The Protein Tyrosine Phosphatase-PEST Is Implicated in the Negative Regulation of Epidermal Growth Factor on PRL Signaling in Mammary Epithelial Cells
Kay Horsch,
Michael D. Schaller and
Nancy E. Hynes
Friedrich Miescher Institute, CH-4002 Basel, Switzerland; and
Department of Cell Biology and Anatomy, University of North Carolina
(M.D.S.), Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: Friedrich Miescher Institute, R-1066.206, Maulbeerstrasse 66, CH-4058 Basel, Switzerland. E-mail: nancy.hynes{at}fmi.ch
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ABSTRACT
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Treatment of HC11 mammary epithelial cells with the lactogenic
hormone PRL promotes differentiation and induction of milk protein gene
expression via stimulation of the Janus kinase (JAK)/signal transducer
and activator of transcription pathway. We have previously shown that
autocrine activation of epidermal growth factor (EGF) receptor
interferes with normal PRL-induced differentiation. Here we show that
PRL activation of JAK2 was dramatically reduced in HC11 cells
pretreated with EGF, demonstrating that the target of EGF receptor
activation is JAK2 kinase. Using an in-gel protein tyrosine phosphatase
(PTP) assay, we observed that the activity of a 125-kDa PTP was
up-regulated in HC11 cells in response to EGF. A specific antiserum was
used to demonstrate that the 125-kDa PTP was PTP-PEST and to
show that EGF treatment of HC11 cells led to an increase in the level
of PTP-PEST. In intact HC11 cells, PTP-PEST was constitutively
associated with JAK2, and in response to EGF treatment there was an
increased level of PTP-PEST in JAK2 complexes. An in
vitro phosphatase assay, using PRL-activated JAK2 as the
substrate and lysates from HC11 cells as the source of PTP-PEST,
revealed that JAK2 could serve as a PTP-PEST substrate. However, in
intact cells the regulation of JAK2 by PTP-PEST was complex, since
transient overexpression of PTP-PEST had a negligible effect on
PRL-induced JAK2 activation. EGFs negative influence on JAK2 activity
was blocked by actinomycin D treatment of HC11 cells, suggesting that
EGF induced a protein that mediated the effects of PTP-PEST on JAK2. In
support of this model, PTP-PEST-containing lysates from EGF-treated
HC11 cells dephosphorylated JAK2 to a greater extent than lysates
prepared from control cells.
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INTRODUCTION
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MAMMARY GLAND differentiation requires the
coordinated action of growth factors and hormones that promote
morphological development and functional differentiation. Mammary
cancer results from genetic changes that alter these normal processes.
To probe the molecular events contributing to normal mammary gland
development and cancer, in vitro cell culture systems have
proven invaluable. The HC11 mouse mammary cell line is a useful model
system for studying mammary cell differentiation, as treatment of the
cells with the lactogenic hormones, PRL and glucocorticoids, leads to
the production of several milk proteins (1, 2, 3). The
intracellular targets of PRL are Janus kinase 2 (JAK2) and signal
transducer and activator of transcription 5 (STAT5). Ligand-induced
activation of PRL receptor (PRLR) leads to stimulation of the JAK2
tyrosine kinase that phosphorylates STAT5 on a conserved tyrosine
residue, leading to STAT5 dimerization, nuclear migration, and
stimulation of target gene transcription, including the gene encoding
the milk protein, ß-casein (4, 5, 6).
We have previously reported that hormonal induction of ß-casein
expression is blocked by the addition of epidermal growth factor (EGF)
to the lactogenic hormone-containing medium (7).
Furthermore, in oncogene-transformed HC11 cells displaying
autocrine activation of the EGF receptor (EGFR), PRL-induced
differentiation is also inhibited (8, 9). Considering the
importance of the EGF-related family of ligands and their receptors in
mammary gland development and in human cancer (10, 11, 12, 13), we
have further explored the mechanism underlying the EGF effect on
differentiation. PRL-induced activation of the JAK2 kinase was
dramatically reduced in cells pretreated with EGF, suggesting that EGF
targets the PRLR-associated kinase. We found that a cytosolic protein
tyrosine phosphatase (PTP), PTP-PEST, is up-regulated in
EGF-treated HC11 cells. PTP-PEST, which is termed after proline
glutamine serine threonine-rich motifs (PEST sequences), is
ubiquitously expressed (14, 15, 16) and is known to have
functions in focal adhesion breakdown, cell spreading, and cell
motility (17, 18). PTP-PEST was able to dephosphorylate
JAK2 in an in vitro phosphatase assay. In vivo,
in HC11 cells we found that JAK2 and PTP-PEST are constitutively
complexed, and EGF treatment of the cells leads to elevated PTP-PEST
expression and an increased association with JAK2. Moreover, EGF
appears to induce an as yet unknown protein, which has a permissive
effect on PTP-PEST, allowing it to target and inhibit JAK2 in the PRLR
complex. The results presented here show that PRL signaling in mammary
cells is controlled by a novel mechanism that appears to involve
PTP-PEST, a PTP that has not previously been implicated in
down-regulation of cytokine signaling. Considering that many breast
tumors display constitutive autocrine activation of the EGFR due to
production of one or more of its ligands (11), this
pathway may contribute to the differentiation block, which is a
characteristic of mammary cancers.
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RESULTS
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STAT5 DNA Binding Is Negatively Regulated by EGF
In HC11 cells, PRL binding to its receptor leads to JAK2-mediated
phosphorylation of STAT5 on a conserved tyrosine residue;
Tyr694 for STAT5a and
Tyr699 for STAT5b (19). The
activated STAT5 proteins dimerize and enter the nucleus, where they
bind to a
-interferon-activated sequence-like site in the
promoter of the ß-casein gene, stimulating its transcription
(4, 5, 6, 20). To investigate the lactogenic hormone-induced
formation of STAT5-DNA complexes in EGF-treated HC11 cells, EMSAs were
performed using nuclear extracts from hormone-treated cells and the
STAT5-binding site from the ß-casein promoter as a probe. The
cultures were treated for 16 h with EGF before adding lactogenic
hormones for 15 min, a time when STAT5 DNA binding is strong and easily
measurable (20). The EMSAs were quantified, revealing that
STAT5-binding activity was decreased by 54% in the EGF-treated
cultures (Fig. 1A
, lane 2 vs.
lane 4). Considering the importance of phosphotyrosine for STAT5 DNA
binding, we examined the possibility that EGF induced a PTP. HC11
cultures were treated with the PTPase inhibitor, orthovanadate. At a
high concentration (1000 µM), orthovanadate
nonspecifically induced STAT5 DNA binding in the absence of lactogenic
hormones (Fig. 1C
, lane 13). However, at 100
µM, orthovanadate had no effect on basal or
lactogenic hormone-induced STAT5 DNA binding (Fig. 1B
, lane 9).
Importantly, the block in STAT5 DNA binding observed in EGF-treated
cultures was essentially reversed in cultures treated with 100
µM orthovanadate. Quantification revealed that
binding of STAT5 from vanadate-treated cultures reached 88% of the
control level (Fig. 1A, lane 5 vs. lane 2). The results
suggest that the negative effect of EGF on STAT5 may ensue through
activation of a PTP, which targets the Tyr residues essential for DNA
binding.

