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INTRODUCTION |
Cytokines regulate a variety of cellular responses, including cell
growth, survival, differentiation, and function and exert their diverse
effects through interaction with specific receptors (1, 2). Receptor
aggregation, as a result of cytokine binding, activates one or more
members of the receptor-associated Janus family of protein-tyrosine
kinases (Jaks)1 (1, 2).
Activated Jaks phosphorylate specific tyrosine residues on the
receptor, which serve as docking sites to recruit a variety of
signaling molecules, such as signal transducers and activators of
transcription (Stats) (1, 3). Stat proteins exist normally as latent
monomers in the cytoplasm and activation of Stat is totally dependent
upon a single tyrosine phosphorylation by Jaks. Once phosphorylated,
the Stats dimerize, translocate to the nucleus, and activate a variety
of cytokine-inducible genes (1, 3).
To date, seven mammalian Stat family members have been identified,
including Stat1, 2, 3, 4, 5A, 5B, and 6. Gene disruptions in mice have
highlighted unique functions for each Stat family member in cytokine
signaling (4). The two closely related Stat5 gene products, Stat5A and
Stat5B, have been of particular interest because of the broad spectrum
of cytokines that induce their activation by tyrosine phosphorylation.
Stat5A and 5B are activated by cytokines that affect the myeloid
lineages, including erythropoietin (Epo), thrombopoietin (Tpo), IL-3,
and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5-7),
and by cytokines that affect lymphoid lineages, including IL-2, IL-7,
IL-9, IL-13, and IL-15 (8-10). In addition, both Stat5 proteins are
activated by growth hormone (GH) and prolactin (11, 12). Gene targeting
studies have demonstrated that Stat5A plays a critical role in
prolactin signaling in the lactating mammary gland, where it is highly
expressed relative to Stat5B (13, 14). In contrast, Stat5B functions in
GH signaling in the liver, where this isoform is highly expressed (14,
15). In addition, studies of Stat5A/5B nullizygous mice have
illustrated a key role of Stat5A and Stat5B in prolactin regulation of
ovarian function (14) and IL-2-induced T cell proliferation (16).
The effect of cytokines is modulated in both magnitude and duration;
therefore, a braking mechanism must be operative in cytokine signaling.
Indeed, several negatively regulatory mechanisms have been identified,
which include receptor endocytosis and lysosomal degradation (17),
dephosphorylation of receptors and Jak kinases by tyrosine phosphatases
(Shp-1 or CD45) (18, 19), and binding of suppressors of cytokine
signaling (SOCS) to receptors and Jaks (20). These events explain
control at the levels of receptor and Jak kinases. However, little is
known about down-regulation of tyrosine-phosphorylated and active Stat
proteins. Although a family of protein inhibitors of activated Stats
(PIAS) has been shown to inhibit Stat function in the nucleus (21), it
is unclear whether a phosphatase or a protease is involved in turnover
of Stats. An ubiquitin-dependent proteasome pathway was
proposed to mediate Stat1 turnover (22). However, the apparent
stabilization of tyrosine-phosphorylated Stat1 by proteasome inhibitors
was due to sustained signaling rather than a direct effect on Stat1 turnover (23). Therefore, it is more likely that the turnover of
tyrosine-phosphorylated Stat1 is mediated by a phosphatase. In fact,
recent studies using cells derived from TC-PTP-deficient mice have
clearly demonstrated that TC-PTP is a Stat1 phosphatase (50). In
previous experiments, we demonstrated that different mechanisms are
involved in regulating the inactivation of various Stat proteins (24).
The proteasome inhibitors MG132 and lactacystin inhibited the turnover
of tyrosine-phosphorylated forms of Stat4, Stat5, and Stat6, without
any effects on the turnover of tyrosine-phosphorylated Stat1, Stat2,
and Stat3. Despite the fact that these two proteasome inhibitors
dramatically stabilize the tyrosine-phosphorylated Stat5, there is no
direct evidence to support a notion that protein degradation is
responsible for the down-regulation of active Stat5 (24). Previous
studies with overexpression systems have suggested that several
phosphatases, including Shp-2 (25), PTP1B (26), TC-PTP (27), and
phosphatase 2A (28), are able to interact and dephosphorylate Stat5A;
however, the physiological role of these phosphatases in
down-regulation of Stat5A is not clear. In this report, we address the
key unanswered question regarding Stat5A down-regulation and identify
Shp-2 as a Stat5A phosphatase.
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EXPERIMENTAL PROCEDURES |
Antibodies, cDNAs, and Inhibitors--
Anti-Statl (C24),
anti-SHP-1 (C-19), and anti-SHP-2 (C-18) antibodies were purchased from
Santa Cruz Biotechnology. Anti-GST (06-332) and anti-phosphotyrosine
antibodies (4G10) were purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). Anti-phosphoStat5 monoclonal antibodies were purchased
from ZYMED Laboratories, Inc. (San Francisco, CA). Antisera against
Stat3, Stat5A, and Stat5B, and Jak2 have been described previously (29,
30). The cDNAs encoding murine Jak2, Stat5A,
Stat5AY-F694, EpoR, and Shp-2 were subcloned into mammalian
expression vectors pRK5 or pcDNA3 (Invitrogen). Protein-tyrosine
kinase inhibitor staurosporine was purchased from Sigma.
