Extracellular Signal-Regulated Kinase (ERK) Interacts with Signal Transducer and Activator of Transcription (STAT) 5a
Tony J. Pircher,
Hanne Petersen,
Jan-
ke Gustafsson and
Lars-Arne Haldosén
Department of Medical Nutrition Karolinska Institute, Novum
S-141 86 Huddinge, Sweden
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ABSTRACT
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Serine phosphorylation of signal transducers and
activators of transcription (STAT) 1 and 3 modulates their DNA-binding
capacity and/or transcriptional activity. Earlier we suggested that
STAT5a functional capacity could be influenced by the mitogen-activated
protein kinase (MAPK) pathway. In the present study, we have analyzed
the interactions between STAT5a and the MAPKs, extracellular
signal-regulated kinases ERK1 and ERK2. GH treatment of Chinese hamster
ovary cells stably transfected with the GH receptor (CHOA cells) led to
rapid and transient activation of both STAT5a and ERK1 and ERK2.
Pretreatment of cells with colchicine, which inhibits tubulin
polymerization, did not inhibit STAT5a translocation to the nucleus and
ERK1/2 activation. In vitro precipitation with a
glutathione-S-transferase-fusion protein containing
the C-terminal transactivation domain of STAT5a showed GH-regulated
association of ERK1/2 with the fusion protein, while this was not seen
when serine 780 in STAT5a was changed to alanine. In vitro
phosphorylation of the glutathione-S-transferase-fusion
proteins using active ERK only worked when the fusion protein contained
wild-type STAT5a sequence. The same experiment, performed with
full-length wild-type STAT5a and the corresponding S780A mutant, showed
that serine 780 is the only substrate in full-length STAT5a for active
ERK. In coimmunoprecipitation experiments, larger amounts of
STAT5a-ERK1/2 complexes were detected in cytosol from untreated
CHOA cells than in cytosol from GH-treated cells, suggesting the
presence of preformed STAT5a-ERK1/2 complexes in unstimulated cells.
Transfection experiments with COS cells showed that kinase-inactive
ERK1 decreased GH stimulation of STAT5-regulated reporter gene
expression. These observations show, for the first time, direct
physical interaction between ERK and STAT5a and also clearly identify
serine 780 as a target for ERK. Furthermore, it is also established
that serine phosphorylation of STAT5a transactivation domain, via the
MAPK pathway, is a means of modifying GH-induced transcriptional
activation.
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INTRODUCTION
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Signal transducers and activators of transcription (STAT) proteins
are a family of latent cytoplasmic transcriptional regulators (1, 2).
Ligand binding to cytokine receptors (including receptors for GH,
several interleukins, PRL, and erythropoietin) and some tyrosine kinase
receptors (including epidermal growth factor receptor and
platelet-derived growth factor receptor) leads to activation of STAT
proteins through tyrosine phosphorylation. In the case of cytokine
receptors, tyrosine phosphorylation is mediated by receptor-bound
members of the Janus kinase (JAK) family (3, 4, 5). Tyrosine
phosphorylation of STAT proteins is obligatory for dimerization,
translocation to the nucleus, and regulation of specific genes.
The importance of serine phosphorylation of STAT proteins has recently
been acknowledged. Studies have mainly focused on serine 727 in
STAT1
and STAT3, which is situated in a mitogen-activated protein
kinase (MAPK) consensus recognition sequence, PMSP (6, 7, 8, 9, 10).
Phosphorylation of serine 727 has been shown to regulate DNA binding
capacity and/or transcriptional activity of these STAT proteins.
Furthermore, treatment of the human fibroblast cell line (U266) with
interferon-ß (IFN-ß)-induced association of STAT1
with ERK2
(11).
We have shown earlier that GH activates JAK2/STAT5 and MAPK pathways
(12, 13). We have also shown that GH activates both isoforms of STAT5,
STAT5a and STAT5b (13). These isoforms differ mainly at the C-terminal
end, one of the differences being the presence of a possible ERK
phosphorylation site (RLSP) in STAT5a (14). Recently, we have examined
the influence of MAPK signaling on the JAK2/STAT5 pathway. Pretreatment
of GH-responsive cells with the MAPK kinase (MEK) inhibitor PD98059
resulted in a decreased amount of GH-induced nuclear STAT5 with
DNA-binding capacity and reduced transcriptional activation of
STAT5-regulated reporter gene (13), whereas GH-stimulated nuclear
translocation of STAT5 was not influenced. Data indicated that STAT5a
is dependent on MAPK activity for full transcriptional activity and
that serine 780 of STAT5a within the putative MAPK recognition sequence
is a likely target for MAPK.
In this study we have further investigated GH activation of the MAPK
pathway, as well as the interaction between this pathway and the
JAK2/STAT5 system. In particular, we have obtained firm evidence for
protein-protein interaction between STAT5a and ERK, established that
serine 780 of STAT5a is a target for ERK, and also directly correlated
ERK activity with STAT5a functional capacity.
