From the Graduate Center for Toxicology, University
of Kentucky, Lexington, Kentucky 40536-0305 and the
Georg
Speyer Haus, Paul Ehrlich Strasse 42-44, 60596 Frankfurt, Germany
Received for publication, August 7, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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The growth hormone family of cytokines transduces
intracellular signals through the Jak2-Stat5 pathway to activate the
transcription of target genes. Amino acids within the C termini of
Stats constitute the transactivation domain but also regulate the time
course of tyrosine phosphorylation and extent of DNA binding. We
mutated Thr757 in the C-terminal of Stat5a
(Thr-Stat5) to Val (Val-Stat5) and Asp (Asp-Stat5) and examined the
effect on nuclear translocation, DNA binding, and prolactin-induced
transcriptional activation of a Stat5-responsive luciferase reporter
gene. Val-Stat5 produced a 5-fold higher increase in transcriptional
activity relative to Thr-Stat5; Asp-Stat5 produced a similar response
to Thr-Stat5. The increased transactivation was ligand induced and was
not due to differences in basal expression of Val-Stat5 or to a
constitutively activated Stat5 protein. Similar rates of loss of DNA
binding ability and phosphorylation of Val- and Thr-Stat5 were observed following a single pulse of prolactin, indicating that the
dephosphorylation pathways were unaltered. The serine-threonine kinase
inhibitor H7 inhibited the transactivation potential of Thr-, Val-, and Asp-Stat5 to a similar extent, eliminating phosphorylation of Thr757 as a regulatory mechanism. The results suggest that
Thr757 modulates the transactivation potential of Stat5 by
a mechanism(s) that is dependent on the formation of Stat5 dimers
and/or their nuclear translocation.
Stat (signal transducers and
activators of transcription) proteins
constitute a family of latent cytoplasmic transcription factors that
are activated by a large number of cytokines and growth factors (1-3).
Ligand binding by the cytokines to their cognate cell surface receptors
leads to a conformational change in the receptor, which in turn results
in activation of the receptor-associated Janus kinases
(Jak).1 The activated Jak
phosphorylate the receptor at specific tyrosine residues within the
cytoplasmic domain, thus recruiting the Stat proteins, which bind via
their Src homology 2 (SH2) domains (2). Jak-mediated tyrosine
phosphorylation of the receptor-bound Stats leads to their dissociation
from the receptor, dimerization by means of reciprocal
SH2-phosphotyrosine interactions and translocation to the nucleus,
where the Stat dimers bind to specific DNA sequences and induce gene
transcription. Seven mammalian members of the Stat family have been
reported and show a high degree of similarity. The highest degree of
similarity occurs in the SH2 domain, whereas the C-terminal domain is
the least conserved region (4). C-terminal deletion mutants and
alternatively spliced variants lacking this region have provided
evidence that this region serves as the transactivation domain (5). The
transactivation domain of Stat5 has been mapped to a region between
amino acids 750-772 (6). Deletion of the C terminus of Stat5 resulted
in a dominant negative phenotype with sustained tyrosine
phosphorylation and DNA binding relative to the wild type but with no
transcriptional activation.
Members of the growth hormone family of cytokines such as prolactin
(PRL) and growth hormone transduce intracellular signals via activation
of the Jak2-Stat5 signal transduction pathway to regulate the
transcription of a variety of genes (7-13). The specificity of
transcriptional regulation by Stat5 is believed to result from its
association with coactivators and corepressors of transcription and
binding of additional transcription factors to the promoter regions of
target genes (14). We have demonstrated that PRL acts via the long form
of the PRL receptor (PRLRL) to facilitate the binding of
Stat5 multimers to two interferon- Reagents and Plasmids--
All reagents were of molecular
biology or cell culture grade. Polynucleotide kinase was obtained from
Life Technologies, Inc. Ovine PRL was kindly provided by Dr. A. F. Parlow of the National Institute of Diabetes and Digestive and Kidney
Diseases, National Hormone and Pituitary Program. Stat5 monoclonal
antibodies raised against ovine Stat5 amino acids 451-469 were
obtained from Transduction Laboratories (Lexington, KY). Polyclonal
antiserum directed against Stat5a was from Zymed
Laboratories Inc. (San Francisco, CA), and anti-phosphotyrosine
antibody (PY20) was from Santa Cruz Biotechnology (Santa Cruz, CA). The
cDNA of PRLRL was a gift from Dr. Paul Kelly (INSERM,
Paris, France). Plasmid pRSV- Construction of Mutants--
Val-Stat5 was generated using the
"megaprimer" polymerase chain reaction method (22) using
5'-GGGAGTACTACACTCCTGTGCTTGCCAAA-3' as the forward primer,
5'-GGGACTAGTCAACATTCAGGAGAGCGAGCCT-3' as the reverse primer,
5'-GGCCACATCCATGACCTCGTCCAGGTC-3' as the mutagenesis primer, and the
2.4-kilobase NotI-SalI fragment of Thr-Stat5 as a
template. Asp-Stat5 was constructed using
5'-GGCCACATCCATGTCCTCGTCCAGGTC-3' as the mutagenesis primer. The
0.4-kilobase polymerase chain reaction product was digested with
SpeI and ScaI and ligated with the 2-kilobase SalI-ScaI fragment of ovine Stat5 and
subsequently ligated to SalI-SpeI digested pXM
vector. Integrity of all plasmids was ascertained by sequencing.