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Figure 1. EGF Inhibits the Induction of STAT5 DNA Binding
Activity
A and B, Competent cultures of HC11 cells that had either been
incubated 16 h with EGF (+) or left untreated (-) were treated
for 15 min with lactogenic hormones (DIP; +) or left untreated (-)
before lysis. In some cultures vanadate (100 µM) was
added 90 min before lysis. In the experiment shown in C, competent HC11
cultures were treated with different concentrations of vanadate 90 min
before lysis. Nuclear extracts were prepared, and EMSAs were performed
using a 30-bp double stranded oligonucleotide encompassing the STAT5
DNA-binding site of the rat ß-casein promoter as a probe. The
specific STAT5 bandshift and the free probe are shown.
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EGF Inhibits Lactogenic Hormone-Induced JAK2 Kinase Activity
To test for effects of PTPs on the JAK/STAT pathway, we examined
the phosphotyrosine content of STAT5 and the other members of this
pathway, JAK2 and PRLR. Lysates were prepared from control cells
treated for 15 min with lactogenic hormones
[10-6 M dexamethasone, 5 µg/ml
insulin, and 5 µg/ml ovine PRL (DIP)] or cells pretreated for
16 h with EGF before lactogenic hormone addition. STAT5, JAK2, and
PRLR were each immunoprecipitated and probed for phosphotyrosine
content. The expression level of PRLR, JAK2, and STAT5 was essentially
the same in control and EGF-treated cultures (Fig. 2A
, lower three panels).
Notably, all three proteins showed a 6080% reduction in their
phosphotyrosine content in EGF-treated compared with control cultures
(Fig. 2A
, upper panels; quantified in Fig. 2B
). The lysates
were also probed directly with phospho-JAK2 antiserum, which recognizes
phospho-Tyr1007 located in the regulatory loop of
JAK2 (21), and with phospo-STAT5-specific serum, which
recognizes the JAK2 phosphorylation sites on STAT5a/b. The
phosphorylation of both proteins was decreased by more than 90% in the
EGF-treated cultures (Fig. 2A
, upper right panels;
quantified in Fig. 2B
).

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Figure 2. EGF Negatively Regulates Lactogenic Hormone
Activation of the JAK/STAT Pathway
A, Competent cultures of HC11 cells that had either been incubated
16 h with EGF (+) or left untreated (-) were treated for 15 min
with lactogenic hormones (DIP; +) or left untreated (-) before lysis.
Whole cell lysates were used for immunoprecipitation of PRLR, JAK2, and
STAT5a. Immunoprecipitates were analyzed by 7.5% SDS-PAGE, blotted
onto a polyvinylidene difluoride membrane, and probed with
phosphotyrosine-specific monoclonal antibody. After stripping, the
membranes were reprobed with the specific antibodies to control for
loading. The same lysates were directly analyzed with phospho-specific
antibodies for JAK2 and STAT5. B, Quantification of the
phosphorylations shown directly above in A. ImageQuant software
(Molecular Dynamics, Inc.) was used to quantify the
phosphorylations and to normalize it to protein. Values shown are
relative to DIP-induced phosphorylation ( ). C, JAK2 kinase activity
in HC11 cultures treated as described in A. Where indicated, vanadate
(100 µM) was added 90 min before lysis.
Immunoprecipitates of JAK2 were incubated in kinase buffer with ATP for
30 min. After the kinase reaction was stopped, the autophosphorylated
immunocomplexes were analyzed by 7.5% SDS-PAGE, blotted onto a
polyvinylidene difluoride membrane, and probed with a
phosphotyrosine-specific monoclonal antibody. After stripping, the
membrane was reprobed with JAK2-specific antiserum. The lowest panel
shows a direct Western blot with phospho-specific JAK2 antibody. D,
Quantification of JAK2 kinase activity ( ) and the corresponding
phospho-JAK2 levels ( ) shown in C. ImageQuant software was used to
quantify the phosphorylations and to normalize it to protein. Values
shown are relative to DIP-induced activity or phosphorylation.
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The preceding results strongly suggest that in HC11 cells JAK2 kinase
is the target for negative regulation by EGF. We performed in
vitro kinase assays with JAK2 (22, 23) to directly
test this possibility. JAK2 was immunoprecipitated from HC11 cultures
treated with various combinations of lactogenic hormones, EGF, and
orthovanadate. The immunoprecipitates were incubated with kinase buffer
in the presence of cold ATP, and JAK2 activity was measured by
determining its phosphotyrosine content in a Western analysis (Fig. 2C
, upper panels). The same lysates were also probed directly
with the phospho-JAK2-specific serum (Fig. 2C
, lower panel).
The activity of JAK2 isolated from cultures of lactogenic hormone
(DIP)-treated cultures was increased 2-fold relative to the basal
activity seen in control or EGF-treated cultures (Fig. 2C
, lane 3
vs. lanes 1 and 2; quantified in Fig. 2D
). Moreover, EGF
treatment severely blocked lactogenic hormone induction of JAK2
activity, returning it to the basal level (Fig. 2C
, lane 4
vs. lane 1; quantified in Fig. 2D
). Importantly,
orthovanadate had no effect on basal JAK2 activity (Fig. 2C
, lane 6);
however, it almost completely reversed (96%) the negative influence of
EGF on lactogenic hormone-induced JAK2 activity (Fig. 2C
, lane 5
vs. lane 3; quantified in Fig. 2D
). These results strongly
support the hypothesis that EGF influences the activity of a PTP that
negatively regulates JAK2 activity.
A 125-kDa PTPase Has Higher Activity in EGF-Treated Cells
To test the involvement of PTPs in the EGF inhibition of JAK/STAT
signaling, we employed an in-gel assay, which reveals the activity of
cytoplasmic PTPs after their separation by SDS-PAGE and in-gel
renaturation (24). Lysates from HC11 cells routinely
displayed 10 active PTPs in the in-gel assay (not shown); the four most
active PTPs migrate as proteins of approximately 50, 62, 64, and 125
kDa (Fig. 3A
). None of these PTPs was
affected by lactogenic hormone treatment. However, the activity of the
125-kDa PTP was elevated in lysates prepared from EGF-treated cells.
Equal loading of the gel was confirmed by Coomassie staining (Fig. 3B
).
Immunoprecipitates of the EGFR were also examined by an in-gel PTP
assay. Only one band of activity was observed, which comigrated with
the activity at 125 kDa. Intriguingly, in comparison to long-term
treatment (Fig. 3A
), its activity was not affected by short-term (15
min) EGF treatment (Fig. 3C
).

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Figure 3. A 125-kDa Protein Tyrosine Phosphatase Has Higher
Activity in EGF-Treated Cells
Competent cultures of HC11 cells that had either been incubated 16
h with EGF (+) or left untreated (-) were treated for 15 min with
lactogenic hormones (DIP; +) or left untreated (-) before lysis.
Protein tyrosine phosphatase activities were analyzed from whole cell
lysates (A) and from immunoprecipitates of EGFR (C) in an in-gel
phosphatase assay, using [32P]poly-(Glu:Tyr) as the
gel-embedded substrate. After SDS-PAGE, SDS was removed from the gel,
proteins were renatured, and the gel was incubated in phosphatase
buffer. The phosphatase reaction was terminated by Coomassie staining
of the gel that also served to control for loading (B). Gels were
dried, and PTP activities were visualized in A and C using a
PhosphorImager (Molecular Dynamics, Inc.). C, Cells were
incubated for 15 min with EGF (+). The asterisk in A
marks the activities of SHP1 and SHP2.
