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Institute for Experimental Cancer Research Tumor Biology Center D-79106 Freiburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-interferon activated sites) in the promoter or upstream enhancer
regions of several milk protein genes (ß-casein, ß-lactoglobulin,
and whey acidic protein) are required for maximal transcription during
lactation and PRL induction (12, 13, 14, 15). Stat5, originally called mammary gland factor (MGF), was discovered as a regulator of the ß-casein gene promoter that binds to the ß-casein gene promoter GAS site (15) and was cloned from sheep tissue as MGF/Stat5 (16). Two highly related Stat5 genes, Stat5a and Stat5b, have been found in mouse and human DNA (17, 18, 19). The encoded proteins are greater than 95% identical, form heterodimers after phosphorylation and activation, and are both able to confer the PRL response to the ß-casein gene promoter (18).
Glucocorticoids, in concert with PRL and insulin, induce the
transcription of the ß-casein gene in vivo and in HC11
mammary epithelial cells in vitro (20, 21). Glucocorticoids
act through the glucocorticoid receptor (GR), a member of the nuclear
receptor family of ligand-inducible transcription factors. Binding of
glucocorticoid hormone leads to activation of the latent receptor,
dimerization, nuclear translocation, specific DNA-binding to
glucocorticoid-response elements, and transcriptional activation
(22, 23). GR also acts as a negative transcriptional regulator and
cross-couples to other transcription factors like AP-1 (Jun/Fos) or
nuclear factor (NF)-
B (24, 25). Nuclear receptors must interact with
additional factors such as SRC-1, TRIP-1, RIP-140/160, TIF-1, SMART,
and N-CoR to mediate both activation and repression of gene expression
(26, 27).
The p300/CBP (CAAT-binding protein) proteins represent a family of
transcription coactivators that potentiate the activity of several
groups of transcription factors by interaction with their activated
forms (28, 29). This was originally observed for the protein kinase
A-activated form of CREB (cAMP response element-binding protein)
(30, 31, 32) and the adenovirus E1A protein (33) and has been extended to
other transcription factors (e.g. ATF, c-Jun, c-Fos, c-Myb,
Sap-1, tax, Myo D, P/CAF, SRC-1, NF-B (28, 29). Most important for
the studies described here, p300/CBP has been shown to be a coactivator
of the GR and also of the interferon-- and interferon-
-induced
members of the Stat family, Stat1 and Stat2 (34, 35, 36, 37). The different
Stat proteins (Stat1, 2, 3, 4, 5a, 5b, 6) are mainly related due to
their similar mode of action by various cytokines (10). Their sequence
homologies and functional relatedness are rather limited. Knock-out
mice, in which Stat1, Stat5a, or Stat5b have been inactivated, indicate
absolutely specialized functions for the different members of the Stat
family (38, 39, 40, 41). Sequence comparison showed that the carboxyl-terminal
transactivation domains are the most dissimilar parts of the different
Stat molecules (42). Various Stat proteins might therefore contact
distinct coactivators or members of the transcription machinery for
transcriptional activation. p300 and CBP are similar in sequence and
functionally interchangeable in most cases. p300 exhibits histone
acetyltransferase activity and associates with a protein with the same
enzymatic activity (P/CAF) (43, 44, 45). Accumulation of histone
acetyltransferase at specific genomic sites has been implicated in the
induction of gene expression through the remodeling of chromatin
structure (46, 47). CBP also plays a role in the negative cross-talk
between the GR and AP-1. Nuclear receptors, including the GR, interact
with CBP and inhibition of GR-mediated AP-1 activity results from
competition for limiting amounts of p300/CBP (35, 36).
The analysis of the mechanism by which PRL and glucocorticoid hormones synergize in their transcriptional control has revealed a direct interaction between Stat5 and the GR (48, 49). This interaction results in an enhancement of Stat5-mediated transcriptional activation and an inhibition of GR-dependent transcription. We further investigated the molecular mechanisms of this transcription factor interaction and the potential role of p300/CBP. Our results show that p300 directly interacts with the transactivation domain of Stat5 and enhances PRL-induced transcriptional activation. This can be observed with Stat5, mStat5a, and mStat5b. Cytokine-activated, but not latent, Stat5 is able to interact with p300/CBP in vitro. The adenoviral p300-binding protein E1A inhibits Stat5- mediated transcriptional induction. The inhibition can be overcome by overexpression of p300. In contrast, Stat5 inhibition of glucocorticoid-mediated transactivation cannot be restored by overexpression of p300. These results indicate that repression of the glucocorticoid response by Stat5 is not the result of competition for limiting amounts of p300, but is most likely due to the direct complex formation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have previously shown that heterologous cell systems, supplied with
the genes encoding the PRL receptor, Stat5, and a ß-casein
promoter-luciferase construct, can be used to study PRL-induced
signaling and transcription, normally restricted to mammary epithelial
cells. To investigate whether the p300 coactivator is involved in the
transcriptional regulation exerted by Stat5, HeLa cells were
transfected with expression vectors for the PRL receptor, MGF/Stat5,
and the ß-casein gene promoter-luciferase construct as a reporter.