Cell Culture and Transfection--
HepG2 cells were cultured in
Dulbecco's modified Eagle's medium/Ham's F-12 medium with 10% fetal
bovine serum as described (15). Cells were transfected using the
calcium phosphate:DNA coprecipitation method (23). A 10-cm plate of
cells was transfected with 5 µg of luciferase reporter constructs
4X0.2pGL3 or 0.5pT109luc, 5 µg of expression vectors for Thr-Stat5
(pXM-Thr-Stat5)/Val-Stat5 (pXM-Val-Stat5)/Asp-Stat5 (pXM-Asp-Stat5), 1 µg of expression vector for the PRLRL
(pL3-PRLRL), and pUC19 as carrier, to a total of 20 µg.
pRSV- Luciferase and Studies with the Serine/Threonine Kinase Inhibitor--
To
investigate the effect of the serine/threonine kinase inhibitor H7,
HepG2 cells were trypsinized 6 h post-transfection, dispensed in
96-well plates, and incubated overnight. H7 (100 µM) was
added to the cells, followed after 1 h by treatment with increasing doses of PRL. Luciferase activity was determined after 6 h of incubation with ligand.
Preparation of Nuclear Extracts and Electromobility Shift Assays
(EMSA)--
Whole cell or nuclear extracts were prepared as described
(24). Cells were transfected with recombinant plasmids and 6-8 h
post-transfection were washed twice with phosphate-buffered saline
(PBS). Fresh medium was added to the plate, and the cells were
incubated for a further 36-40 h. PRL (0.5 µg/ml) was then added
directly to the cells in the 10-cm culture dish and removed at the
indicated times. For nuclear extracts, cells were washed with PBS,
scraped and collected in cold PBS, pelleted, and resuspended in cold
buffer A (10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 1 mM NaF, 0.5 mM DTT, 0.2 mM PMSF, 1 µg/ml pepstatin, 5 µg/ml aprotinin, 2 µg/ml leupeptin, and 5 µg/ml antipain). The
suspension was incubated on ice for 10 min and centrifuged for 10 s at high speed. The pellet was resuspended in 20-100 µl of cold
buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, 1 mM
Na3VO4, 10 mM NaF, 0.5 mM DTT, 0.2 mM PMSF, and the protease
inhibitors at the above mentioned concentrations), incubated on ice for
20 min, and centrifuged at 4 °C for 2 min, and the extract was
aliquoted and stored at
Double-stranded consensus Stat5 oligonucleotide
(5'-agatTTCTAGGAAttcaatcc-3') was used as probe for EMSA. The probe was
radiolabeled with [ Western Blots--
Proteins from whole cell extracts or nuclear
extracts from unstimulated or PRL-stimulated (0.5 µg/ml) cells were
immunoprecipitated with polyclonal antiserum directed against Stat5a or
PY20, prior to immunoblotting with either PY20 or a Stat5a (1:3,000
dilution) antibody. An anti-rabbit or anti-mouse IgG horseradish
peroxidase antibody (1:5,000) was used as a secondary antibody, and the
proteins were visualized by the ECL detection system (Amersham
Pharmacia Biotech). Bands were digitized and quantified by
computer-assisted imaging using MCID/M4 software supplied by Imaging
Research Inc. (St. Catharines, Ontario, Canada).
Val-Stat5 Exhibits a Higher Transactivation Potential than
Thr-Stat5 or Asp-Stat5--
Within the C terminus of Stat5 is a
stretch of 22 amino acids (750) indispensable for PRL-induced
transactivation (6, 26). The conserved sequence,
FDL(D/E)(E/D)V(T/S)DVARHVEELLRRPMD, in this transactivation
domain in the Stat5a and Stat5b isoforms of various species contains a
single threonine/serine as a potential phosphorylation site (6). We
mutated the Thr757 in Stat5a to a Val or an Asp to
determine the effect of loss of this putative phosphorylation site and
replacement with an acidic amino acid residue, respectively, on the
transactivation potential of Thr-Stat5. When cells were transfected
with Val-Stat5 and stimulated with PRL, a dose-dependent
increase in the transcription of the reporter construct 4X0.2pGL3 was
observed. This induction was 5-5.5 times greater than that in cells
transfected with Thr-Stat5 (Fig.