Cells and Transfection--
32D(EpoR wt) and 32D(EpoR H) cells
were cultured in RPMI 1640 containing 10% fetal bovine serum and
supplemented with murine IL-3 (25 units/ml). COS-7 cells and mouse
embryonic fibroblasts (MEF) derived from wild-type or Shp-2 mutant mice
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. For transfection, COS-7 cells were seeded
in 100 or 60-mm tissue culture dishes 24 h before transfection,
and subconfluent cells were transfected with various combinations of
cDNAs in mammalian expression vectors by LipoFectamine (Invitrogen)
according to the manufacturer's instructions. For
co-immunoprecipitation experiments, cells were lysed in lysis buffer
without phosphatase inhibitors and phosphate salts (20 mM
Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM EDTA, 1%
Triton X-100, aprotinin 3 µg/ml, pepstain 2 µg/ml, leupeptin 1 µg/ml) or phosphate buffer (10 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM EDTA, 0.1 mM
Na3VO4, 50 mM NaF, 30 mM Na4P2O7, 1% Triton X-100, aprotinin 3 µg/ml, pepstain 2 µg/ml, leupeptin 1 µg/ml) 48 h after transfection. For dephosphorylation experiments, MEF cells were stimulated with cytokines and subsequently lysed with SDS-PAGE sample buffer at different time points following cytokine removal.
Pulse Chase Experiments--
Log-phase 32D(EpoR H) cells were
starved in RPMI 1640 containing 1% fetal bovine serum overnight. Cells
were washed twice with phosphate-buffered saline and cultured in
methionine-free Dulbecco's modified Eagle's medium at 2 × l07/ml containing 10% dialyzed fetal bovine serum with 100 µCi/ml of [35S]methionine for 3 h. Then, murine
IL-3 (25 units/ml) and Epo (20 units/ml) were added for 15 min. Cells
were washed three times with phosphate-buffered saline and resuspended
at 1 × 106/ml in fresh medium without cytokines. At
the times indicated, whole-cell lysate was generated, and
immunoprecipitation and SDS-PAGE was carried out. Then the proteins
were transferred to nitrocellulose membrane, and the membrane was
exposed to film.
Peptide Binding and Mass Spectrometry Analysis--
The
tyrosine-phosphorylated peptides (TPVLAKAVDG(pY)VKPQIKQ) and the
control non-tyrosyl-phosphorylated (TPVLAKAVDGYVKPQIKQ) derived from
Stat5A were synthesized and conjugated to Sepharose 4B beads through
the primary amino groups at the N terminus of the peptides. 32D (EpoR
wt) cells (3 × 108) were lysed in lysis buffer
without phosphatase inhibitors and phosphate salts (20 mM
Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM EDTA, 1%
Triton X-100, aprotinin 3 µg/ml, pepstain 2 µg/ml, leupeptin 1 µg/ml) at 4 °C. Cell extracts were precleared with Sepharose beads
and subsequently incubated with 300 µl of the tyrosine-phosphorylated peptides-conjugated or the non-tyrosine-phosphorylated control peptides-conjugated Sepharose beads at 4 °C for 1 h. After
washing three times with cold lysis buffer, the proteins binding to the peptide-conjugated Sepharose beads were eluted in SDS sample buffer at
100 °C for 5 min and were subjected to SDS-PAGE and silver staining.
The protein specifically bound to the tyrosine-phosphorylated peptides
underwent an in-gel tryptic digestion. The peptides derived from the protein band were subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis (31). Afterward, the mass spectrometry data were subjected to
a search of the NCBInr protein data base with ProteinProspector programs at prospector.ucsf.edu.
PTPase Assays--
After binding to cell lysate from 32D (EpoR
wt) cells (1 × 107) and a subsequent five-time
washing with cold lysis buffer, peptide-conjugated Sepharose beads were
incubated in 50 µl of PTPase buffer (50 mM Tris, pH 7.4, 0.2 mM phosphotyrosine peptide substrate) at 22 °C for
1 h of 100 ml of malachite green solution (Upstate Biotechnology, Inc.) was added to each reaction, which was then incubated at 22 °C
for 5 min prior to measurement of OD660 nm to quantify the
level of free phosphate cleaved by the PTPases from the substrate.
In Vitro Binding of GST Fusion Proteins--
GST fusion proteins
GST-SH2, containing both SH2 domains of Shp-2 and GST-PTP, containing
the PTP catalytic domain of Shp-2, in pGEX vectors were expressed in
Escherichia coli and lysed in lysis buffer without
phosphatase inhibitors and phosphate salts (20 mM Tris-HCl,
pH 7.6, 50 mM NaCl, 5 mM EDTA, 1% Triton
X-100, aprotinin (3 µg/ml), pepstain (2 µg/ml), leupeptin (1 µg/ml)) by sonication and purified by glutathione-Sepharose beads.
After washing with cold lysis buffer, the glutathione-Sepharose beads containing comparable GST fusion proteins were incubated with cell
lysate from COS-7 cells co-transfected with Stat5A and Jak2. After
washing three times, the bound proteins were eluted in SDS sample
buffer at 100 °C for 5 min and were subjected to SDS-PAGE and
Western blot analysis with anti-GST or anti-Stat5A antibodies.