The cells were treated with PRL, and luciferase activities were
measured. PRL treatment of the cells caused an approximately 5-fold
stimulation of the reporter gene (Fig. 1A
, lanes 1 and 2). Cotransfection of
increasing amounts of p300 increased the PRL-dependent transactivation
(lanes 3 to 8). p300 specifically enhanced the Stat5-induced
transcription; basal activity of the ß-casein promoter was not
affected.
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and COS7 cells (data not shown). Stat5 induction resulted in about an
18-fold stimulation of the reporter gene in 293 cells (Fig. 1B, lanes 1
and 2). Increasing amounts of p300 further enhanced the PRL-induced
activity of the ß- casein reporter gene up to 120-fold (lanes
3–8).
Two closely related variants of Stat5 are encoded by the mouse genome
(mStat5a and mStat5b) (18). These proteins are greater than 95%
identical and differ mainly in their carboxyl-terminal transactivation
region. They can form homo- and heterodimers, and both variants are
able to confer the PRL response to the ß-casein gene promoter,
underlining their functional similarity (13, 18). MGF/Stat5, used in
the former experiments, is the sheep homolog of Stat5 and very similar
in sequence to mStat5a. To investigate whether p300 is involved in the
transcriptional regulation exerted by both variants of Stat5, HeLa
cells were transfected with ß-casein reporter plasmid and expression
vectors for the PRL receptor, Stat5a (Fig. 1C), and Stat5b (Fig. 1D).
Both Stat5 homologs stimulated the activity of the reporter gene about
6-fold (lanes 1 and 2). A p300 expression vector was included in the
transfection protocol (lanes 3 and 4). In the absence of PRL
stimulation of the cells, p300 expression did not affect basal
luciferase activity (lanes 3). Treatment with PRL caused a luciferase
induction. This induction was 2-fold higher in the p300-transfected
cells (lanes 4) when compared with the the controls (lanes 2). The
enhancement of PRL induction by p300 was observed for Stat5a as well as
for Stat5b.
E1A Inhibits PRL-Induced Transactivation by Stat5
The adenovirus E1A protein binds to p300 and CBP and inactivates
their function as coactivators of CREB-mediated transcription (50, 51).
The selective inhibition of p300 function allowed us to investigate
whether p300 is necessarily required for the PRL-induced
transactivation by Stat5. HeLa cells were transfected with expression
vectors for the PRL receptor, Stat5, and the ß-casein promoter
reporter construct. Treatment of the cells with PRL resulted in the
induction of luciferase activity (Fig. 2A, lanes 1 and 2). Inclusion of an
expression vector for E1A in the transfection scheme resulted in the
suppression of PRL induction (lanes 3 and 4). This suppression was
overcome by the augmentation of p300 expression. Enhanced expression of
p300 not only restored PRL-induced transactivation, but resulted in
higher induction levels than in the control cells (compare lanes 2 and
8).
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CR1 of E1A is
lacking the p300 and the Rb-binding functions; the mutant CR2 lacks
the Rb-binding function, but retains its p300-binding function (52).
The structure of these molecules is schematically shown in Fig. 2.
CR1 expression did not interfere with Stat5 induction of
transcription (Fig. 2B, lanes 5 and 6), and CR2 inhibited
PRL-induced transcription (lanes 7 and 8) to a similar extent as the
E1A wild-type molecule (lanes 1 and 2). A third variant of E1A, lacking
amino acids 64–68 and unable to bind p300 but still able to bind Rb,
was tested. This variant did not affect PRL inducibility of the
reporter construct (data not shown). This indicates that the
p300-binding site within the E1A molecule is required for suppression
of Stat5-mediated transcription.