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RESULTS
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Kinetics of ERK and STAT5a Activation after GH Stimulation
Data from our previous study indicated that MAPK could influence
STAT5a functional capacity (13). In our present study we have
investigated whether ERK1/2 and STAT5a follow the same activation
kinetics after GH stimulation and whether GH activates both ERK1 and
ERK2. CHOA cells were incubated with GH for different times, and
cytosol and nuclear extracts were analyzed using Western blotting with
antibodies against ERK1/2, active ERK1/2, and STAT5a. The antibody
against active ERK1/2 only recognizes ERK1/2 where threonine 183 and
tyrosine 185 are phosphorylated. Phosphorylation of these two amino
acids by MEK results in activation of ERK1/2 (15, 16, 17). In cytosolic
extract, active ERK1 and 2 were detected after 4 min of GH stimulation
(Fig. 1A
, middle panel). The
highest levels of active ERK1/2 were seen after 8 min of stimulation,
after which the active forms slowly disappeared up until 20 min of GH
stimulation. GH induced higher levels of active ERK2 than ERK1. The
antiactive ERK antibody has been reported to have the same affinity for
active forms of ERK1 and ERK2 (18). No significant reduction of STAT5a
or ERK1/2 protein levels in cytosol (as a result of nuclear
translocation) was detected upon GH stimulation (Fig. 1A
, upper
and lower panel), indicating that GH activated a minor pool of
ERK1/2 and STAT5a. Analysis of nuclear extracts showed that both ERK1
and 2 were present in the nucleus in the absence of GH stimulation,
while STAT5a translocated to the nucleus after GH stimulation (Fig. 1B
, upper and lower panel). Active forms of ERK1/2 were clearly
detected after 4 min of GH stimulation (Fig. 1B
, middle
panel). The highest levels of active ERK1/2 in nuclear extracts
were seen after 812 min. The highest level of nuclear STAT5a protein
was seen after 48 min, after which a slow decrease occurred, a
pattern that resembled that of the DNA-binding capacity of nuclear
STAT5, as analyzed by gel shift technique (Fig. 1C
). From the above
data it can be concluded that GH activated both ERK forms and that ERK1
and 2 and STAT5a had similar activation kinetics after GH stimulation
of CHOA cells.

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Figure 1. Activation of ERK1, ERK2, and STAT5a by GH in CHOA
Cells
CHOA cells, grown in serum-free medium, were treated with or without
100 nM GH for indicated times. Cytosolic extract (panel A)
and nuclear extracts (B) were analyzed by Western blot technique using
polyclonal antibodies against STAT5a, ERK1/2, and active MAPK. C,
Nuclear extracts were analyzed with a gel shift method using a
STAT5-binding oligonucleotide as a probe.
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Activation of ERK and Nuclear Translocation of STAT5a after
Depolymerization of Microtubuli
MAPKs were initially described as microtubuli-associated proteins
(19, 20). Later investigations showed that MAPKs are also present in
cytosolic compartments not associated with microtubuli and also in the
nucleus (21, 22). We have investigated whether intact microtubuli are
required for GH activation of ERK1/2 in CHOA cells (Fig. 2
). Furthermore, we have analyzed whether
translocation of STAT5a from cytosol to nucleus is dependent on intact
microtubuli. CHOA cells were pretreated with or without 10
µM colchicine before treatment with GH. This
concentration of colchicine has been shown to effectively depolymerize
microtubuli in CHOA cells (E. L. K. Goh, Pircher, T.J. and
Lobie, P.E., manuscript submitted). Cytosolic and nuclear extracts from
treated cells were analyzed by Western blot using antibodies against
active ERK and STAT5a. Analysis of nuclear extract indicated that
depolymerization of microtubuli did not inhibit GH-induced activation
of ERK1/2 and also did not inhibit nuclear translocation of STAT5a.
Analysis of cytosolic extract showed no decrease in ERK1/2 activation
by GH after treatment with colchicine (data not shown). Thus, intact
microtubuli were not required for GH-induced activation of ERK1/2 and
GH-induced nuclear translocation of STAT5a.

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Figure 2. Depolymerization of Microtubuli with Colchicine
CHOA cells, grown in serum-free medium, were pretreated for 60 min with
or without 10 µM colchicine before GH treatment for
different times. Nuclear extracts were analyzed with Western blot
technique using polyclonal antibodies against STAT5a and active MAPK.
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Influence of ERK on STAT5a Functional Capacity
We showed earlier that inhibition of MEK with PD98059 resulted in
decreased GH-induced activation of STAT5-regulated reporter gene
expression and proposed that decreased MAPK activity was responsible
for this effect of PD98059 (13). In the present study we have analyzed
the direct influence of ERK activity on STAT5-regulated transcription.
COS cells were transfected with expression vectors for GH receptor,
STAT5a, and wild-type or kinase mutant ERK1. In kinase mutant
ERK1-transfected cells, GH-induced reporter gene expression was
significantly reduced as compared with COS cells transfected with
wild-type ERK1 (Fig. 3
). Thus, STAT5a
functional capacity was influenced by active ERK1.

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Figure 3. ERK-Dependent Expression of STAT5-Regulated
Reporter Gene
COS cells were transfected with the STAT5-regulated reporter construct,
SPI-GLE1-luc, and expression vectors for GHR, STAT5a, wild-type ERK1
(ERK-wt), or kinase inactive ERK1 (ERK-kin-) or control (pcDNA) and
ß-galactosidase. After 12 h treatment with 100 nM
hGH, cells were harvested and extracts were assayed for luciferase and
ß-galactosidase activities. Values shown represent normalized
luciferase activities in arbitrary units. Results represent the
mean ± SD of triplicate estimations. Results
presented are representative of three experiments.