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RESULTS |
Down-regulation of the Tyrosine-phosphorylated Stat5A Is via
Dephosphorylation--
Stat5A is activated by IL-3 in
IL-3-dependent myeloid cells 32D (EpoR wt), which also
express wild-type erythropoietin receptors (EpoR wt), and removal of
IL-3 leads to the disappearance of the tyrosine-phosphorylated Stat5
(24, 29). However, only a small fraction of Stat5 is activated by
tyrosine phosphorylation following treatment of 32D (EpoR wt) cells
with IL-3, which makes it difficult to investigate down-regulation of
the tyrosine-phosphorylated Stat5 (24). To overcome this problem, we
employed a variant of IL-3-dependent 32D myeloid cell line,
32D (EpoR H), which expresses a truncated form of the EpoR. Due to
elimination of the carboxyl-terminal region of the EpoR cytoplasmic
domain, which is required for negative regulation of Epo signaling by
recruiting Shp-1, Jak2 activation is more sustained in 32D (EpoR H),
relative to 32D (EpoR wt), cells upon exposure to Epo (32). Stimulation
of these cells, 32D (EpoR H) with both IL-3 and Epo, is expected to
induce tyrosine phosphorylation of relatively larger amounts of Stat5.
32D (EpoR H) cells were starved, pulse-labeled with
[35S]methionine, and then stimulated with both IL-3 and
Epo. After removal of the cytokines, levels of phosphorylated Stat5A
were examined at different time points. IL-3 plus Epo induced tyrosine
phosphorylation of substantial amounts of Stat5A protein as indicated
by immunoblotting with the anti-pTyr antibodies (Fig.
1A). In addition, IL-3 plus Epo induced tyrosine phosphorylation of substantial amounts of Stat5B
protein in 32D (EpoR H) cells (data not shown). Interestingly, 4 h
after cytokine removal, tyrosine-phosphorylated Stat5A disappeared, as
indicated by the absence of the phosphorylated band (Fig.
1A). However, the total amount of Stat5A, which is the sum
of the phosphorylated and non-phosphorylated forms, remained constant
throughout the duration of the experiment as indicated by the
invariable densities of [35S]methionine-labeled Stat5A
bands in the autoradiography (Fig. 1A). Quantitation of the
band densities further confirmed that levels of Stat5A proteins
remained unchanged throughout the duration of the experiment (Fig.
1B). These data provide direct evidence that the
down-regulation of tyrosine-phosphorylated Stat5A is mediated by a
protein-tyrosine phosphatase.

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Fig. 1.
Down-regulation of tyrosine-phosphorylated
Stat5A is via dephosphorylation. A, analysis of
down-regulation of tyrosine-phosphorylated Stat5A with
[35S]methionine pulse-labeling. 32D (EpoR H) cells were
starved overnight, pulse-labeled with [35S]methionine and
stimulated with both IL3 and Epo. After removal of the
[35S]methionine and the cytokines, the cells were
collected and lysed at the indicated time points. Cell lysates were
immunoprecipitated with anti-Stat5A antibodies and precipitated
proteins were subjected to SDS-PAGE and subsequently were transferred
to a nitrocellulose membrane. The membrane was blotted with
anti-phosphotyrosine ( -pTyr) antibody (upper) or
visualized by autoradiography (lower). B,
quantitation of the band density of total Stat5A. The band densities of
total Stat5A shown in A, (lower) was quantified
by densitometry. The figure shown is representative of three
independent experiments.
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Shp-2 Specifically Associates with the Tyrosine-phosphorylated
Peptide Derived from Stat5A--
Phosphorylation of a single tyrosine
residue (Tyr-694) of Stat5A results in its activation (29); therefore,
the phosphatase responsible for Stat5A dephosphorylation must recognize
Tyr-694 of Stat5A. The amino acid sequence surrounding a target
phosphotyrosine plays a critical role in determining the specificity
and avidity of the interaction between a phosphatase and its substrate
(33). Therefore, we utilized tyrosine-phosphorylated peptides
corresponding to the sequence surrounding Tyr-694 of Stat5A to identify
the phosphatase capable of interacting with tyrosine-phosphorylated Stat5A. Tyrosine-phosphorylated peptides and their corresponding non-phosphorylated control peptides were conjugated to Sepharose beads.
As the phosphorylated Tyr-694 (pTyr-694) on one Stat5A molecule is able
to interact with the SH2 domain on another Stat5A molecule, resulting
in a homodimerization, we first examined the ability of the
phosphorylated and non-phosphorylated Stat5A peptides to specifically
associate with Stat5A itself, the known Stat5A physiological partner.
Cell extracts from 32D (EpoR wt) myeloid cells were incubated with
peptide-conjugated Sepharose beads. After extensive washing, bound
proteins were eluted from the beads and subjected to SDS-PAGE and
Western blot analysis for Stat5A. Stat5A associated with the
tyrosine-phosphorylated Stat5A peptides but not the non-phosphorylated
control peptides (Fig. 2A). In contrast, Stat1 molecules, which do not dimerize with Stat5A, did not
bind to the tyrosine-phosphorylated peptides even though they were
present at readily detectable levels (Fig. 2A).

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Fig. 2.
Shp-2 associates with the phosphopeptides
corresponding to the tyrosine phosphorylation site within Stat5A.