The Carboxyl-Terminal Transactivation Domain of Stat5 Is Required
for E1A-Dependent Suppression of Induction
We have previously reported that the transactivation domain of
Stat5 is located in the very carboxyl-terminal region. This
transactivation domain is autonomously active when fused to a
heterologous DNA-binding domain. The strength of the transactivation
domain was found to be weaker than those of Stat6 and VP16 (53).
Exchange of the Stat5 transactivation domain with that of VP16 resulted
in a molecule that retained its cytokine regulation, but which had a
stronger transactivation potential (53, 54). We investigated whether
the transactivation domain of Stat5 participates in the inhibitory
effect of E1A on the transcriptional activation. A molecule lacking the
endogenous transactivation domain of Stat5, but comprising the VP16
transactivation domain (Stat5750VP16), was used for this
purpose.
PRL induction of cells transfected with Stat5750VP16
leads to a much stronger transcriptional activation of the
ß-casein reporter (Fig. 2C, lanes 1 and 2) compared with the
wild-type Stat5 (Fig. 2B, lanes 1 and 2). Coexpression of E1A with
Stat5750VP16 (Fig. 2C, lanes 3 and 4), CR1 (lanes 5 and 6), or
CR2 (lanes 7 and 8) did not interfere with the
transcriptional activation by Stat5750VP16. Even a 10-fold increase
of E1A vector in the cotransfection scheme did not cause inhibition
(data not shown). These experiments show that E1A inhibition of
transcriptional activation is dependent upon the presence of the Stat5
transactivation domain and can be circumvented by the fusion of a
truncated Stat5 to the VP16 transactivation domain. These experiments
indicate that p300 acts as a coactivator of Stat5 through the
transactivation domain.
p300 Interacts with the Carboxyl-Terminal Transactivation Domain of
Stat5
Coactivators are thought to be transcription factors that
stabilize complexes between regulated activator proteins and the basal
transcription factors through direct protein-protein interactions (26).
Since p300 acts as a Stat5 coactivator, we investigated their potential
interaction in coimmunoprecipitation experiments. COS7 cells were
transfected with expression vectors encoding PRL receptor, Stat5, and
p300. Transfected cells were treated with PRL, nuclear extracts were
prepared, and proteins were immunoprecipitated with p300-specific
antibodies. The immunoprecipitates were analyzed by Western blotting
and developed with an antiserum specific to the carboxyl terminus
(lanes 1–3) or an antiserum specific for a more amino-terminal region
of Stat5 (lanes 4–7). The band in Fig. 3, lane 2, indicates that the
p300-specific antiserum coimmunoprecipitates Stat5, most likely due to
a direct protein-protein interaction. When control antibodies, with
specificity for the yeast protein GAL4, were used in the
immunoprecipitation reaction, no Stat5 was detected (lane 3).
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indicate that the
carboxyl-terminal region of Stat5 is involved in the p300 interaction.
To corroborate this conclusion, we performed immunoprecipitations from
nuclear extracts of cells transfected with Stat5750, a variant in
which the carboxyl-terminal transactivation domain (amino acids
751–794) of Stat5 has been deleted (55). Only a very weak band can be
detected in Fig. 3 (lane 7), indicating that the carboxyl-terminal
transactivation domain of Stat5 is necessary for the interaction with
p300.
A second, independent method was employed to confirm this conclusion, a
mammalian two-hybrid assay. For this purpose we fused the GAL4
DNA-binding domain (amino acids 1–147) to the carboxyl-terminal
transactivation domain of Stat5 (amino acids 722–794) (55). This
fusion construct (GAL4-Stat5TA) was cotransfected with a
Galp3TK-Luciferase into COS7 cells. The reporter construct
is regulated by three palindromic GAL4-binding sites and a minimal
thymidine kinase (TK) promoter. A 4-fold induction of transcription was
observed (Fig. 4, lanes 1 and 2).
Addition of increasing amounts of a p300-VP16 expression vector,
encoding a p300 fused to the VP16 transactivation domain (56)
significantly enhanced the activity of the reporter gene (lanes 2- 5).
Transfection of p300-VP16 in the presence of a control vector, encoding
only the GAL4 DNA-binding domain (GAL4-DBD), had no affect on the
activity of the reporter construct (lanes 6–9). These results indicate
that the transactivation domain of Stat5 directly interacts with p300
and that the transactivation domain of Stat5 is necessary and
sufficient for this interaction.