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GH-Regulated Interaction between ERK and C-Terminal Part of
STAT5a
We have previously suggested that serine 780, within the
C-terminal part of STAT5a, is a MAPK substrate (13). To further
strengthen this notion, we produced
glutathione-S-transferase (GST)-fusion proteins either
containing wild-type sequence of the C-terminal 64 amino acids of
STAT5a or the same 64 amino acids but with serine 780 mutated to
alanine (Fig. 4A
). Figure 4B
shows a
Coomassie-stained SDS-PAGE gel with purified GST-fusion proteins. For
both wild-type and serine-mutated GST-fusion constructs, two main bands
could be seen. These proteins were also detected by Western blot
analysis with anti-STAT5a antibody directed against the last 18
C-terminal amino acids of STAT5a (data not shown). GST-fusion proteins
were used for precipitation and in vitro phosphorylation
assays. CHOA cells were treated with or without GH for 10 min.
Cytosolic extracts from these cells were incubated with GST and
wild-type or serine mutant C-terminal STAT5a GST fusion proteins.
Precipitates were separated on SDS-PAGE and analyzed by Western blot
with anti-ERK1/2 antibody (Fig. 4C
). GST fusion proteins and, to a
smaller extent, GST precipitated ERK1 in extracts from stimulated or
unstimulated cells. ERK2 coprecipitated with wild-type and, to a
smaller extent, with mutant GST fusion protein in extracts from
stimulated or unstimulated cells. GH treatment of CHOA cells induced an
increase in association of ERK1 and 2 to wild-type GST fusion protein,
an effect that could not be seen with serine mutant GST fusion protein
or GST. GST and GST fusion proteins were also incubated with
commercially available active ERK and radiolabeled ATP. Incubations
were analyzed using SDS-PAGE/autoradiography. As can be seen in Fig. 4D
, only GST fusion protein containing wild-type C-terminal sequence of
STAT5a was phosphorylated. These data suggest a GH-regulated
interaction of ERK1/2 with the C-terminal part of STAT5a and that
serine 780, within that part, is a target for ERK.

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Figure 4. In Vitro Interaction between
GST-STAT5a and Recombinant Active MAPK
A, C-terminal STAT5a GST-fusion proteins constructed. B, Coomassie
staining of SDS-PAGE gel loaded with purified GST (C) and STAT5a
GST-fusion proteins (WT and SA). Two different amounts of GST-fusion
proteins were loaded. C, Cytosolic extract from CHOA cells, treated
with and without 100 nM GH, was precipitated with either 4
µg of GST (C), wild-type (WT), or S780A (SA) C-terminal STAT5a fusion
proteins. Precipitations were analyzed by Western blot using
anti-ERK1/2 antibody. D, The GST-fusion constructs were also incubated
in vitro with active MAPK and ATP(32P) for
30 min at 30 C. Phosphorylated entities were detected by
SDS-PAGE/autoradiography.
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ERK Phosphorylation of Recombinant Full-Length STAT5a in
Vitro
Analysis of the amino acid sequence of STAT5a showed that more
possible serine/threonine phosphorylation sites exist in addition to
the C-terminal MAPK phosphorylation site. To investigate whether these
sites are targets for ERK and also to determine whether MAPK can
phosphorylate serine 780 in the full-length protein, we studied ERK
phosphorylation of full-length STAT5a in vitro. We expressed
recombinant His-tagged wild-type STAT5a and serine 780 alanine mutant
of STAT5a (STAT5aS780A) in insect cells using the baculovirus system
(Fig. 5A
). Western blot analysis of
extracts from infected cells with anti-STAT5a antibody confirmed
the expression of recombinant proteins (Fig. 5B
). We have previously
shown that recombinant MGF (STAT5) is tyrosine phosphor-ylated
in insect cells by an unknown endogenous tyrosine kinase and that this
MGF has DNA-binding capacity with the proper binding specificity (23).
We analyzed DNA-binding capacity and specifity for recombinant
wild-type and mutant STAT5a. Figure 5D
shows the result of gel shift
analysis of recombinant STAT5aS780A in which an oligonucleotide
containing the STAT5 binding site from ß-casein promoter was used as
probe. The recombinant mutant STAT5a had DNA-binding capacity, which
was lost when insect cells were treated with the protein kinase
inhibitor staurosporine (data not shown). The recombinant serine mutant
STAT5a also exhibited the same DNA binding specificity as earlier
determined for recombinant MGF and STAT5 from CHO cells (23, 24).
Identical results were obtained when recombinant wild-type STAT5a was
analyzed in the same way (data not shown). This indicates that both
recombinant STAT5a forms are expressed properly folded. Full-length
STAT5a and STAT5aS780A were incubated together with active ERK and
radiolabeled ATP, after which the incubations were analyzed by
SDS-PAGE/autoradiography. Figure 5C
shows that only recombinant
wild-type STAT5a was phosphorylated. We have also used wild-type STAT5a
and serine mutant STAT5a, expressed in COS cells, in in
vitro phosphorylation experiments. These experiments also showed
that only wild-type STAT5a was phosphorylated (data not shown). Taken
together, these experiments show that ERK phosphorylated full-length
STAT5a and that serine 780 was the only MAPK phosphorylation site in
STAT5a under these in vitro conditions.

