PRL-Induced ER
Gene Expression Is Mediated by Janus Kinase 2 (Jak2) While Signal Transducer and Activator of Transcription 5b (Stat5b) Phosphorylation Involves Jak2 and a Second Tyrosine Kinase
Jonna Frasor,
Uriel Barkai,
Liping Zhong,
Asgerally T. Fazleabas and
Geula Gibori
Departments of Physiology and Biophysics (J.F., U.B., L.Z., G.G.)
and Obstetrics and Gynecology (A.T.F.), University of Illinois at
Chicago, Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Geula Gibori, Ph.D., 835 South Wolcott, M/C 901, University of Illinois at Chicago, Chicago, Illinois 60612. E-mail: ggibori{at}uic.edu
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ABSTRACT
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In the rat corpus luteum of pregnancy, PRL stimulation of ER
expression is a prerequisite for E2 to have any luteotropic effect.
Previous work from our laboratory has established that PRL stimulates
ER
expression at the level of transcription and that the
transcription factor Stat5 (signal transducer and activator of
transcription 5) mediates this stimulation. Since it is well
established that PRL activates Stat5 through the tyrosine kinase, Janus
kinase 2 (Jak2), the role of Jak2 in PRL regulation of ER
expression
was investigated. In primary luteinized granulosa cells, the general
tyrosine kinase inhibitors, genistein and AG18, and the Jak2
inhibitor, AG490, prevented PRL stimulation of ER
mRNA levels,
suggesting that PRL signaling to the ER
gene requires Jak2 activity.
However, using an antibody that recognizes the
tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it
was found that AG490 could inhibit PRL-induced Stat5a phosphorylation
only and had little or no effect on Stat5b phosphorylation. These
effects of AG490 were confirmed in COS cells overexpressing Stat5b.
Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of
ER
promoter activity and Stat5b phosphorylation while a
constitutively active Jak2 could stimulate both in the absence of PRL.
Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b
nuclear translocation and DNA binding. Therefore, it seems that in the
presence of AG490, Stat5b remains phosphorylated, is located in the
nucleus and capable of binding DNA, but is apparently transcriptionally
inactive. These findings suggest that PRL may activate a second
tyrosine kinase, other than Jak2, that is capable of
phosphorylating Stat5b without inducing transcriptional activity. To
investigate whether another signaling pathway is involved, the src
kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K),
LY294002, were used. Neither inhibitor alone had any major effect
on PRL regulation of ER
promoter activity or on PRL-induced
Stat5b phosphorylation. However, the combination of AG490 and
LY294002 largely prevented PRL-induced Stat5b phosphorylation.
These findings indicate that PRL stimulation of ER
expression
requires Jak2 and also that PRL can induce Stat5b phosphorylation
through two tyrosine kinases, Jak2 and one downstream of PI3K.
Furthermore, these results suggest that the role of Jak2 in activating
Stat5b may be through a mechanism other than simply inducing Stat5b
phosphorylation.
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INTRODUCTION
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IN RODENTS, PRL plays a unique role in both
the rescue and continued function of the corpus luteum during
pregnancy. One well established function of PRL in regulating luteal
function is its ability to stimulate ER expression, thereby maintaining
luteal responsiveness to E2 (1, 2). Previous
investigations in our laboratory have demonstrated that PRL can
stimulate both ER
and ERß expression (3).
Furthermore, this stimulation occurs at the level of transcription and
is mediated by the transcription factor Stat5 (signal transducer and
activator of transcription 5) (4).
The PRL receptor (PRL-R) is a member of the cytokine/hematopoietic
receptor superfamily. These receptors are characterized by four
conserved cysteines, a Trp-Ser-X-Trp-Ser (WSXWS) motif in their
extracellular domain, and no intrinsic kinase activity (5, 6). It is well established that the PRL-R is associated with the
tyrosine kinase Janus kinase 2 (Jak2) and activates Jak2 rapidly upon
exposure to PRL (7, 8, 9, 10, 11). Signaling through Jak2 has been
shown to be necessary for both PRL-induced proliferation of Nb2 cells
and regulation of gene transcription through the transcription factor
Stat5 (12, 13, 14). Jak2 has been shown to activate Stat5
through phosphorylation of both Stat5a and Stat5b on specific tyrosine
residues in the C terminus (15, 16). The ability of
PRL to utilize Jak2 appears to be specific since overexpression of Jak1
or Jak3 does not amplify PRL signaling to milk proteins (17, 18).
In addition to the Jak/Stat pathway, PRL has been shown to activate
other signaling pathways including PKC (19, 20, 21, 22, 23, 24),
phosphatidylinositol 3-kinase (PI3K) (25, 26, 27), MAPK
(26, 27, 28, 29, 30, 31, 32, 33), and the src family of tyrosine kinases
(36, 37, 38). Recently, a role for PKC
in PRL regulation of
relaxin expression in the rat corpus luteum has been identified
(39, 40, 41). Also, PRL is known to stimulate PI3K activity in
both Nb2 and CHO cells stably transfected with the PRL-R (25, 26). PRL stimulation of PI3K activity is necessary for the
activation of PKB kinase and the prevention of apoptosis in rat
decidual cells (42) and Nb2 cells (27).
Although the function of src tyrosine kinase activity in PRL signaling
has not been well studied, it may play a role in PRL activation of the
PI3K pathway (43, 44).
Our previous studies have indicated that PRL utilizes the transcription
factor Stat5 to mediate regulation of ER
gene transcription
(4). It is well established that PRL activates Stat5
through phosphorylation on specific tyrosine residues in the C terminus
of Stat5a and Stat5b (Y694 and Y699) by the tyrosine kinase Jak2
(15, 16). Although PRL has been shown to activate many
different signaling pathways, Stat5 activation has been shown only to
occur by PRL through Jak2. Therefore, it seems likely that the tyrosine
kinase Jak2 is involved in PRL regulation of ER
expression. The
purpose of this investigation was to examine the role of Jak2 in
PRL-stimulated, Stat5- mediated regulation of ER
expression. We have
found that while Jak2 activity is required for both PRL stimulation of
ER
expression and Stat5a and Stat5b phosphorylation, a second
tyrosine kinase, downstream of PI3K, can also mediate PRL-induced
Stat5b phosphorylation specifically, but not Stat5a phosphorylation.
This second tyrosine kinase, however, does not appear to be involved in
either Stat5b transcriptional activity or ER
expression.
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RESULTS
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To investigate whether PRL stimulation of ER
mRNA expression is
tyrosine kinase dependent, primary luteinized granulosa cells were
treated with PRL in the presence of two different general tyrosine
kinase inhibitors, genistein or AG18. Interestingly, both of these
inhibitors have been used to prevent PRL stimulation of gene expression
(45, 46, 47) but failed to prevent PRL inhibition of
20
-hydroxysteroid dehydrogenase expression (10).
When cells were treated with genistein, both basal and PRL-stimulated
levels of ER
mRNA were decreased, as determined by semiquantitative
RT-PCR (Fig. 1
, A and B). Similar results
were observed when primary luteinized granulosa cells were treated with
AG18, although the lowest dose used was less effective (Fig. 1
, C and
D). These findings indicate that PRL stimulation of ER
expression is
dependent upon the activation of a tyrosine kinase.