A, association of Stat5A with the tyrosine-phosphorylated
Stat5A peptides. 32D (EpoR wt) cells (1 × 107) were
lysed in lysis buffer without phosphatase inhibitors and phosphate
salts at 4 °C. Cell lysates were incubated with Sepharose beads
conjugated with the tyrosine-phosphorylated peptides (pTyr-694 Pep) or
the non-phosphorylated control peptides (Tyr-694 Pep). Bound proteins
were subjected to SDS-PAGE, transferred to a nitrocellulose membrane,
and blotted with anti-Stat5A or anti-Stat1 antibodies
(upper). The presence of Stat5A and Stat1 in 32D (EpoR wt)
cell lysates was confirmed by immunoprecipitation and Western blot
analysis with anti-Stat5A or anti-Stat1 antibodies, respectively
(lower). B, association of protein-tyrosine
phosphatase activity with the tyrosine-phosphorylated Stat5A peptides.
32D (EpoR wt) cells (1 × 107) were lysed in lysis
buffer without phosphatase inhibitors and phosphate salts at 4 °C.
Cell lysates were incubated with Sepharose beads conjugated with the
tyrosine-phosphorylated peptides (pTyr-694 Pep) or the
non-phosphorylated control peptides (Tyr-694 Pep). After extensive
washing, the peptide-conjugated Sepharose beads were subjected to an
in vitro protein-tyrosine phosphatase assay. C,
association of a 70-kDa protein with the tyrosine-phosphorylated Stat5A
peptides. 32D (EpoR wt) cells (5 × 108) were lysed in
lysis buffer with (phosphate) or without phosphatase inhibitors and
phosphate salts (Tris-HCl) at 4 °C. Cell lysates were precleared
with unconjugated Sepharose beads and subsequently incubated with
Sepharose beads conjugated with the tyrosine-phosphorylated peptides
(pTyr-694 Pep) or the non-phosphorylated control peptides (Tyr-694 Pep)
at 4 °C for 1 h. After washing, bound proteins were eluted and
subjected to SDS-PAGE followed by silver staining. A protein that
migrated at apparent molecular weight of 70 kDa (arrow)
specifically bound to the tyrosine-phosphorylated Stat5A peptides only
in Tris-HCl lysis buffer. D, confirmation of the 70-kDa
protein as Shp-2 by Western blot analysis. 32D (EpoR wt) cells (1 × 107) were lysed in lysis buffer without phosphatase
inhibitors and phosphate salts at 4 °C. Cell lysates were incubated
with Sepharose beads conjugated with the tyrosine-phosphorylated Stat5A
peptides (pTyr-694 Pep) or the non-phosphorylated control peptides
(Tyr-694 Pep). Bound proteins were subjected to Western blot analysis
with anti-Shp-2 or anti-Shp-1 antibodies (upper). The
presence of Shp-2 and Shp-1 in 32D (EpoR wt) cell lysates were
confirmed by the immunoprecipitation and Western blot analysis with
anti-Shp-2 or anti-Shp-1 antibodies, respectively
(lower).
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Next, we examined whether tyrosine-phosphorylated Stat5A
peptide-conjugated Sepharose beads could recruit a protein-tyrosine phosphatase. Cell extracts from 32D (EpoR wt) myeloid cells were preincubated with unconjugated Sepharose beads to remove nonspecific binding proteins. Subsequently, precleared supernatants were incubated with Sepharose beads conjugated with tyrosine-phosphorylated or non-phosphorylated Stat5A peptides. After extensive washing, the beads
were subjected to an in vitro protein phosphatase assay. Significantly more protein phosphatase activity was associated with
beads conjugated with tyrosine-phosphorylated peptides relative to
those conjugated with non-phosphorylated control peptides (Fig. 2B). Therefore, peptides containing the phosphorylated
tyrosine residue within Stat5A bind a protein-tyrosine phosphatase.
To identify the potential Stat5A phosphatase, we initiated a large
scale screening for proteins that specifically bind to Stat5A
phosphopeptides. To permit the potential phosphatase to bind to the
tyrosine-phosphorylated peptides, 32D (EpoR wt) myeloid cells were
lysed in lysis buffer without phosphatase inhibitors and phosphate
salts. All procedures were carried out at 4 °C to avoid
dephosphorylation of the phosphorylated peptides. Cell extracts were
precleared with unconjugated Sepharose beads and subsequently incubated
with Sepharose beads conjugated with tyrosine-phosphorylated peptides
or with non-phosphorylated control peptides. After extensive washing,
proteins bound to the beads were eluted and subjected to SDS-PAGE and
silver staining. A protein migrating at an apparent molecular mass of
~70 kDa specifically bound to the tyrosine-phosphorylated, but not
control, peptides (Fig. 2C). Interestingly, binding of the
70 kDa protein could only be detected in the Tris-HCl buffer, which had
neither phosphate salts nor phosphatase inhibitors (Fig. 2C). This suggests that the interaction between the 70 kDa
protein and the tyrosine-phosphorylated peptides was not likely due to an interaction between an SH2 domain and a phosphotyrosine in vitro, although weak interaction of an SH2 domain with a
phosphotyrosine may be blocked by excess amount of phosphate or its
analogs. Following an in-gel tryptic digestion, the peptides derived
from the 70 kDa protein were subjected to matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry
analysis (31). The resulting mass spectrometry data (not shown) were
used to search the NCBInr protein data bases with ProteinProspector
software (prospector.ucsf.edu), identifying the 70-kDa protein as a
known SH2 domain-containing protein-tyrosine phosphatase, Shp-2. The identity of this protein was further confirmed by immunoblot analysis using a specific anti-Shp-2 antibody (Fig. 2D). Notably, the
other mammalian SH2 domain-containing protein-tyrosine phosphatase, Shp-1, bound to neither the tyrosine-phosphorylated peptides nor the
non-phosphorylated control peptides even though both Shp-1 and Shp-2
proteins were highly expressed in the 32D (EpoR wt) cells (Fig.