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Figure 5. In Vitro Phosphorylation of
Full-Length Recombinant STAT5a with Active MAPK
A, Recombinant STAT5a proteins. B, Western blot of extracts from
Sf9-insect cells infected with recombinant baculovirus showing
expression of wild-type STAT5a (WT) and STAT5aS780A (SA). Extract from
noninfected cells was used as control (C). C, In vitro
phosphorylation of recombinant baculovirus- expressed proteins with
active MAPK. D, Effect of competition with GAS-like oligonucleotides on
binding of recombinant STAT5aS780A to ß-casein probe. Excesses of
various GAS-like oligonucleotides were used as competitors. See
Materials and Methods for key.
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Coprecipitation of STAT5a and ERK
To show interaction between ERKs and STAT5a in cells with
endogenous levels of these proteins, we treated CHOA with GH for 8 min
and prepared cytosolic extracts. Equal amounts of polyclonal
anti-ERK1/2 or anti-ERK2 antibody were added to extracts for
precipitation. Precipitates were analyzed by Western blot with
antibodies against STAT5a or ERK1/2. The anti-ERK1/2 antibody
precipitated ERK1 and ERK2, with ERK1 being precipitated better than
ERK2 (Fig. 6A
). It was only upon longer
exposure that weak STAT5a coimmunoprecipitation could be detected in
extracts from treated or untreated CHOA cells (data not shown). We have
tested two different anti-ERK1/2 antibodies from different
manufacturers with the same results. Both of these antibodies are
directed against the C-terminal part of ERK, amino acids 339353 and
333367, respectively. The anti-ERK2 antibody precipitated, in our
hands, repetitively both ERK1 and ERK2. This antibody is raised against
full-length murine ERK2. With this antibody STAT5a coimmunoprecipitated
in extracts from both treated and untreated cells. A considerably lower
amount of STAT5a was coimmunoprecipitated in extract from treated
cells, indicating that a complex between STAT5a and ERK1 and/or ERK2
was present in unstimulated CHOA cells that started to dissociate upon
GH stimulation. This finding was further investigated by transfection
of COS cells with expression vector for GH receptor and different
combinations of expression vectors for wild-type or serine 780 mutant
of STAT5a and hemagglutinin (HA)-tagged wild-type or kinase mutant of
ERK1. COS cells were then treated with GH for 8 min. Cytosolic extract
was prepared, and anti-HA antibody was added for immunoprecipitation.
Precipitates were analyzed by Western blot with anti-STAT5a antibody.
In Fig. 6B
it can be seen that wild-type and serine 780 mutant of
STAT5a coimmunoprecipitated with both wild-type and kinase mutant ERK1.
The amount of wild-type STAT5a coimmunoprecipitated in extracts from
COS cells transfected with wild-type STAT5a and wild-type ERK1 was less
than in the other extracts. Thus, both STAT5a forms were complexed with
both ERK1 forms, but dissociation of complex upon GH stimulation only
occurred in COS cells transfected with wild-type STAT5a and wild-type
ERK1.

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Figure 6. GH Modulated Co-Precipitation of STAT5a with ERK2
from CHOA Cells
A, CHOA cells, serum deprived over night, were treated with and without
100 nM GH. Cytosolic extracts were incubated with
polyclonal antibody against ERK or ERK2. Antibody-bound material was
precipitated with protein A-Sepharose and analyzed by Western blotting
with antibodies against STAT5 and ERK1/2. B, COS cells were transfected
with the expression vectors for GH receptor and STAT5a or STAT5aS780A
and HA-tagged wild-type ERK1 (HA-ERK-wt) or HA-tagged kinase inactive
ERK1 (HA-ERK-kin-). After 10 min of GH stimulation, cytosolic extracts
were prepared. Anti-HA antibody was added to extracts, after which
bound material was precipitated with protein A-Sepharose and analyzed
by Western blotting using antibodies against STAT5a.
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DISCUSSION
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In our previous study we showed that inhibition of MEK with
PD98059 before treatment of cells with GH resulted in inhibition of
GH-induced MAPK activation, reduction of GH-induced STAT5 in nuclear
extract with DNA binding capacity, and decreased GH-induced activation
of a STAT5-regulated reporter construct (13). Experiments in that study
also indicated that STAT5a, which has a MAPK recognition sequence
within its C-terminal domain, could be a target for MAPK. Since this is
an important issue for the understanding of GH signaling, we have
specifically addressed this notion using several experimental
approaches.
STAT5a and ERK1/2 coimmunoprecipitated from extracts of CHOA cells,
indicating that STAT5a and ERK1/2 can complex in these cells. An
unexpected finding was that a lower amount of STAT5a-ERK1/2 complex was
found in extract from GH-stimulated cells, which indicates that this
complex is preformed before and dissociates after GH stimulation of
CHOA cells. In U266 cells the reverse finding has been reported (11).