1A). The response of cells
transfected with Asp-Stat5 and the 4X0.2pGL3 construct was similar to
that of cells transfected with Thr-Stat5. The basal luciferase activity
remained unaltered (Fig. 1B), indicating that neither of the
mutants was constitutively active. In transfection experiments with the
reporter construct of the ntcp promoter containing the two
native GAS elements coupled to the thymidine kinase promoter, Val-Stat5
also showed a significantly increased transactivation potential
relative to that of Thr-Stat5 and Asp-Stat5 (Fig. 1C).
Effect of the Serine/Threonine Kinase Inhibitor H7 on the
Transactivation Potential of Thr-, Val-, and Asp-Stat5--
Protein
kinase inhibitors of the H series are the most commonly used inhibitors
of serine/threonine phosphorylation (27). H7 is one of the three widely
used inhibitors and inhibits protein kinase C, in addition to cAMP- and
cGMP-dependent protein kinase (cAPK and cGPK,
respectively). To determine whether phosphorylation of
Thr757 of Stat5 plays a role in signal transduction, HepG2
cells pretreated with vehicle or H7 for 1 h were treated for
6 h with increasing doses of PRL before determining the luciferase
activity. H7 decreased the PRL-induced transcription of the reporter
gene by ~90% at 1 µg/ml PRL. This observation is consistent with
previous reports that serine/threonine phosphorylation of Stat
molecules is required for the activation of its DNA binding and
subsequent transactivation potential (5, 16, 28). If the
phosphorylation of Thr757 were essential for Stat5
activation, then the Val-Stat5 and Asp-Stat5 mutants should not be
sensitive to the inhibitor. However, the effect of H7 was similar in
the wild type and both Val-Stat5 and Asp-Stat5 transfected cells (Fig.
2).
Increased Nuclear Localization of Val-Stat5 as Compared with
Thr-Stat5--
We carried out further studies comparing Thr-Stat5 and
Val-Stat5 to investigate the mechanism of the enhanced response to Val-Stat5. The increase in luciferase activity in cells transfected with Val-Stat5 versus Thr-Stat5 was not due to differences
in their expression, because similar levels of immunoreactive Val- and
Thr-Stat5 protein were present in unstimulated whole cell extracts
(Fig. 3A, lanes 1 and 2). In contrast, increased levels of immunoreactive
Stat5 were observed in nuclear extracts from Val-Stat5-transfected
cells relative to those obtained from Thr-Stat5-transfected cells
following stimulation with PRL for 1 or 6 h (Fig. 3A,
compare lanes 4 and 6 with lanes 3 and
5). The increased Stat5 levels in the nuclear extracts from
Val-Stat5 transfected cells were observed as early as 5 min after PRL
induction (Fig. 3B).
Increased Transactivation Potential of Val-Stat5 Correlates with
Increased Nuclear Translocation and DNA Binding Ability--
To
investigate the mechanism(s) for the enhanced transactivation potential
of Val-Stat, nuclear extracts from transfected cells were probed for
DNA binding ability. In the absence of transfected Stat5, no specific
DNA binding ability was observed in HepG2 cells transfected with
PRLRL and treated with PRL (Fig.
4A, lane 2 versus lane 4), indicating nondetectable levels of
endogenous Stat5. These results are consistent with our studies that
showed no PRL-mediated induction of luciferase activity in HepG2 cells
transfected with PRLRL and an ntcp-promoter
luciferase construct under these same conditions (15). Consistent with
observations of low basal levels of luciferase activity, no specific
DNA binding ability was observed in unstimulated cells (Fig.
4B, lanes 1 and 2). Specific
DNA-protein complex formation was observed in both Thr-Stat5- and
Val-Stat5-transfected cells as early as 15 min following stimulation
with PRL, with DNA-binding of Val-Stat5 being higher than that of
Thr-Stat5 at all time points up to 24 h (Fig. 4, B and
C). The observed bands were confirmed as Stat5 by incubation
with the anti-Stat5 antibody that supershifted the Stat5 complex (Fig.
4C, lanes 11 and 12). The specificity
of the Stat5 bands was also confirmed by coincubation with 100-fold
excess unlabeled Stat5 oligomer in the EMSA reaction, which markedly
reduced the signal (Fig. 4C, lanes 13 and
14).
The increase in specific DNA binding in cells transfected with
Val-Stat5 did not seem to be due to increased affinity for the cognate
DNA-recognition sequence but to increased nuclear concentrations.
