Dominant Negative Variants of the SHP-2 Tyrosine Phosphatase Inhibit Prolactin Activation of Jak2 (Janus Kinase 2) and Induction of Stat5 (Signal Transducer and Activator of Transcription 5)-Dependent Transcription
Susanne Berchtold1,
Sinisa Volarevic1,
Richard Moriggl,
Mladen Mercep and
Bernd Groner
Institute for Experimental Cancer Research (S.B., R.M., B.G.)
Tumor Biology Center and Department of Biology University of
Freiburg 79106 Freiburg, Germany
Novartis Inc. (S.V.,
M.M.) CH 4002 Basel, Switzerland
 |
ABSTRACT
|
---|
PRL plays a central role in the regulation
of milk protein gene expression in mammary epithelial cells and in the
growth and differentiation of lymphocytes. It confers its activity
through binding to a specific transmembrane, class I hematopoietic
receptor. Ligand binding leads to receptor dimerization and activation
of the tyrosine kinase Jak (janus kinase) 2, associated with the
membrane-proximal, intracellular domain of the receptor. Jak2
phosphorylates and activates Stat5, a member of the Stat (signal
transducers and activators of transcription) family. PRL receptor also
activates SHP-2, a cytosolic tyrosine phosphatase. We investigated the
connection between these two signaling events and derived a dominant
negative mutant of SHP-2 comprising the two SH2 domains
[SHP-2(SH2)2]. An analogous variant of the SHP-1 phosphatase
[SHP-1(SH2)2] was used as a control. The dominant negative mutant of
SHP-2 was found to inhibit the induction of tyrosine phosphorylation
and DNA-binding activity of m-Stat5a, m-Stat5b, and the
carboxyl-terminal deletion variant m-Stat5a
749, as well as the
transactivation potential of m-Stat5a and m-Stat5b. The dominant
negative mutant SHP-1(SH2)2 had no effect. The kinase activity of Jak2
is also dependent on a functional SHP-2 phosphatase. We propose that
SHP-2 relieves an inhibitory tyrosine phosphorylation event in Jak2
required for Jak2 activity, Stat5 phosphorylation, and transcriptional
induction.
 |
INTRODUCTION
|
---|
The polypeptide hormone PRL is produced in the anterior
pituitary, regulates the activity of milk protein gene promoters in
mammary epithelial cells (1, 2, 3), and plays an important role in the
growth and differentiation of lymphocytes (4, 5). It exerts its action
via the PRL receptor and the activation of intracellular signaling
molecules, e.g. the Jak (janus kinase)-Stat (signal
transducers and activators of transcription) pathway. The PRL receptor
belongs to the hematopoietin receptor superfamily (6) and does not
possess intrinsic tyrosine kinase activity but is associated with the
cytoplasmic tyrosine kinase Jak2 (5, 7, 8). Ligand binding leads to
dimerization of the receptor and activation of Jak2 (8). Jak2
phosphorylates the PRL receptor as well as the transcription factor
Stat5. Upon phosphorylation Stat5 forms homodimers, translocates to the
nucleus, and specifically binds to the promoter regions of target
genes, thus activating transcription (9, 10).
Two Stat5 homologs have been identified, Stat5a and Stat5b,
encoded by two closely related genes that are expressed in most
tissues (11, 12, 13, 14). Stat5a was originally found in the mammary gland of
lactating animals (15, 16). Stat5 was shown to be an in
vitro substrate for Jak2 (3). Stat5a and Stat5b form homo- or
heterodimers upon tyrosine phosphorylation by Jak2 (12), and both can
induce transcription of the ß-casein gene promoter. Acquisition of
specific DNA-binding activity precedes the transcriptional induction of
milk protein genes (15, 2). Stat5a and Stat5b are also activated by
other cytokines, growth factors, or hormones [e.g. GH,
erythropoietin, thrombopoietin, interleukin (IL)-2, IL-3, IL-5, IL-7,
IL-9, IL-15, granulocyte macrophage-colony stimulating factor (GM-CSF)
and epidermal growth factor (11, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)] . The multitude of the
inducing signals implicates Stat5 as an important regulator of immune
function.
In addition to Jak2 and Stat5 induction, PRL receptor
activation also results in the stimulation of the protein tyrosine
phosphatase SHP-2 (27). This cytosolic tyrosine phosphatase, also known
as SH-PTP2 (28), SH-PTP3 (29), PTP2C (30), PTP1D (31), and Syp (32),
contains two SH2 domains at its amino terminus and a carboxyl-terminal
catalytic domain. SHP-2 is closely related to SHP-1 (33), also called
PTP1C (34), SH-PTP1 (35), HCP (36), and SHP (37). SHP-1 is expressed
predominantly in hematopoietic and in epithelial cells. Mice with
mutations in SHP-1 exhibit the motheaten phenotype (38) and have
hematopoietic abnormalities, i.e. SHP-1 plays a central role
in hematopoiesis. SHP-2 is ubiquitously expressed. It has been shown to
associate with ligand- activated growth factor, hormone, and cytokine
receptors, similar to the receptors for platelet-derived growth factor,
epidermal growth factor (39, 40, 41), insulin (42), and erythropoietin (43, 44). These interactions are mediated by the SH2 domains of SHP-2. Upon
treatment of cells with IL-3 and GM-CSF, SHP-2 was also shown to be
phosphorylated and to interact with Grb2 and PI-3-kinase (45).
It is generally thought that phosphatases attenuate or block tyrosine
phosphorylation-mediated signals and play an inhibitory role (33, 46, 47). Since tyrosine phosphorylation itself can also cause the
inhibition of kinase activity, e.g. through phosphorylation
of tyrosine 527 in c-src (48), it is conceivable that
the phosphatases could also play a positive role in cytokine signaling.