IFNß stimulation of these cells activated MAPK and caused MAPK to
complex with STAT1a. With COS cells we showed that wild-type and serine
780 mutant of STAT5a complex with ERK1, either as a wild-type or kinase
mutant form. It was only with the combination of wild-type STAT5a
(containing serine 780) and wild-type ERK1 (active kinase) that less
complex was seen in these GH-stimulated cells. This experiment
indicates that complex formation neither requires the presence of
serine 780 in full-length STAT5a nor an activated ERK. It is likely
that the interacting regions of the two proteins are the C-terminal
part of STAT5a, with its ERK recognition sequence, and the kinase
region of ERK. It could be that other regions of the two proteins are
also involved.
It has earlier been shown that the catalytic and substrate recognition
domains of ERK1 are situated in the N-terminal and C-terminal parts of
ERK1, respectively (25). When we used two different anti-ERK1/2
antibodies, raised against amino acids 333367 and 339353 of the
C-terminal part of ERK, for coimmunoprecipitation of ERK and STAT5a,
only weakly detectable signals of coprecipitated STAT5a were found.
This was not the case with the antibody raised against full-length
recombinant ERK2. One explanation for this finding could be that the
C-terminal substrate recognition domain of ERK binds an unknown part of
STAT5a, which results in steric hindrance of the binding site of the
two different anti-ERK1/2 antibodies. One of the nuclear targets of
ERK, Elk-1, which is a member of the ternary complex factor subfamily
of ETS-domain transcription factors (26), has been demonstrated to bind
ERK2 by a domain that is distinct from, and located N-terminally to,
its phosphoacceptor motif (27). Targeting via this domain is essential
for the efficient and rapid phosphorylation of Elk-1 in
vitro and full rapid activation in vivo. The existence
of a similar ERK-binding domain in STAT5a remains to be determined. In
the experiments in which we used wild-type and serine 780 mutated
C-terminal STAT5a GST-fusion constructs, GH-induced activation of ERKs
resulted in increased binding of ERK1 and ERK2 to wild-type GST-fusion
construct only. This indicates that activation of ERK increases the
affinity of its kinase domain or enzyme active site for its recognition
amino acid sequence of STAT5a in which serine 780 seems to be of key
importance for successful interaction. The GST-fusion protein
constructs contained the last 64 C-terminal amino acids of STAT5a. With
both wild-type and serine 780-mutated GST fusion protein constructs,
some background binding to ERK1 and ERK2 was seen that was higher than
ERK binding to GST only. At present, it is too early to propose that
this 64-amino acid long sequence contains an ERK-targeting domain.
Further studies are needed to resolve this issue.
In extract from GH-treated COS cells, transfected with wild-type forms
of both STAT5a and ERK1, less STAT5a-ERK1 complex was detected than in
extracts from GH-treated COS cells transfected with wild-type ERK1 and
serine 780-mutated STAT5a. Thus, it is possible that phosphorylation of
serine 780 decreases the affinity for interaction between STAT5a and
ERK1. If complex formation between STAT5a and ERK1 is due to an
interaction at two sites, phosphorylation of serine 780 probably
decreases the affinity for interaction at both these sites.
MAPK-interacting kinase 1 (Mnk1) and ribosomal S6 kinase (RSK), both of
which are activated by ERK1/2 after mitogen stimulation, have been
shown to bind more strongly to inactive than active ERK, implying that
they dissociate from ERK after mitogen stimulation (28, 29).
The finding of a preformed STAT5a-ERK complex before GH stimulation and
its dissociation after GH stimulation makes it likely that serine
phosphorylation of STAT5a occurs in cytosol. Our time course study
showed that GH-induced activation of ERK1 and ERK2 and STAT5a had
similar kinetics. It is not possible to conclude from this experiment
which of the phosphorylations occurs first. Other experiments are
needed to answer this question. Serine phosphorylation of STAT1, STAT3,
and STAT5 can occur without tyrosine phosphorylation (6, 8, 30, 31).
Serine phosphorylation of STAT3 has been shown to negatively modulate
STAT3 tyrosine phosphorylation (9), indicating possibilites for
modulation of STAT activation by serine kinases.
ERKs were initially described as microtubuli-associated proteins (19, 20). Later investigations have shown that ERKs are also present in
cytosolic compartments not associated with microtubuli and also in the
nucleus (21). It has been determined that one-third of the total MAPK
is associated with microtubular cytoskeleton in NIH 3T3 fibroblasts, a
pool that constitutes half of the detectable MAPK activity after
mitogenic stimulation (20). In our present study, disruption of
microtubuli with colchicine did not decrease GH-induced ERK1 and ERK2
activation and also did not inhibit STAT5a nuclear translocation. Thus,
it is probable that STAT5a is not complexed with microtubuli-associated
ERK. It has recently been shown that GH-induced reorganization of actin
cytoskeleton is not required for STAT5-mediated transcriptional
activation (32). This finding and the results presented in the present
paper suggest that STAT5 nuclear transport occurs via a
cytoskeleton-independent mechanism.
