrosine phosphorylation states of
Stat5 proteins in prolactin-treated COS-7 cells by anti-phosphotyrosine immunoblotting also corroborated the phosphoamino acid analyses and
showed that Stat5 wild-type proteins and the various Ser-to-Ala mutants
were tyrosine-phosphorylated equally well in response to prolactin
stimulation (Fig. 3, A and B, upper
panels). Similarly, immunoblotting confirmed that mutants
Stat5a-Y694F and Stat5b-Y699F did not become tyrosine-phosphorylated on
additional sites. Immunoblotting also verified that each of the mutants
was expressed at comparable levels in COS-7 cells. We conclude from
these experiments that the PSP motif is a major serine phosphorylation
site in both Stat5a and Stat5b.
Analysis of DNA Binding Activities of Stat5 Mutants--
The
ability of wild-type proteins and mutants Stat5a-S725A and Stat5b-S730A
to bind to an oligonucleotide probe corresponding to the prolactin
response element of the Assessment of Transactivation Potential of Mutants Stat5a-S725A and
Stat5b-S730A--
Expression plasmids encoding the prolactin receptor,
wild-type or mutant Stat5 proteins and a Effect of Protein Kinase Inhibitors on Constitutive and
Prolactin-regulated Serine Phosphorylation of Stat5a and Stat5b in Nb2
Lymphocytes--
To examine Stat5 serine phosphorylation in cells with
endogenous prolactin receptors and to begin pharmacological
characterization of the prolactin-activated Stat5 serine kinase,
quiescent Nb2 lymphocytes were pretreated with a series of kinase
inhibitors for 30 min before they were incubated with or without
prolactin (100 nM) for another 15 min. The inhibitors used
included staurosporine (500 nM), PD98059 (100 µM), H7 (100 µM), and wortmannin (100 nM). Immunoblotting for phosphotyrosine and phosphoserine
levels were done in parallel on immunoprecipitated Stat5a (Fig.
6A) and Stat5b (Fig.
6B) proteins. Control cells incubated without inhibitors revealed a similar distinction in serine phosphorylation between Stat5a
and Stat5b in Nb2 cells as that observed in COS-7 cells, with the PSP
motif constitutively serine-phosphorylated in Stat5a and highly
inducible in Stat5b. Furthermore, whereas none of the inhibitors
significantly antagonized PRL-induced tyrosine or serine phosphorylation of Stat5a or Stat5b in Nb2 cells, the MAPK/ERK kinase
inhibitor PD98059 significantly reduced basal serine phosphorylation levels in both Stat5a and Stat5b (Fig. 6, A and
B), and significantly inhibited PRL-induced activation of
MAPK (Fig. 6C). H7 had a similar but less marked effect on
basal Stat5 phosphoserine levels (Fig. 6, A and
B). In contrast, staurosporine, which also markedly
inhibited PRL-induced MAPK activation under these conditions (Fig.
6C), had little or no effect on constitutive Stat5a or
Stat5b phosphoserine levels (Fig. 6, A and B).
The fact that staurosporine pretreatment (500 nM) for 30 min did not block subsequent PRL-induced Stat5 tyrosine phosphorylation
may suggest that maximally effective conditions have not been achieved
for this combined inhibitor of tyrosine and serine kinases. However, we
have observed that pretreatment of Nb2 cells for 30 min with
staurosporine up to 2 µM consistently blocks PRL-induced
MAPK activation and tyrosine phosphorylation of Stat1 and Stat3 but not
Stat5 and Jak2.2 It is
possible that longer preincubation times with staurosporine will block
constitutive Stat5 serine phosphorylation. On the other hand, in light
of the comparable sensitivity of MAPK to PD98059 and staurosporine
under the conditions tested, the constitutively active Stat5 serine
kinase appeared, at least in relative terms, more resistant to
staurosporine than to PD98059. Additional studies are needed to extend
this initial pharmacological characterization of Stat5 serine kinase
activities, and the anti-Stat5-phosphoserine antibodies will allow the
development of specific in vitro enzyme assays. At present,
we conclude that constitutive Stat5 serine phosphorylation is sensitive
to PD98059, whereas the PRL-activated serine kinase is not sensitive to
PD98059, staurosporine, H7, or wortmannin.