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The 125-kDa PTP Is PTP-PEST
Considering that PTP-PEST is approximately 125 kDa and has been
reported to associate with EGFR (25), this PTP was
examined more closely. PTP-PEST was specifically immunoprecipitated
from lysates of HC11 cells, which had been treated with EGF and/or the
lactogenic hormones. The results show that the activity of the
immunoprecipitated PTP-PEST (Fig. 4A
, upper panel) has the
same electrophoretic mobility as the 125-kDa activity identified in the
previous experiment (Fig. 3
). Furthermore, PTP-PEST activity was
increased 2-fold in all cultures treated with EGF, but not in cultures
treated with lactogenic hormones alone (Fig. 4
, A and B, lanes 2 and 4 vs.
lane 3). There was also more PTP-PEST in the immunoprecipitates from
EGF-treated compared with control HC11 cultures (Fig. 4A
, lower
panel), suggesting that the increased PTP-PEST activity seen in
the EGF-treated cultures might reflect increased PTP-PEST
expression.
To examine whether PTP-PEST was the only active PTP migrating at 125
kDa, PTP-PEST was immunodepleted from lysates of EGF-treated HC11
cells. The supernatant from the first PTP-PEST immunoprecipitation
(Fig. 4C
, lane 2) had low levels of activity (not visible in this
exposure). After two rounds of immunoprecipitation, PTP-PEST was
depleted from the lysates (Fig. 4C
, lane 5), and there was no
detectable PTP activity in the supernatants (Fig. 4C
, lanes 4 and 6).
The supernatants of extracts precleared with nonimmune serum showed
high levels of activity at 125 kDa (Fig. 4C
, lane 8). Thus, PTP-PEST is
the only active PTP migrating at 125 kDa.
PTP-PEST Is Complexed with JAK2
Since PRL-stimulated JAK2 is negatively regulated by EGF treatment
of the HC11 cells, we next examined complexes of JAK2 for associated
PTP-PEST. JAK2 was immunoprecipitated from lysates of HC11 cells, which
had been treated with EGF and/or the lactogenic hormones, and the
immunoprecipitates were analyzed for complexed PTP-PEST by a Western
blotting analysis (Fig. 5A
, upper
panel) and for PTP-PEST activity in the in-gel PTP assay (Fig. 5B
, lower panel). We detected PTP-PEST in the JAK2
immunoprecipitates from each of the cellular lysates regardless of
culturing conditions. Furthermore, there was 2-fold more PTP-PEST
protein, and activity in the JAK2 immunoprecipitates from lysates of
EGF-treated cultures compared with control or lactogenic
hormone-treated cultures (Fig. 5
, A and B, lanes 2 and 4; quantified in
Fig. 5D
). An in-gel assay using total cellular lysates from the same
experiment is shown in the top panel of Fig. 5B
. A
constitutive complex of JAK2 and PTP-PEST was also found by performing
the reverse experiment, where JAK2 was detected in immunoprecipitates
of PTP-PEST (Fig. 5C
, upper panel). In the
immunoprecipitates from EGF-treated cells, which contained more
PTP-PEST, there was more associated JAK2.

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Figure 5. JAK2 and PTP-PEST Are Constitutively Associated in
HC11 Cells
Competent cultures of HC11 cells that had either been incubated for
16 h with EGF (+) or left untreated (-) were treated for 15 min
with lactogenic hormones (DIP; +) or left untreated (-) before lysis.
A, Immunoprecipitates of JAK2 were probed by Western analysis for
associated PTP-PEST (upper panel). The filter was
stripped and reprobed for JAK2 (lower panel). B, JAK2
was immunoprecipitated and associated PTP-PEST activity was analyzed by
an in-gel PTP assay (lower panel) as described in Fig. 3 . The upper panel shows the activity of the whole cell
extracts. C, Immunoprecipitates of PTP-PEST were probed by Western
analysis for associated JAK2 (upper panel). The filter
was stripped and reprobed for PTP-PEST (lower panel). D,
ImageQuant software (Molecular Dynamics, Inc.) was used to
quantify the total activity of PTP-PEST ( ), JAK2-associated PTP-PEST
activity ( ) from B, and JAK2-associated PTP-PEST protein ( ) from
A. The values represent the average fold induction of two independent
experiments. E, PTP-PEST or JAK2 were immunodepleted by three rounds of
immunoprecipitation with specific antibodies, and control (pre-IP) and
depleted lysates were compared in Western blots for PTP-PEST
(lower panel) or JAK2 (upper panel).
Nonimmune (ni) serum was used as a control (two left
panels). To determine the quantitative amounts of PTP-PEST and
JAK2 associated with each other, Western blots of three depletion
experiments were analyzed using ImageQuant software (Molecular Dynamics, Inc.). The numbers below the blots show
the averages as the percentage of protein relative to the nondepleted
lysates.
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To determine the fraction of PTP-PEST, which was associated with JAK2
and vice versa, we performed immunodepletion experiments on
HC11 whole cell lysates using specific antisera for both proteins, then
probed the supernatants for each protein. The results showed that 64%
of JAK2 was complexed and coimmunoprecipitated with PTP-PEST; on the
other hand, 54% of the cellular PTP-PEST could be coimmunoprecipitated
with JAK2 (Fig. 5E
).
EGF Slowly Inhibits Lactogenic Hormone-Induced ß-Casein Promoter
Activity and JAK2/STAT5 Phosphorylation
In the preceding experiments HC11 cells were treated for 16 h
with EGF. We next examined the kinetics of EGFs effect on the
JAK2/STAT5 pathway using two approaches. First, we examined ß-casein
promoter activity using HC11 cells stably transfected with a rat
ß-casein promoter-luciferase construct, HC11-Lux cells
(26). Multiple transcription factors, including STAT5 and
the GR, cooperate to induce ß-casein transcription (27).
For HC11 cells to become transcriptionally responsive to lactogenic
hormones they must be grown to and maintained at confluence for a few
days, when they are referred to as competent cultures (2, 28). Treatment of control cultures of competent HC11-Lux cells
for 4 h with lactogenic hormones results in an approximately
4-fold induction of luciferase activity (29). In the
following experiment competent HC11-Lux cells were treated for various
times with EGF, and 4 h before harvesting the lactogenic hormone
mix was added. Control cultures and cultures treated 1 h with EGF
displayed essentially the same luciferase activity in response to
lactogenic hormones (Fig. 6
, last two pairs of bars). The negative effect of
EGF on transcriptional activation is relatively slow. Starting from
2 h of EGF treatment, there was a gradual decrease in hormonal
induction of luciferase activity, which was maximal from 8 h
onward (Fig. 6
).

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Figure 6. Kinetics of EGF Inhibition of Lactogenic
Hormone-Induced ß-Casein Promoter Activity
Competent cultures of HC11 cells stably transfected with a ß-casein
promoter luciferase expression plasmid (HC11-Lux cells) were induced
for 4 h with lactogenic hormones (DIP) before lysis. To examine
the kinetics of EGFs effect on ß-casein promoter activity, 10 ng/ml
EGF were added to the cells for the indicated times before lysis.