SHP-2 was shown to be essential for interferon
/ß-induced gene
transcription (49), and recent experiments (27) have shown that its
activation through the PRL receptor contributes to ß-casein promoter
activation. Upon PRL treatment, it is phosphorylated and forms a
complex with the PRL receptor and Jak2. As both Stat5 and SHP-2 play a
role in the efficient transcriptional induction of the ß-casein gene,
we investigated the relationship between the phosphorylation of Stat5
and the activity of SHP-2. Dominant negative variants of SHP-1 and
SHP-2 have been derived, and their effects on tyrosine phosphorylation,
in vitro DNA-binding activity, and transactivation of
m-Stat5a and Stat5b were analyzed. We also analyzed a dominant negative
mutant of m-Stat5a (m-Stat5a
749) (50). Active SHP-2 was found to be
essential for efficient tyrosine phosphorylation of all Stat5 variants
analyzed and the transcriptional induction of the ß-casein-luciferase
construct. The kinase activity of Jak2 was also found to be dependent
on functional SHP-2.
 |
RESULTS
|
---|
Derivation of a Dominant Negative Variant of SHP-2 by Deletion of
the Phosphatase Domain
The phosphorylation on tyrosine 694 of Stat5 has been shown
to be a crucial requirement for the acquisition of DNA-binding
activtity and the transcriptional induction through Stat5 (17). The
phosphorylation of Stat5 upon PRL receptor activation is accomplished
by the receptor-associated Jak2 kinase. SHP-2 is a tyrosine phosphatase
that is associated with Jak2 and is activated upon PRL receptor
engagement in a trimeric complex (27). Although Stat5 dephosphorylation
clearly results in the loss of the DNA-binding activity and the
transactivation potential (50), phosphatase action has also been
implicated in the positive regulation of PRL action (27).
To investigate the role of SHP-2 in Stat5 activation, we derived
a dominant negative variant of SHP-2. This truncated molecule comprises
both SH2 domains (amino acids 1219) but lacks the catalytic domain
(SHP-2(SH2)2)). As a control, a similar construct was generated from
the related SH2 domain containing tyrosine phosphatase, SHP-1. This
phosphatase shares high homology with SHP-2 [SHP-1(SH2)2] and is
shown in Fig. 1B
. The proteins still can
interact with their substrates via their SH2 domains, but do not
display phosphatase activity. SHP-1 and SHP-2 normally bind to the
membrane-proximal domain of cytokine receptors, i.e. their
site of action is close to the inner periplasmic membrane. To retain
this cellular localization, a tag consisting of the amino-terminal 15
amino acids of c-src was fused to the N termini of the SHP-1
and SHP-2 constructs (51, 52). The 15 amino acids provide the
constructs with a myristylation signal that targets them to the
membrane. The dominant negative functions of these molecules were
established in transfected T cells and 293 cells (S. Volarevic and M.
Mercep, submitted for publication). The N-terminal c-src
sequence improved the efficiency of the dominant negative effect. In
addition, a chimeric molecule containing wild-type SHP-2 and the
myristylation site of c-src was created. These constructs
are shown in Fig. 1B
. To visualize the expression of the
SHP-constructs, they were transfected into COS7 cells, extracts were
prepared, and Western blotting experiments were performed with an
antibody directed against the c-src tag sequence (Fig. 1C
).
SHP-2(SH2) was expressed slightly less efficiently (lanes 69) than
SHP-2 (lanes 25) or SHP-1(SH2)2 (lanes 1013). An increase in the
signals for SHP-2 in cells treated with PRL (lanes 3 and 5) can be
observed. The appearance of double bands suggests that secondary
modifications might have been induced.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 1. Structure of Stat5 Variants and SHP-1 and 2
Constructs
A, Structure of m-Stat5a, the carboxyl-terminal deletion mutant
m-Stat5a 749, and m-Stat5b. Amino acid positions (aa), the SH2
domain, the tyrosine residue (Y), which is phosphorylated upon
activation, and the C-terminal transactivation domain (TAD) are
indicated. B, Structure of SHP-2 and the carboxyl-terminal deletion
mutants SHP-2(SH2)2 and SHP-1(SH2)2. Amino acid positions, SH2 domains,
the catalytic domain, and the N-terminal src-tag with the myristylation
site are indicated. C, Expression of SHP-2, SHP-2(SH2)2, and
SHP-1(SH2)2 in transfected cells. COS7 cells were transfected with the
PRL receptor and increasing amounts of SHP-2, SHP-2(SH2)2, or
SHP-1(SH2)2 and induced with PRL (+) or left untreated (-). Whole-cell
extracts were prepared and Western blots were performed. The membrane
was probed with an antibody recognizing the src-tag of
the phosphatase constructs.
|
|
SHP-2 Activity Is Essential for the Induction of Tyrosine
Phosphorylation and DNA-Binding Activity of m-Stat5a after PRL Receptor
Activation
To study the influence of SHP-2 activity on Stat5 induction, COS7
cells were transfected with the PRL receptor, m-Stat5a and SHP-2,
SHP-2(SH2)2, or SHP-1(SH2)2. Cells were stimulated for 1 h with
PRL, and whole-cell extracts were prepared. The tyrosine
phosphorylation of m-Stat5a was analyzed in Western blots with a
phosphotyrosine-specific antibody (Fig. 2A
, upper panel). PRL
treatment of the cells caused a strong increase in tyrosine
phosphorylation of m-Stat5a (Fig. 2A
, lanes 1 and 2). Expression of
wild-type SHP-2 had no effect on the induction of tyrosine
phosphorylation of m-Stat5a (lanes 3 and 4). Tyrosine phosphorylation
of m-Stat5a upon PRL treatment was strongly reduced with 1 µg
SHP-2(SH2)2 DNA (lane 6) and undetectable when 2 and 4 µg of
SHP-2(SH2)2 DNA were introduced (lanes 7 and 8). SHP-1(SH2)2 did not
affect tyrosine phosphorylation of m-Stat5a, even at high levels of
expression (lanes 1012). The amount of m-Stat5a expressed in the
transfected cells was similar in all samples (Fig. 2A
, lower
panel).

View larger version (54K):
[in this window]
[in a new window]
|
Figure 2. Tyrosine Phosphorylation and DNA Binding of
m-Stat5a Is Inhibited by SHP-2(SH2)2
COS7 cells were transfected with the PRL receptor m-Stat5a and
increasing amounts of SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2. Cells were
stimulated with PRL for 1 h (+) or left untreated (-), and
whole-cell extracts were prepared. A, Western blotting analysis of
m-Stat5a. Proteins from whole-cell extracts were separated by SDS gel
electrophoresis, blotted onto nitrocellulose, and incubated with a
phosphotyrosine-specific antibody ( -P-Tyr). The membrane was
reprobed with an antibody recognizing both Stat5a and Stat5b
( -Stat5, lower panel). B, DNA-binding activities of
m-Stat5a. Bandshift experiments were performed with whole-cell extracts
using the 32P-labeled Stat5-binding site of the bovine
ß-casein promoter as a probe.