Incubation of the nuclear extracts of Thr-Stat5 and Val-Stat5 with
increasing concentrations of unlabeled Stat5 consensus oligomer
competed for specific Thr- and Val-Stat5 DNA binding with similar
efficiency (Fig. 5A). There
was no major change in the binding specificity, because incubation of
the nuclear extracts with either the Stat3 oligomer (upper strand,
5'-gatccTTCTGGGAAtcctagatc-3') or an oligomer containing a GAS element
essentially identical to that activated by interferon- Increased Nuclear Levels of Val-Stat5 Are Not a Consequence of
Delayed Dephosphorylation--
Dephosphorylation of Stat5
Tyr694, rather than Stat5 protein degradation, has been
shown to be the primary determinant of down-regulation of its DNA
binding ability (6, 24, 32). The region between amino acids 750 and 794 exhibits a role in down-regulating the DNA binding activity of Stat5.
We postulated that this region might regulate the interaction between
Thr-Stat5 and a phosphatase, which by dephosphorylation of
Tyr694, would lead to down-regulation of Stat5 DNA binding
ability. Accordingly, mutation of Thr757 to Val could
disrupt the interaction of the phosphatase with Stat5, leading to a
delayed dephosphorylation of Tyr694 and/or other
serine/threonine residues. This would increase the steady-state levels
of the translocated Stat5 multimers and facilitate binding to the GAS
elements, thus allowing for an increased transactivation potential. To
test this possibility, cells transfected with either Thr-Stat5 or
Val-Stat5 were treated with a single pulse of PRL for 30 min, after
which the medium was removed and replaced with ligand-free
medium. Nuclear extracts prepared at various times thereafter were then
subjected to EMSA. These nuclear extracts were also immunoprecipitated
with anti-phosphotyrosine antibody (PY-20) and probed with Stat5a
antibody by Western analysis. Both Thr- and Val-Stat5 exhibited a rapid
decrease in their DNA binding ability, such that by 2 h, no DNA
binding ability could be detected (Fig.
6A). The DNA-protein complexes
formed were quantitated and plotted as a function of time. As shown in
Fig. 6B, the DNA-protein complexes decayed with very similar
kinetics. Similar rates of loss were also observed when the nuclear
extracts were first immunoprecipitated with anti-phosphotyrosine
antibody and the levels of phosphorylated Stat5 were visualized with a
polyclonal Stat5a antibody by Western analysis (Fig. 6, C
and D).
Signal transduction via the Stat5 pathway requires the recruitment
of the latent cytoplasmic transcription factor to the phosphorylated tyrosine residue in the cytoplasmic domain of the receptor,
phosphorylation of a key tyrosine (Tyr694) in Stat5 by the
receptor-associated Jak, followed by the interaction of the SH2 domain,
and formation of dimers by each of the phosphotyrosines in the two
Stats in a reciprocal manner. The dimers are then translocated to the
nucleus, where they bind to the GAS elements via their DNA-binding
domain and direct an increase in specific transcriptional activity by
the recruitment of the general transcription factors via their
transactivation domain. A number of studies have demonstrated that
amino acids 750-772 in the C termini of Stat5 are essential for the
increase in transcriptional activity, and may also act to down-regulate
activity by increasing the dephosphorylation of Tyr694.
Other studies have demonstrated that although tyrosine phosphorylation is obligatory to Stat5 transcriptional activation, serine
phosphorylation can modulate the binding to DNA (5). Thus, mutation of
Ser725 to Ala in Stat5a resulted in sustained DNA binding
activity relative to that of wild type Stat5a but with no change in
transcriptional activation (21). The increased DNA binding observed in
the present experiments is similar to the increased binding observed
with C-terminal deletion mutants and naturally occurring splice
variants of Stat5a and Stat5b (6, 30). However, unlike the C-terminal deletion variants of Stat5a and Stat5b that exert a dominant negative phenotype, mutation of Thr757 to Val resulted in a higher
transactivation potential, whereas simulation of a phosphorylated site
by mutation to Asp had no effect on activity. These data suggest that
Thr757 is not an essential site for phosphorylation in the
induction process mediated by the wild type version of Stat5. The
observation that incubation with the serine/threonine kinase inhibitor
H7 equally inhibited transactivation by wild type Stat5a and its mutants further supports the argument that Thr757 is not a
site for phosphorylation of Stat5a. The similar inhibitory effect of H7
on Thr-, Val-, and Asp-Stat5-mediated transcription suggests that other
phosphorylation sites that contribute to Stat5 function are targets for
the serine/threonine kinase inhibitor. However, our results are
consistent with reports that Stat5a/5b isoforms are phosphorylated at
tyrosine and serine, but not threonine residues, following activation
with PRL or interleukin-2 in a variety of cell lines (33-35).
Because dephosphorylation of Stat5 Tyr694 is the primary
regulator of down-regulation of its DNA binding ability and amino acids 750-794 have been shown to down-regulate the DNA binding activity of
Stat5, we postulated that Thr757 might interact with a
phosphatase that mediated the dephoshorylation of Tyr694.