From our results it is not possible to estimate the amount of STAT5a
complexed with ERK1 and/or ERK2. Further experiments are needed to
describe the molecular events occurring after GH stimulation and the
role of a preformed STAT5a-ERK1/2 complex. The prevailing dogma for
activation of STATs states that, after JAK-mediated phosphorylation of
tyrosine residues situated on the intracellular region of the cytokine
receptor, STATs are recruited to the receptor via binding to these
phosphorylated tyrosines via their SH2 domains. After receptor binding
STATs are activated via JAK-mediated tyrosine phosphorylation. If
preformed STAT5a-inactive ERK1/2 complexes are present in the receptor
proximal environment and if tyrosine phosphorylation precedes serine
phosphorylation, ERK1/2 could also be recruited to receptor upon ligand
stimulation. It has been shown previously that ERK coimmunoprecipitated
with IFN-
/ß receptor (11). It has been shown recently that STAT3,
when bound to the IFNAR1 chain of the type I IFN receptor, itself bound
phosphatidylinositol 3-kinase and thus functioned as a docking molecule
(33).
Our in vitro kinase experiments with C-terminal STAT5a-GST
fusion protein and recombinant full-length STAT5a showed that serine
780 is the only target for ERK under these conditions. This does not
exclude that serine 780 can be phosphorylated by other serine kinases.
Epidermal growth factor or interleukin-6 treatment of the same cell
line has been reported to induce phosphorylation of serine 727 in STAT3
by ERK-dependent or ERK-independent pathways, respectively (9). This
shows that different hormones, acting on the same cell, can induce
serine phosphorylation through activation of different serine kinases.
Furthermore, the same hormone can activate different pathways in
different cells to induce serine phosphorylation of STAT3 (34, 35). It
is possible that serine phosphorylation of STAT5a can also occur at
other sites than serine 780. Recently, Yamashita et al. (36)
identified serine 725 of STAT5a, within a PSP motif, as being
constitutively phosphorylated in COS and Nb2 cells. Their data also
suggested the existence of a second major serine phosphorylation site
in STAT5a, which was not identified. The constitutive phosphorylation
of STAT5a was shown to be suppressed by the MEK inhibitor PD98059.
Interestingly, in STAT5b, which is approximately 90% homologous to
STAT5a but lacks the C-terminal ERK recognition sequence (14, 37),
phosphorylation of the corresponding serine (730) was stimulated by PRL
but was not suppressed by PD98059. Mutational analysis showed that
phosphorylation of serine 725 in STAT5a and serine 730 in STAT5b was
not essential for DNA binding and transcriptional activation. Other
studies have shown that STAT5a and b are both serine phosphorylated by
ERK-independent pathways, constitutively in a mouse mammary epithelial
cell line (38) and after interleukin-2 treatment of T lymphocytes (39, 40).
In our earlier study we showed that inhibition of MEK resulted in
abolished GH-induced ERK activation and decreased capacity of STAT5a to
activate transcription. From these data we concluded that ERK was
involved. It cannot be totally excluded that another mechanism was
responsible for the effect of MEK inhibition. Transfection experiments
with COS cells, in the present study, showed that the presence of the
inactive form of ERK1 decreased the capacity of STAT5a to activate
reporter gene transcription. Thus, this experiment confirms the
involvement of ERK in regulation of STAT5a functional capacity.
In our earlier study we proposed that ERK phosphorylates serine 780 in
STAT5a. In the present study experiments were performed that lend
further support to this notion. From results described in our present
work the following model for interaction between ERK and STAT5a in CHOA
cells can be proposed (Fig. 7
). In
unstimulated cells STAT5a is complexed with inactive ERK. ERK binds to
STAT5a via its C-terminal substrate recognition domain to an unknown
region on STAT5a and via its active site to the C-terminal ERK
recognition sequence in STAT5a. Upon GH stimulation, MEK activates ERK
through phosphorylation of specific threonine and tyrosine residues in
ERK. Active ERK phosphorylates serine 780 in STAT5a, resulting in
decreased affinity between the two proteins and dissociation of the
complex.

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Figure 7. Proposed Model for STAT5a Interaction with ERK in
CHOA Cells
In unstimulated cells STAT5a is complexed with inactive ERK. ERK binds
to STAT5a via its C-terminal substrate recognition domain to an unknown
region on STAT5a and via its active site to the C-terminal ERK
recognition sequence in STAT5a. Upon GH stimulation, MEK activates ERK
through phosphorylation of specific threonine and tyrosine residues in
ERK. Active ERK phosphorylates serine 780 in STAT5a, resulting in
decreased affinity between the two proteins and dissociation of the
complex.
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Several STAT factors have now been described to be serine
phosphorylated (2). In some cases, ERK has been shown to be the kinase
involved (9, 11). More studies are needed to identify other kinases
taking part in serine phosphorylation of STAT factors as well as
additional sites on STATs that can be serine/threonine phosphorylated.
The biological importance of cross-talk between ERK and STAT5a is at
present unknown. In our present study, MAPK pathway was activated by
GH, simultaneously with activation of JAK2/STAT5 pathway. As MAPK
pathway is also activated by several other hormones and growth factors,
this could be a means for these factors to modulate GH-regulated gene
expression. This remains to be determined. The consequence of serine
phosphorylation of STAT5a C-terminal transactivation domain is at
present also unknown. In our earlier study we found that inhibition of
serine phosphorylation did not inhibit nuclear translocation of STAT5a
but only decreased the amount of STAT5 in nuclei with DNA-binding
capacity (13). We suggested that this could be due to increased
phosphotyrosine dephosphorylation of non-serine-phosphorylated STAT5a.