|
|
Tyrosine phosphorylation is required for dimerization, nuclear
translocation, and DNA binding of Stat proteins. To determine whether
SHP-2(SH2)2 has an effect on the DNA-binding activity of m-Stat5a,
bandshift experiments were carried out. The high-affinity Stat5-binding
site present in the rat ß-casein promoter was used as a probe (Fig. 2B
). In nonstimulated cells, no specific DNA-binding activity was
observed (lane 1). PRL induction led to a strong DNA-binding activity
of m-Stat5a (lane 2). Coexpression of low amounts of wild-type SHP-2
did not influence DNA binding of m-Stat5a. Only very high
concentrations caused a weak inhibition of DNA binding (lane 8). In
contrast, coexpression of SHP-2(SH2)2 dramatically reduced DNA binding
of m-Stat5a even at low concentrations (lane 10). When higher
concentrations were used, DNA binding of m-Stat5a was completely
abolished (lanes 1214). SHP-1(SH2)2 did not alter DNA binding of
m-Stat5a (lanes 16, 18, and 20). Despite the high homology of the
SH2 domains of these phosphatases, specificity in the regulation of
m-Stat5a tyrosine phosphorylation and DNA-binding activity is
restricted to SHP-2.
SHP-2(SH2)2 Inhibits Tyrosine Phosphorylation and DNA-Binding
Activity of m-Stat5a
749 and of m-Stat5b
The deletion mutant m-Stat5a
749, shown in Fig. 1A
, has been
derived earlier and shows peculiar properties with respect to its
regulation of tyrosine phosphorylation (50). The induction of tyrosine
phosphorylation of m-Stat5a
749 upon PRL treatment of cells was
indistinguishable from that of the wild-type molecule. The
down-regulation, mediated by dephosphorylation, however, was strongly
delayed (50), and we suggested that the interaction with a phosphatase
is impaired. We therefore investigated the influence of SHP-2 on the
tyrosine phosphorylation of this mutant (Fig. 3
). Whole-cell extracts were prepared
from COS7 cells transfected with m-Stat5a
749, the PRL receptor, and
the SHP-1 and 2 constructs. Upon PRL induction of the cells,
m-Stat5a
749 was analyzed and tyrosine phosphorylation of
m-Stat5a
749 was found (Fig. 3A
, lanes 1 and 2). Tyrosine
phosphorylation of m-Stat5a
749 was not affected by wild-type SHP-2
(lanes 3 and 4). Cotransfection of SHP-2(SH2)2 led to a dose-dependent
inhibition of tyrosine phosphorylation of m-Stat5a
749 (lanes 57).
Compared with the wild-type m-Stat5a, the deletion mutant seemed
slightly less sensitive toward the inhibitory effect of SHP-2(SH2)2.
SHP-1(SH2)2 had no effect on tyrosine phosphorylation of m-Stat5a
749
(lane 9), and the molecule was similarly expressed in all samples
(Fig. 3A
, lower panel).
To corroborate the findings we correlated tyrosine phosphorylation of
m-Stat5a
749 with its DNA-binding activity (Fig. 3B
). PRL induction
resulted in strong DNA binding of m-Stat5a
749 (lanes 1 and 2).
Expression of wild-type SHP-2 (lane 3) had no influence on DNA binding,
whereas expression of SHP-2(SH2)2 inhibited DNA binding of
m-Stat5a
749 (lanes 46). This corresponds to decreased tyrosine
phosphorylation (Fig. 3A
, lanes 57). SHP-1(SH2)2 did not alter DNA
binding of m-Stat5a
749 (lanes 7 and 8). This demonstrates that
tyrosine phosphorylation and DNA binding of m-Stat5a
749 are
positively regulated by SHP-2.
m-Stat5a and m-Stat5b exhibit a high sequence homology but are encoded
by different genes. They differ mainly in their C-terminal sequences
(12). We investigated whether SHP-2 also regulates tyrosine
phosphorylation and DNA binding of m-Stat5b. Extracts from COS7 cells
transfected with m-Stat5b, the PRL receptor, and the SHP-1 and 2
constructs were prepared, and tyrosine phosphorylation of m-Stat5b was
visualized (Fig. 4A
). Induction of
tyrosine phosphorylation (lanes 1 and 2) was not affected by the
cotransfection of wild-type SHP-2 (lanes 36) or SHP-1(SH2)2 (lanes
1114). Coexpression of SHP-2(SH2)2 resulted in an inhibition of
tyrosine phosphorylation of m-Stat5b (lanes 710). Again, higher
amounts of SHP-2(SH2)2 were required to inhibit tyrosine
phosphorylation of m-Stat5b when compared with m-Stat5a. Bandshift
experiments were also carried out (Fig. 4B
). DNA-binding activity of
m-Stat5b was induced by PRL (lanes 1 and 2). Expression of SHP-2 or
SHP-1(SH2)2 did not alter the DNA-binding activities of m-Stat5b (lanes
36 and 1113). Expression of SHP-2(SH)2 led to a dose-dependent
inhibition of DNA binding of Stat5b (lanes 710). These data show that
functional SHP-2 activity is required for the efficient induction of
tyrosine phosphorylation and DNA-binding activity of all Stat5 variants
analyzed.

View larger version (48K):
[in this window]
[in a new window]
|
Figure 4. SHP-2(SH2)2 Inhibits Tyrosine Phosphorylation and
DNA Binding of m-Stat5b
COS7 cells were transfected with m-Stat5b, the PRL receptor, and SHP-2,
SHP-2(SH2)2, or SHP-1(SH2)2. Cells were stimulated with PRL for 1
h or left untreated. Whole cell extracts were prepared. A, Tyrosine
phosphorylation of m-Stat5b. Whole-cell extracts were analyzed in
Western blotting experiments. The membrane was incubated with the
anti-phosphotyrosine antibody ( -P-Tyr) and reprobed with the
anti-Stat5 antibody ( -Stat5, lower panel). B, DNA
binding of m-Stat5b. Whole-cell extracts were introduced into bandshift
experiments using the Stat5-binding site of the bovine ß-casein
promoter as a probe.
|
|
Transactivation of the ß-Casein Gene Promoter by PRL Is Inhibited
by the Dominant Negative Variant of SHP-2
We studied the effect of SHP-2 on PRL-induced transcription of the
ß-casein gene promoter. Wild-type SHP-2, SHP-2(SH2)2, or SHP-1(SH2)2
was introduced into COS7 cells together with m-Stat5a, the PRL
receptor, and the ß-casein gene promoter luciferase construct. A
ß-galactosidase gene was included to normalize for transfection
efficiency. Luciferase activities were determined in extracts from
cells cultured in the absence and presence of PRL (Fig. 5
). Relative luciferase activity
represents the ratio of luciferase to ß-galactosidase activity.