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(lane 1, upper
panel). No binding of latent Stat5 was observed to the GST-CBP
fusion molecules (lanes 2–7, upper panel). To investigate
whether phosphorylation and dimerization of Stat5 is necessary for the
interaction with p300/CBP, binding experiments were also performed with
whole-cell extracts from cells treated with PRL and containing the
activated form of Stat5. The fusion protein comprising the region of
CBP between amino acids 451 and 721 was found to interact with the
activated form of Stat5 (lane 3, lower panel). This
indicates that dimerization of Stat5 is required for the interaction
with the distinct part of p300/CBP and is consistent with our
coimmunoprecipitation experiments shown in Fig. 3 in which activated
Stat5 was present.
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). In the absence of transfected GR, no
luciferase activity was detected, whether or not the cells were treated
with dexamethasone (lanes 1–3). A strong induction was observed when
the GR, but no Stat5, was transfected and the cells were induced with
dexamethasone (lanes 4 and 5) or dexamethasone plus PRL (lane 6).
Increasing amounts of Stat5 in the transfection scheme did not affect
dexamethasone inducibility of the MMTV-LTR as long as Stat5 was not
activated by PRL (lanes 8, 10, 12, 14, and 16). PRL addition to the
cells, i.e. Stat5 activation, suppressed the dexamethasone
inducibility of the MMTV-LTR in a Stat5 dose-dependent manner (lanes 9,
11, 13, 15, and 17). These results could be explained as the
consequence of competition of the GR and Stat5 for limiting amounts of
p300/CBP or by the interference of GR function through complex
formation with Stat5.
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Introduction of the GR into COS7 cells resulted in the strong
inducibility of a MMTV-LTR luciferase reporter gene by dexamethasone
(Fig. 7, lanes 1 and 2). Expression and
induction of Stat5 suppresses the MMTV-LTR luciferase induction (lanes
3 and 4). Addition of increasing amounts of p300 enhanced the
glucocorticoid-dependent transactivation in a dose- dependent
manner (lanes 5, 7, and 9). This indicates that endogenous p300
expression is limiting the extent of GR induction. The suppression of
dexamethasone induction of the MMTV-LTR by the activated Stat5 was not
relieved, however, by the increase in p300. Although the absolute
values of luciferase activity increased slightly in the presence of 5
µg and 10 µg of p300 vector in the transfection, the repression of
induction by activated Stat5 persisted (lanes 4, 6, 8, and 10). We
conclude that repression of GR-mediated induction by activated Stat 5
does not result from competition for limiting amounts of p300, a
mechanism suggested in the functional interaction between nuclear
receptors and AP-1 (35, 36). We propose that repression of GR function
by Stat5 is a consequence of their complex formation.
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, lanes 6 and 7). Activation of Stat5
through tyrosine phosphorylation is a necessary prerequisite for the
inhibitory effect. We also investigated whether the transactivation
domain of Stat5 participates in the inhibitory effect. A mutant lacking
the carboxyl-terminal transactivation domain, Stat5750, inhibited
the glucocorticoid-induced transactivation of the MMTV-LTR reporter
plasmid as well as wild-type Stat5 (Fig. 8; compare lanes 4 and 5 with
8 and 9). This result supports our finding that interaction of Stat5
with p300 is not involved in the repression process.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A direct connection between the glucocorticoid- and PRL-induced signal
transduction pathways was recently found that involves complex
formation between the GR and Stat5 (48, 58, 59). This interaction
enhances PRL-induced Stat5-activated transcription, but suppresses
glucocorticoid-responsive transcriptional activation. We have further
investigated this interaction and examined the role of the coactivator
p300/CBP in the induction process. Members of the p300/CBP protein
family were originally detected through their ability to bind to E1A
and CREB (31, 33) and subsequently identified as coactivators for
nuclear hormone receptors (35, 36). p300/CBP act as integrators for
several signal transduction pathways (28, 29), e.g. they may
mediate the cross-talk between nuclear receptors and AP-1 (35, 36) and
connect IFN- and Ras/AP-1 signaling (60).