To examine whether the tyrosine kinase required for PRL action is Jak2,
the inhibitor, AG490, was used. AG490 was originally described as a
specific Jak2 inhibitor (48). However, recent
investigations indicate that AG490 can inhibit Jak3 as well as
activation of the MAPK pathway, although MAPK appears to be downstream
of Jak2 or Jak3 (49, 50, 51, 52). Since PRL has not been shown to
activate Jak3 and since no other specific inhibitors of Jak2 have been
identified, we used AG490 to block PRL activation of Jak2. Primary
luteinized granulosa cells were treated with PRL for 12 h, either
alone or in the presence of 25 µM AG490, and the effect
on ER
mRNA expression was examined using real-time quantitative
RT-PCR (Fig. 2A
). It was found that AG490
completely prevented PRL stimulation of ER
mRNA expression in this
model, suggesting that the tyrosine kinase required for PRL action on
ER
expression is Jak2. Interestingly, AG490 alone (data not shown)
as well as genistein or AG18 alone appeared to decrease ER
mRNA
expression, suggesting that some level of signaling through Jak2 may be
required to maintain the basal level of ER
expression in primary
luteinized granulosa cells.

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Figure 2. Effect of AG490, a Jak2 Kinase Inhibitor, on PRL
Stimulation of ER Expression and Stat5 Phosphorylation
A, Primary luteinized granulosa cells were treated for 12 h with
PRL (1 µg/ml) alone or in combination with AG490 (25
µM). RNA was isolated and quantitative, real-time RT-PCR
was carried out for ER as described in Materials and
Methods. The data for each treatment group were normalized to
the level of ER in the untreated control group and presented here as
the mean and SEM for the six different samples in each
group. B, Primary luteinized granulosa cells were treated with PRL (1
µg/ml) for 5 min following a 30-min pretreatment with vehicle or
AG490 (25 µM). Stat5a and Stat5b were immunoprecipitated
with specific antibodies, and Western blotting was performed using an
antibody, which recognizes the phosphorylated form (P-Stat5) of Stat5a
and Stat5b (Y694/Y699), and then with the same antibodies used for
immunoprecipitation. Data presented here are representative of three
independent experiments. C, COS cells were cultured in six-well plates
and transfected with 0.5 µg/well ER -luc and expression vectors for
ß-gal (0.5 µg/well), Stat5b (1 µg/well), and either
PRL-RL (2 µg/well) or PRL-RCA (2 µg/well).
Twenty-four hours after the start of transfection, cells were treated
with AG490 (25 µM) for an additional 24 h.
Luciferase activity was measured in each well and normalized to the
ß-gal activity within that well. The experiment was repeated three
times in triplicate. Within each experiment the data were normalized to
control values. Data presented here represent the combined mean and
SEM for three experiments. D, COS cells were transfected
and treated as described. Western blotting was performed on WCE using
the same antibodies as in panel B. Data presented here are
representative of three independent experiments.
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Since we have previously shown that PRL regulation of ER
expression
is mediated by Stat5 (4), we next investigated whether the
effect of AG490 was due to its ability to block activation of Stat5.
Primary luteinized granulosa cells were treated with PRL for 5 min,
either alone or after a 30-min pretreatment with AG490. Stat5a and
Stat5b were then immunoprecipitated, and phosphorylation was examined
by Western blotting. Both Stat5a and Stat5b become highly
phosphorylated on Y694 and Y699, respectively, in response to PRL in
these cells; however, only PRL-induced phosphorylation of Stat5a was
prevented by AG490 whereas phosphorylation of Stat5b was unaffected
(Fig. 2B
). These results suggest that Jak2 appears to be sufficient for
PRL activation of Stat5a, whereas a second tyrosine kinase may be
involved in PRL activation of Stat5b.
It is possible that the inhibitory effect of AG490 on PRL-induced ER
mRNA expression may be due to the prevention of Stat5a phosphorylation.
However, as we have previously shown, both Stat5a and Stat5b can
mediate ER
expression to an equal extent (4).
Furthermore, we have found that Stat5b is highly expressed in luteal
cells whereas Stat5a is expressed at nearly undetectable levels (data
not shown). This suggests then that Stat5b may, in fact, be the major
regulator of ER
expression in vivo. Therefore, we decided
to further investigate the role of Jak2 in Stat5b phosphorylation and
transcriptional activity using COS cells, since these cells express
undetectable levels of endogenous Stat5 by Western analysis. In
addition, these experiments may eliminate the possibility that the
effect of AG490 on ER
mRNA expression was the result of blocking
Stat5a phosphorylation. COS cells were transfected with an
approximately 700-bp fragment of the ER
promoter, which has been
linked to the luciferase reporter gene and previously shown to be
responsive to PRL (4). These cells were also transfected
with an expression vector for Stat5b and for either the long form of
the PRL-R (PRL-RL), which in the absence of PRL
treatment served as the control, or with an expression vector for a
constitutively active PRL-R (PRL-RCA). This
active receptor has previously been shown to mediate gene transcription
of PRL-regulated genes (4, 53). Cells were then treated
with vehicle or 25 µM AG490 for 24 h.
PRL-RCA stimulation of ER
promoter-driven
luciferase activity was prevented by AG490 (Fig. 2C
). The effect of
AG490 was also examined on Stat5b phosphorylation in COS cells and, as
was the case in primary cells, AG490 had no inhibitory effect on Stat5b
phosphorylation (Fig. 2D
). These findings suggest that AG490 can
prevent PRL stimulation of ER
promoter activity and apparent Stat5b
transcriptional activity without affecting Stat5b phosphorylation.
Because it is well accepted that PRL activates Stat5b through Jak2, we
decided to confirm that Jak2 is involved in PRL stimulation of ER
promoter activity and Stat5b phosphorylation. To do this, we used
expression vectors for a kinase-negative and a constitutively active
Jak2 (KN-Jak2, CA-Jak2). The KN-Jak2 was created by mutation to the
kinase domain, rendering it inactive, while the CA-Jak2 was made by
deletion of a pseudokinase domain, which acts to negatively regulate
Jak2 kinase activity (54, 55). When COS cells were
transfected with the ER
promoter, Stat5b, and
PRL-RCA, the KN-Jak2 could completely prevent
PRL-RCA stimulation of ER
promoter activity
(Fig. 3A
). The effect of KN-Jak2 on
Stat5b phosphorylation was also examined. In contrast to AG490,
KN-Jak2 could, in large part, reverse the effect of
PRL-RCA on Stat5b phosphorylation (Fig. 3B
). When
COS cells were transfected with only the ER
promoter and Stat5b,
CA-Jak2 could stimulate promoter activity in the absence of any PRL
signal transduction (Fig. 3C
). In addition, CA-Jak2 induced a high
degree of Stat5b phosphorylation in the absence of PRL (Fig. 3D
). These
findings indicate that Jak2 is involved in PRL stimulation of ER
expression and can mediate Stat5b phosphorylation.