Assessment of Phosphorylation Sites Contributing to the Unique
Stat5b Gel Mobility Shift--
As observed previously (26, 27) and
seen in Fig. 3, A and B (lower
panels), Stat5b but not Stat5a undergoes a pronounced mobility
shift in SDS-PAGE after activation. Phosphorylation often causes gel
mobility retardation of proteins in SDS-PAGE, and the mobility shift of
Stat5b disappeared after in vitro phosphatase treatment of
Stat5b (28, 34). The lack of an inducible shift of Stat5a could not
simply be attributed to constitutive phosphorylation of
Ser725, because the Stat5a-S725A mutant showed wild-type
migration on SDS-PAGE (Fig. 3). On the other hand, mutation of
Ser730 to alanine only led to a small, 15-20% reduction
in the shift of mutants Stat5b-S730A and Stat5b-S715A,S730A (Fig.
3B; data not shown). In contrast, mutation of conserved
tyrosine residue Tyr699 completely abolished the
prolactin-induced mobility shift (Fig. 3B). To test the
possibility that the unique mobility shift of Stat5b could be
attributable to a combined phosphorylation of Tyr699 and a
Stat5b-specific tyrosine or serine that required prephosphorylation of
Tyr699, we individually substituted with alanines each of
the five unique serine residues that are conserved across species in
Stat5b but not present in Stat5a (Ser35, Ser45,
Ser132, Ser427, and Ser748), as
well as mutated Tyr679 to phenylalanine (see Fig.
1B). Tyr679 is the only conserved tyrosine
residue in Stat5b that is not present in Stat5a, and phosphorylation of
a second tyrosine residue of Stat3, Tyr656, was recently
demonstrated (35). However, each of these Stat5b mutants behaved
normally with regard to inducible mobility retardation (data not
shown). We therefore conclude that inducible phosphorylation of
conserved serine residues Ser35, Ser45,
Ser132, Ser427, and Ser748 and
tyrosine residue Tyr679, which are unique to Stat5b and not
present in Stat5a, does not contribute to the unique Stat5b gel
mobility shift. Furthermore, because the phosphoamino acid analysis
showed stoichiometrically significant residual levels of constitutive
serine phosphorylation of the Stat5a-S725A mutant but not Stat5b-S730A,
it is possible that a second constitutively phosphorylated serine
residue in Stat5a may be responsible for this structural difference
between Stat5a and Stat5b. Further work is needed to resolve this
distinction between Stat5a and Stat5b mobility.
The present study identified the conserved PSP motif of Stat5
transcription factors as a major phosphorylation site that can be
differentially modulated between Stat5a and Stat5b. Whereas serine
residue Ser725 of Stat5a was constitutively phosphorylated
in both COS-7 cells and Nb2 lymphocytes and could not be further
phosphorylated by prolactin treatment, the corresponding
Ser730 residue of Stat5b was only weakly phosphorylated
under basal conditions but was highly responsive to prolactin.
Interestingly, constitutive serine phosphorylation of Stat5a
Ser725 was suppressed when cells were pretreated with the
MAPK/ERK kinase inhibitor PD98059. However, the drug did not inhibit
prolactin-induced phosphorylation of the PSP motif of either Stat5a or
Stat5b. Collectively, these observations suggested that two pathways
regulate Stat5 serine phosphorylation, one sensitive to PD98059 and one
that is insensitive and stimulated by prolactin. Furthermore, the
prolactin-activated Stat5 serine kinase was also resistant to several
other inhibitors, including staurosporine, H7, and wortmannin. It
remains to be established whether the prolactin-activated pathway and
the constitutive pathway regulate two distinct Stat5 serine kinases, or
whether the two pathways converge on one Stat5 serine kinase that by
itself is insensitive to PD98059, staurosporine, H7, and wortmannin.
Recent studies of Stat3 have also suggested two serine phosphorylation
pathways, a growth factor-activated, MAPK-dependent pathway
and an interleukin 6-activated, MAPK-independent pathway (24). There is
currently no direct evidence suggesting that Stat5 proteins can be
substrates for MAPK. In fact, several reports and the present study
argue against a role for MAPK in prolactin and interleukin 2 stimulation of Stat5 transcription factors (27-29). Furthermore,
because PD98059 and staurosporine both inhibited MAPK, but only PD98059
blocked constitutive Stat5 serine phosphorylation, we also propose that
the constitutive Stat5 serine phosphorylation pathway does not require
MAPK. Finally, previous in vitro peptide kinase assays have
shown that the PSP sequence of Stat5 is a relatively poor substrate for
MAPK when compared with the PXSP-sequence of Stat1 and Stat3
(21, 22). Thus, it appears likely that individual Stats are
phosphorylated by different serine kinases with at least some degree of selectivity.