2D). Therefore, Shp-2 specifically associates with the
tyrosine-phosphorylated peptides corresponding to the
tyrosine-phosphorylation site within Stat5A.
Shp-2 Associates with the Tyrosine-phosphorylated Stat5A in
Vivo--
The next critical issue we addressed was whether Shp-2
interacts with tyrosine-phosphorylated Stat5A in cells. Shp-2 and
Stat5A were co-expressed in COS-7 cells in the presence or absence of Jak2 kinase. As expected, overexpression of Jak2 induced tyrosine phosphorylation of Stat5A (Fig.
3A, upper panel),
and the tyrosine phosphorylation was abolished when the critical
Tyr-694 of Stat5A was mutated to phenylalanine (Fig. 3A,
upper panel). To determine whether Stat5A and Shp-2 form a
complex, Shp-2 immunoprecipitates from lysates of COS-7 transfectants
were subjected to SDS-PAGE and Western blot analysis with anti-Stat5A
antibodies. Stat5A stably associated with Shp-2 in the presence of Jak2
and, to a much lesser extent, in the absence of Jak2 (Fig.
3A, lower panel). Stat5A failed to co-precipitate
with Shp-2 when Tyr-694 of Stat5A was mutated to phenylalanine (Fig.
3A, lower panel). Therefore, Shp-2 associates
with Stat5A in a manner that is dependent on phosphorylation of the
critical Tyr-694 of Stat5A.

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Fig. 3.
Shp-2 associates with the
tyrosine-phosphorylated Stat5A in vivo.
A, association of Shp-2 with Stat5A depending on
phosphorylation of Tyr-694. cDNAs of wild-type Stat5A or a mutant
form of Stat5A, in which Tyr-694 was substituted with phenylalanine
(Y-F694), and cDNAs of Shp-2 were co-transfected into COS-7 cells
in the presence or absence of Jak2-encoding cDNAs. After
transfection, COS-7 cells were lysed in the lysis buffer without
phosphatase inhibitors and phosphate salts. Cell lysates were
immunoprecipitated with anti-Shp-2 or anti-Stat5A antibodies.
Precipitated proteins were subjected to SDS-PAGE and transferred to
nitrocellulose membranes. The proteins immunoprecipitated with
anti-Stat5A were blotted with anti-phosphotyrosine
( -pTyr) or anti-Stat5A ( -Stat5A) (upper)
whereas the proteins immunoprecipitated with anti-Shp-2 antibodies were
blotted with anti-Stat5A ( -Stat5A) (lower).
B, interaction between endogenous Shp-2 and Stat5A depending
on phosphorylation of Stat5A. 32D (EpoR wt) cells cultured in medium
containing IL-3 (IL-3) or starved ( ) overnight were lysed in lysis
buffer with (phosphate) or without (Tris-HCl) phosphatase inhibitors
and phosphate salts at 4 °C. Cell lysates were immunoprecipitated
with anti-Shp-1, anti-Shp-2, or anti-Stat5A. Precipitated proteins were
subjected to Western blot analysis. The proteins immunoprecipitated
with anti-Stat5A were blotted with anti-phosphotyrosine ( -pTyr) or
anti-Stat5A ( -Stat5A) antibodies (upper) whereas the
proteins immunoprecipitated with anti-Shp-1 or anti-Shp-2 antibodies
were blotted with anti-Stat5A ( -Stat5A) antibodies
(lower). The figure shown is representative of three
independent experiments.
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We next examined whether endogenous Shp-2 associates with
tyrosine-phosphorylated Stat5A under more physiological conditions. The
IL-3-dependent 32D (EpoR wt) cells were cultured in
IL-3-containing media or starved in media without IL-3 and subsequently
lysed. As expected, tyrosine-phosphorylated Stat5A could be readily
detected in the presence of IL-3 but not in its absence (Fig.
3B, upper panel). Interestingly,
co-immunoprecipitation of Shp-2 and Stat5A was readily detectable in
lysates of IL-3-treated cells, whereas this interaction was barely
detected in starved cells (Fig. 3B, lower panel).
The association of Stat5A with Shp-2 could only be detected in Tris-HCl
buffer without phosphate salts and phosphatase inhibitors (Fig.
3B, lower panel). Additionally, Stat5A did not associate with Shp-1 under the same conditions (Fig. 3B,
lower panel). These results demonstrate that
tyrosine-phosphorylated Stat5A specifically associates with endogenous
Shp-2 under physiologically relevant conditions.