Our data suggest that Stat5b is not transcriptionally active although
still phosphorylated in the presence of AG490. Therefore, we next
examined the effect of AG490 on Stat5b nuclear translocation and DNA
binding. COS cells were transfected with Stat5b and either
PRL-RL or PRL-RCA and then
treated with AG490 for 24 h. As a control, cells were also
transfected with KN-Jak2 or CA-Jak2. To determine whether Stat5b was
located in the nucleus, Western blotting was performed for Stat5b on
nuclear extracts from these transfected cells (Fig. 4A
). In the absence of
PRL-RCA, Stat5b could not be detected in the
nuclear fractions. PRL-RCA induced Stat5b nuclear
translocation, and this was prevented by KN-Jak2, but not AG490, while
the CA-Jak2 could induce Stat5b nuclear translocation in the absence of
PRL. In parallel groups, Western blotting was performed on whole-cell
extracts (WCE) to ensure that Stat5b was expressed at equivalent levels
(data not shown). To examine whether AG490 could prevent Stat5b DNA
binding, the same nuclear extracts were incubated with a labeled probe
corresponding to a consensus Stat5 response element.
PRL-RCA induced the formation of one DNA-protein
complex, which has been shown to contain Stat5 (15) (Fig. 4B
). AG490 treatment had no inhibitory effect on Stat5b DNA binding
while KN-Jak2 could prevent the formation of this complex. In addition,
the same complex was formed by CA-Jak2 in the absence of
PRL-RCA. These results indicate that in the
presence of AG490, Stat5b remains phosphorylated, located in the
nucleus, capable of binding DNA, and yet unable to activate
transcription of the ER
gene promoter.

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Figure 4. Role of Jak2 in Stat5b Nuclear Translocation and
DNA Binding
COS cells were transfected and treated as described in Figs. 2 and 3 .
Nuclear extracts were prepared and Western blotting was performed on 20
µg of nuclear protein using a specific antibody for Stat5b (A). Data
presented here are representative of three independent experiments.
Alternatively, 10 µg nuclear protein were incubated with 50K cpm of
end-labeled oligonucleotide corresponding to the ß-casein Stat5
response element (B). The DNA-protein complexes were separated on a
4.5% polyacrylamide gel. The gel was then exposed to a PhosphoImager
for analysis. These results are representative of three independent
experiments.
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As a control, the effects of Jak2 activity on Stat5a phosphorylation
and nuclear translocation were also examined in COS cells. As was the
case in primary luteinized granulosa cells, AG490 could prevent
PRL-RCA-induced phosphorylation of Stat5a (Fig. 5A
). In addition, KN-Jak2 also prevented
Stat5a phosphorylation in COS cells (Fig. 5B
). Nuclear extracts were
also examined for the presence of Stat5a, and it was found that both
AG490 and KN-Jak2 could prevent, while CA-Jak2 could stimulate, Stat5a
translocation to the nucleus (Fig. 5C
).

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Figure 5. Role of Jak2 in Stat5a Phosphorylation and Nuclear
Translocation
COS cells were cultured, transfected, and treated as described for
Figs. 2 and 3 except that Stat5a (1 µg/well) was transfected in place
of Stat5b. Western blotting was performed on WCE for phosphorylated
Stat5 and Stat5a (A and B). Western blotting was also performed on
nuclear extracts for Stat5a (C). Data presented here are representative
of two independent experiments.
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One possible explanation for the lack of effect of AG490 on Stat5b
could be that PRL can induce Stat5b phosphorylation through two
different signaling pathways. Perhaps when Jak2 is active, Stat5b is
transcriptionally active, but when Jak2 is inactivated by AG490, a
second tyrosine kinase can induce Stat5b phosphorylation but not its
ability to regulate gene transcription. This possibility is similar to
that observed for Stat5b activation by the tyrosine kinase src. It has
been shown that src can induce Stat5b phosphorylation, nuclear
translocation, and DNA binding. However, when Stat5b is activated by
src it is not capable of stimulating gene promoter activity
(56). PRL is known to activate src family members in the
T-cell line Nb2, rat liver, chicken embryo fibroblasts, and embryonic
astrocytes (37, 38, 57, 58). It could be possible that PRL
can activate src in the presence of AG490 and that src could be
responsible for a phosphorylated but transcriptionally inactive Stat5b.
To examine this, COS cells were transfected with ER
-luc, Stat5b, and
either PRL-RL or PRL-RCA.
Cells were then treated for 24 h with PP2, a specific inhibitor of
src, and the effects on ER
promoter activity and Stat5b
phosphorylation were examined. PP2 was found to have no effect on
PRL-RCA-induced ER
promoter activity (Fig. 6A
). In addition, Stat5b phosphorylation
was unaffected by PP2, either alone or in combination with AG490 (Fig. 6B
). Although we have not investigated whether
PRL-RCA can activate src in COS cells or whether
PP2 can prevent this activation, our findings suggest that this
tyrosine kinase may not be involved in
PRL-RCA-induced Stat5b phosphorylation or
transcriptional activity.

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Figure 6. Effect of PP2, a c-Src Kinase Inhibitor, on
PRL-RCA Stimulation of ER Promoter Activity and Stat5b
Phosphorylation in COS Cells
A, COS cells were cultured and transfected as described in Fig. 2 .
Twenty-four hours after the start of transfection, cells were treated
with PP2 (25 µM) for an additional 24 h. Luciferase
activity was measured in each well and normalized to the ß-gal
activity within that well (A). The experiment was repeated three times
in triplicate. Within each experiment the data were normalized to
control values. Data presented here represent the combined mean and
SEM for three experiments. B, COS cells were transfected
and treated as described above. In addition, cells were treated with 25
µM AG490 or 25 µM PP2, either alone or in
combination. WCE were prepared, and Western blotting was performed
using an antibody that recognizes the phosphorylated form of Stat5 and
then with a specific antibody for Stat5b. Data presented here are
representative of three independent experiments.
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In addition to src, PRL has also been shown to activate the PI3K
signaling pathway in various cell types (25, 26, 42).
Therefore, the PI3K inhibitor LY294002 was used to treat COS cells that
had been transfected with ER
-luc, Stat5b, and
PRL-RL or PRL-RCA. LY294002
had little effect on either ER
promoter activity or Stat5b
phosphorylation in the presence of PRL-RCA (Fig. 7
). Surprisingly, the combination of
AG490 and LY294002 was capable of markedly reducing Stat5b
phosphorylation. These findings suggest that Stat5b can be
phosphorylated by two tyrosine kinases, Jak2 and a second tyrosine
kinase, which is downstream of PI3K.

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Figure 7. Effect of LY294002, a PI3K Inhibitor, on
PRL-RCA Stimulation of ER Promoter Activity and Stat5b
Phosphorylation in COS Cells
A, COS cells were cultured and transfected as described in Fig. 2 .
Twenty-four hours after the start of transfection, cells were treated
with LY294002 (25 µM) for an additional 24 h.
Luciferase activity was measured in each well and normalized to the
ß-gal activity within that well (A). The experiment was repeated
three times in triplicate. Within each experiment the data were
normalized to control values. Data presented here represent the
combined mean and SEM for three experiments. B, COS cells
were transfected and treated as described above. In addition, cells
were treated with 25 µM LY294002, either alone or in
combination with 25 µM AG490. WCE were prepared and
Western blotting was performed using an antibody that recognizes the
phosphorylated form of Stat5 and then with a specific antibody for
Stat5b. Data presented here are representative of three independent
experiments.
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DISCUSSION
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The results of these studies indicate that PRL stimulation of
ER
transcription requires Jak2 tyrosine kinase activity.