It is still controversial whether a serine phosphorylation site other
than Ser727 exists in Stat3 (17, 24). Interestingly, in the
present study phosphoamino acid analyses showed stoichiometrically
significant levels of residual constitutive serine phosphorylation of
the Stat5a-S725A mutant but not of the corresponding Stat5b-S730A mutant. This suggested the existence of a second major serine phosphorylation site unique to Stat5a, although minor phosphorylation of several bystander serine residues has not been ruled out.
Nonetheless, the observed differential phosphorylation of Stat5a and
Stat5b supports the notion that these two homologous genes have evolved into structurally and functionally discernible proteins (26, 27).
Stat5a and Stat5b differ the most in their COOH-terminal trans-activation domains, which also contain the phosphorylated PSP
motif (see Fig. 1B). Stat5a and Stat5b can undergo both
homodimerization and heterodimerization and bind to similar DNA
response elements (26). On the other hand, Stat5a and Stat5b may also
form distinct complexes to DNA (26). Consistent with distinct, but
overlapping functions of Stat5a and Stat5b, the phenotypes of mice
deficient in either gene are relatively normal (7, 8). However, Stat5b is unable to compensate for Stat5a deficiency in the mammary gland, resulting in a loss of prolactin-induced milk production (7). Conversely, Stat5b null mice have specific growth hormone-signaling defects, resulting in stunted growth and liver dysfunction (8). The
current work adds to a series of dissimilarities and suggests regulatory and structural differences between the highly homologous Stat5a and Stat5b proteins.
The present demonstration of regulated serine phosphorylation of Stat5
proteins extends documented serine phosphorylation to include four of
the seven known Stat transcription factors. In addition, Stat4 is a
candidate serine kinase target based on indirect evidence using serine
kinase inhibitors (36). A general pattern of serine phosphorylation of
Stat transcription factors is therefore emerging, although further work
is needed to firmly establish the biological role of serine modulation.
An initial report demonstrated a positive effect of Stat1 and Stat3
serine phosphorylation on transcriptional activation (17). Subsequent work has suggested a positive effect of serine phosphorylation on DNA
binding (16), although this has been disputed (37). Finally, a recent
paper suggested that serine phosphorylation negatively regulates
tyrosine phosphorylation of Stat3 (24). The present data did not
support a role for serine phosphorylation of the PSP motif in
modulating DNA binding or transcriptional activity of Stat5, nor was
there any negative effect of serine phosphorylation on Stat5 tyrosine
phosphorylation. The current evidence for a second major serine
phosphoacceptor site in Stat5a suggests that additional work is needed
to understand the functional role of serine phosphorylation of Stat
transcription factors. Motivated in part by recent studies, which have
suggested a role of constitutively activated or serine phosphorylated
Stat3 in the progression of several cancers, including chronic
lymphocytic leukemias, breast tumors, and transformed fibroblasts (38,
39), we are currently examining the effect of serine phosphorylation on
the transcriptional activities of Stat5a and Stat5b on additional Stat5-responsive genes as well as in cells other than COS-7.
In summary, we have demonstrated that serine residues
Ser725 of Stat5a and Ser730 of Stat5b are major
phosphorylation sites. Unexpectedly, phosphorylation control of this
PSP motif differed between the highly homologous Stat5a and Stat5b
proteins, and phosphoamino acid analysis suggested the existence of a
second, major serine phosphorylation site unique to Stat5a. Although
further studies are needed to establish the functional role of
regulated Stat5 serine phosphorylation, the data suggested the
existence of two pathways leading to serine phosphorylation of the PSP
motif of Stat5, one PD98059-resistant, prolactin-activated pathway and
one PD98059-sensitive, constitutive pathway. As more is learned about
the involvement of Stat transcription factors in pathogenesis and
progression of disease, the ability to pharmacologically manipulate
individual Stat family members is expected to have important clinical consequences.
*
This work was supported by National Institutes of Health
Grant RO1 DK52013-01A1 and Uniformed Services University of the Health Sciences Grant RO74HF.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Pathology,
Uniformed Services University of the Health Sciences, School of
Medicine, Bethesda, MD 20814. Tel.: 301-295-3801; Fax: 301-295-1640; E-mail: hrui{at}usuhs.mil.
The abbreviations used are:
MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated
kinase; PRL, prolactin; PAGE, polyacrylamide gel electrophoresis.