View larger version (52K):
[in this window]
[in a new window]
|
Figure 5. Transcriptional Induction of the ß-Casein Gene
Promoter Is Inhibited by SHP-2(SH2)2
COS7 cells were cotransfected with the ß-casein gene promoter
luciferase construct, the PRL receptor m-Stat5a, and SHP-2 (lanes
36), SHP-2(SH2)2 (lanes 710), or SHP-1(SH2)2 (lanes 1114). A
ß-galactosidase gene was included to normalize for transfection
efficiency. Cells were induced with PRL for 15 h (+) or left
untreated (-). Luciferase activities were determined after 48 h.
They were in a range from about 201200. Transcriptional activation by
m-Stat5a after induction with PRL in the absence of any phosphatase
construct was set as 100% (lane 2). All other luciferase activities
were compared with this value. Mean values and SDs of three
independent experiments are shown.
|
|
m-Stat5a induction strongly activates the ß-casein promoter after PRL
treatment of the cells (lanes 1 and 2). Expression of wild-type SHP-2
had no effect on the m-Stat5a-mediated transactivation (lanes 36).
Expression of SHP-2(SH2)2 caused a dose-dependent suppression of
transactivation by m-Stat5a (lanes 710). Transcriptional induction of
the ß-casein luciferase construct was not influenced by SHP-1(SH2)2
(lanes 1114). These data indicate that SHP-2 is also essential for
the efficient transactivation of the ß- casein promoter through
m-Stat5a. Inhibition of transactivation by the dominant negative
SHP-2(SH2)2 variant correlates with the inhibition of tyrosine
phosphorylation and DNA binding of m-Stat5a.
The Kinase Activity of Jak2 Is Suppressed by the Dominant Negative
SHP-2 Variant
The induction of Jak2 is an immediate early event upon PRL
receptor activation and precedes the phosphorylation of Stat5. To
determine at which level SHP-2(SH2)2 interferes with the activation of
Stat5, we tested the possibility that Jak2 tyrosine kinase activity is
subject to regulation by SHP-2. For this reason we measured kinase
activity of Jak2 in extracts from cells transfected with SHP-2 and the
dominant negative SHP-2(SH2)2. Cell lysates were immunoprecipitated
with a Jak2-specific antibody, and the precipitates were collected on
protein A Sepharose beads. After washing, Jak2 bound to the beads was
introduced into an in vitro kinase reaction, and the
proteins were separated by SDS-gel electrophoresis, blotted onto
nitrocellulose, and visualized by autoradiography (Fig. 6A
). Kinase activity was observed in
cells not transfected with SHP-2 (lane 1), and expression of wild-type
SHP-2 did not alter the kinase activity of Jak2 (lanes 24). Kinase
activity of Jak2 was strongly inhibited by the expression of
SHP-2(SH2)2 (lane 5). Similar amounts of Jak2 were present in the
individual extracts as analyzed by Western blotting (Fig. 6B
). These
results indicate that SHP-2 positively regulates the kinase activity of
Jak2 and that proper function of SHP-2 is necessary for the efficient
signal transduction through the Jak2-Stat5 pathway.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 6. Kinase Activity of Jak2 Is Inhibited by SHP-2(SH2)2
COS7 cells were transfected with expression plasmids encoding Jak2 and
SHP-2 (lanes 24) or SHP-2(SH2)2 (lane 5). Lysates were prepared and
immunoprecipitated with a Jak2-specific antibody. The
immunoprecipitates were incubated with [32P] ATP,
separated by SDS gel electrophoresis, and blotted onto nitrocellulose.
One of four experiments giving similar results is shown. A,
Phosphorylated proteins were visualized by autoradiography. B, Jak2 was
visualized by Western blotting analysis with a Jak2-specific
antibody.
|
|
 |
DISCUSSION
|
---|
PRL plays a central role in the induction of milk protein
synthesis. Binding to its receptor leads to receptor dimerization and
activation of the associated kinase, Jak2. Jak2 then phosphorylates the
receptor as well as several signaling components including Stat5. The
transcription factor dimerizes and translocates to the nucleus where it
induces transcription of target genes, e.g. the ß-casein
gene. PRL receptor activation also results in tyrosine phosphorylation
and activation of the phosphatase SHP-2. It has been suggested that
SHP-2 could also be a substrate for Jak2 (27). Since activation of the
Jak-Stat pathway is dependent upon specific tyrosine phosphorylation
events and SHP-2 is a tyrosine phosphatase, it seems somewhat
counterintuitive that this enzyme should be involved in the regulation
of this pathway in a positive fashion. However, the results of Ali
et al. (27) and the data reported here suggest that
PRL-induced tyrosine phosphorylation, DNA binding, and transactivation
of Stat5 are dependent on SHP-2 function, supporting the notion that
SHP-2 acts in a positive fashion in the signaling through growth factor
receptors (46).
A deletion mutant of SHP-2 comprising only the SH2 domains and lacking
the catalytic domain exhibits a dominant negative phenotype upon the
wild-type molecule. Most likely, it functions by blocking the
substrate-binding sites through its SH2-domains. This dominant negative
effect is enhanced by an amino-terminal myristylation signal that
causes a membrane-proximal localization of the phosphatase variants.
SHP-2(SH2)2 efficiently inhibited the PRL induction of tyrosine
phosphorylation, DNA binding, and transactivation of m-Stat5a. This
corroborates previous results (3) and the current model, which states
that tyrosine phosphorylation is a prerequisite for DNA binding and
transactivation by Stat5.
Inhibition of tyrosine phosphorylation by the action of dominant
negative SHP-2 was also observed for the C-terminal deletion mutant
m-Stat5a
749. However, higher levels of SHP-2(SH2)2 were required.
This may be due to an additional negative regulatory mechanism whose
function is dependent upon sequences in the C terminus of Stat5. We
showed that the C terminus of Stat5 is required for efficient
dephosphorylation of the activated molecule, possibly through the
interaction with a nuclear tyrosine phosphatase (50). C-Terminal
deletion mutants show prolonged tyrosine phosphorylation and DNA
binding. They behave similar to naturally occurring splice variants of
Stat5, which also are lacking carboxyl-terminal amino acids (50, 53).
Also, to inhibit tyrosine phosphorylation and DNA binding of m-Stat5b,
higher amounts of SHP-2(SH2)2 were needed when compared with m-Stat5a.
After PRL induction, m-Stat5b also shows prolonged tyrosine
phosphorylation and DNA-binding activity when compared with m-Stat5a
(50).