Promoter activity was measured in triplicate using total cell lysates
and the luciferase assay system (Promega Corp.). In the
last lane the activity of control cultures that were not treated with
EGF is shown.
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To directly examine the kinetics of EGF inhibition on JAK2/STAT5
activation, we next determined the phosphotyrosine content of JAK2 and
STAT5 activity, using phospho-STAT5 antiserum, in HC11 cells pretreated
for various times with EGF, then stimulated for 15 min with lactogenic
hormones. There was a gradual decrease in the phosphotyrosine content
of both JAK2 and STAT5, which was not evident at 4 h, but was
decreased by approximately 40% and 75% after, respectively, 8 and
16 h of EGF treatment (Fig. 7A
;
quantified in Fig. 7B
). These results agree fairly well with the
kinetics of the decrease in ß-casein promoter activity observed in
the previous experiment, although it should be noted that the
luciferase activity was clearly decreased by 2 h whereas we
generally begin to see decreased levels of phosphorylation on JAK2 and
STAT5 after more than 4 h of EGF treatment. Considering the
multiple transcription factors that cooperate to induce ß-casein
transcription (27), it is possible that EGF has additional
targets on this promoter.

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Figure 7. Kinetics of the EGF Effect on JAK/STAT Activity and
PTP-PEST Induction
A, Competent cultures of HC11 cells were treated for the indicated
times with EGF before treatment for 15 min with lactogenic hormones
(DIP). In the upper panels JAK2 immunoprecipitates were
probed in a Western analysis with a phosphotyrosine-specific monoclonal
antibody, then the filter was stripped and reprobed for JAK2. In the
lower panels a Western analysis for STAT5a and
phospho-STAT5 was performed. B, Phosphorylation of JAK2 and STAT5 from
two independent experiments, as described in A, were quantified using
ImageQuant software and normalized to the protein levels. The graph
represents the average phosphorylation of JAK2 and STAT5 relative to
the DIP control. C, Competent HC11 were treated with 10 ng/ml EGF for
the indicated times. Whole cell lysates were prepared and analyzed by
Western blotting with PTP-PEST-specific antiserum (upper
panel) or by an in-gel PTP assay (lower panel)
as described in Fig. 3 . D, The activity and protein level of PTP-PEST from two separate experiments were quantified using ImageQuant
software. The graph shows the averages for PTP-PEST protein and
activity as a percentage relative to the control value.
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EGF Inhibition of JAK2/STAT5 Activation Requires de
Novo RNA Synthesis
The results in Figs. 6
and 7
show that the effect of EGF on
JAK2/STAT5 phosphorylation and transcriptional induction is slow.
Furthermore, it has previously been demonstrated that EGF blocks
lactogenic hormone-induced STAT5 DNA binding with similar, slow
kinetics (30). Taken together the results suggest that the
negative effect of EGF is indirect, requiring de novo RNA
and protein synthesis. To test this, cells were pretreated with the RNA
synthesis inhibitor actinomycin D (ActD), EGF was added to the HC11
cultures, and the lactogenic hormones were added to the medium for the
final 15 min. In these experiments EGF was added for 5 h because,
as shown in Fig. 8A
, there was only a
slight loss of STAT5 compared with a strong decrease in 16-h
ActD-treated cultures (not shown). Quantification of the Western blots
in Fig. 8A
revealed that in EGF-treated cultures phospho-STAT5 was 36%
of the control level, whereas in ActD-pretreated cells phospho-STAT5
remained at the control level (114% vs. 100%; Fig. 8A
).

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Figure 8. EGF Inhibition of Lactogenic Hormone-Induced
JAK2/STAT5 Activation Requires de Novo RNA Synthesis
Competent cultures of HC11 cells were treated for 5 h with EGF
(+), lactogenic hormones (DIP) were added for 15 min, and lysates were
prepared. Where indicated ActD was added 90 min before EGF addition to
inhibit de novo RNA synthesis. A, Western analysis for
STAT5 activation was performed using phospho-STAT5-specific antibodies
(upper panel). The lower panel shows a
Western blot for total STAT5a. Note that after 6.5 h of ActD
treatment there was a slight decrease in the level of STAT5 in the
cells. STAT5 phosphorylation and protein level were quantified using
ImageQuant software. STAT5 phosphorylation levels after normalization
to protein are shown below the blots as the percentage
of tyrosine phosphorylation relative to the DIP control and
represent the average of two independent experiments. B, Western
analysis for PTP-PEST was performed using specific antibodies
(upper panel). The lower panel shows the
in-gel activity of PTP-PEST. C, The activity and protein level of
PTP-PEST from three independent experiments, as shown in B, were
quantified using ImageQuant software. The graph shows the average
± SD of the activity ( ) and protein level ( )
relative to the nontreated control.
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The results presented above show that EGF stimulates the transcription
of a gene(s) in HC11 cells that is necessary to mediate negative
regulation of the JAK/STAT pathway. One possibility is PTP-PEST itself,
as increased amounts of this protein were observed in EGF-treated HC11
cells (Figs. 4
and 5
). A Western blotting analysis revealed that the
level of PTP-PEST gradually increased after EGF addition (Fig. 7C
, upper panel). Quantification of the results revealed that
after 4 h of treatment there was a minimal increase in PTP-PEST
protein and activity (Fig. 7D
), both of which were further increased by
1.7- and 2.2-fold in cultures treated for, respectively, 8 and 16
h with EGF. Furthermore, in cells pretreated with ActD, the EGF-induced
increase in PTP-PEST protein and activity was blocked (Fig. 8B
;
quantified in Fig. 8C
), suggesting that PTP-PEST could be an
ActD-sensitive EGF target responsible for negative regulation of
JAK2.
Overexpression of PTP-PEST in HC11 Cells Is Not Sufficient to
Inhibit JAK2 Activity
The slow effects of EGF on both JAK2 inhibition and on PTP-PEST
induction prompted us to directly ask whether overexpression of
PTP-PEST inhibits JAK2. To this end PTP-PEST was transiently
overexpressed in competent HC11 cultures. The adaptor protein
p130cas, which has previously been identified as
a PTP-PEST substrate (31, 32), was also examined in these
experiments. In HC11 cultures with elevated PTP-PEST levels resulting
either from long-term EGF treatment (Fig. 9A
) or from transient overexpression
(Fig. 9B
), the phosphotyrosine content of p130cas
was dramatically reduced compared with the level in control cultures
(Fig. 9
, A and B, lanes 3 vs. lanes 1 and 2). In contrast,
transient overexpression of PTP-PEST did not have a significant effect
on lactogenic hormone-induced STAT5 phosphorylation [Fig. 9C
(a), lane
4] or JAK2 phosphorylation [Fig. 9C
(b), lane 4]. In the same
experiment, HC11 cells transfected with the control vector and treated
for 16 h with EGF failed to respond to lactogenic hormones,
displaying essentially no phosphorylation of STAT5 [Fig. 9C
(a), lane
3] and JAK2 [Fig. 9C
(b), lane 3]. The level of PTP-PEST expression
and activity was similar in both cultures [Fig. 9C
(c and d), lanes 3
and 4]. Importantly, there was an increase in the level of PTP-PEST
complexed with JAK2 in both cultures [Fig. 9C
(e), lanes 3 and 4],
ruling out the possibility that ectopically overexpressed PTP-PEST was
not associated with JAK2. Thus, in HC11 cells,
p130cas was sensitive to high levels of PTP-PEST
expression either induced by long-term EGF treatment or resulting from
transient overexpression. In striking contrast, activity of the
JAK/STAT pathway was only blocked in the EGF-treated cultures.