We found that p300 enhances PRL-induced transcriptional acitvation by Stat5a and Stat5b. These two variants of the Stat5 protein are encoded by highly related genes and show a sequence identity of greater than 95% at the amino acid level. The most pronounced sequence diversity is located in their carboxyl- terminal transactivation region (18, 55), the region required for p300-mediated coactivation. Both proteins can form homo- and heterodimers and are able to confer the PRL response to the ß-casein gene promoter, i.e. they are functionally very similar (13, 18). This is supported by our finding that Stat5a and Stat5b, despite their sequence divergence in the carboxyl-terminal region, can cooperate with p300.
The involvement of p300 in Stat5-induced transcription was confirmed in experiments in which we observed that E1A inhibits Stat5-induced transcriptional activation. E1A is thought to bind directly to p300 and thereby prevents it from functioning as a coactivator of other transcription factors (61), but E1A has also been shown to be able to interact with other crucial cellular regulators such as the transcriptional repressor Dr1, YY1, the general transcription factor TBP, and Rb (62, 63, 64, 65). It also participates in the control of transcription, DNA synthesis, and cell cycle regulation and differentiation. Mutational analysis of the E1A protein has shown that some activities of E1A are distinct from others and are carried out independently by different regions of the protein (66). Our observations that E1A-mediated inhibition of Stat5-induced transcriptional activation can be overcome by overexpression of p300 and that inhibition by E1A requires the presence of an intact p300-binding site of E1A indicate that the effect of E1A on Stat5 action is based on its physical interaction with p300.
293 cells are adenovirus transformed and express endogenous E1A. It
might have been anticipated that E1A expression would not allow
Stat5-mediated transactivation. In fact, we observed an 18-fold
induction of the ß-casein reporter gene. We conclude that the
residual, functional amounts of p300 are sufficient for transcriptional
activation by Stat5 (Fig. 1B, lanes 1 and 2). Increasing amounts of
p300 further enhanced the PRL-induced activity of the ß-casein
reporter gene up to 120 fold (lanes 3–8). The higher induction in 293
cells as compared with HeLa cells might result from a modification of
p300 by E1A. An E1A-induced phosphorylation of p300 has been shown to
influence the interaction with transcription factors (DRF) and enhance
the induction of responsive promoters (67).
Our data might have interesting implications for the pathology of adenovirus infections. If E1A expression, upon viral infection, interferes with Stat5-induced transcription, processes dependent upon crucial cytokines and growth factors, such as interleukin (IL)-2, IL-3, IL-5, IL-7, IL-9, IL-10, IL-15, PRL, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), GH, TPO, and epidermal growth factor (EGF) might be affected. These cytokines have been shown to exert at least part of their signaling potential through the activation of Stat5 (17, 19, 68, 69, 70, 71, 72, 73, 74, 75, 76).
The inhibition of Stat5-induced transcription by E1A requires the Stat5
transactivation domain. Coimmunoprecipitation experiments and mammalian
two-hybrid assays revealed a direct interaction between p300 and Stat5.
Interactions between p300 and two other members of the STAT family,
Stat1 and Stat2, have recently been described, and the
carboxyl-terminal regions of Stat1 and Stat2 have been found to be
involved in those interactions (34, 37). In Stat1 there is an
additional contact region in the amino terminus (37). The
carboxyl-terminal regions of Stat1 and Stat2 both function as
transactivation domains, but are distinct in character. The carboxyl
terminus of Stat2, a highly acidic region, does not undergo serine
phosphorylation. The carboxyl- terminal transactivation domain of Stat5
could form an amphipathic -helix with clusters of acidic residues on
one face and hydrophobic residues on the other (55). Despite these
differences, p300 acts as a coactivator for all three Stat factors.
Our observation that p300 is a coactivator of Stat5 reveals at least three interesting protein-protein interactions in the lactogenic hormone scheme: p300 interacts with Stat5 and the GR; Stat5 and the GR interact with each other. Since the GR enhances Stat5 action at the level of the ß-casein gene promoter and Stat5 antagonizes the GR at the level of the MMTV-LTR, we analyzed the effect of p300 on Stat5-mediated repression on glucocorticoid response. p300 enhances the GR-dependent transcriptional activation of the MMTV-LTR in the absence of activated Stat5, but does not counteract Stat5-mediated repression of induction. Analysis of Stat5 variants in transfection assays indicate that the activation of Stat5 through tyrosine phosphorylation is required for the repression of Stat5 on GR function. However, the transactivation domain of Stat5, which is also the domain responsible for the interaction with p300/CBP, is not required. Competition for p300 has been invoked in the interaction between Stat1 and AP-1 (60) and is probably also responsible for the E1A inhibition of Stat5 transactivation described here. Repression of GR transactivation by Stat 5, however, is not the consequence of competition for limiting amounts of p300, but most likely results from the complex formation between the two transcription factors.