Furthermore, PRL activation of Stat5a appears to be dependent on Jak2
activity only since both AG490 and KN-Jak2 could prevent PRL-induced
phosphorylation of Stat5a. PRL activation of Stat5b, however, appears
to be multifaceted (summarized in Table 1
). Both PRL-induced phosphorylation of
Stat5b and Stat5b-mediated ER
transcription could be inhibited by
KN-Jak2 and stimulated by CA-Jak2. In contrast, the Jak2 inhibitor,
AG490, prevented Stat5b transcriptional activity without affecting its
phosphorylation, nuclear translocation, or DNA binding activity. As a
control, AG490 was found to prevent Stat5a phosphorylation and nuclear
translocation. Only when the combination of AG490 and the PI3K
inhibitor, LY294002, was used could PRL-induced Stat5b phosphorylation
be prevented, suggesting that Jak2 and a tyrosine kinase downstream of
PI3K may be involved in Stat5b phosphorylation. However, this second
tyrosine kinase does not appear to be involved in PRL stimulation
of ER
promoter activity.
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Table 1. Effect of Jak2 Expression Vectors and Kinase
Inhibitors on PRL Induction of Stat5b Phosphorylation, Nuclear
Translocation, DNA Binding and Transcriptional
Activity
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Several potential questions and implications arise from these findings.
First, two tyrosine kinases appear to be capable of mediating
PRL-induced Stat5b phosphorylation on the same tyrosine residue, Y699.
The first kinase is Jak2, which is well established, whereas the second
tyrosine kinase would be a novel kinase apparently downstream of PI3K.
The Btk/Tec family of tyrosine kinases have been shown to be activated
by PI3K, presumably by virtue of their pleckstrin homology domain,
which interacts with phosphoinositide products of PI3K and induces
membrane localization (59, 60, 61, 62). Furthermore, one member of
this family, Bmx, can induce Stat1, Stat3, and Stat5 phosphorylation
and DNA binding activity (63). However, Bmx could also
activate Stat5-mediated transcription (63). Whether any
members of the Tec family of kinases are involved in PRL signaling
remains to be investigated.
Second, it is not clear whether PRL activation of PI3K is dependent on
Jak2 activity. The KN-Jak2 could, in large part, prevent Stat5b
phosphorylation, suggesting that the tyrosine kinase downstream of PI3K
is also prevented by KN-Jak2. In contrast, AG490 did not prevent Stat5b
phosphorylation, suggesting that activation of this second tyrosine
kinase is not Jak2 dependent. Previous studies in this area suggest
that PRL can activate PI3K through two pathways, one involving Jak2 and
the insulin receptor substrate and the other involving the src kinase
fyn and the adaptor protein Cbl (43, 44, 64, 65, 66). It is
possible that the activation of PI3K depends on the presence of Jak2
protein and not its activity. If the mutation to KN-Jak2 significantly
changes its conformation, then the formation of a signaling complex
could be disrupted and prevent PI3K activation. AG490, on the other
hand, is a tyrphostin tyrosine kinase inhibitor, which
presumably would only block the substrate binding site and perhaps not
affect the ability of Jak2 to interact with PI3K.
Third, when Jak2 is active, Stat5b can regulate gene transcription. In
the absence of Jak2 activity, Stat5b can no longer stimulate ER
promoter activity even though it is phosphorylated, located in the
nucleus, and capable of binding DNA. These data suggest that Jak2
activity is required for PRL regulation of gene transcription in some
fashion beyond its ability to induce Stat5b phosphorylation on Y699.
The ability of src kinase to activate Stat5b phosphorylation and DNA
binding without transcriptional activity support this possibility
(56). Whether Jak2 may be involved in the activation of
signaling molecules in addition to Stat5, such as coactivators or other
transcription factors, has not been investigated.
And finally, the ability of PRL to activate Stat5b phosphorylation
through two kinases appears to be specific for Stat5b and not Stat5a.
Very few instances of differential regulation of Stat5a and Stat5b have
been reported; however, one in particular is of interest. Insulin can
induce tyrosine phosphorylation of both Stat5a and Stat5b
(67). AG490 prevented phosphorylation of Stat5a only and
had no effect on Stat5b phosphorylation. When an insulin receptor
kinase inhibitor was used, Stat5b phosphorylation induced by insulin
was completely prevented. Although additional data suggested that
Stat5b is a direct target of the insulin receptor (67, 68), one of the downstream pathways of insulin is PI3K. Perhaps
a similar tyrosine kinase downstream of both PRL and insulin activation
of PI3K is involved in Stat5b-specific phosphorylation.
Taken together, our results indicate that PRL regulation of ER
expression requires Jak2 activity. In addition, our data suggest the
ability of PRL to induce Stat5b phosphorylation and transcriptional
activity through Jak2 whereas Stat5b phosphorylation, but not
transcriptional activity, may be mediated by novel tyrosine kinase
downstream of PI3K.
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MATERIALS AND METHODS
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Materials
PMSG, human CG (hCG), DMEM/F12 (1:1), DMEM, horseradish
peroxidase-conjugated secondary antibodies, LY294002, and all
other reagent grade chemicals were obtained from Sigma
(St. Louis, MO). Genistein and AG18 were obtained from ICN Biomedicals, Inc. (Aurora, OH). AG490 and PP2 were from
Calbiochem (San Diego, CA). Protogel, a 30%
acrylamide/bis-acrylamide mixture (37.5:1) was from National
Diagnostics (Atlanta, GA). T4 polynucleotide kinase and TRIzol were
purchased from Life Technologies, Inc. (Gaithersburg, MD).
The Advantage RT-for-PCR kit and the chemiluminescence
ß-galactosidase (ß-gal) substrate were from CLONTECH Laboratories, Inc. (Palo Alto, CA). FBS was from HyClone Laboratories, Inc. (Logan, UT). ExTaq DNA polymerase, ExTaq PCR
buffer, and deoxynucleotide triphosphate (dNTP) were obtained
from Panvera (Madison, WI). 32P-Labeled
nucleotides were from Amersham Pharmacia Biotech
(Arlington Heights, IL). DNA Master SYBR Green I was purchased
from Roche Molecular Biochemicals (Indianapolis, IN).
Trypsin-EDTA, antibiotics, and Amphotericin were from Mediatech
(Herndon, VA). Antibodies to Stat5a, Stat5b, and phosphorylated
Stat5a/5b were from Upstate Biotechnology, Inc. (Lake
Placid, NY). Protein A/G agarose beads and the enhanced
chemiluminescence detection reagents were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The luciferase assay
substrates and reporter lysis buffer were purchased from Promega Corp. (Madison, WI). Ovine PRL (PRL-18, 30 IU/mg) was obtained
from NIDDK, NIH (Bethesda, MD).
Primary Luteinized Granulosa Cell Culture and
Transfection
Immature female Sprague Dawley rats were obtained from Sasco
Animal Labs (Madison, WI) and housed under controlled conditions of
light and temperature with free access to standard rat chow and water.
All experiments were conducted in accordance with the principles and
procedures of the NIH Guide for the Care and Use of Laboratory Animals
and were approved by the Institutional Animal Care and Use Committee.