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Figure 9. Transient Overexpression of PTP-PEST in HC11 Cells
A, p130Cas was immunoprecipitated from lysates of
HC11 cells that had either been treated 16 h with EGF (+) or left
untreated (-). The immunoprecipitates were probed for phosphotyrosine
content (upper panel) and reprobed for
p130Cas (lower panel). B and C, HC11 were
transiently transfected with PTP-PEST expression vector or empty
control vector, pcDNA3.1, and incubated for an additional 2 d to
allow for competency. B, p130Cas was immunoprecipitated,
probed for phosphotyrosine, and reprobed for p130Cas.
Expression of PTP-PEST was examined by Western blot analysis with
anti-PTP-PEST antibodies. C, Lower panel, Where
indicated, EGF was included for 16 h (+) in the medium of the
transiently transfected HC11 cultures, and the cells were treated for
15 min with lactogenic hormones (+DIP) before lysis. a and b, Western
analysis was performed using phospho-specific antibodies for STAT5 (a)
and JAK2 (b). c, Expression of PTP-PEST was examined by Western blot
analysis with anti-PTP-PEST antibodies. e, JAK2 was immunoprecipitated,
and associated PTP-PEST activity was analyzed by in in-gel phosphatase
assay with [32P]poly-(Glu:Tyr) as the gel-embedded
substrate. d, The assay of total lysates.
|
|
PTP-PEST-Containing Lysates from EGF-Treated HC11 Cells
Dephosphorylate JAK2 More Efficiently than Lysates from Control
Cells
The previous experiment shows that ectopically overexpressed
PTP-PEST complexed with JAK2; however, it had essentially no effect on
the in vivo activity of the kinase. These results suggest
that EGF induces another protein that mediates the negative regulation
of JAK2. To experimentally address this, we developed an in
vitro JAK2 phosphatase assay, as described in the following
experiment. Lysates of HC11 cells treated for various times with EGF
and precleared of JAK2 were used as the source of PTP-PEST (Fig. 10A
). To show specificity, PTP-PEST was
immunodepleted from the indicated extracts (Fig. 10A
, lanes 2 and 6).
Immunoprecipitates of activated JAK2, prepared from lactogenic
hormone-treated HC11 cells, were used as the in vitro
substrate. It should be noted that we employed a JAK2-specific
antiserum that does not coimmunoprecipitate PTP-PEST for preparation of
the substrate. JAK2 was incubated with the HC11 cell lysates, as
described in Materials and Methods, and its
phosphorylation was determined by a Western analysis (Fig. 10B
). The
level of phosphotyrosine in JAK2 immunoprecipitates incubated only with
lysis buffer served as an input control (Fig. 10B
, lane 1). JAK2
incubated with lysates from non-EGF-treated HC11 cells had 52% less
phosphotyrosine compared with the control (Fig. 10
, B and C, lane 3
vs. lane 1). PTP-PEST was responsible for this, since
immunodepleted lysates did not dephosphorylate JAK2 (Fig. 10
, B and C,
lane 2). Importantly, the lysates prepared from EGF-treated cultures
were more active toward JAK2 then the control lysate (Fig. 10
, B and C,
lanes 4 and 5 vs. lane 3). In comparison to phosphorylation
after incubation with the control lysate (48% taken as 100%), JAK2
showed 52% and 87% decreases in phosphotyrosine after treatment with
lysates from, respectively, 8- and 16-h EGF-treated cultures.
Immunodepletion of PTP-PEST from the lysates prepared from EGF-treated
cultures also restored JAK2 phosphorylation (Fig. 10
, B and C, lane 6),
although the level reached only 80% of the control value. Finally, the
addition of orthovanadate to the in vitro assay reversed the
effects of PTP-PEST (Fig. 10
, B and C, lane 7). Taken together the
results show that JAK2 is a PTP-PEST substrate. Moreover, they support
our working model that in HC11 cells EGF slowly induces the expression
of a protein that mediates the dephosphorylation of PRL-activated JAK2
by PTP-PEST (Fig. 11
). The formal
proof for this model will depend upon our identification of this
protein. Furthermore, depleting HC11 cells of PTP-PEST, for example via
RNA-mediated reduction (33) of PTP-PEST mRNA, will
also be required to prove that PTP-PEST is the responsible PTP.

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Figure 10. PTP-PEST Dephosphorylates JAK2
The source of PTP-PEST for the in vitro assay was HC11
whole cell lysates made from cultures treated for the indicated times
with EGF. These were immunodepleted of JAK2, and where indicated
PTP-PEST was also immunodepleted (lanes 2 and 6) or 2 mM
orthovanadate was added (lane 7). PTP-PEST protein level and activity
in the lysates are shown in A. B, Active JAK2, the substrate in the
in vitro phosphatase assay, was immunoprecipitated from
HC11 whole cell lysates prepared from competent cultures treated for 15
min with lactogenic hormones. [PTP-PEST is not coimmunoprecipitated
with the JAK2 antiserum (SC-278) used in this experiment.] Immune
complexes were collected with protein A-Sepharose, washed, and used as
a substrate in the vitro dephosphorylation assay, where they were
treated with the lysates shown in A. In lane 1, JAK2 complexes were
incubated with phosphatase buffer only. Phosphatase reactions were
stopped by washing the immune complexes and boiling in sample buffer.
JAK2 immunoprecipitates were Western blotted and probed for
phosphotyrosine with a specific monoclonal antibody (upper
panel). After stripping, the membrane was reprobed with JAK2
antiserum (lower panel). C, The relative phosphorylation
level of JAK2 from two independent experiments was determined using
ImageQuant software, and the averages are shown as a percentage
relative to the buffer-treated control. , JAK2 dephosphorylation
using PEST-depleted lysates.
|
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Figure 11. Working Model for EGF-Induced Inhibition of PRL
Signaling in HC11 Mammary Epithelial Cells
A, PRL-induced activation of the JAK-STAT pathway promotes
transcription of milk protein genes and mammary differentiation.