An alternative explanation for the different levels of SHP-2 (SH2)2
required to inhibit individual Stat5 variants could be related to their
potential to induce the recently discovered Stat-induced Stat
inhibitor, SSI-1 (54). This inhibitor is part of a mechanism
responsible for switching off the cytokine signal and its function is
dependent on the transactivation potential of Stat variants.
SHP-1 has been shown to associate with the ß-subunit of the IL-3
receptor and the erythropoietin receptor and seems to play a role in
Jak2 dephosphorylation and termination of signaling (55, 56, 57). SHP-1 and
Jak2 can interact directly through a binding domain present in the N
terminus of SHP-1 and independent of SH2 domain-phosphotyrosine
interactions, resulting in the induction of the enzymatic activity of
the phosphatase in in vitro protein tyrosine phosphatase
assays (58). No effect of SHP-1 was observed in the PRL induction of
Stat5. SHP-2 has been assigned a positive role in several signal
transduction pathways. It associates with the corresponding receptors
and is tyrosine phosphorylated upon stimulation with e.g.
erythropoietin, IL-3/GM-CSF, or IL-6/CNTF (ciliary neurotrophic
factor)/LIF (leukemia-inhibitory factor) (43, 45). SHP-2 forms a
trimeric complex with Jak2 and the PRL receptor upon stimulation with
PRL (27). Phosphorylation leads to activation of the phosphatase.
The dominant negative mutant SHP-2(SH2)2 inhibits the kinase activity
of Jak2, indicating a positive role for SHP-2 in Jak2 activation. It is
possible to envisage a mechanism of regulation similar to one found for
the src family of protein tyrosine kinases, involving tyrosine
phosphate residues with positive and with negative regulatory potential
(48). Upon PRL induction, receptor-associated Jak2 molecules are
brought into proximity and phosphorylate each other and the
intracellular domain of the receptor. Phosphorylated receptor and
phosphorylated Jak2 could serve as docking sites for SHP-2, and
phospholipids in the membrane as well as tyrosine kinase activity could
cause its activation. Activation results in enhanced enzymatic activity
as well as in the generation of new binding sites for adaptor proteins,
e.g. Grb2 (41, 59). The activated phosphatase could then
dephosphorylate an inhibitory tyrosine in Jak2, resulting in a
conformational change and in full activation of the kinase. Jak2 is
tyrosine phosphorylated in the kinase domain and has 14 potential
phosphorylation sites (J. Ihle, personal communication and Ref.60)
seven of which are conserved among the members of the Jak kinase
family. The extent of autophosphorylation is likely to be a regulatory
mechanism for kinase activity. Fully active Jak2 could then
phosphorylate the Stat proteins. Alternatively, SHP-2 might influence
Jak2 activity indirectly by dephosphorylating and inactivating another
phosphatase involved in Jak2 regulation. Our observations are also
consistent with a model in which SHP-2 increases the potential of
individual substrates to become phosphorylated. The distinction of
autokinase activity vs. substrate kinase activity of Jak2
and their regulation by SHP-2 will be dependent on the characterization
of protein complexes formed upon PRL receptor activation.
We propose that SHP-2 acts upstream of Stat5, most likely by activating
Jak2 through dephosphorylation of an inhibitory tyrosine residue. We
were able to correlate the function of SHP-2 tyrosine phosphatase with
the activation of Jak2 tyrosine kinase activity, Stat5 dimerization and
translocation to the nucleus, and specific gene transcription. The fact
that SHP-2 also interacts with other signaling components, such as Grb2
and phosphatidylinositol 3'-kinase (45), shows that this phosphatase is
a general attenuator of divergent signaling pathways. Additional
tyrosine phosphatases, with nuclear sites of action, are probably
involved in the recycling of activated Stat molecules and signal
termination (50, 61).
 |
MATERIALS AND METHODS
|
---|
Plasmids
The (-344 to -1) ß-casein gene promoter luciferase
construct, the expression vectors for m-Stat5a, m-Stat5b, and the
deletion mutant m-Stat5a
749 (schematically shown in Fig. 1A
), and
the PRL receptor have been described previously (12, 50, 62). pHM75
encodes the ß-galactosidase gene driven by the human cytomegalovirus
(CMV) promoter.
To clone the SHP-1 and -2 genes, mRNA was isolated from Jurkat cells
using a Dynabeads kit (Dynal, Oslo, Norway). The cDNA was generated by
using a reverse transcription kit (Perkin Elmer, Norwalk, CT). The cDNA
was then used as a template for PCR. SHP-2 and SHP-2(SH2)2 were made by
PCR amplification using a 5'-primer encoding the first 12 amino acids
of SHP-2 and a 3'-primer encoding the last 10 amino acids of SHP-2 (for
SHP-2) or a 3'-primer encoding amino acids 209219 for SHP-2(SH2)2.
The PCR fragments were cloned into SalI-BamHI
sites of the pBluescript II SK vector (Stratagene, La Jolla, CA).
SHP-1(SH2)2 was PCR amplified using a 5'-primer encoding for the first
10 amino acids of SHP-1 and a 3'-primer encoding amino acids 209219
of SHP-1. The PCR fragment was cloned into
SalI-XbaI sites of the pBluescript II SK vector
and sequenced. Constructs were then cloned into
SalI-BamHI sites (SHP-2) or
SalI-XbaI (SHP-1(SH2)2) of the CMV5 vector (63).
An oligonucleotide coding for the first 15 amino acids of
c-src was fused to the amino terminus. All cloning junctions
and PCR fragments were sequenced using the T7 dideoxy sequencing kit
(Pharmacia, Piscataway, NJ).
Cell Culture and Transfections
COS7 cells were maintained in DMEM medium containing 10% FCS
and 2 mM glutamine and were transfected and induced with
PRL (5 µg/ml) as described previously (17). Transfections were
performed using the calcium phosphate precipitation technique.
Luciferase and ß-galactosidase activities were determined as
described previously (3, 62). For transfections, 2 µg PRL receptor, 2
µg m-Stat5 expression vectors, 14 µg of the SHP-constructs, 2
µg of the luciferase reporter construct, and 1 µg pHM75 were
used.
Antibodies and Immunoblotting Analysis
Antibodies against Jak2 (C-20, rabbit, polyclonal), Stat5 (C-17,
rabbit, polyclonal), recognizing both m-Stat5a and m-Stat5b,
c-src (N-16, rabbit, polyclonal), and phosphotyrosine (PY69,
mouse, monoclonal) were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Proteins were separated on 7.5 or 12.5% SDS-polyacrylamide
gels and blotted onto nitrocellulose filters. The membranes were
blocked with 2% BSA in TBST (20 mM Tris-HCl, pH 7.5, 150
mM NaCl, 0.05% Tween 20) overnight. Incubation with
specific antibodies was performed for 1 h at room temperature.