Follicular development was induced in immature rats (2426 d of age)
by injection of 15 IU PMSG ip. An ovulatory dose of hCG (10 IU, ip) was
given 48 h later. Luteinized granulosa cells were harvested from
large preovulatory follicles 78 h after hCG injection. The ovarian
bursa and surrounding fat were removed, and the ovaries were incubated
sequentially in DMEM/F12 (1:1) containing 6 mM EGTA and
then 0.5 M sucrose. Granulosa cells were harvested by
puncturing the follicles with 25-g needles, washed in DMEM/F12, and
then cultured in six-well plates at a density of 0.25 x
106 cells per well for RNA extraction and at a
density of 1 x 106 cells per 60-mm plate
for protein extraction. The culture media was DMEM/F12 supplemented
with 1% FBS, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and
0.25 µg/ml Amphotericin. The cells were cultured
for 72 h at 37 C in a 5% CO2, humidified
atmosphere. After 72 h, media were changed and cells were cultured
overnight before the start of treatment. After treatment, cells were
washed twice with cold PBS and stored at -80 until processed.
RNA Isolation and Reverse Transcription
RNA from cell cultures was isolated using Trizol according to
the manufacturers instructions. Reverse transcription was carried out
using reagents from the Advantage RT-for-PCR kit. One microgram of
total RNA was incubated with 1 µl oligo (dT)18
and 0.5 µl random hexamer at 70 C for 2 min. Four microliters of 5x
reaction buffer, 1 µl dNTP (10 mM each), 0.5 µl RNase
inhibitor, and 1 µl Moloney murine leukemia virus reverse
transcriptase were added to each sample, and the total volume was
brought to 20 µl with diethyl pyrocarbonate-treated
H2O. The reaction was carried out for
1 h at 42 C followed by 5 min at 94 C. The reverse transcribed
product was then diluted to a final volume of 100 µl by adding
DEPC-treated H2O. Five-microliter aliquots of the
diluted product were used for either semiquantitative or quantitative,
real-time RT-PCR.
Semiquantitative RT-PCR
For each sample to be analyzed by semiquantitative RT-PCR,
5-µl identical aliquots of diluted reverse transcribed mRNA were used
for the gene of interest and for the ribosomal protein L19, which
served as an internal control. Diluted RT product (5 µl, representing
50 ng of total RNA) was combined with 20 pmol primers, 1x PCR buffer,
150 µM dNTP, 0.8 U ExTaq, and
-32P-dCTP (2.5 µCi of 3,000 µCi/mmol) in a
final volume of 40 µl. The primers used have previously been
described (3, 69). The samples were overlaid with light
mineral oil, and PCR was carried out in two parts. First, five cycles
were carried out with an annealing and extension temperature of 69 C
for 5 min followed by denaturing at 95 C for 1 min. The second set
consisted of a varying number of cycles with annealing at 65 C for 25
sec, extension at 72 C for 30 sec, and denaturing at 95 C for 25 sec.
PCR reactions were carried out in a Perkin-Elmer/Cetus Thermal Cycler
(Perkin-Elmer Corp., Norwalk, CT). For ER
, 25 step 2
cycles were used and 18 cycles for L19. The conditions were such that
the amplification of the products was in the exponential phase and the
assay was linear with respect to the amount of input RNA. Reaction
products for ER
were combined with the corresponding L19 products
and electrophoresed on an 8% polyacrylamide nondenaturing gel. After
autoradiography, data were analyzed using a Molecular Dynamics, Inc. PhosphorImager and ImageQuant version 3 software
(Molecular Dynamics, Inc., Sunnyvale, CA). The intensity
of the ER
signal was normalized to that of the ribosomal protein L19
internal control.
Real-time, Quantitative RT-PCR
To generate standard curves for quantitative PCR, rat
ER
cDNA, which was kindly provided by Dr. Maruyama, was diluted to
concentrations ranging from 103 to
107 copies/µl. Five-microliter aliquots of
standards or diluted RT products were combined with 2 µl 10x DNA
Master SYBR Green I, 1.6 µl MgCl2 (3
mM final concentration), and specific primers for rat ER
(0.5 µM 77 final concentration). Reactions were carried
out in glass capillary tubes in a total volume of 20 µl. The DNA
Master SYBR Green I mix contains Taq DNA polymerase,
reaction buffer, dNTP, 10 mM
MgCl2, and SYBR Green I dye, which is a specific
fluorescence dye for double-stranded DNA. PCR reactions were performed
in the Roche Lightcycler instrument, and the accompanying
software was used for data analysis (Roche Molecular Biochemicals). After a 2-min denaturation, PCR cycles were
carried out as follows: 0 sec at 95 C, 10 sec at the annealing
temperature, and 15 sec at 72 C. For ER
, 40 cycles at an annealing
temperature of 63 C was used. At the end of each cycle, the amount of
double-stranded DNA was monitored by measuring the level of SYBR Green
I fluorescence. After the completion of all cycles, a level of
fluorescence was selected at which all of the standards and samples
were within the linear range of amplification. The crossing point, or
the number of cycles necessary for each sample or standard to obtain
the selected level of fluorescence, was calculated using the
Roche Lightcycler software. Based on these crossing
points, a standard curve was generated, and the number of ER
copies
was calculated for each sample. The data presented represent the number
of copies of ER
in 1 µl of diluted RT product, which corresponds
to 10 ng of starting RNA.
Culture and Transfection of COS Cells
COS cells were routinely cultured in DMEM medium supplemented
with 10% FBS, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and
0.25 µg/ml Amphotericin. Cultures were carried out at 37
C in a 5% CO2, humidified atmosphere. For
transient transfections, 100K cells were seeded per well in six-well
plates and cultured as described above for 24 h. In general, cells
were 50% confluent at the start of transfection. For transfections,
DNA was combined with water (90 µl/well) and 2.5 M
CaCl2 (10 µl/well) and mixed. In general, a
total of 45 µg DNA were transfected per well, and the total amount
of DNA was equalized with empty vector when necessary. An equal
volume of 2x
N,-N-bis[2-hydroxyethyl]-2-amino-ethanesulfonic
acid-buffered saline was added, and DNA was allowed to
precipitate at room temperature for 10 min. The DNA was then added
dropwise to each well, and cells were cultured for 24 h at 3%
CO2. Twenty-four hours after the start of
transfection, media were changed to standard culture media supplemented
with 1% FBS, and cells were cultured for an additional 24 h at
5% CO2 in the presence or absence of various
inhibitors. After treatment, cells were washed twice with cold PBS and
stored at -80 C for reporter assays.
Reporter Assays
Luciferase and ß-gal activities were measured by first
preparing cell lysates in 1x reporter lysis buffer. Luciferase
activity driven by the ER
promoter was measure by combining lysate
with firefly luciferase assay substrate and measuring luminescence for
10 sec on a Lumat LB 9507 luminometer (EG&G Berthold, Oak Ridge,
TN). As a control, cells were cotransfected with an expression
vector for ß-gal. ß-gal Activity was measured in a separate aliquot
of lysate by incubating with a luminescent ß-gal substrate for 1
h at room temperature and then measuring luminescence for 5 sec. The
luciferase activity was normalized to the ß-gal activity within the
same well.