However, when we compared the rates of loss of DNA binding ability and
dephosphorylation of Thr- and Val-Stat5 following a pulse dose of oPRL,
they were both very similar. These observations indicate that the
increased transactivation potential of Val-Stat5 is not a consequence
of delayed dephosphorylation of this mutant protein but rather because
of an increased rate of formation and/or nuclear translocation of Stat5 dimers.
Several mechanisms could be involved in the enhanced transactivation by
Val-Stat5. Overcoming negative regulation could be one of the
mechanisms by which Val-Stat5 elicits an increased response. The SOCS
(suppressors of cytokine
signaling) family of inhibitors either bind the activated
Jaks or compete with Stat5 for binding to the receptor to bring about
inhibition (reviewed in Ref. 36). Because the mutation is in
Thr757 of Stat5 and not in the receptor or Jak2, such a
mechanism is unlikely. Delayed dephosphorylation was experimentally
ruled out as a possible reason for increased Val-Stat5 transactivation. The coactivators and corepressors of transcription like CBP/p300 are
known to interact with the C-terminal of the activated Stat5 (37).
However, in cotransfection experiments, we found no evidence of
increased interaction of Val-Stat5 with p300, compared with Thr-Stat5
(data not shown). The most likely mechanism of enhanced transactivation
by Val-Stat5, therefore, is either a more effective association between
Val-Stat5 and Jak2 to facilitate phosphorylation by Jak2, increased
stability and translocation of the Val-Stat5 dimers following
activation, or both. Crystallographic studies have shown that in Stat
dimers, the C termini are in very close proximity, whereas the N
termini are far apart, forming a saddle-like structure around the DNA
(38). The present data suggest that replacement of the OH group of
Thr757 with the methyl group of Val results in increased
hydrophobic bonding between the C termini of Val-Stat5, leading to more
stable dimers. This hypothesis also explains the sustained high levels of phosphorylated Val-Stat5 in the nucleus. The faster nuclear translocation could result from faster dimerization as a result of
better interaction between Jak2 and Val-Stat5, or alternatively, an
enhanced rate of actual translocation into the nucleus. Mutation of
Thr757 to a Val, however, does not appear to disrupt the
interaction between Stat5 and the nuclear phosphatases that are
involved in deactivation and resetting of the Jak-Stat pathway. Whether
a specific PIAS (protein inhibitor of
activated Stats) exists for Stat5, as has been
shown for Stat1 and Stat3 (38), is not known. If identified, altered
interaction with such a PIAS might also account for the increased
transactivation potential of Val-Stat5.
Recently, Callus and Mathey-Prevot (39) further characterized
the C termini of murine Stat5a and confirmed that deletion of the last
57 amino acids results in the loss of ability to transactivate. Within
this region, deletion of a 12-amino acid block (749) also dramatically decreased the transactivation potential of a chimerae of
the DNA-binding domain of Gal4 with the C-terminal transactivation domain of Stat5. However, when the full-length Stat5 containing this
deletion was cotransfected into cells expressing the PRL receptor and a
Stat5-responsive luciferase reporter gene, the luciferase activity in
response to PRL was not affected. This was found to be due to enhanced
DNA binding activity of the 12-amino acid deletion mutant, which these
authors also postulated might reflect an enhanced rate of dimerization.
Mutation of Ser756 (analogous to Thr757 in the
present study) within this 12-amino acid block to Gly resulted in a
25% decrease in the transactivation following PRL treatment, even
though neither the DNA binding nor phosphorylation of
Tyr694 were altered. The basis for the modest decrease in
transactivation in the S756G mutant was postulated to reflect subtle
structural changes in the 12-amino acid region. Importantly, mutation
of the hydrophobic Phe751 and Leu753 to Ala
reduced transactivation about 70%, indicating the importance of
hydrophobicity of this region to transactivation. These findings support the hypothesis that the increased hydrophobicity of Val-Stat5 is a key factor in its enhanced transactivation potential.