To localize the structural domains of Shp-2 involved in association
with Stat5A, we examined the ability of GST fusion proteins, containing
either the two SH2 domains of Shp-2 (GST-SH2) or the protein-tyrosine
phosphatase domain (PTP) of Shp-2 (GST-PTP) (Fig. 4A), to bind
tyrosine-phosphorylated Stat5A. Glutathione-Sepharose beads-loaded with
the same amounts of GST-SH2 or GST-PTP fusion proteins were incubated
with lysates of COS-7 cells that had been co-transfected with Stat5A
and Jak2. The amount of tyrosine-phosphorylated Stat5A bound to the GST
fusion proteins was assessed by Western blot analysis. The Shp-2 PTP
domain fusion proteins associated with tyrosine-phosphorylated Stat5A
whereas the Shp-2 SH2 domain fusion protein failed to (Fig.
4B). This same Shp-2 SH2 domain fusion protein has been
shown to be able to bind growth hormone receptor (34). Therefore, the
PTP catalytic domain, rather than the SH2 domains, of Shp-2 is
primarily involved in association with tyrosine-phosphorylated
Stat5A.

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Fig. 4.
The PTP catalytic domain rather than the SH2
domains of Shp-2 is primarily involved in association with
tyrosine-phosphorylated Stat5A. A, a schematic diagram
of GST fusion proteins containing the two SH2 domains (GST-SH2) or the
PTP catalytic domain (GST-PTP) of Shp-2. B, association of
tyrosine-phosphorylated Stat5A with GST-PTP fusion proteins. GST-SH2 or
GST-PTP fusion proteins were incubated with lysates of COS-7 cells that
had been co-transfected with Stat5A and Jak2. The amount of
tyrosine-phosphorylated Stat5A bound to the GST fusion proteins was
assessed by Western blot analysis with anti-Stat5A antibodies
(upper). The amount of GST fusion proteins was assessed by
Western blot analysis with anti-GST antibodies (lower). The
figure shown is representative of two independent experiments.
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Shp-2 Dephosphorylates Tyrosine-phosphorylated Stat5A in
Vivo--
To determine whether Shp-2 dephosphorylates
tyrosine-phosphorylated Stat5A in cells, we tested the ability of Shp-2
to attenuate Epo-induced tyrosine phosphorylation of Stat5A. Stat5A and
EpoR cDNAs were co-transfected into COS-7 cells with or without
Shp-2. 48 h later, cells were stimulated with Epo for 30 min and
subsequently lysed. The level of Epo-induced tyrosine phosphorylation
of Stat5A was examined. Overexpression of Shp-2 significantly
attenuated Epo-induced tyrosine phosphorylation of Stat5A (Fig.
5A). To further determine the
role of Shp-2 in dephosphorylation of Stat5A, we examined
dephosphorylation of Stat5A in wild-type and Shp-2-deficient (Shp-2
/
) fibroblast cells derived from embryos of mice
with a targeted deletion of exon 3 of Shp-2 (35). These MEFs were
engineered to stably express Epo receptors and Stat5A molecules, and
the cells were stimulated with Epo for 15 min. After the cytokine was
removed, a tyrosine kinase inhibitor, staurosporine, was added to the
cells. Levels of tyrosine-phosphorylated Stat5A were assessed at
different time points following cytokine removal. Decreased levels of
tyrosine-phosphorylated Stat5A were observed within 30 min, and
tyrosine-phosphorylated Stat5A was barely detectable within 1 h of
cytokine removal in wild-type MEFs (Fig. 5B). In contrast,
the rate of dephosphorylation of Stat5A was dramatically delayed in
Shp-2
/
MEFs, in which tyrosine-phosphorylated Stat5A
was detectable even at 4 h following cytokine removal (Fig.
5B). To determine that the observed delay in Stat5A
dephosphorylation in Shp-2
/
MEFs is due to the lack of
Shp-2 protein, we performed reconstitution analysis. Shp-2 was
transiently introduced into the Epo receptor and Stat5A expressing
Shp-2
/
MEFs via transient transfection. Reintroduction
of Shp-2 into Shp-2
/
MEFs was sufficient to restore
Stat5A dephosphorylation (Fig. 5B). Therefore, Shp-2 is
indeed required for the dephosphorylation of Stat5A. To demonstrate
that the delayed dephosphorylation of Stat5A in Shp-2
/
MEFs was not due to the delayed down-regulation of Jak2 activity, we
examined the turnover rate of active Jak2 kinases. The turnover rate of
active Jak2 kinases, indicated by the disappearance of tyrosine-phosphorylated forms, was comparable in both wild-type and
Shp-2
/
MEFs (Fig. 5C). Thus, the delayed
dephosphorylation of Stat5A in Shp-2
/
MEFs was not due
to the delayed turnover of Jak2 activity. To determine whether delayed
dephosphorylation of Stat5A in Shp-2
/
cells was
Stat5A-specific, the rate of dephosphorylation of Stat3 was also
assessed. Wild-type and Shp-2
/
MEFs, which express both
Stat3 and Oncostatin M (OSM) receptors, were stimulated for 15 min with
OSM, a cytokine which potently activates Stat3 (36). The levels of
tyrosine-phosphorylated Stat3 were assessed at different time points
following cytokine removal. The rate of dephosphorylation of
tyrosine-phosphorylated Stat3 was comparable in wild-type and
Shp-2
/
MEFs (Fig. 5D), which demonstrates
that Shp-2 is not involved in the dephosphorylation of Stat3 molecules.