2
H. Yamashita and H. Rui, manuscript in preparation.
Department of Pathology, Uniformed Services
University of the Health Sciences, Bethesda, Maryland 20814, the
§ Department of Integrative Biology, Pharmacology, and
Physiology, University of Texas at Houston, Houston, Texas 77030, and
the ¶ Intramural Research Support Program, Division of Basic
Sciences,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-casein reporter gene in COS-7 cells, suggesting that serine kinase
control of Stat5 activity differs from that of Stat1 and Stat3.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and Stat3 and is
important for interferon-induced nuclear translocation, DNA binding,
and maximal transcriptional activation (16-18). Substitution of
Ser727 of Stat1 with alanine abolished interferon-
and induced an antiviral state and growth arrest of cells (19, 20).
and Stat3 phosphorylation (17, 18, 23). On the other hand,
MAPK-independent serine phosphorylation of Stat3 also has been reported
(24, 25). Stat5a and Stat5b were recently shown to become
phosphorylated on serine residues after activation by prolactin or
interleukin 2 (26, 27), but the phosphoacceptor sites have not yet been
identified. Stat5 transcription factors lack the conserved PMSP motif
of Stat1 and Stat3. However, serine residues Ser725 of
Stat5a and Ser730 of Stat5b are candidate phosphorylation
sites because they are located within a positionally homologous PSP
motif. In contrast to Stat1 and Stat3, Stat5 proteins do not appear to
be substrates of extracellular signal-regulated kinases (ERKs)
(27-29), raising the possibility that Stat5 proteins are regulated
differently than other Stats.
-casein gene
regulation by several laboratories (30-32). We now report that the
proline-juxtaposed serine residues Ser725 of Stat5a and
Ser730 of Stat5b are major phosphorylation sites, based on
phosphoamino acid analysis and the generation of site-directed
anti-Stat5-phosphoserine antibodies. Unexpectedly, phosphorylation
control of this PSP motif differed between the highly homologous Stat5a
and Stat5b proteins, and phosphoamino acid analysis suggested the
existence of a second major serine phosphorylation site unique to
Stat5a. The data also indicated that two distinct pathways lead to
serine phosphorylation of the PSP motif of Stat5, one
PD98059-resistant, prolactin-activated pathway, and one
PD98059-sensitive, constitutive pathway. Because phosphorylation of the
PSP motif of Stat5a or Stat5b was not essential for DNA binding or
transcriptional activation of a -casein reporter gene in COS-7
cells, it is possible that regulation of Stat5 activity by serine
kinases differs from that of Stat1 and Stat3.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
344 to 1) -casein gene promoter linked
to the luciferase reporter gene (pZZ1; kindly provided by Bernd Groner,
Institute for Experimental Cancer Research, Freiburg, Germany) and the
plasmid pCH110 containing the -galactosidase gene under the control
of the simian virus 40 promoter have been described previously (30, 31). Plasmid p3PRLR containing a 2.7-kb human prolactin receptor cDNA (kindly provided by Paul A. Kelly, Intitut National de la Santé et de la Recherche Médicale, Paris, France) was
constructed by cloning into the EcoRI site of pcDNA3
expression vector (Invitrogen). Mutants of Stat5a and Stat5b were
prepared from double-stranded DNAs using the QuikChange site-directed
mutagenesis kit (Stratagene) with oligonucleotide primers designed to
alter serine residues to alanines or tyrosines to phenylalanines. The
following mutants of mouse Stat5a were generated by this method: S710A
(TCC to GCC), S725A (TCC to GCC), and Y694F (TAC to TTC). These mutants
of Stat5b were also made: S35A (TCA to GCA), S45A (TCA to GCA), S132A
(AGT to GCT), S427A (TCT to GCT), S715A (TCC to GCC), S730A (TCC to GCC), S748A (TCC to GCC), Y679F (TAT to TTT), and Y699F (TAC to TTC).
Before use, the DNA sequence of each mutant was verified.
-galactosidase assays, 2 µg of pZZ1 and 0.2 µg of pCH110
encoding the -galactosidase gene were added. One day after
transfection, cells were stimulated with 10 nM human
prolactin (NIDDK-hPRL-SIAFP-B2, AFP-2969A), which was supplied by the
National Hormone and Pituitary Program, National Institute of Diabetes
and Digestive and Kidney Diseases, the National Institute of Child
Health and Human Development, and the U.S. Department of Agriculture.
70 °C.
-casein gene was used.