In summary, the present studies show that amino acid residues present
in the C-terminal transactivation domain of wild type Stat5a,
specifically Thr757, play a critical role in regulating its
function as a transcription factor. The presence of the hydroxyl group
in Thr757 appears to limit the rate of phosphorylation
and/or nuclear translocation of Stat5, because substitution with a Val
at this position markedly increased its ability to form
transcriptionally active dimers. The increased hydrophobicity of
Val-Stat5 appears to be a major contributor to its DNA binding and
transactivation activity, consistent with the essential nature of other
nearby hydrophobic amino acids.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-activated sequences (GAS)-like elements in the promoter of the rat liver
ntcp
(Na+/taurocholate
cotransporting polypeptide) gene to increase
its transcription (15). Obligatory tyrosine phosphorylation at
Tyr694 and subsequent serine/threonine
phosphorylation has been demonstrated for hepatic Stat5 activation
following growth hormone stimulation (9, 10, 16). Stat5a and Stat5b are
highly homologous genes that share about 96% amino acid sequence
homology (17-20). The amino acids within the C-terminal region in
Stat5a and Stat5b isoforms from various species are conserved and
contain a serine/threonine at position 757, which could serve as a
potential phosphorylation site. Two serine residues (Ser779
and Ser725) in the C-terminal domain of Stat5a have been
shown to be phosphorylation sites. Although mutation of
Ser779 to Ala had no effect on PRL-induced transcriptional
activation of a
-casein reporter construct, mutation of
Ser725 to Ala resulted in prolonged DNA binding activity
following PRL treatment (21). Thus, serine phosphorylation may serve to
modulate Stat5a transactivation. The present studies were designed to
evaluate the role of the conserved Thr757 in
transcriptional regulation of ntcp and used site-directed mutagenesis to alter Thr757 to Val and Asp and compare the
transactivation properties of these mutants to those of wild type
Stat5. We also studied the effect of the serine/threonine kinase
inhibitor, H7, on the transactivation potential of wild type Stat5 and
its mutants. The present studies show that the mutation of
Thr757 to Val markedly increased the transactivation
potential of Stat5, whereas mutation to Asp did not significantly alter
this potential.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase was kindly provided by
Dr. Noonan of the University of Kentucky. The luciferase reporter
plasmid 4X0.2SpGL3 was constructed as described (15) by linking four
GAS elements (TTCTTGGAA) to the ntcp minimal promoter (
158
to +47) and ligating this fragment in the HindIII site of pGL3 basic vector (Promega, Madison, WI). Plasmid 0.5pT109luc was
constructed by inserting the 500-base pair Hindlll fragment of the
ntcp promoter (
1237 to
758) upstream of the herpes
simplex virus thymidine kinase minimal promoter (HSVtk) in pT109Luc
(American Type Culture Collection, Manassas, VA) (15). Plasmid DNA was extracted and purified using the Qiagen midicolumns or by CsCl gradient
centrifugation twice. The inhibitor,
1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl (H7) was obtained
from Alexis Biochemicals (San Diego, CA).
-galactosidase (5 µg) was included in each transfection to
monitor transfection efficiency. Single transfections were conducted in
triplicate, and the mean was calculated for each data point. The data
are the means (± S.E.) for three independent transfections.
-Galactosidase Assays--
Cells were treated
with varying concentrations of PRL 6-8 h post-transfection. Luciferase
and
-galactosidase assays were performed 40-44 h post-transfection,
and the normalized luciferase response was determined as relative light
units divided by
-galactosidase activity
(A415 nm/min). The
hormone-dependent fold induction was calculated relative to
normalized luciferase response obtained in the absence of PRL treatment.
80 °C until further use. For whole cell
extracts, cells were collected in ice-cold PBS. After a brief
centrifugation, cells were suspended in ice-cold extraction buffer
containing 400 mM NaCl, 50 mM KCl, 20 mM HEPES buffer, pH 7.9, 1 mM EDTA, pH 8.0, 20% glycerol, 1 mM Na3VO4, 1 mM NaF, 1 mM DTT, 0.2 mM PMSF, and
protease inhibitors at the same concentrations as above. The cells were
lysed with three freeze-thaw cycles (15 min at 37 °C, 15 min at
70 °C), followed by centrifugation at 20,800 × g
at 4 °C for 15 min. The supernatant was removed and stored at
70 °C. The protein concentration in the nuclear and whole cell
extracts was estimated by the procedure of Lowry et al.
(25). The protease inhibitors, DTT and PMSF were added to the buffers
just before use.