Immunoprecipitation and Western Blotting
WCE from primary luteinized granulosa cells and COS cells were
prepared by lysing cells in RIPA buffer (1x PBS, 1% Nonidet, 0.5%
sodium deoxycholate, 0.1% SDS) containing 1 µM sodium
orthovanadate, 10 µg/ml PMSF, and 30 µl/ml aprotinin. For
immunoprecipitation, 500 µg of WCE were incubated with 4-µl
anti-Stat5a or anti-Stat5b antibodies for 1 h at 4 C. Protein A/G
agarose beads were added, and the mixture was incubated overnight at 4
C on a rocking platform. The beads were washed four times in PBS,
resuspended in 2x electrophoresis buffer, and boiled for 5 min. For
Western blots performed on WCE, protein was diluted in an equal volume
of 2x electrophoresis buffer and boiled for 5 min. Twenty microliters
of immunoprecipitated protein or 20 µg of WCE were separated on a
10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Western
blotting was performed by blocking nonspecific binding with 5% dry
milk in Tris-buffered saline buffer containing 0.05% Tween 20 for
1 h. Blots were then incubated with the primary antibody overnight
at 4 C on a rocking platform. After a series of washes, blots were
incubated with a secondary antibody linked to horseradish peroxidase
for 1 h. After extensive washing, blots were analyzed using an
enhanced chemiluminescence detection system and exposed to x-ray
film.
EMSA
Nuclear extracts were prepared from cell cultures by a published
method (70). A probe corresponding to the bovine
ß-casein Stat5 response element (15) was labeled by
incubating 0.5 pmol annealed oligonucleotides with 11 U T4 kinase and
25 µCi
-32P ATP (3,000 µCi/mmol). The
specific activity of the probes was greater than 8,000 cpm/fmol.
Ten-microgram nuclear extracts were incubated with 50K cpm of labeled
probes in 1x binding buffer (12 mM HEPES, pH 7.9, 40
mM KCl, 5 mM MgCl2, 0.12
mM EDTA, 0.06 mM EGTA, 0.5 mM
dithiothreitol, 10% glycerol) at room temperature for 30 min. The
samples were then run on a 4.5% nondenaturing polyacrylamide gel in
0.25x Tris-buffered EDTA buffer at 200 V for 23 h. The gels
were dried and analyzed by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to O. Silvennoinen for the Jak2 expression
vectors, Jean Djiane and Paul Kelly for the PRL-R expression vectors,
and Toshio Kitamura for the Stat5 expression vectors.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants HD-11119 and HD-12356 (to
GG).
Abbreviations: ß-gal, ß-Galactosidase; CA-Jak2,
constitutively active Jak2; dNTP, deoxynucleotide triphosphate; Jak,
Janus kinase; KN-Jak2; kinase-negative Jak2; PRL-R, PRL receptor;
PRL-RCA, constitutively active PRL-R;
PRL-RL, long form of PRL-R; STAT, signal
transducer and activator of transcription; WCE, whole-cell
extract.
Received for publication March 26, 2001.
Accepted for publication July 24, 2001.
 |
REFERENCES
|
---|
-
Gibori G, Richards JS, Keyes PL 1979 Prolactin control of
receptor for estradiol in corpora lutea of pregnant rats. Adv Exp Med
Biol 112:5358[Medline]
-
Gibori G, Richards JS 198 Dissociation of two distinct
luteotropic effects of prolactin: regulation of luteinizing
hormone-receptor content and progesterone secretion during pregnancy.
Endocrinology 102:767774
-
Telleria CM, Zhong L, Deb S, Srivastava RK, Park KS, Sugino
N, Park-Sarge OK, Gibori G 1998 Differential expression of the estrogen
receptors
and ß in the rat corpus luteum of pregnancy: regulation
by prolactin and placental lactogens. Endocrinology 139:24322442[Abstract/Free Full Text]
-
Frasor J, Park K, Byers M, Telleria CM, Kitamura T, Yu-Lee
LY, Djiane J, Park-Sarge OK, Gibori G, Differential roles for Stat5a
and Stat5b in prolactin stimulation of estrogen receptor
and ß
transcription. Mol Endocrinol, in press
-
Bazan JF 1989 A novel family of growth factor receptors: a
common binding domain in the growth hormone, prolactin, erythropoietin
and IL-6 receptors, and the p75 IL-2 receptor ß-chain. Biochem
Biophys Res Commun 164:78895[Medline]
-
Rozakis-Adcock M, Kelly PA 1991 Mutational analysis of the
ligand-binding domain of the prolactin receptor. J Biol Chem 266:1647216477[Abstract/Free Full Text]
-
Dusanter-Fourt I, Muller O, Ziemiecki A, Mayeux P, Drucker B,
Djiane J, Wilks A, Harpur AG, Fischer S, Gisselbrecht S 1994 Identification of JAK protein tyrosine kinases as signaling molecules
for prolactin. Functional analysis of prolactin receptor and
prolactin-erythropoietin receptor chimera expressed in lymphoid cells.
EMBO J 13:25832591[Abstract]
-
Campbell GS, Argetsinger LS, Ihle JN, Kelly PA, Rillema JA,
Carter-Su C 1994 Activation of JAK2 tyrosine kinase by prolactin
receptors in Nb2 cells and mouse mammary gland explants. Proc Natl Acad
Sci USA 91:52325236[Abstract]
-
Rui H, Kirken RA, Farrar WL 1994 Activation of
receptor-associated tyrosine kinase JAK2 by prolactin. J Biol Chem 269:53645368[Abstract/Free Full Text]
-
Zhong L, Parmer TG, Robertson MC, Gibori G 1997 Prolactin-mediated inhibition of 20
-hydroxysteroid dehydrogenase
gene expression and the tyrosine kinase system. Biochem Biophys Res
Commun 235:587592[CrossRef][Medline]
-
David M, Petricoin 3rd EF, Igarashi K, Feldman GM, Finbloom
DS, Larner AC 1994 Prolactin activates the interferon-regulated p91
transcription factor and the Jak2 kinase by tyrosine phosphorylation.