Complex formation between Stat5 and the GR has been observed in COS7
cells overexpressing GR and Stat5 (48) but also in HC11 mammary
epithelial cells expressing physiological levels of GR and Stat5 (59).
Decreased binding of the GR to glucocorticoid- response elements in
the presence of activated Stat5 might be a possible mechanism for
repression. This, however, could not be observed in in vitro
bandshift experiments with extracts from transfected cells (data not
shown). A further possible mechanism for the Stat5-mediated inhibition
of the GR transactivation function is that a complex of Stat5 and GR
might cause sterical hindrance and interfere with contacts between the
GR and coactivators or the basal transcription machinery. It will be of
interest to investigate whether complex formation between GR and Stat5
also influences the transrepression function of the GR on AP-1 and
NF-B-responsive genes. p300/CBP seems to be an important integrator
in these different signaling pathways involved in inflammation reaction
and immune response. The observed negative cross-talk of Stat5 on GR
function might also have implications for T lymphocyte development and
differentiation as well as in erythropoiesis. GR is thought to control
thymocyte apoptosis and proliferation of erythroblasts (77).
GR activation has a synergistic effect on the transcription of the ß-casein gene promoter activated by Stat5 (48). When p300 was added, a further increase in transcription, above the GR-mediated effect, was observed (not shown). These results indicate that the GR/Stat5 complex recruits high levels of p300. The ß-casein reporter construct used in our experiments contains, in addition to the Stat5-binding sites, several binding sites for regulator proteins such as C/EBP and YY1 (15). C/EBPß has been shown to interact and collaborate with p300, and relief of YY1 transcriptional repression has been described to be mediated by p300 (78, 79). This indicates that there are several contact sites for p300 in the ß-casein promoter and that p300 might coordinate the cooperation between different regulatory factors that bind to the ß-casein promoter. The recruitment of this coactivator and its associated histone acetyl transferase activity may result in a chromatin configuration that allows the efficient assembly and enhanced stabilization of the preinitiation complex, and its local concentration may eventually determine the extent of transcriptional induction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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750
(pXM-MGF750), the point mutant Stat5 Y694F (pXM-MGF Y694F), and the
long form of the PRL receptor (pcDNAI-PrlR) were described previously
(13, 16, 55). To construct Stat5750VP16, the transactivation domain
of VP16 (amino acids 411–489) was amplified by PCR and fused in frame
to the 3'-end of pXM-MGF750 (53). The human GR expression vector
pRSVhGRa (GR) (80), the MMTV-LTR-luciferase reporter (81), and the
expression vectors for pCMV (cytomegalovirus promoter
enhancer)-p300 and p300-VP16 have been described (33, 56). The
same is true for the RSV LTR (Rous sarcoma virus long terminal
repeat)-E1A 12S constructs and the mutants E1ACR1 and E1ACR2
(52), and the reporter plasmid Galp3TK-luciferase, which
contains three copies of the UASG cloned upstream of the TK
promoter (82). The plasmids encoding the GAL4-Stat5TA fusion comprise
the DNA-binding and dimerization domains of yeast GAL4 protein (amino
acids 1–147) and the carboxyl-terminal transactivation domain of Stat5
(amino acids 722–794). GAL4-DBD and GAL4-Stat5TA have been described
previously (55).
Cell Culture and Transfection
HeLa cells, 293 cells, and COS7 cells were maintained in DMEM
containing 10% FCS, 2 mM glutamine, and 50 µg/ml
gentamycin. Transient transfection experiments were performed using the
calcium phosphate precipitation technique (13). Half-confluent cells in
10-cm dishes were cotransfected with the ß-casein luciferase reporter
constructs (2 µg) and expression vectors for Stat5 (2 µg) or its
variants, the PRL receptor (250 ng), and p300 or E1A expression
plasmids, as indicated in the figure legends. To monitor the
GR-dependent transcriptional activation, the MMTV-LTR-luciferase
reporter construct (4 µg) was cotransfected with an expression vector
encoding the GR (2 µg), the PRL receptor (250 ng), and the indicated
amounts of Stat5 and p300. Plasmid (0.5 µg) encoding the
ß-galactosidase gene driven by the CMV promoter was included to
monitor transfection efficiency. The DNA was adjusted to 10 µg with
empty vector-DNA. One day after transfection, the cells were treated
with 5 µg/ml ovine PRL and/or 10-7 M
dexamethasone for 16 h before harvesting.