PTP-PEST, which is constitutively associated with JAK2, does not block
JAK2/STAT5 activation. B, Transient overexpression of PTP-PEST in HC11
leads to an increase in the PTP-PEST/JAK2 association; however, PRL
induction of JAK2 activity is essentially normal. C, EGF treatment of
HC11 cells leads to an increase in PTP-PEST expression and PTP-PEST
JAK2 association. EGF also induces the expression of a putative
mediator protein X, which we propose is needed to modify the complex,
allowing PTP-PEST to efficiently target JAK2 and down-regulate the
response of the cells to PRL-induced differentiation.
|
|
 |
DISCUSSION
|
---|
Upon ligand binding and cytokine receptor dimerization, the
associated JAK kinases are activated and phosphorylate-specific
tyrosine residues in the STATs leading to their dimerization, nuclear
translocation, and DNA binding (34) (35). In
comparison to stimulation of the JAK/STAT pathway, processes leading to
its down-regulation are less well understood. The fact that continuous
JAK activity is essential for sustained STAT phosphorylation
(36) and the transient nature of this phosphorylation
suggest that tyrosine dephosphorylation plays an important role in
negative regulation of this pathway. However, the PTPs and their
specific targets are not well described (37). The results
presented here suggest that PTP-PEST plays a novel role in
down-regulation of PRL signaling in mammary epithelial cells. Our
results imply that EGF, which we have previously shown to inhibit
PRL-induced STAT5 activation (7, 8), exerts its effects
via PTP-PEST down-regulation of JAK2 kinase activity. Furthermore, we
demonstrate for the first time that there is constitutive PTP-PEST/JAK2
association in intact cells and that in vitro PTP-PEST
dephosphorylates JAK2. The effects of EGF on HC11 cells are 2-fold. EGF
stimulates PTP-PEST expression, leading to an increase in the
PTP-PEST/JAK2 complex. More importantly, EGF appears to induce an as
yet unknown protein, which has a permissive effect on PTP-PEST,
allowing it to target and inhibit JAK2 in the PRLR complex. Our current
working model is shown in Fig. 11
.
Results from the in vitro phosphatase assay revealed
that JAK2 is a PTP-PEST substrate. Importantly, the extent of JAK2
dephosphorylation increased when lysates were prepared from EGF-treated
cultures, supporting our model of an EGF-induced mediator protein. We
have, of course, considered alternative explanations for the results,
the simplest one being that EGF induces the expression of another PTP
that down-regulates JAK2 activity. Although impossible to completely
rule out, there is little evidence to support this possibility. Despite
the fact there are multiple PTPs in the HC11 lysates, the in
vitro assay shows that JAK2 is particularly sensitive to PTP-PEST.
However, we have noted that PTP-PEST immunodepletion from lysates of
EGF-treated HC11 cells restored the phosphotyrosine content of JAK2 to
only 80% of the control level, compared with 100% restoration using
PTP-PEST immunodepleted control lysates (Fig. 10
). Thus, it is possible
that additional PTPs are induced by EGF treatment of HC11 cells;
however, they were not detected in the in gel PTP assay (Fig. 3
and
data not shown). The facts that there was a specific, constitutive
association of JAK2 and PTP-PEST in HC11 cells and that additional
PTP-PEST, either induced endogenously by EGF or after transient
transfections, became complexed with JAK2 suggest that the association
of the two proteins is biologically relevant. Thus, we favor the
hypothesis that an EGF- induced protein is required to mediate the
effect of PTP-PEST on JAK2. Future experiments will be aimed at
identifying novel JAK2-complexed proteins that augment the ability of
PTP-PEST to dephosphorylate the kinase.
The results of the experiments using orthovanadate allow us to draw
some general conclusions about control of JAK2 in mammary cells. First,
treatment of HC11 cells with the inhibitor had no effect on JAK2
activity (Fig. 2C
) or STAT5 DNA binding (Fig. 1
), in control or
lactogenic hormone-treated cultures. This suggests that the kinase is
not held in check by the complexed PTP-PEST or any other PTP and shows
that JAK2 activity is strictly regulated by PRL binding to its
receptor. Even after JAK2 activation PTP-PEST did not negatively
regulate the kinase, as orthovanadate treatment of HC11 cultures had no
effect on lactogenic hormone-mediated JAK2 stimulation. Only in
EGF-treated cultures could orthovanadate treatment restore JAK2
activity and STAT5 DNA binding to their normal levels. This contrasts
with the in vitro assay where we observed PTP-PEST-mediated
JAK2 dephosphorylation to 48% of the control level when lysates were
prepared from non-EGF-treated HC11 cells, perhaps because some
restraints were lost during preparation of the lysates. Taken together,
the results suggest that in intact cells the activity of PTP-PEST
toward JAK2 is kept tightly controlled. Only after EGF treatment and
presumably association of a mediating protein with the complex is
PTP-PEST able to dephosphorylate JAK2.
Negative regulators of the JAK/STAT pathway have
previously been described. These include PTPs as well as non-PTPs
(37); of the latter the most well known is a family of
proteins known as suppressors of cytokine signaling (SOCS) or
cytokine-inducible SH2-containing protein (CIS). These proteins are
transcriptionally induced by STATs in response to a variety of
cytokines. The SOCS/CIS proteins bind either directly to JAK kinases,
inhibiting their activity, or to activated receptors, thereby
preventing the recruitment and phosphorylation of STATs
(37). It has been shown that PRL-induced STAT activity can
be down-regulated after transfection of various SOCS/CIS family members
(38). Furthermore, PRL up-regulates the transcription of
diverse SOCS/CIS mRNAs in the T47D breast tumor cell line
(38) and in mammary glands of lactating mice
(39). Thus, it is very likely that SOCS/CIS proteins have
general roles in controlling the STAT5 response to lactogenic signaling
in mammary cells. However, for various reasons they do not appear to be
involved in the process that we describe here. SOCS/CIS have not yet
been described to be up-regulated in response to EGF. More importantly,
SOCS/CIS-mediated effects on cytokine signaling do not require PTP
activity, nor are they sensitive to treatment with orthovanadate, which
contrasts with what we have observed in our experiments.
Turning to phosphatases, specific PTPs have been implicated in
the negative regulation of JAK2 and/or STAT5. CD45, a transmembrane PTP
highly expressed in cells of the hemopoietic lineage, has recently been
shown to dephosphorylate JAK2, leading to negative regulation of
multiple cytokines (40). Overexpression of the cytosolic
PTP1B in COMMA-1D cells, from which HC11 cells were originally isolated
(1), led to a decrease in PRL-induced STAT5
phosphorylation (41). In these experiments JAK2 activity
was not affected by PTP1B expression, suggesting that STAT5 was the
direct target. The SH2 domain-containing protein tyrosine phosphatases
(SHPs), SHP1 and SHP2, have also been implicated in
down-regulation of cytokine receptor signaling (34). SHP1
has been reported to associate with and dephosphorylate JAK2
(42). Furthermore, in response to GH, SHP1 associates with
STAT5b in livers of male rats, where it may contribute to termination
of GH signaling (43). STAT5 has also been reported to
serve as a SHP2 substrate in an in vitro assay and trapping
substrate mutants of SHP2 complex with STAT5 (44, 45). We
detected SHP1 and SHP2 activity using the in-gel PTP assay on HC11
whole cell extracts (Fig. 3A
, asterisk). However, neither
SHP1 nor SHP2 was complexed with JAK2, nor was their activity increased
in EGF-treated HC11 cells. In contrast, the activity of SHP2 was
increased after treatment with the cytokine IL-6 (Horsch, K.,
unpublished data), showing that the in-gel PTP assay is sensitive
enough to see changes in activity of these PTPs. Taken together, these
results suggest that neither of the SH2-containing PTPs plays a major
role in deactivation of JAK2 signaling in the HC11 cells in response to
lactogenic hormones or EGF.