Polyacrylamide gels (4%) containing 5% glycerol and 0.25 × Tris
borate/EDTA were prerun in 0.25 × Tris borate/EDTA buffer at
4-10 °C for 1.5 h at 300 V. After loading of samples, the gels
were run at room temperature for ~3 h at 250 V. Gels were dried by
heating under vacuum and exposed to x-ray film (X-Omat, Eastman Kodak
Co.).
-Galactosidase Assays--
One day after
transfection, COS-7 cells were stimulated with prolactin for 16 h
and harvested. Cells were washed twice with PBS and lysed in
Triton/glycylglycine lysis buffer (1% Triton X-100, 25 mM
glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM dithiothreitol) and centrifuged
at 12,000 × g for 5 min at 4 °C. Supernatants were
used for luciferase and -galactosidase assays. For luciferase
assays, 100 µl of cell lysate were mixed with 360 µl of assay
buffer containing 25 mM glycylglycine, pH 7.8, 15 mM potassium phosphate, pH 7.8, 15 mM
MgSO4, 4 mM EGTA, 2 mM ATP, and 1 mM dithiothreitol. Luciferase activity of each sample was
determined by measuring luminescence after injection of 200 µl of 1 mM luciferine. Assays for -galactosidase were carried
out using Galacto-Light Plus (Tropix, Inc.). Two hundred microliters of
reaction buffer were added to 20 µl of cell lysate and incubated at
room temperature for 60 min. Measurements were made by injecting 200 µl of light emission accelerator II (Tropix, Inc.), and each sample
was counted for 5 s. To correct for differences in transfection
efficiencies, luciferase activities were normalized to the
-galactosidase values in each individual sample.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Overview of mutation sites in Stat5a and
Stat5b. A, alignment of Stat5 protein sequences
corresponding to regions surrounding Ser727 of Stat1 and
Stat3. B, structure of mouse Stat5a and Stat5b. The highly
homologous DNA binding domains, Src homology domain 3 regions
(SH3), and Src homology domain 2 regions (SH2)
are indicated (96% amino acid identity). Nonhomologous regions of
Stat5a and Stat5b are hatched. The COOH-terminal regions of
Stat5a and Stat5b containing the SH2 docking site and trans-activation
domain only display 80% amino acid identity.
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Fig. 2.
Phosphoamino acid analysis of wild-type
proteins (WT) and mutants of Stat5a (A) and
Stat5b (B). Autoradiographs of immunoprecipitated
wild-type and mutant forms of Stat5a (A) and Stat5b
(B) from [32P]orthophosphate-labeled COS-7
cells incubated with (+) or without ( ) PRL (10 nM) for 30 min at 37 °C are shown in the upper panels of
A and B. The lower panels show
phosphoamino acid analysis of wild-type and mutant forms of Stat5a
(A) and Stat5b (B) with (+) or without () PRL
stimulation. 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. Representative data from three
independent experiments are shown. Migrational positions of
phosphoserine (pSer), phosphothreonine (pThr),
and phosphotyrosine (pTyr) are indicated on the
right.
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Fig. 3.
Phosphotyrosine and phosphoserine content of
wild-type (WT) and mutants of Stat5a (A) and
Stat5b (B) by immunoblotting. COS-7 cells transfected
with the prolactin receptor and Stat5a or Stat5b mutants were starved
with serum-free Dulbecco's modified Eagle's medium for 16 h and
incubated with medium ( ) or 10 nM PRL (+) at 37 °C for
60 min. Lysates were immunoprecipitated with anti ()-Stat5a
(A) or -Stat5b (B). Parallel samples were
blotted for phosphotyrosine (-PY, upper
panels), site-specific phosphoserine (-PS725 and
-PS730, middle panels), -Stat5a (A,
lower panel), or -Stat5b (B, lower panel).
-casein gene promoter was evaluated (Fig.
4, A and B). Using
prolactin-responsive COS-7 cells, mutants Stat5a-S725A and Stat5b-S730A
were found to be fully capable of forming DNA complexes with a
32P-labeled -casein promoter probe and were at least as
efficient as wild-type proteins over the duration of the 20-h test
period (Fig. 4, A and B). Similarly, mutants
Stat5a-S710A and Stat5b-S715A were also fully capable of binding DNA,
whereas no induction of DNA binding was observed with the tyrosine
mutants Stat5a-Y694F and Stat5b-Y699F (data not shown), consistent with
the critical role of these tyrosine residues for Stat5 activation (30).
We therefore concluded that the serine phosphorylation state of the PSP
motif is not essential for Stat5 DNA binding.
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Fig. 4.