-32P]ATP using polynucleotide
kinase at the 5'-OH (blunt) ends. The labeled probe was gel eluted in
1× NET buffer (0.1 M NaCl, 1 mM EDTA, 1 mM Tris-HCl, pH 7.6) after polyacrylamide (12%) gel
electrophoresis. Briefly, 10 µg of protein was incubated for 20 min
at room temperature with 10 fmol of purified probe in a 15-µl buffer
consisting of 5 mM Tris-HCl, pH 7.9, 15 mM
HEPES-KOH, pH 7.9, 0.08 M KCl, 3.5 mM
MgCl2, 5 mM EDTA, 5 mM DTT, 10%
glycerol, 0.1% Tween 20, and 0.133 mg/ml (dI-dC):poly(dI-dC). Unbound
probe was separated from protein-bound probe in a 4% polyacrylamide
gel containing 2.5% glycerol and 0.25× TBE (25 mM
Tris-HCl, 25 mM boric acid, and 0.25 mM EDTA,
pH 8.0). The gel was dried and exposed to film at
80 °C. For
competition assays, a 10-50-fold molar excess of unlabeled oligomer
was added to the incubation mixture. For supershift studies, the medium
was incubated with Stat5 monoclonal antibody for 15 min at 4 °C and
30 min at room temperature, before the addition of the labeled probe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparison of the transactivation potentials
of mutants versus wild type Stat5. HepG2 cells
were transfected with 4X0.2pGL3 (A and B) or
0.5pT109luc (C), expression vectors for Thr-Stat5 ( ),
Val-Stat5 (
), or Asp-Stat5 (
), PRLRL, and
-galactosidase. Cells were treated with increasing concentrations of
PRL and luciferase activity assayed after 40-44 h. A,
Val-Stat5 exhibited greater transactivation potential than Thr-Stat5 or
Asp-Stat5. B, estimation of basal luciferase activity showed
that the mutants were not constitutively active. C,
Val-Stat5 also showed a higher transactivation potential in cells
transfected with the ntcp promoter containing the two native
GAS elements.
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Fig. 2.
Effect of a serine/threonine kinase inhibitor
on the transactivation potential of Thr-, Val-, and Asp-Stat5.
HepG2 cells were transfected with 4X0.2pGL3, expression vectors for
Thr-Stat5 (open bars), Val-Stat5 (black bars),
and Asp-Stat5 (hatched bars), PRLRL, and
-galactosidase. Cells were pretreated with 100 µM H7
and then exposed to increasing concentrations of PRL for 6 h
before quantitation of luciferase activity.
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Fig. 3.
Western blots of basal and PRL-induced levels
of Thr-Stat5 and Val-Stat5. A, whole cell extracts (200 µg of protein) from unstimulated Thr-Stat5 and Val-Stat5 transfected
cells (lanes 1 and 2, respectively) or nuclear
extracts (200 µg of protein) prepared from Thr-Stat5-transfected
(lanes 3 and 5) and Val-Stat5-transfected
(lanes 4 and 6) cells 1 and 6 h after
stimulation with 0.5 µg/ml PRL, respectively, were immunoprecipitated
with 5 µg of polyclonal Stat5a antibody. Western blots were performed
on immunoprecipitated proteins using polyclonal Stat5a antibody.
B, nuclear extracts prepared from Thr-Stat5 or Val-Stat5
transfected cells without (lanes 1 and 2) or with
exposure to 0.5 µg/ml PRL for 2 min (lanes 3 and
4), 5 min (lanes 5 and 6), and 15 min
(lanes 7 and 8) were immunoprecipitated with 2 µg of anti-phosphptyrosine antibody (PY-20), and Western blots were
performed using mouse monoclonal Stat5 antibody. Samples in lanes
1, 3, 5, and 7 are from
Thr-Stat5-transfected cells, and those in lanes 2,
4, 6, and 8 are from
Val-Stat5-transfected cells.
View larger version (57K):
[in a new window]
Fig. 4.
Increased nuclear translocation and DNA
binding ability of Val-Stat5 versus Thr-Stat5.
HepG2 cells were transfected with expression vectors for Thr-Stat5 or
Val-Stat5 and/or PRLRL and were either untreated or treated
with 0.5 µg/ml PRL for varying time periods. A,
32P-labeled Stat5 consensus oligonucleotide was incubated
with nuclear extracts from untransfected cells (lane 1) or
cells transfected with PRLRL (lane 2) or
Thr-Stat5 (lane 3) or both PRLRL and Thr-Stat5
(lane 4). B, 32P-labeled Stat5
consensus oligonucleotide was incubated with nuclear extracts from
Thr-Stat5-transfected (lanes 1, 3, 5,
and 7) or Val-Stat5-transfected (lanes 2,
4, 6, and 8) cells that were either
untreated (lanes 1 and 2) or treated for 15 min
(lanes 3 and 4), 30 min (lanes 5 and
6), or 60 min (lanes 7 and 8) with
PRL, respectively. C, 32P-labeled Stat5
consensus oligonucleotide was incubated with nuclear extracts from
Thr-Stat5- or Val-Stat5-transfected cells that were either untreated
(lanes 1 and 2) or treated for 1 h
(lanes 3 and 4), 6 h (lanes 5 and
6), 12 h (lanes 7 and 8), and
24 h (lanes 9 and 10), respectively, with
PRL (0.5 µg/ml). Nuclear extracts prepared from Thr-Stat5-transfected
(lanes 11 and 13) or Val-Stat5-transfected
(lanes 12 and 14) cells 1 h after PRL
treatment were treated with 1 µg of polyclonal rabbit Stat5a antibody
to induce a supershift (SS) complex (lanes 11 and
12) or exposed to a 100-fold molar excess of unlabeled Stat5
consensus oligonucleotide (lanes 13 and
14).