An emerging theme in signal transduction is the
cross-regulation of different classes of membrane receptors. Receptor
tyrosine kinases and cytokine receptors in some instances cooperate to
induce signaling pathways (44, 46). Negative regulation
has also been observed, and considering the mammary gland specifically,
there is a complex interaction between EGFR and PRLR. On the one hand,
PRLR activation has a negative influence on EGF-induced signaling. In
mammary cells continuously exposed to PRL, EGFR has been shown to be
phosphorylated on specific threonine residues, a modification that had
a negative effect on its kinase activity, leading to impaired
EGF-induced biological responses (47). On the other hand,
as shown here, continuous exposure of HC11 mammary cells to EGF dampens
their response to PRL. It is interesting to consider the negative
regulation mediated by PRLR on EGFR and vice versa in light
of their roles in the development of the mammary gland. EGFR is
expressed at all stages (48, 49) and appears to have a
particularly important role in proliferation of ductal epithelial
cells, as wa-2 mice, which harbor a mutation in the EGFR
kinase domain, exhibit sparse ductal growth (10). PRLR
does not have a major role in the proliferative phase; however, it
plays an essential role in differentiation of the gland into a
milk-producing organ (50). The negative effects of EGF on
PRL signaling, which we have described, here might be a part of a more
general control of differentiation-inducing stimuli during the normal
proliferative stage of development. During the rapid growth of the
gland that occurs at sexual maturity and continues during pregnancy,
there appear to be numerous mechanisms inhibiting differentiation
and milk protein gene expression. These range from negative
regulatory transcription factors (4, 27, 51) that block
expression of differentiation specific genes to the mechanism we
describe here whereby EGF directly impinges upon one of the major
differentiation-inducing signaling molecules, JAK2.
 |
MATERIALS AND METHODS
|
---|
Materials
The antibodies used were EGFR-specific rabbit polyclonal
antibody SC-03 (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA); STAT5a and STAT5b polyclonal antisera (29);
phospho-STAT5-specific monoclonal antibody, which recognizes both
isoforms (Upstate Biotechnology, Inc., Lake Placid, NY)
(52); JAK2-specific polyclonal antibodies SC-278
(Santa Cruz Biotechnology, Inc.) and 06-255 (Upstate Biotechnology, Inc.), phospho-JAK2-specific rabbit polyclonal
antiserum (44-426, BioSource Technologies, Inc.,
Camarillo, CA) which recognizes Tyr1007/1008 in
the activation site; PTP-PEST-specific polyclonal antiserum
(53); phosphotyrosine-specific monoclonal antibody
(Upstate Biotechnology, Inc.); and
p130cas polyclonal antiserum SC-860 (Santa Cruz Biotechnology, Inc.). PRLR-specific polyclonal antiserum,
generated by immunizing rabbits with a peptide encompassing amino acids
466478 of mouse PRLR, was provided by Dr. Charles Streuli
(Manchester, UK). The growth factors and hormones used were recombinant
human EGF, dexamethasone, insulin, and ovine PRL (all from
Sigma, St. Louis, MO). The inhibitors used were ActD
(Sigma) and sodium orthovanadate (Sigma).
Cell Culture and Transfections
HC11 mammary epithelial cells were grown to confluence and
maintained for 2 d in growth medium (RPMI 1640 supplemented with
10% FCS, 10 ng/ml EGF, and 5 µg/ml insulin). These are referred to
as competent cultures (28). To induce differentiation, EGF
was removed from the medium a minimum of 24 h before addition of
the lactogenic hormone mix (DIP). Transient transfections were
performed as follows. HC11 cells were grown to 70% confluence, and
control empty vector or a plasmid encoding the PTP-PEST cDNA
(53) was introduced into cells using the Fugene
transfection reagent (Roche Molecular Biochemicals,
Indianapolis, IN) according to the manufacturers protocol. Two days
after transfection the cells were confluent, EGF was removed from the
medium for 24 h, then the cultures were treated for 15 min with
lactogenic hormones, and lysates were prepared in Nonidet P-40 buffer
[1% Nonidet P-40, 50 mM Tris (pH 7.5), 120 mM
NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol (DTT), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5
mM phenylmethylsulfonylfluoride, 5 mM sodium
fluoride, 20 µg M ß-glycerophosphate, and 2
mM sodium orthovanadate].
Luciferase Assays
HC11 cells stably transfected with a ß-casein promoter
luciferase construct (p-334/-1ßc-Lux) (29) were grown to
and maintained for 2 d at confluence, then treated 4 h with
lactogenic hormone-containing medium. To examine the kinetics of EGFs
effect on induction of luciferase activity, the cultures were incubated
with 10 ng/ml EGF for various times before or after the addition of
lactogenic hormones, which in all cases were present for 4 h.
Luciferase activity was determined on triplicate samples using the
luciferase assay system (Promega Corp., Madison, WI) as
described by the manufacturer. Total light emission was measured during
the first 3 sec of the reaction using a luminometer (Berthold
Microlumat LB96P).
EMSA
Nuclear extracts were prepared by scraping cells into
cytoplasmic extraction buffer [10 mM KCl, 20
mM HEPES (pH 7.0), 1 mM
MgCl2, 0.1% Triton X-100, 20% glycerol, 0.1
M EGTA, 0.5 mM DTT, 2 mM sodium
orthovanadate, 50 µM ß-glycerophosphate, 50
mM sodium fluoride, 2 mM
phenylmethylsulfonylfluoride, 5 µg/ml leupeptin, and 5 µg/ml
aprotinin] and shearing with 20 strokes using a Dounce homogenizer
(Wheaton, pestle B, Kontes Co., Vineland, NJ). Nuclei were pelleted by
centrifugation at 800 x g for 5 min and then treated
with nuclear extraction buffer (cytoplasmic extraction buffer and 300
mM NaCl) for 30 min on ice. Extracts were
clarified by centrifugation for 15 min at 16,000 x g.
EMSAs were performed by incubating 10 µg nuclear protein extract with
the STAT5 DNA-binding site from the rat ß-casein promoter
(5'-AGATTTCTAGGAATTCA ATCC-3') (5) for 30 min on ice in 20
µl EMSA buffer [10 mM HEPES (pH 7.6), 2
mM
NaH2PO4, 0.25
mM EDTA, 1 mM DTT, 5
mM MgCl2, 80
mM KCl, 2% glycerol, and 100 µg/ml
poly(dI-dC)]. Specific binding was analyzed on a 4% native
polyacrylamide gel, prerun for 2 h at 200 V in 0.25x TBE [22.5
mM Tris borate (pH 8.0) and 0.5
mM EDTA]. The samples were loaded and
electrophoresed for 1 h at 200 V, and the gels were dried and
autoradiographed.
Immunoprecipitation and Western Blot Analysis
Whole cell lysates were obtained by solubilizing cells in
Nonidet P-40 buffer [1% Nonidet P-40, 50 mM Tris (pH
7.5), 120 mM NaCl, 5 mM EDTA, 1 mM
EGTA, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin,
0.5 mM phenylmethylsulfonylfluoride, 5 mM
sodium fluoride, 20 µM ß-glycerophosphate, and 2
mM sodium orthovanadate] for 5 min on ice. The lysates
were clarified by centrifugation at 16,000 x g for 15
min. For immunoprecipitations, equal amounts of protein were incubated
with specific antibodies for 12 h on ice. Immune complexes were
collected with protein A-Sepharose (Sigma) by rotating the
complexes for 30 min at 4 C and washing three times with lysis buffer
and once with TNE buffer (50 mM Tris, 140
mM NaCl, and 5 mM EDTA).