After washing twice in TBST, the filters were incubated with an
appropriate secondary antibody coupled to horseradish peroxidase.
Immunoreactive bands were visualized using an epichemiluminescence
Western blotting system (Amersham, Arlington Heights, IL) according to
the manufacturers protocol.
Immunoprecipitations and in Vitro Kinase Assays
Cells were lysed in lysis buffer (0.5% Nonidet P 40, 10%
glycerol, 50 mM Tris, pH 8.0, 200 mM NaCl, 0.1
mM EDTA, 1 mM Na3VO4,
0.5 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 3 µg/ml aprotinin, 1 µg/ml leupeptin) for 10 min at
4 C. The insoluble material was removed by centrifugation. Cleared
lysates were incubated for 2 h at 4 C with the Jak2-specific
antibody. The immunoprecipitates were isolated with protein
A-Sepharose. Samples were washed once with lysis buffer and twice with
kinase buffer (50 mM NaCl, 5 mM
MgCl2, 5 mM MnCl2, 0.1
mM Na3VO4, 10 mM HEPES,
pH 7.4). The in vitro kinase reactions were performed in 30
µl kinase buffer in the presence of 10 µM ATP and 1
µCi [32P]ATP. In vitro phosphorylated
proteins were separated by SDS-PAGE, blotted onto nitrocellulose
filters, and visualized by autoradiography.
Preparation of Whole-Cell Extracts and Electric Mobility Shift
Assay
Whole-cell extracts were prepared by suspending the cell pellet
in a hypertonic buffer containing 400 mM NaCl, 50
mM KCl, 20 mM HEPES, pH 7.9, 1 mM
EDTA, 20% glycerol, 1 mM dithiothreitol, 0.1
mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin.
After three times freeze-thawing, the lysates were centrifuged for 10
min at 4 C and 14,000 rpm (20,800 x g). The
supernatants were used for Western blotting or bandshift experiments.
Bandshift assays were performed as previously described (3). The
high-affinity Stat5-binding site of the bovine ß-casein promoter
(5'-AGATTTCTAGGAATTCAAATC-3') was used as a probe. This oligonucleotide
was end-labeled with polynucleotide kinase to a specific activity of
8,000 cpm/fmol.
 |
ACKNOWLEDGMENTS
|
---|
We thank Atsushi Miyajima (Tokyo, Japan) and Carol Stocking
(Hamburg, Germany) for the provision of plasmids, Christian Beisenherz,
Edith Pfitzner, Elisabeth Stöcklin, and Manuela Wissler
(Freiburg, Germany) for helpful discussions, and Ines Fernandez for
editorial assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Bernd Groner, Institute for Experimental Cancer Research, Tumor Biology Center, D-79106 Freiburg, Breisacher Strasse 117, Germany.
1 The first two authors contributed equally to this work 
Received for publication August 15, 1997.
Revision received January 2, 1998.
Accepted for publication January 7, 1998.
 |
REFERENCES
|
---|
-
Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B 1988 Prolactin regulation of the ß-casein expression and of a
cytosolic 120-kd protein in a cloned mouse mammary epithelial cell
line. EMBO J 7:20892095[Abstract]
-
Standke GJR, Meier VS, Groner B 1994 Mammary gland factor
activated by prolactin in mammary epithelial cells and acute-phase
response factor activated by IL-6 in liver cells share DNA binding and
transactivation potential. Mol Endocrinol 8:469477[Abstract]
-
Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin
induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for
DNA binding and induction of transcription. EMBO J 13:43614369[Abstract]
-
Yu-Lee L, Hrachovy JA, Stevens AM, Schwarz LA 1990 Interferon-regulatory factor 1 is an immediate-early gene under
transcriptional regulation by prolactin in Nb2 cells. Mol Cell Biol 10:30873094[Medline]
-
Dusanter-Fourt I, Muller O, Ziemiecki A, Mayeux P, Drucker B,
Djiane J, Wilks A, Harpur AG, Fisher S, Gisselbrecht S 1994 Identification of Jak protein tyrosine kinases as signaling molecules
for prolactin. Functional analysis of prolactin receptor and
prolactin-erythropoietin receptor chimera expressed in lymphoid cells.
EMBO J 13:25832591[Abstract]
-
Bazan F 1989 A novel family of growth factor receptors: a
common binding domain in the growth hormone, prolactin,
erythropoietin and IL-6 receptors, and p75 IL-2
receptor-chain. Biochem Biophys Res Commun 164:788795[Medline]
-
Rui H, Kirken RA, Farrar WL 1994 Activation of
receptor-associated tyrosine kinase Jak2 by prolactin. J Biol Chem 269:53645368[Abstract/Free Full Text]
-
Lebrun JJ, Ali S, Sofer L, Ullrich A, Kelly PA 1994 Prolactin-induced proliferation of Nb2 cells involves tyrosine
phosphorylation of the prolactin receptor and its associated tyrosine
kinase Jak2. J Biol Chem 269:1402114026[Abstract/Free Full Text]
-
Ihle JN 1996 Stats: Signal transducers and activators of
transcription. Cell 84:331334[Medline]
-
Heim MH 1996 The Jak-stat pathway: specific signal
transduction from the cell membrane to the nucleus. Eur J Clin
Invest 26:112[Medline]
-
Azam M, Erdjument-Bromage H, Kreider BL, Xia M, Quelle F, Basu
R, Saris C, Tempst P, Ihle JN, Schindler C 1995 Interleukin-3 signals
through multiple isoforms of Stat5. EMBO J 14:14021411[Abstract]
-
Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning and expression of Stat5 and an additional homologue (Stat5b)
involved in prolactin signal transduction in mouse mammary tissue. Proc
Natl Acad Sci USA 92:88318835[Abstract]
-
Rippberger JA, Fritz S, Richter K, Hocke GM, Lottspeich F, Fey
GH 1995 Transcription factors Stat3 and Stat5b are present in rat liver
nuclei in an acute phase response and bind interleukin 6 response