To determine the cellular compartment, in which Shp-2 dephosphorylates
Stat5A, we examined Stat5A dephosphorylation in cytoplasmic and nuclear fractions derived from wild-type and Shp-2-deficient MEFs treated with
Epo followed by removal of the cytokine. Interestingly, the Shp-2-deficiency delayed dephosphorylation of Stat5A mainly in the
cytoplasm (Fig. 5E). We conclude from these studies that
Shp-2 is a Stat5A phosphatase and is specifically involved in
dephosphorylation of the tyrosine-phosphorylated Stat5A in
cytoplasm.

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Fig. 5.
Shp-2 is involved in Stat5A dephosphorylation
in vivo. A, overexpression of Shp-2
attenuating Epo-induced tyrosine phosphorylation of Stat5A. Epo
receptor and Stat5A cDNAs were co-transfected into COS-7 cells in
the presence or absence of Shp-2. Forty-eight hours after transfection,
COS-7 cells were stimulated with Epo for 30 min and subsequently lysed.
Cell lysates were immunoprecipitated with anti-Stat5A antibodies.
Precipitated proteins were subjected to Western blot analysis with
anti-phosphotyrosine ( -pTyr) or anti-Stat5A
( -Stat5A) antibodies. B, dramatically delayed
dephosphorylation of Stat5A in Shp-2-deficient fibroblasts. Epo
receptor and Stat5A cDNAs were stably expressed in wild-type
(Shp-2+/+) or Shp-2-deficient (Shp-2 / ) MEF
or in Shp-2-deficient MEF, in which Shp-2 was re-introduced back via
transient transfection (Shp-2 / + Shp-2). These MEFs
were stimulated with Epo for 15 min, and subsequently Epo was removed.
At different time points following cytokine removal, cells were
collected and lysed. Cell lysates were subjected to Western blot
analysis with anti-phosphoStat5 ( -pStat5) or anti-Stat5A
( -Stat5A) antibodies. C, Shp-2 deficiency having no
effect on the turnover rate of active Jak2 kinase. Wild-type
(Shp-2+/+) or Shp-2-deficient (Shp-2 / ) MEFs
were stimulated with Epo as described in B. Cell lysates
were immunoprecipitated with anti-Jak2 antibodies. Precipitated
proteins were subjected to Western blot analysis with
anti-phosphotyrosine ( -pTyr) or anti-Jak2 ( -Jak2) antibodies.
D, comparable rates of dephosphorylation of Stat3 in
wild-type and Shp-2 mutant MEFs. Wild-type (Shp-2+/+) or
Shp-2 mutant (Shp-2 / ) MEFs were stimulated with
Oncostatin M (OSM) for 15 min, and subsequently OSM was removed. At
different time points following cytokine removal, cells were collected
and lysed. Cell lysates were immunoprecipitated with anti-Stat3
antibodies. Precipitated proteins were subjected to Western blot
analysis with anti-phosphotyrosine ( -pTyr) or anti-Stat3 antibodies.
E, delayed dephosphorylation of Stat5A in the cytoplasma of
Shp-2-deficient MEFs. Wild-type (Shp-2+/+) or
Shp-2-deficient (Shp-2 / ) MEFs were stimulated with Epo
as described in B. Cytoplasmic and nuclear extracts from
these MEFs were subjected to Western blot analysis with
anti-phosphoStat5 ( -pStat5) or anti-Stat5A ( -Stat5A) antibodies.
The figure shown is representative of three independent
experiments.
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DISCUSSION |
Shp-2 is an SH2 domain-containing tyrosine phosphatase that is
widely expressed in all tissues (37, 38), similar to Stat5A (12). Shp-2
appears to be involved in multiple signaling pathways as a positive or
a negative regulator (38, 39). Targeted deletion of Shp-2 in mice
resulted in early embryonic lethality (35). Several putative substrates
of Shp-2 have been identified, including SHPS-1/SIRP
(40), PZR (41),
PDGF-R
(42), Gab1 (43), and IRS1 (44). Shp-2 has been shown to
interact with Jak kinases (45, 46) and, in Shp-2 mutant cells, tyrosine
phosphorylation of Jak1 but not Jak2 was significantly enhanced upon
IFN-
stimulation (47). Nonetheless, phosphorylated Jak kinases
appear not to be substrates of Shp-2 (48). Consistently, we show here
that Shp-2 deficiency did not influence the turnover of
tyrosine-phosphorylated Jak2 upon Epo stimulation (Fig. 5B).
Jak2 might be the substrate of Shp-1 (18) and the interaction between
Jak2 and Shp-1 is direct and independent of SH2 domain-phosphotyrosine
interaction (49). We show in the present studies that Shp-2, but not
Shp-1, specifically interacts with Stat5A in vivo and this
interaction is tyrosine phosphorylation-dependent. The
functional relevance of this interaction was established in experiments
that showed that overexpression of Shp-2 impaired Epo-induced tyrosine
phosphorylation of Stat5A and that Shp-2 deficiency dramatically
delayed dephosphorylation of Stat5A following cytokine removal.