in the human
interferon regulatory factor 1 gene promoter (31) (upper strand
5'-taattTTCCCCGAAgtaca-3') did not alter the Stat5 consensus oligomer
binding to the proteins (Fig. 5B). In coexpression
experiments, increasing the amounts of Thr-Stat5 plasmid partially
decreased the transactivation produced by Val-Stat5, suggesting that
the two proteins compete for the same DNA binding sequence (data not
shown). A direct measurement of the "off time" of the preformed
DNA-wild type or mutant protein complex using equilibrium displacement
experiments was inconclusive because of the inherent low affinity of
the Stat5 protein for the DNA recognition site and the rapid rate of
dissociation. Thus, a 50-fold molar excess of competing unlabeled Stat5
oligomer displaced greater than 90% of the specifically bound complex
within 1 min (data not shown).
View larger version (60K):
[in a new window]
Fig. 5.
Thr-Stat5 and Val-Stat5 exhibit similar Stat5
DNA binding affinity. Nuclear extracts were prepared from HepG2
cells transfected with expression vectors for Thr-Stat5 or Val-Stat5
and PRLRL and treated for 1 h with PRL (0.5 µg/ml).
A, nuclear extracts from Thr-Stat5-transfected (lanes
1, 3, 5, and 7) or
Val-Stat5-transfected (lanes 2, 4, 6,
and 8) cells were incubated with 1000-fold (lanes
1 and 2), 500-fold (lanes 3 and
4), 50-fold (lanes 5 and 6), and
10-fold (lanes 7 and 8) molar excess of unlabeled
Stat5 oligomer before competition with 32P-labeled Stat5
consensus oligonucleotide. B, nuclear extracts from
Thr-Stat5-transfected (lanes 1, 3, 5,
and 7) or Val-Stat5-transfected (lanes 2,
4, 6, and 8) cells were incubated with
a 50-fold molar excess of unlabeled Stat3 (lanes 1 and
2), IRF-1 (lanes 3 and 4), or Stat5
(lanes 5 and 6) consensus oligonucleotides before
competition with 32P-labeled Stat5 consensus
oligonucleotide. Lanes 7 and 8 represent
experiments in which nuclear extracts were treated with 1 µg of
polyclonal Stat5a antibody to induce a supershift (SS)
complex.
View larger version (36K):
[in a new window]
Fig. 6.
Thr-Stat5 and Val-Stat5 exhibit similar rates
of decay of DNA binding ability and dephosphorylation. HepG2 cells
were transfected with expression vectors for Thr-Stat5 or Val-Stat5 and
PRLRL. Cells were stimulated with a single 30-min pulse of
PRL (0.5 µg/ml) after which the medium was replaced. A,
32P-labeled Stat5 consensus oligonucleotide was incubated
with nuclear extracts from Thr-Stat5-transfected (lanes 1,
3, 5, and 7) or Val-Stat5-transfected
(lanes 2, 4, 6, and 8)
cells immediately following PRL stimulation (lanes 1 and
2), or 30 (lanes 3 and 4), 60 (lanes 5 and 6), and 120 min (lanes 7 and 8) following incubation in PRL-free medium.
B, a plot of the optical density of the nuclear Stat5a
complex versus time in Thr-Stat5-transfected ( ) and
Val-Stat5-transfected (
) cells shown in A. C,
nuclear extracts (100 µg) from Thr-Stat5- or Val-Stat5-transfected
cells were immunoprecipitated with 5 µg of anti-phosphotyrosine
antibody (PY-20) followed by Western analysis using polyclonal Stat5a
antibody. The numbering of the lanes is the same as for A. D, a plot of the optical density of Stat5a versus
time in Thr-Stat5-transfected (
) and Val-Stat5-transfected (
)
cells shown in C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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.
§ Present address: Millennium Pharmaceuticals Inc., 270 Albany St., Cambridge, MA 02139.
¶ Present address: Dept. of Molecular Biology, 2800 Plymouth Rd., Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48205.
** To whom correspondence should be addressed: 306 Health Sciences Research Bldg., Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536-0305. Tel.: 859-257-3760; Fax: 859-323-1059; E-mail: maryv@pop.uky.edu.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007156200
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ABBREVIATIONS |
---|
The abbreviations used are:
Jak, Janus kinases;
SH, Src homology;
PRL, prolactin;
PRLRL, long form of the
prolactin receptor;
GAS, -interferon activated sequences;
H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine·2HCl;
PBS, phosphate-buffered saline;
DTT, dithiothreitol;
PMSF, phenylmethylsulfonyl fluoride;
EMSA, electromobility shift
assays.
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REFERENCES |
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