elements. J Biol Chem 270:2999830006[Abstract/Free Full Text]
-
Kazansky AV, Raught B, Lindsey M, Wang Y, Rosen JM 1995 Regulation of mammary gland factor/Stat5a during mammary gland
development. Mol Endocrinol 9:15981609[Abstract]
-
Schmitt-Ney M, Doppler W, Ball RK, Groner B 1991 Beta-casein
gene promoter activity is regulated by the hormone-mediated relief of
transcriptional repression and a mammary-specific nuclear factor. Mol
Cell Biol 11:37453755[Medline]
-
Wakao H, Schmitt-Ney M, Groner B 1992 Mammary specific nuclear
factor is present in lactating rodent and bovine mammary tissue and
composed of a single polypeptide of 89 kDa. J Biol Chem 267:1636516370[Abstract/Free Full Text]
-
Gouilleux F, Pallard C, Dusanter-Fourt I, Wakao H, Haldosen
LA, Norstedt G, Levy D, Groner B 1995 Prolactin, growth hormone,
erythropoietin and granulocyte-macrophage colony stimulating factor
induce MGF-Stat5 DNA binding activity. EMBO J 14:20052013[Abstract]
-
Gouilleux F, Moritz D, Humar M, Moriggl R, Berchtold S, Groner
B 1995 Prolactin and interleukin 2 receptors in T lymphocytes signal
through a MGF/Stat5-like transcription factor. Endocrinology 136:57005708[Abstract]
-
Johnston JA, Bacon CM, Finbloom DS, Rees RC, Kaplan D, Shibuya
K; Ortaldo JR, Gupta S, Chen YQ, Giri JD, O'Shea JJ 1995 Tyrosine
phosphorylation and activation of Stat5, Stat3, and Janus kinases by
interleukins 2 and 15. Proc Natl Acad Sci USA 92:87058709[Abstract]
-
Lin JX, Migone TS, Tsang M, Friedmann M, Wheatherbee JA, Zhou
L, Yamauchi A, Bloom ET, Mietz J, John S, Leonard WJ 1995 The role of
shared receptor motifs and common Stat proteins in the generation of
cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13 and
IL-15. Immunity 2:331339[Medline]
-
Pallard C, Gouilleux F, Bénit L, Cocault L, Souyri M,
Levy D, Groner B, Gisselbrecht S 1995 Dusanter-Fourt, I. Thrombopoietin
activates a Stat5-like factor in hematopoietic cells. EMBO J 14:28472856[Abstract]
-
Ruff-Jamison S, Chen K, Cohen S 1995 Epidermal growth factor
induces the tyrosine phosphorylation and nuclear translocation of Stat5
in mouse liver. Proc Natl Acad Sci USA 92:42154218[Abstract]
-
Fujii H, Nakagawa Y, Schindler U, Kawahara A, Mori H,
Gouilleux F, Groner B, Ihle JN, Minami Y, Miyazaki T, Taniguchi T 1995 Activation of Stat5 by interleukin 2 requires a carboxyl-terminal
region of the interleukin 2 receptor chain but is not essential for the
proliferative signal transmission. Prolc Natl Acad Sci USA 92:54825486
-
Mui AL, Wakao H, OFarrell AM, Harada N, Miyajima A 1995 Interleukin-3, granulocyte-macrophage colony stimulating factor and
interleukin-5 transduce signals through two Stat5 homologs. EMBO J 14:11661175[Abstract]
-
Wakao H, Harada N, Kitamura T, Mui AL, Miyajima A 1995 Interleukin 2 and erythropoietin activate Stat5/MGF via distinct
pathways. EMBO J 14:25272535[Abstract]
-
Wood TJ, Sliva D, Lobie PE, Pircher TJ, Gouilleux F, Wakao H,
Gustafsson JA, Groner B, Norstedt G, Haldosen LA 1995 Mediation of
growth hormone-dependent transcriptional activation by mammary gland
factor/Stat5. J Biol Chem 16:94489453[CrossRef]
-
Ali S, Chen Z, Lebrun JJ, Vogel W, Kharitonenkov A, Kelly PA,
Ullrich A 1996 PTP1D is a positive regulator of the prolactin signal
leading to -casein promoter activation. EMBO J 15:135142[Abstract]
-
Freeman Jr RM, Plutzky J, Neel BG 1992 Identification of a
human src homology 2-containing protein-tyrosine phosphatase: a
putative homolog of Drosophila corkscrew. Proc Natl Acad Sci USA 89:1123911243[Abstract]
-
Adachi M, Sekiya M, Miyachi T, Matsuno K, Hinoda Y, Imai K,
Yachi A 1992 Molecular cloning of a novel protein-tyrosine phosphatase
SH-PTP3 with sequence similarity to the src-homology region 2. FEBS
Lett 314:335339[CrossRef][Medline]
-
Ahmad S, Banville D, Zhao Z, Fischer EH, Shen SH 1993 A widely
expressed human protein-tyrosine phosphatase containing src homology 2
domains. Proc Natl Acad Sci USA 90:21972201[Abstract]
-
Vogel W, Lammers R, Huang J, Ullrich A 1993 Activation of a
phosphotyrosine phosphatase by tyrosine phosphorylation. Science 259:16111614[Medline]
-
Feng GS, Hui CC, Pawson T 1993 SH2-containing phosphotyrosine
phosphatase as a target of protein-tyrosine kinases. Science 259:16071611[Medline]
-
Scharenberg AM, Kinet JP 1996 The emerging field of
receptor-mediated inhibitory signaling: SHP or SHIP? Cell 87:961964[Medline]
-
Shen, SH, Bastien L, Posner BI, Chretien P 1991 A
protein-tyrosine phosphatase with sequence similarity to the SH2
domains of protein-tyrosine kinases. Nature 352:736739[CrossRef][Medline]
-
Plutzky J, Neel BG, Rosenberg RD 1992 Isolation of a src
homology 2-containing tyrosine phosphatase. Proc Natl Acad Sci USA 89:11231127[Abstract]
-
Yi T, Mui ALF, Krystal G, Ihle JN 1993 Hematopoietic cell
phosphatase associates with the interleukin-3 (IL-3) receptor b-chain
and down-regulates IL-3-induced tyrosine phosphorylation and
mitogenesis. Mol Cell Biol 13:75777586[Abstract]
-
Matthews RJ, Bowne DB, Flores E, Thomas ML 1992 Characterization of hematopoietic protein tyrosine phosphatases:
description of a phosphatase containing an SH2 domain and another
enriched in proline-, glutamic acid-, serine-, and threonine-rich
sequences. Mol Cell Biol 12:23962405[Abstract]
-
Shultz LD, Schweitzer PA, Rajan TV, Yi T, Ihle JN, Matthews J,
Thomas ML, Beier DR 1993 Mutations at the murine motheaten locus are
within the hematopoietic cell protein-tyrosine phosphatase