Proc Natl Acad Sci USA 91:71747178[Abstract]
-
Lebrun JJ, Ali S, Sofer L, Ullrich A, Kelly PA 1994 Prolactin-induced proliferation of Nb2 cells involves tyrosine
phosphorylation of the prolactin receptor and its associated
tyrosine kinase JAK2. J Biol Chem 269:1402114026[Abstract/Free Full Text]
-
Goupille O, Daniel N, Bignon C, Jolivet G, Djiane J 1997 Prolactin signal transduction to milk protein genes: carboxy-terminal
part of the prolactin receptor and its tyrosine phosphorylation are not
obligatory for JAK2 and STAT5 activation. Mol Cell Endocrinol 127:155169[CrossRef][Medline]
-
Pezet A, Buteau H, Kelly PA, Edery M 1997 The last proline of
Box 1 is essential for association with JAK2 and functional activation
of the prolactin receptor. Mol Cell Endocrinol 129:199208[CrossRef][Medline]
-
Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces
phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA
binding and induction of transcription. EMBO J 13:43614369[Abstract]
-
Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning and expression of Stat5 and an additional homologue (Stat5b)
involved in prolactin signal transduction in mouse mammary tissue. Proc
Natl Acad Sci USA 92:88318835[Abstract]
-
Gao J, Hughes JP, Auperin B, Buteau H, Edery M, Zhuang H,
Wojchowski DM, Horseman ND 1996 Interactions among Janus kinases and
the prolactin (PRL) receptor in the regulation of a PRL response
element. Mol Endocrinol 10:847856[Abstract]
-
Han Y, Watling D, Rogers NC, Stark GR 1997 JAK2 and STAT5, but
not JAK1 and STAT1, are required for prolactin-induced
ß-lactoglobulin transcription. Mol Endocrinol 11:11801188[Abstract/Free Full Text]
-
Franklin RB, Ekiko DB, Costello LC 1992 Prolactin stimulates
transcription of aspartate aminotransferase in prostate cells. Mol Cell
Endocrinol 90:2732[CrossRef][Medline]
-
Rillema JA, Waters SB, Tarrant TM 1989 Studies on the possible
role of protein kinase C in the prolactin regulation of cell
replication in Nb2 node lymphoma cells. Proc Soc Exp Biol Med 192:140144[Abstract]
-
DeVito WJ, Avakian C, Stone S, Okulicz WC 1993 Prolactin-stimulated mitogenesis of cultured astrocytes is mediated by
a protein kinase C-dependent mechanism. J Neurochem 60:832842[Medline]
-
Fan G, Rillema JA 1993 Prolactin stimulation of protein kinase
C in isolated mouse mammary gland nuclei. Horm Metab Res 25:564568[Medline]
-
Villanueva LA, Mendez I, Ampuero S, Larrea F 1996 The
prolactin inhibition of follicle-stimulating hormoneinduced
aromatase activity in cultured rat granulosa cells is in part tyrosine
kinase and protein kinase-C dependent. Mol Hum Reprod 2:725731[Abstract]
-
Fenton SE, Sheffield LG 1997 Prolactin inhibits EGF-induced
DNA synthesis in mammary epithelium via early signaling mechanisms:
possible involvement of protein kinase C. Exp Cell Res 236:285293[CrossRef][Medline]
-
al-Sakkaf KA, Dobson PR, Brown BL 1996 Activation of
phosphatidylinositol 3-kinase by prolactin in Nb2 cells. Biochem
Biophys Res Commun 221:779784[CrossRef][Medline]
-
Ratovondrahona D, Fournier B, Odessa MF, Dufy B 1998 Prolactin
stimulation of phosphoinositide metabolism in CHO cells stably
expressing the PRL receptor. Biochem Biophys Res Commun 243:127130[CrossRef][Medline]
-
Al-Sakkaf KA, Mooney LM, Dobson PR, Brown BL 2000 Possible
role for protein kinase B in the anti-apoptotic effect of prolactin in
rat Nb2 lymphoma cells. J Endocrinol 167:8592[Abstract/Free Full Text]
-
Carey GB, Liberti JP 1995 Stimulation of receptor-associated
kinase, tyrosine kinase, and MAP kinase is required for
prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2
lymphoma. Arch Biochem Biophys 316:179189[CrossRef][Medline]
-
Buckley AR, Rao YP, Buckley DJ, Gout PW 1994 Prolactin-induced
phosphorylation and nuclear translocation of MAP kinase in Nb2 lymphoma
cells. Biochem Biophys Res Commun 204:11581164[CrossRef][Medline]
-
Camarillo IG, Linebaugh BE, Rillema JA 1997 Differential
tyrosyl-phosphorylation of multiple mitogen-activated protein kinase
isoforms in response to prolactin in Nb2 lymphoma cells. Proc Soc Exp
Biol Med 215:198202[Abstract]
-
Buckley AR 2000 Transcriptional regulation of pim-1 by
prolactin: independence of a requirement for Jak2/Stat signaling.
J Neuroimmunol 109:4046[CrossRef][Medline]
-
Nohara A, Ohmichi M, Koike K, Jikihara H, Kimura A, Masuhara
K, Ikegami H, Inoue M, Miyake A, Murata Y 1997 Prolactin stimulates
mitogen-activated protein kinase in human leiomyoma cells. Biochem
Biophys Res Commun 238:473477[CrossRef][Medline]
-
Das R, Vonderhaar BK 1996 Activation of raf-1, MEK, and MAP
kinase in prolactin responsive mammary cells. Breast Cancer Res Treat 40:141149[Medline]
-
Das R, Vonderhaar BK 1996 Involvement of SHC, GRB2, SOS and
RAS in prolactin signal transduction in mammary epithelial cells.
Oncogene 13:11391145[Medline]
-
Piccoletti R, Bendinelli P, Maroni P 1997 Signal transduction
pathway of prolactin in rat liver. Mol Cell Endocrinol 135:169177[CrossRef][Medline]
-
Sorensen P, Sheffield LG 1997 Involvement of c-src in
ß-casein expression by mammary epithelial cells. Biochem Biophys Res
Commun 241:710713[CrossRef][Medline]
-
Clevenger CV, Medaglia MV 1994 The protein tyrosine kinase
P59fyn is associated with prolactin (PRL) receptor and is activated by
PRL stimulation of T-lymphocytes. Mol Endocrinol 8:674681[Abstract]
-
Berlanga JJ, Fresno Vara JA, Martin-Perez J, Garcia-Ruiz JP 1995 Prolactin receptor is associated with c-src kinase in rat liver.
Mol Endocrinol 9:14611467[Abstract]
-
Peters CA, Maizels ET, Hunzicker-Dunn M 1999 Activation of PKC
delta in the rat corpus luteum during pregnancy. Potential role of
prolactin signaling. J Biol Chem 274:3749937505[Abstract/Free Full Text]
-
Peters CA, Maizels ET, Robertson MC, Shiu RP, Soloff MS,
Hunzicker-Dunn M 2000 Induction of relaxin messenger RNA expression in
response to prolactin receptor activation requires protein kinase C
signaling. Mol Endocrinol 14:576590[Abstract/Free Full Text]
-
Peters CA, Cutler RE, Maizels ET, Robertson MC, Shiu RP,
Fields P, Hunzicker-Dunn M 2000 Regulation of PKC
expression by
estrogen and rat placental lactogen-1 in luteinized rat ovarian
granulosa cells. Mol Cell Endocrinol 162:181191[CrossRef][Medline]
-
Tessier C, Prigent-Tessier A, Ferguson-Gottschall S,
Gibori G 2000 PRL and TGFß 1 play opposite roles in the
regulation of apoptosis in the rat decidua. Proc
32nd Annual Meeting of the Society for the Study
of Reproduction, Pullman, WA, 2000, p 218 (Abstract)
-
al-Sakkaf KA, Dobson PR, Brown BL 1997 Prolactin induced
tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol
3-kinase activation in Nb2 cells. J Mol Endocrinol 19:347350[Abstract/Free Full Text]
-
Hunter S, Burton EA, Wu SC, Anderson SM 1999 Fyn associates
with Cbl and phosphorylates tyrosine 731 in Cbl, a binding site for
phosphatidylinositol 3-kinase. J Biol Chem 274:20972106[Abstract/Free Full Text]
-
Fan G, Rillema JA 1992 Effect of a tyrosine kinase inhibitor,
genistein, on the actions of prolactin in cultured mouse mammary
tissues. Mol Cell Endocrinol 83:5155[CrossRef][Medline]
-
Daniel N, Waters MJ, Bignon C, Djiane J 1996 Involvement of a
subset of tyrosine kinases and phosphatases in regulation of the
ß-lactoglobulin gene promoter by prolactin. Mol Cell Endocrinol 118:2535[CrossRef][Medline]
-
Dajee M, Kazansky AV, Raught B, Hocke GM, Fey GH, Richards JS 1996 Prolactin induction of the
2-macroglobulin gene in rat ovarian
granulosa cells: stat 5 activation and binding to the interleukin-6
response element. Mol Endocrinol 10:171184[Abstract]
-
Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot Z,
Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roifman CM 1996 Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor.