This does not exclude that other mechanisms, such as STAT5a dimer
stability, its interaction with proteins belonging to the
transcriptional machinery, or proteasome-dependent breakdown of STAT5a,
are influenced by serine phosphorylation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
CHO cells, stably transfected with rat GH receptor cDNA (CHOA),
were grown to 50% confluence in 100-mm culture dishes using Hams
F-12 medium (Life Technologies, Gaithersburg, MD) containing 10% FCS,
50 U/ml penicillin, and 50 µg/ml streptomycin. After washing with
PBS, cells were incubated in serum-free medium for approximately
16 h before treatment with human GH (hGH), a generous gift from
Pharmacia & Upjohn, Stockholm, Sweden. For mictrotubular disruption,
cells were pretreated with 10 µM colchicine (Sigma
Chemical Co., St. Louis, MO) 60 min before GH treatment.
Preparation of Cellular Extracts
After treatment with hGH for indicated times, cultured cells
were rinsed with ice-cold PBS and frozen on dry ice. The cells were
then scraped into an RSB buffer (10 mM Tris, pH 7.4, 10
mM NaCl, 6 mM MgCl2, 1
mM dithiothreitol, 0.1 mM
Na3VO4) and disrupted with a Dounce
homogenizer. The supernatant obtained after centrifugation was used as
cytosolic extract. The nuclear pellet obtained after centrifugation was
resuspended in 3 volumes of extraction buffer (20% glycerol, 20
mM HEPES, pH 7.9, 420 mM NaCl, 1.5
mM MgCl2, 0.2 mM EDTA, 0.2
mM phenylmethylsulfonyl fluoride, 1 mM
dithio-threithol, 0.1 mM Na3VO4)
and incubated on ice for 30 min. The supernatant obtained after
centrifugation was used as nuclear extract. Protein concentration was
measured by the Bradford method.
Western Blotting
Western blotting was performed as previously described (13). The
membranes were incubated with primary polyclonal antibodies,
anti-ERK1/2 [dilution, 1:500; StressGen (Biotechnologies Corp.,
Victoria, Canada) and Transduction Laboratories (Lexington,
KY)], anti-STAT5a (dilution, 1:750; Santa Cruz Biotechnology, Santa
Cruz, CA) and antiactive ERK (dilution, 1:20000; Promega, Madison, WI).
The membranes were analyzed with the enhanced chemiluminescence
method (Amersham, Arlington Heights, IL).
GST-Fusion Proteins
The expression plasmids for wild-type STAT5a, pME18S-STAT5a, and
serine 780-mutated STAT5a, pME18S-STAT5aS780A (13), were used to PCR
amplify the C-terminal end of STAT5a and STAT5aS780A, including the
codons for the last 64 amino acids (aa 730-aa 794), using the synthetic
oligonucleotides, 5'-TCGAGAATTCCCCTCAACCTCACTACAACATG-3' (containing
EcoRI cloning site) and
5'-TCAGCTCGAGTCAGGACAGGGAGCTTCTAGC-3' (containing XhoI
cloning site). The oligos were synthesized by Cybergene (Huddinge,
Sweden). The PCR products were ligated into the
EcoRI/XhoI sites of the GST fusion vector,
pGEX-4T3 (Pharmacia Biotech). Expression and purification of GST-fusion
proteins were done according to the manufacturers instructions
(Pharmacia Biotech). GST-fusion proteins were used in coprecipitation
and in vitro kinase experiments.
Coprecipitation with GST-Fusion Protein
Four micrograms of GST-fusion proteins were incubated for 45 min
at room temperature with cytosolic extract from CHOA cells, treated
with or without 100 nM GH for 10 min, in a HEPES buffer (20
mM HEPES, pH 7.6, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM Na3VO4, 10
mM NaF, 0.2 mM phenylmethylsulfonyl fluoride,
10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 µg/ml pepstatin A).
A 40-µl slurry (50:50) of glutathione-Sepharose (Pharmacia Biotech)
was added to incubations, and after 30 min the precipitations were
washed three times with PBS containing 0.1% Triton X-100. The
precipitation were boiled in SDS solubilization buffer after which
samples were separated by SDS-PAGE and analyzed by Western
blotting technique with a polyclonal antibody against ERK1/2.
Baculo Virus-Expressed Proteins
The plasmids pME18S-STAT5a and pME18S-STAT5aS780A were PCR
amplified using the synthetic oligonucleotides,
5'-ATCGGAATTCAAATGGCGGGCTGGAT-TCAG-3' (containing the EcoRI
cloning site) and 5'-TCAGTCTAGATCAGGACAGG-GAGCTTCTA-3' (containing the
XbaI cloning site). The oligos were synthesized by Cybergene
(Huddinge, Sweden). The PCR products were ligated into the
EcoRI/XbaI cloning site of the pFastBac HTB
vector (Bac-to-Bac baculovirus expression system, Life Technologies).