Luciferase and ß-Galactosidase Assays
Two days after transfection cells were harvested, washed twice
in cold PBS, and lysed in 25 mM glycylglycine, pH 7.8, 1
mM dithiothreitol (DTT), 15 mM
MgSO4, 4 mM EGTA, and 1% Triton for 10 min at
4 C. Samples were centrifuged for 5 min at 14,000 rpm. Cleared
supernatants were used for luciferase and ß-galactosidase assays. For
ß-galactosidase determination, 20 µl of extracts were added to 200
µl of reaction buffer containing 100 mM Na-phosphate, pH
8.0, 1 mM MgCl2, and 1 x Galacton
(Tropix, Bedford, MA) and incubated for 30 min at room temperature.
Measurements were made by injecting 300 µl of accelerator solution
(10% Emerald luminescent amplifier and 0.2 N NaOH), and the samples
were counted for 20 sec in the Luminometer 953 (Berthold, Pforzheim,
Germany). Luciferase activities (100 µl extracts and 300 µl
accelerator solution) were quantified in the same apparatus and
normalized to the ß-galactosidase activities. At least three
independent experiments were performed.
Coimmunoprecipitation Assay
COS7 cells were transfected with expression plasmids encoding
Stat5 or Stat5750, p300, and the PRL receptor. One day after
transfection, the cells were treated with 5 µg/ml ovine PRL for
1 h before harvesting. Nuclear extracts from the induced cells
were prepared. Protein (200 µg) was incubated with 4 µg of
p300-specific monoclonal antibodies (Upstate Biotechnology, Lake
Placid, NY) or as a control, with antibodies against the GAL4
DNA-binding domain (Santa Cruz Biotechnology, Santa Cruz) overnight at
4 C at constant agitation. Protein A/G-Sepharose-coupled beads
(Pierce, Rockford, IL) were added for 1 h. The beads were
pelleted and washed five times with incubation buffer (20
mM HEPES,pH 7.9, 100 mM NaCl, 10 mM
KCl, 0.1 mM NaVO4, 1 mM EDTA, 1
mM DTT). The immunoprecipitates were separated by SDS-PAGE,
and the Western blots were developed with antiserum specific against
the carboxyl terminus of Stat5a ( Stat5C) or an antiserum against
amino acids 661–677 of Stat5a ( Stat5N).
Mammalian Two-Hybrid Assay
Half-confluent HeLa cells in 10-cm dishes were cotransfected
with the reporter plasmid Galp3TK-luciferase (2.5 µg) and
expression vectors for GAL4-Stat5TA (1 µg), the GAL4-DBD (1 µg),
and p300-VP16. Plasmid (0.5 µg) encoding the ß-galactosidase gene
driven by the CMV promoter was included to monitor transfection
efficiency. Luciferase assays were performed as described.
GST-Fusion Protein Interaction Assay and Western Blot
Analysis
The expression vectors for the GST-CBP fusion proteins have been
described previously (83). GST-fusion proteins were purified from
bacterial extracts using glutathione-Sepharose beads as specified by
the manufacturer (Pharmacia, Freiburg, Germany). Whole-cell extracts
from HeLa cells stably expressing the PRL receptor and Stat5a were
prepared as described previously (13). The cells were either untreated,
to obtain the latent form of Stat5a, or treated with 5 µg/ml ovine
PRL for 1 h before harvesting, to obtain the activated form of
Stat5a. For binding assays, 150 µg protein from whole-cell extracts
were incubated with 10–20 µg of GST-fusion proteins bound on beads
at 4 C for 1 h under constant agitation in 500 µl 50
mM Tris-HCl (pH 8), 100 mM NaCl, 0,3
mM DTT, 10 mM MgCl2, 10% glycerol
and 0,1% Nonidet P-40 (NP40). The protein complexes were washed five
times in binding buffer. The bound proteins were eluted from the beads
with SDS sample buffer and analyzed by SDS-PAGE. Western blot analyses
were done using the antiserum specific against the carboxyl terminus of
Stat5a.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 30, 1998. Revision received June 4, 1998. Accepted for publication July 2, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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