Immunoprecipitated proteins were boiled in sample buffer and analyzed
by SDS-PAGE. Immunodepletion was performed by two or three consecutive
incubations of lysates with the specific antiserum. Proteins were
electroblotted onto polyvinylidene difluoride membranes (Roche Molecular Biochemicals) and detected with specific antiserum
after blocking the filters with 10% horse serum (Life Technologies, Inc., Grand Island, NY) in TTBS [50
mM Tris (pH 7.5), 150 mM
NaCl, and 0.05% Tween 20]. Proteins were visualized with
peroxidase-coupled secondary antibody using the ECL detection system
(Amersham Pharmacia Biotech). For reprobing, membranes
were stripped in 62.5 mM Tris (pH 6.8), 2% SDS,
and 100 mM ß-mercaptoethanol for 30 min at 60
C.
In Vitro JAK2 Kinase Assay
The cells were lysed in Nonidet P-40 extraction buffer, and
1.5-mg aliquots were precleared with protein A-Sepharose for 30 min on
ice, followed by incubation with a JAK2-specific polyclonal antibody
for 2 h on ice. Immune complexes were collected with protein
A-Sepharose by rotation for 30 min at 4 C and washed four times with
cold lysis buffer and twice with kinase buffer [50 mM Tris
(pH 7.5), 0.1% Nonidet P-40, 100 mM NaCl, 10
mM MgCl2, 5 mM MnCl, 100
µM sodium orthovanadate, and 1 mM DTT].
Kinase buffer (100 µl) with or without 15 µM ATP was
added to the immunoprecipitates, which were incubated for 30 min at 37
C on a Thermo-Shake (Eppendorf, Hamburg, Germany) (22, 23). The kinase reaction was stopped by washing with cold lysis
buffer and boiling the complexes in sample buffer. Immunoprecipitated
proteins were subjected to SDS-PAGE, blotted onto polyvinylidene
difluoride membranes, and probed with a phosphotyrosine-specific
monoclonal antibody. After stripping, membranes were reprobed with
JAK2-specific antiserum.
In-Gel Phosphatase Assay
We employed an in-gel PTP assay essentially as described
previously (22). For detection of PTP activity,
SDS-polyacrylamide gels containing 2 x 105
cpm/ml 32P-labeled substrate were prepared. The
substrate (poly-[glutamic-acid:tyrosine]; 20,00050,000 kDa;
Sigma) was dissolved in kinase buffer [30 mM
MgCl2, 1 mM
MnCl2, 1 mM sodium vanadate, 10
mM DTT, 0.05% Triton X-100, 50 mM imidazole
(pH 7.2), and 0.2 mM ATP] and incubated with
[32P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ) and recombinant
glutathione-S-transferase-c-Src (provided by Dr. F. Boehmer,
University of Jena, Jena, Germany) for 18 h by rotation at room
temperature. The phosphatase substrate was purified via a Sepharose
G-50 column (Amersham Pharmacia Biotech). Nonidet P-40
cell lysates (50 µg) were electrophoresed, and in-gel PTP activity
was visualized as follows. The gels were sequentially shaken for the
indicated times at room temperature in 250 ml of the following buffers:
90 min in PB1 [50 mM Tris (pH 8.0) and 20%
isopropanol); twice for 30 min each time in PB2 [50
mM Tris (pH 8.0) and 0.3% ß-mercaptoethanol];
90 min in PB3 [50 mM Tris (pH 8.0), 0.3%
ß-mercaptoethanol, 6 M guanidine hydrochloride,
and 1 mM EDTA], three times for 30 min each time
in PB4 [50 mM Tris (pH 8.0), 0.3%
ß-mercaptoethanol, 1 mM EDTA, and 0.04% Tween
40], and 15 h in PB5 [50 mM
2-morpholinoethanesulfonic acid (pH 6.0), 0.3% ß-mercaptoethanol, 1
mM EDTA, 0.04% Tween 40, and 4
mM DTT]. After these incubations, gels were
stained with Coomassie brilliant blue, destained in 40% ethanol/10%
acetic acid, dried, and analyzed using a PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA).
In Vitro PTP Assay
JAK2 was immunoprecipitated from 500 µg Nonidet P-40 lysates
prepared from competent cultures of HC11 cells treated for 15 min with
lactogenic hormones. The JAK2 antiserum used, Santa Cruz SC-278, does
not coimmunoprecipitate PTP-PEST. Immune complexes were collected with
protein A-Sepharose, washed with inhibitor-free Nonidet P-40 buffer
(Nonidet P-40 buffer lacking phenylmethylsulfonylfluoride, NaF, sodium
vanadate, and ß-glycerophosphate), and used as substrate in the
in vitro dephosphorylation assay. Lysates were prepared in
inhibitor-free Nonidet P-40 buffer from competent cultures of HC11
cells, either left untreated or treated for various times with 10 ng/ml
EGF. Lysate (500 µg) from which JAK2 was immunodepleted was used as
the source of PTP-PEST for the in vitro assays. As controls,
lysates from which PTP-PEST had also been immunodepleted or lysates
containing 2 mM sodium orthovanadate were
employed. For in vitro dephosphorylation, 300 µl lysate
plus 300 µl phosphatase buffer [50 mM
2-morpholinoethanesulfonic acid (pH 6.0), 0.3% ß-mercaptoethanol, 1
mM EDTA, 0.04% Tween 40, and 4
mM DTT] were added to the JAK2-protein
A-Sepharose complexes, and these were rotated at 4 C for 30 min. To
determine the initial JAK2 phosphotyrosine content, the JAK2-protein
A-Sepharose complexes were rotated with 300 µl Nonidet P-40 lysis
buffer plus 300 µl phosphatase buffer. The reaction was terminated by
pelleting the JAK2 immunocomplexes and washing the pellets three times
with Nonidet P-40 buffer containing phosphatase inhibitors and once
with TNE. Immunoprecipitated JAK2 was released by boiling in sample
buffer, subjected to SDS-PAGE, and blotted onto polyvinylidene
difluoride membranes, which were probed with a phosphotyrosine-specific
monoclonal antibody. After stripping the membrane, the filters were
reprobed with JAK2-specific antiserum.
 |
ACKNOWLEDGMENTS
|
---|
We thank C. Streuli for the PRLR antiserum, F. Boehmer for Src
kinase, and M. Wartmann for initial results with the HC11-Lux cells. We
thank K. Burridge, T. Holbro, C. Streuli, A. Badache, B. Hemmings, and
the members of the Hynes laboratory for helpful suggestions and
discussions.
 |
FOOTNOTES
|
---|
This work was supported by the Novartis Research Foundation.
Abbreviations: ActD, Actinomycin D; CIS, cytokine-inducible
SH2-containing protein; DIP, lactogenic hormone mixture
(10-6 M dexamethasone, 5 µg/ml insulin, and
5 µg/ml ovine PRL); DTT, dithiothreitol; EGF, epidermal growth
factor; EGFR, epidermal growth factor receptor; JAK, Janus kinase;
PEST, proline glutamine serine threonine-rich sequence; PRLR, PRL
receptor; SOCS, suppressors of cytokine signaling; SHP, SH2
domain-containing protein tyrosine phosphatase; STAT, signal transducer
and activator of transcription.
Received for publication March 5, 2001.
Accepted for publication August 27, 2001.
 |
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