Clones were identified and both strands were sequenced by automatic
sequencing. Further steps, including transformation of the plasmids
into DH10 Bac cells containing bacmid and helper DNA, isolation of
recombinant bacmid DNA, transfection into Sf9-insect cells, and protein
expression, were done according to the manufacturers instruction
(Life Technologies). His-tagged recombinant proteins were purified with
nickel resin and used in coprecipitation and in vitro kinase
experiments.
In Vitro Phosphorylation
Purified GST-fusion proteins or recombinant wild-type or mutated
STAT5a were incubated in a kinase buffer (Promega), 1 µCi
[
-32P]ATP (Amersham)]/sample and 50 ng active ERK
(Promega) in a final incubation volume of 50 µl. The incubation was
allowed to proceed for 20 min at 30 C, after which the reaction was
terminated with SDS-solubilization buffer, run on an SDS-gel, and
visualized by autoradiography and PhosphorImager (Fuji Photo Film Co.,
Ltd, Japan).
Immunoprecipitation
Cellular extracts were diluted to 500 µl with TMB-buffer (50
mM Tris, pH 7.4, 10 mM MgCl2, 0.1%
BSA, 1 mM Na3VO4, 10 mM
NaF, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 10 µg/ml aprotinin and 2 µg/ml pepstatin A) and
incubated with either 5 µg polyclonal anti-ERK1/2 (Stress Gene and
Transduction Laboratories), 5 µg polyclonal anti-ERK2 (ERK2
immunoprecipitation antibody; Upstate Biotechnology, Inc., Lake Placid,
NY) or 5 µg monoclonal phosphotyrosine antibody (PY20;
Transduction Laboratories) for 45 min at room temperature. After
incubation for 30 min with 30 µl (50:50 slurry) of protein
A-Sepharose (Pharmacia Biotech), immune complexes were washed three
times with TMB buffer and further processed for Western blot analysis
as previously described (13).
Gel Electrophoresis Mobility Shift Assay
Gel electrophoresis mobility shift assay was performed
according to standard protocol previously described (24). An
oligonucleotide containing the STAT5 binding,
-interferon activated
sequence (GAS)-like element from the ß-casein promoter
(TGCTTCTTGGAATT) was used as a probe (18). Competition studies were
performed with the following double-stranded oligonucleotides
representing various GAS-like promoter elements from cytokine-regulated
genes (41): human guanylate binding protein (GBP)-ATTACTCTAAA; human
FC
receptor type 1 (Fc
R1)-TTTCCCAGAAA; human IFN consensus
sequence binding protein (ICSBP)-TTTCTCCGAAA; human IFN-induced protein
53 (IFP-53)-ATTCTCAGAAA; human IFN-regulatory factor 1
(IRF-1)-TTTCCCCGAAA; mouse Ly 6E antigen (Ly6E)-ATTCCTGTAAG; and mouse
monokine induced by IFN-
(Mig)-CTTACTATAAA.
Cellular Transfection and Luciferase Assay
Transfection of COS cells and luciferase assay were performed as
previously described (13). In short, 50% confluent 30-mm dishes with
COS cells were transfected with 1 µg of STAT5-regulated reporter
plasmid pSPI-GLE1-luc (12) and expression plasmids for STAT5a (0.2
µg; pME18S-STAT5a), GHR (0.2 µg; pLM108), and wt-ERK1 (0.3 µg;
pcDNA1/Neo-HA-ERK1-wt) or the kinase inactive ERK1 (0.3 µg; T192A,
pcDNA1/Neo-HA-ERK1-kin-) or control plasmid (0.3 µg; pcDNA) and
ß-gal (0.05 µg; pCMV-gal). Luciferase and ß-galactosidase
activities were measured in 96-well plates in the luminometer Arthos
Lucy1 (Arthos Labtec Instruments, Salzburg, Austria) after
automatic injection of reagent.
For precipitation studies 50% confluent 100-mm dishes with COS cells
were transfected with the expression vectors (1 µg of each plasmid)
for the GH receptor and STAT5a or STAT5aS780A and HA-tagged wild-type
ERK1 (HA-ERK-wt) or HA-tagged kinase inactive ERK1 (HA-ERK-kin-) and
pcDNA. Transfections were performed with DOTAP (Boheringer
Mannheim, Mannheim, Germany). The last 16 h before harvest the COS
cells were grown in serum-free DMEM, after which the cells were treated
with GH for 8 min, and the cytosolic extract was prepared as
described.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Alice F. Mui for STAT5a expression vector, Dr.
Jacques Pouysségur and Dr. Pär Gerwins for ERK1-wild-type
and ERK1-kinase-inactive expression plasmids, and Dr. Tim Wood for
SPI-GLE1-luciferase reporter gene.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Lars-Arne Haldosén, Department of Medical Nutrition, Karolinska Institute, Novum, S-141 86 Huddinge, Sweden. E-mail:
Lars-Arne.Haldosen{at}mednut.ki.se
This work was supported by grants from the Swedish Cancer Society
(Grant 3020-B9704XBB), the Swedish Medical Research Council (Grant
03X-06807), Karolinska Institutet, and Magnus Bergvalls
Stiftelse.
Received for publication May 28, 1998.
Revision received December 10, 1998.
Accepted for publication January 8, 1999.
 |
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