Prolactin-inducible DNA binding activities of
wild-type (WT) and mutants of Stat5a (A) and
Stat5b (B). COS-7 cells transfected with the prolactin
receptor and Stat5a or Stat5b wild-type (WT) or serine to
alanine mutants (Stat5a-S725A and Stat5b-S730A) were starved with
serum-free Dulbecco's modified Eagle's medium for 16 h and
incubated with (+) or without ( ) PRL (10 nM) for 0-20 h,
and cell extracts were prepared for gel shift analysis with
32P-labeled -casein promoter probe. Markers
indicate migrational positions of Stat5a or Stat5b.
-casein gene
promoter-luciferase reporter gene were cotransfected into COS-7 cells.
A constitutively expressed -galactosidase gene was also included to
compensate for differences in transfection efficiencies. Luciferase
activities were measured in extracts of cells incubated in the absence
or presence of prolactin for 16 h (Fig.
5, A and B). The
wild-type Stat5a and Stat5b proteins both mediated a highly consistent
2.5-fold induction of the reporter gene after prolactin stimulation,
whereas mutants Stat5a-Y694F and Stat5b-Y699F were completely inactive as previously reported (30). Each of the three Stat5a serine mutants,
S725A, S710A, and S710A,S725A, showed signals comparable to those of
the wild-type Stat5a (Fig. 5A). Likewise, mutant
Stat5b-S730A was also equally as efficient as wild-type Stat5b to
induce luciferase activity (Fig. 5B). Basal transcription
levels were also not affected by mutation of the serine residue of the
PSP motif of either Stat5a or Stat5b. In contrast, the inducible
luciferase activities were reduced in prolactin-treated cells
transfected with Stat5b-S715A or Stat5b-S715A,S730A. Indeed, neither
Stat5b-S715A nor Stat5b-S715A,S730A mediated significant
prolactin-induced luciferase activity. Further studies are needed to
establish why mutant Stat5b-S715A exhibits reduced ability to activate
transcription of the -casein gene. More importantly, we conclude
that phosphorylation of the conserved PSP motif of Stat5a and Stat5b is
not critical for their inducible transactivation of a -casein
reporter gene in COS-7 cells. It remains possible that serine
phosphorylation influences the transcriptional activity of Stat5 toward
other responsive genes or in the presence of cofactors other than those
available in COS-7 cells.
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Fig. 5.
Prolactin-inducible -casein gene
activation by wild-type (WT) and mutants of Stat5a
(A) and Stat5b (B). COS-7 cells were
transfected with a -casein-luciferase reporter gene, the prolactin
receptor, mutants of Stat5a and Stat5b, and a -galactosidase gene
under the control of the simian virus 40 promoter. Cells were treated
with (+) or without () PRL (10 nM) for 16 h.
Luciferase and -galactosidase activities in cell extracts were
determined, and the ratios of the luciferase to -galactosidase
activities are shown. The mean values of four independent experiments
are presented, and S.E. values are indicated by bars. Note
interassay consistency; no normalization to controls was made.
Differences between unstimulated and prolactin-stimulated levels were
compared by one-way analysis of variance followed by Scheffe's
multiple range test (***, p < 0.001; **,
p < 0.01; *, p < 0.05).
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Fig. 6.
Effect of protein kinase inhibitors on
prolactin-induced serine phosphorylation of Stat5a and Stat5b in Nb2
lymphocytes. A and B, quiescent Nb2 cells were
preincubated with dimethylsulfoxide (Ctrl), staurosporine
(Stsp, 500 nM), PD98059 (100 µM),
H7 (100 µM), or wortmannin (100 nM) for 30 min before stimulation with (+) or without ( ) prolactin for 15 min.
Lysates were immunoprecipitated with anti ()-Stat5a (A)
or -Stat5b (B). Parallel samples were blotted for
phosphotyrosine (-PY, upper panels),
site-specific phosphoserine (-PS725 and
-PS730, middle panels), -Stat5a
(A, lower panel), or -Stat5b (B,
lower panel). C, inhibition of prolactin-induced
activation of MAPK by PD98059 and staurosporine. Quiescent Nb2 cells
were preincubated with dimethylsulfoxide (Ctrl), PD98059
(100 µM), or staurosporine (Stsp, 500 nM) for 30 min before stimulation with (+) or without ()
prolactin for 15 min. Samples from whole cell lysates were separated by
SDS-PAGE and blotted with antibodies to phosphorylated MAPK
(upper panel). Parallel samples were blotted with anti-pan
ERK antibodies (lower panel).
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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