Nonetheless, in Shp-2-deficient cells, Stat5A is still
dephosphorylated, albeit delayed. It is possible that there is another
phosphatase(s) involved in the dephosphorylation of Stat5A. Previous
studies with overexpression systems have suggested that other
phosphatases, PTP1B (26), TC-PTP (27), and phosphatase 2A (28), in
addition to Shp-2 (25), are able to interact and dephosphorylate
Stat5A. However, the physiological role of these phosphatases in
down-regulation of Stat5A is not clear. In fact, recent studies using
TC-PTP-deficient cells have clearly demonstrated that TC-PTP is a
Stat1, but not a Stat5 phosphatase (50). Although our findings provide
direct evidence that Shp-2 is a Stat5A phosphatase in vivo,
the possibility that there is another Stat5A phosphate(s) still exists.
What is the effect of Shp-2 deficiency-caused delay of Stat5A
dephosphorylation on the function of Stat5? We examined the induction
of CIS or OSM, two genes that are regulated by
Stat5, in Shp-2-deficient MEFs. Interestingly,
delayed-dephosphorylation of Stat5A in Shp-2-deficient MEFs did not
enhance or extend the induction of the two Stat5 target genes (data not
shown). This result is not a total surprise. Continuous treatment of
cells with IL-3 or Epo prolongs the phosphorylation of Stat5, but fails to extend the induction of Stat5 target genes (29). In addition, our
previous studies (24) have shown that a proteasome inhibitor, MG132,
blocks dephosphorylation of both Stat5A and Stat5B, but does not
detectably affect the induction of CIS or OSM (data not shown).
Therefore, Stat5 must cooperate with additional transcription factor(s), whose activity is only transiently up-regulated by cytokines, to induce Stat5 target genes. Prolonged phosphorylation of
Stat5 alone is not sufficient to extend induction of Stat5 target genes.
The molecular basis for the Stat5A and Shp-2 interaction is not fully
understood. SH2 domains of Shp-2 have been shown to be important in
many cases for subcellular localization of the phosphatase to its
substrates (51, 52). However, our findings here demonstrate that the
PTP catalytic domain of Shp-2 interacts directly with the sole
phosphotyrosine residue of Stat5A while the SH2 domains of Shp-2 appear
not to be directly involved in the interaction between the enzyme and
this substrate (Fig. 4). An important factor that contributed to our
successful identification of Shp-2 as a Stat5A phosphatase might be the
right choice of buffer condition used in this study. Phosphate salt,
sodium pyrophosphate (30 mM), but not sodium vanadate, is
sufficient to block the interaction between Shp-2 and phosphorylated
Stat5A (data not shown). However, in the presence of phosphatase
inhibitors and phosphate salts, only the catalytically inactive
Cys-to-Ser mutant Shp-2, not wild-type, interacts with
tyrosine-phosphorylated Stat5 (25). In addition, at a low temperature,
ionic and hydrophilic interaction might stabilize the association of
the phosphotyrosine of Stat5A with the catalytic domain of Shp-2, which
might also contribute to our identification of Shp-2.
In previous studies, we identified a relatively small and potential
amphipathic helical region of the carboxyl domain of Stat5A that is
important in the control of Stat5A dephosphorylation (24). Therefore,
the small region of the carboxyl domain and the phosphotyrosine of
Stat5A participate in contact with Shp-2. Interestingly, this region is
also a transcriptional activation domain (24). Nevertheless, fractionation of wild-type and Shp-2-deficient MEFs treated with Epo
followed by a removal of the cytokine reveals a delay of Stat5A dephosphorylation mainly in the cytoplasmic fraction of Shp-2-deficient MEFs (Fig. 5E). Moreover, we have demonstrated that DNA
binding is not required for the down-regulation of
tyrosine-phosphorylated Stat5A, and that the tyrosine-phosphorylated
amino-terminal deletion mutant of Stat5A, which is defective in nuclear
translocation, is recycled normally as wild-type Stat5A (24) (data not
shown). Our findings agree with other studies, which have also shown
that down-regulation of phosphorylated Stat5A can occur in the
cytoplasm (25). Therefore, it is more likely that the small region of the carboxyl domain of Stat5A independently recruits a transcriptional complex and Shp-2, although the possibility that Shp-2 exists in the
transcriptional activation complex could not be excluded. Given the
fact that Shp-2 is mainly distributed in the cytoplasm (53), it is
reasonable that dephosphorylation of Stat5A primarily occurs in
cytoplasm. However, the possibility that a yet unidentified nuclear
phosphatase dephosphorylates Stat5A in the nucleus still exists. In
this regard, a recently identified Stat1 phosphatase, TC-PTP,
dephosphorylates Stat1 in both nucleus and cytoplasm (50).
Our previous experiments have indicated that different mechanisms are
involved in the turnover of different tyrosine-phosphorylated Stat
molecules. The difference is conferred by the carboxyl domains of Stat
proteins because the Stat5A phenotype of stability in the presence of
proteasome inhibitor (MG132) can be transferred onto Statl by simply
replacing its carboxyl terminus with that of Stat5A (24). Our current
findings that Shp-2 deficiency delays down-regulation of Stat5A but not
Stat3 (Fig. 5C) further support the notion that the
dephosphorylation of various tyrosine-phosphorylated Stats is executed
by different phosphatases. The carboxyl domains of different Stats
might determine which phosphatase is recruited for their
dephosphorylation. It will be interesting to examine the effect of
Shp-2 deficiency on the down-regulation of other Stats, especially
Stat4 and Stat6.