(Hcph) gene. Cell 73:14451454[Medline]
-
Kazlauskas A, Feng GS, Pawson T, Valius M 1993 The 64-kDa
protein that associates with the platelet-derived growth factor
receptor beta subunit via Tyr-1009 is the SH2-containing
phosphotyrosine phosphatase Syp. Proc Natl Acad Sci USA 90:69396943[Abstract]
-
Lechleider RJ, Freeman RM, Neel BG 1993 Tyrosyl
phosphorylation and growth factor receptor association of the human
corkscrew homologue, SHP-2. J Biol Chem 268:1343413438[Abstract/Free Full Text]
-
Li W, Nishimura R, Kashishian A, Batzer AG, Kim WJH, Cooper
JA, Schlessinger J 1994 A new function for a phosphotyrosine
phosphatase-linking GRB2-SOS to a receptor tyrosine kinase. Mol Cell
Biol, 14:509517[Abstract]
-
Kharitonenkov A, Schnekenburger J, Chen Z, Knyazev Pl, Ali S,
Zwick E, White M, Ullrich A 1995 Adapter function of the
protein-tyrosine phosphatase 1D in insulin receptor/insulin receptor
substrate-1 interaction. J Biol Chem 270:2918919193[Abstract/Free Full Text]
-
Tauchi T, Feng GS, Shen R, Hoatlin M, Bagby Jr GC, Kabat D, Lu
L, Broxmeyer HE 1995 Involvement of SH2-containing phosphatase Syp
in erythropoietin receptor signal transduction pathways. J Biol
Chem 270:56315635[Abstract/Free Full Text]
-
Tauchi T, Damen JE, Toyama K, Feng GS, Broxmeyer HE, Krystal G 1996 Tyrosine 425 within the activated erythropoietin receptor binds
Syp, reduces the erythropoietin required for Syp tyrosine
phosphorylation, and promotes mitogenesis. Blood 87:44954501[Abstract/Free Full Text]
-
Welham MJ, Dechert U, Leslie KB, Jirik F, Schrader JW 1994 Interleukin (IL)-3 and granulocyte/macrophage colony-stimulating
factor, but not IL-4 induce tyrosine phosphorylation, activation, and
association of SHPTP2 with Grb2 and phosphatidylinositol 3'-kinase.
J Biol Chem 269:2376423768[Abstract/Free Full Text]
-
Tonks NK, Neel BG 1996 From form to function-signaling by
protein tyrosine phosphatases. Cell 87:365368[Medline]
-
Okumura M, Thomas ML 1995 Regulation of immune function by
protein tyrosine phosphatases. Curr Opin Immunol 7:312319[CrossRef][Medline]
-
Superti-Furga G, Courtneidge SA 1995 Structure-function
relationship in Src family and related protein tyrosine kinases.
BioEssays 17:321330[Medline]
-
David M, Zhou G, Pine R, Dixon JE, Larner AC 1996 The SH2
domain-containing tyrosine phosphatase PTP1D is required for
interferon/-induced gene expression. J Biol Chem 271:1586215865[Abstract/Free Full Text]
-
Moriggl R, Gouilleux-Gruart V, Jähne R, Berchtold S,
Gartmann C, Liu X, Hennighausen L, Sotiropoulos A, Groner B, Gouilleux
F 1996 Deletion of the carboxy-terminal transactivation domain of
MGF-Stat5 results in sustained DNA binding and a dominant negative
phenotype. Mol Cell Biol 16:56915700[Abstract]
-
Cross FR, Garber EA, Pellman D, Hanafusa HA 1984 short
sequence in the p60src N terminus is required for p60src myristylation
and membrane association and for cell transformation. Mol Cell Biol 4:18341842[Medline]
-
Pellman D, Garber EA, Cross FR, Hanafusa H 1985 An N-terminal
peptide from p60src can direct myristylation and plasma membrane
localization when fused to heterologous proteins. Nature 314:374377[Medline]
-
Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN 1996 Naturally occurring dominant negative variants of Stat5. Mol Cell Biol 16:61416148[Abstract]
-
Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A,
Nishimoto N, Kajita T, Taga T, Yoshizaki K, Akira S, Kishimoto T 1997 Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924929[CrossRef][Medline]
-
Yi T, Cleveland JL, Ihle JN 1992 Protein tyrosine phosphatase
containing SH2 domain: characterization, preferential expression in
hematopoietic cells and localization to human chromosome 12p12p-13. Mol
Cell Biol 12:836846[Abstract]
-
Klingmüller U, Lorenz U, Cantley LC, Neel BG, Lodish HF 1995 Specific recruitment of SHP-1 to the erythropoietin receptor
causes inactivation of JAK2 and termination of proliferation signals.
Cell 80:729738[Medline]
-
Chen HE, Chang S, Trub T, Neel BG 1996 Regulation of
colony-stimulating factor 1 receptor signaling by the SH2
domain-containing tyrosine phosphatase SHPTP1. Mol Cell Biol 16:36853697[Abstract]
-
Jiao H, Berrada K, Yang W, Tabrizi M, Platanias LC, Yi T 1996 Direct association with and dephosphorylation of Jak2 kinase by the
SH2-domain-containing protein tyrosine phosphatase SHP-1. Mol Cell Biol 16:69856992[Abstract]
-
Zhao ZH, Shen SH, Fischer EH 1993 Stimulation by phospholipids
of a protein-tyrosine-phosphatase containing 2 SRC homology-2 domains.
Proc Natl Acad Sci USA 90:42514255[Abstract]
-
Feng J, Witthuhn BA, Matsuda T, Kohlhuber F, Kerr IM, Ihle JN 1997 Activation of Jak2 catalytic activity requires phosphorylation of
Y1007 in the kinase activation loop. Mol Cell Biol 17:24972501[Abstract]
-
Haspel RL, Salditt-Georgieff M, Darnell Jr JE 1996 The rapid
inactivation of nuclear tyrosine phosphorylated Stat1 depends upon a
protein tyrosine phosphatase. EMBO J 15:62626268[Abstract]
-
Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF)
is a novel member of the cytokine regulated transcription factor gene
family and confers the prolactin response. EMBO J 13:21822191[Abstract]
-
Andersson S, Davis DN, Dählback H, Jörnvall H,
Russell DW 1989 Cloning, structure, and expression of the
mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid
biosynthetic enzyme. J Biol Chem 264:82228229[Abstract/Free Full Text]