Nature 379:645648[CrossRef][Medline]
-
Kirken RA, Erwin RA, Taub D, Murphy WJ, Behbod F, Wang L,
Pericle F, Farrar WL 1999 Tyrphostin AG-490 inhibits cytokine-mediated
JAK3/STAT5a/b signal transduction and cellular proliferation of
antigen-activated human T cells. J Leukoc Biol 65:891899[Abstract]
-
Wang LH, Kirken RA, Erwin RA, Yu CR, Farrar WL 1999 JAK3,
STAT, and MAPK signaling pathways as novel molecular targets for the
tyrphostin AG-490 regulation of IL-2-mediated T cell response.
J Immunol 162:38973904[Abstract/Free Full Text]
-
Zhu T, Lobie PE 2000 Janus kinase 2-dependent activation of
p38 mitogen-activated protein kinase by growth hormone. Resultant
transcriptional activation of ATF-2 and CHOP, cytoskeletal
re-organization and mitogenesis. J Biol Chem 275:21032114[Abstract/Free Full Text]
-
De Vos J, Jourdan M, Tarte K, Jasmin C, Klein B 2000 JAK2
tyrosine kinase inhibitor tyrphostin AG490 downregulates the
mitogen-activated protein kinase (MAPK) and signal transducer and
activator of transcription (STAT) pathways and induces apoptosis in
myeloma cells. Br J Haematol 109:823828[CrossRef][Medline]
-
Gourdou I, Gabou L, Paly J, Kermabon AY, Belair L, Djiane J 1996 Development of a constitutively active mutant form of the
prolactin receptor, a member of the cytokine receptor family. Mol
Endocrinol 10:4556[Abstract]
-
Paukku K, Valgeirsdottir S, Saharinen P, Bergman M, Heldin CH,
Silvennoinen O 2000 Platelet-derived growth factor (PDGF)-induced
activation of signal transducer and activator of transcription (Stat) 5
is mediated by PDGF ß-receptor and is not dependent on c-src, fyn,
jak1 or jak2 kinases. Biochem J 345:759766[CrossRef][Medline]
-
Saharinen P, Takaluoma K, Silvennoinen O 2000 Regulation of
the Jak2 tyrosine kinase by its pseudokinase domain. Mol Cell Biol 20:33873395[Abstract/Free Full Text]
-
Kazansky AV, Kabotyanski EB, Wyszomierski SL, Mancini MA,
Rosen JM 1999 Differential effects of prolactin and src/abl kinases on
the nuclear translocation of STAT5B and STAT5A. J Biol Chem 274:2248422492[Abstract/Free Full Text]
-
Fresno Vara JA, Carretero MV, Geronimo H, Ballmer-Hofer K,
Martin-Perez J 2000 Stimulation of c-Src by prolactin is independent of
Jak2. Biochem J 345:1724[CrossRef][Medline]
-
Mangoura D, Pelletiere C, Leung S, Sakellaridis N, Wang DX 2000 Prolactin concurrently activates src-PLD and JAK/Stat signaling
pathways to induce proliferation while promoting differentiation in
embryonic astrocytes. Int J Dev Neurosci 18:693704[CrossRef][Medline]
-
Laffargue M, Ragab-Thomas JM, Ragab A, Tuech J, Missy K,
Monnereau L, Blank U, Plantavid M, Payrastre B, Raynal P, Chap H 1999 Phosphoinositide 3-kinase and integrin signalling are involved in
activation of Bruton tyrosine kinase in thrombin-stimulated platelets.
FEBS Lett 443:6670[CrossRef][Medline]
-
Varnai P, Rother KI, Balla T 1999 Phosphatidylinositol
3-kinase-dependent membrane association of the Brutons tyrosine
kinase pleckstrin homology domain visualized in single living
cells. J Biol Chem 274:1098310989[Abstract/Free Full Text]
-
Qiu Y, Robinson D, Pretlow TG, Kung HJ 1998 Etk/Bmx, a
tyrosine kinase with a pleckstrin-homology domain, is an effector of
phosphatidylinositol 3'-kinase and is involved in interleukin 6-induced
neuroendocrine differentiation of prostate cancer cells. Proc Natl Acad
Sci USA 95:36443649[Abstract/Free Full Text]
-
August A, Sadra A, Dupont B, Hanafusa H 1997 Src-induced
activation of inducible T cell kinase (ITK) requires
phosphatidylinositol 3-kinase activity and the Pleckstrin homology
domain of inducible T cell kinase. Proc Natl Acad Sci USA 94:1122711232[Abstract/Free Full Text]
-
Saharinen P, Ekman N, Sarvas K, Parker P, Alitalo K,
Silvennoinen O 1997 The Bmx tyrosine kinase induces activation of the
Stat signaling pathway, which is specifically inhibited by protein
kinase C
. Blood 90:43414353[Abstract/Free Full Text]
-
Yamauchi T, Kaburagi Y, Ueki K, Tsuji Y, Stark GR, Kerr IM,
Tsushima T, Akanuma Y, Komuro I, Tobe K, Yazaki Y, Kadowaki T 1998 Growth hormone and prolactin stimulate tyrosine phosphorylation of
insulin receptor substrate-1, -2, and -3, their association with p85
phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly
PI3-kinase activation via JAK2 kinase. J Biol Chem 273:1571915726[Abstract/Free Full Text]
-
Berlanga JJ, Gualillo O, Buteau H, Applanat M, Kelly PA, Edery
M 1997 Prolactin activates tyrosyl phosphorylation of insulin receptor
substrate 1 and phosphatidylinositol-3-OH kinase. J Biol Chem 272:20502052[Abstract/Free Full Text]
-
Hunter S, Koch BL, Anderson SM 1997 Phosphorylation of cbl
after stimulation of Nb2 cells with prolactin and its association with
phosphatidylinositol 3-kinase. Mol Endocrinol 11:12131222[Abstract/Free Full Text]
-
Storz P, Doppler H, Pfizenmaier K, Muller G 1999 Insulin
selectively activates STAT5b, but not STAT5a, via a JAK2-independent
signalling pathway in Kym-1 rhabdomyosarcoma cells. FEBS Lett 464:159163[CrossRef][Medline]
-
Chen J, Sadowski HB, Kohanski RA, Wang LH 1997 Stat5 is a
physiological substrate of the insulin receptor. Proc Natl Acad Sci USA 94:22952300[Abstract/Free Full Text]
-
Sugino N, Zilberstein M, Srivastava RK, Telleria CM,
Nelson SE, Risk M, Chou JY, Gibori G 1998 Establishment and
characterization of a simian virus 40-transformed temperature-sensitive
rat luteal cell line. Endocrinology 139:19361942[Abstract/Free Full Text]
-
Andrews NC, Faller DV 1991 A rapid micropreparation technique
for extraction of DNA-binding proteins from limiting numbers of
mammalian cells. Nucleic Acids Res 19:2499[Medline]