 |
INTRODUCTION |
Protein tyrosine phosphorylation and dephosphorylation are
fundamental cellular signaling mechanisms that control cell growth and
differentiation (1). In mammalian cells, the SH2 domain-containing tyrosine phosphatases, which include SHP-1 and SHP-2, share a general
structure: two SH2 domains at the N terminus followed by a catalytic
domain and a C-terminal region containing two putative sites of
tyrosine phosphorylation. SHP-2 (previously known as SH-PTP2, PTP1D,
Syp, PTP2C, and SHPTP3) is expressed ubiquitously and is suggested to
play a positive role in the regulation of proximal events downstream of
receptor protein-tyrosine kinases (2, 3). Genetic investigation of
SHP-2 indicates that this phosphatase is crucial for gastrulation
during mammalian development, as mice homozygous for the mutant allele
die in utero at mid-gestation (4-6). In contrast, SHP-1
(previously known as SH-PTP1, PTP1C, HCP, and SHP) is expressed
predominantly in hematopoietic cells, where it seems to function
primarily as a negative regulator of multiple cytokine and growth
factor signaling pathways (2, 3).
It has been postulated that SHP-2 associates with activated
EGF1 and PDGF receptors via
its SH2 domains and becomes rapidly tyrosine-phosphorylated upon ligand
stimulation, which may alter the catalytic activity or regulate its
interaction with other SH2 domain-containing proteins/regulators (7,
8). In the case of PDGF stimulation, tyrosine phosphorylation of SHP-2,
which occurs within its C terminus, creates a binding site for the
Grb2·Sos complex, thereby leading to activation of the
Ras/mitogen-activated protein (MAP) kinase cascade (9, 10). Previous
studies have indicated that enzymatic activity or the SH2 domains of
SHP-2 are required for MAP kinase activation evoked by EGF, insulin,
insulin-like growth factor-1, and fibroblast growth factor (11-14).
However, the mechanisms and the active sites of SHP-2 in the regulation
of MAP kinase activation are not clear.
Little is known about the participation and regulation of SHP-2 in the
activity of G protein-coupled receptors that lack intrinsic tyrosine
kinase activity. It was reported recently that
-thrombin induced
tyrosine phosphorylation of SHP-2 in fibroblasts (15). A catalytically
inactive mutant of SHP-2 strongly inhibited the stimulatory effects of
-thrombin on c-fos transcription and DNA synthesis. This
inhibition could be reversed by cotransfection of SHP-2, but not SHP-1
(15). However, the mechanism by which the G protein-coupled receptor
regulates SHP-2 is unknown. In this study, we found that SHP-2 (but not
SHP-1) was specifically activated by UK14304, an agonist of the
2A-adrenergic receptor, and by lysophosphatidic acid
(LPA) in Madin-Darby canine kidney (MDCK) cells. We have demonstrated
for the first time that the activation of SHP-2 by these Gi
protein-coupled receptors is directly mediated by Fyn kinase and that
there is a specific physical interaction of Fyn with SHP-2 in MDCK cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Protein A- and glutathione-Sepharose and
[
-32P]ATP were purchased from Amersham Pharmacia
Biotech. The Src family kinase assay kit and monoclonal
anti-phosphotyrosine antibody (4G10) were purchased from Upstate
Biotechnology, Inc. Raytide, pp60c-Src tyrosine
kinase, and PP1 were purchased from Calbiochem. UK14304 was purchased
from Research Biochemical Inc. Polyvinylidene difluoride (PVDF)
membranes were obtained from Millipore. Polyclonal antibodies against
SHP-2 (C-18), SHP-1, Src (N-16 and SRC-2), Fyn (FYN3), Yes, Lck, and
Lyn; monoclonal anti-glutathione S-transferase (GST) antibody; and GST fusion proteins containing the SH2 or SH3 domains of
SHP-2, Fyn, and Grb2 were obtained from Santa Cruz Biotechnology. Monoclonal anti-SHP-1, anti-SHP-2, anti-EGF receptor, anti-Grb2, anti-phosphotyrosine (PY20), and anti-Fyn antibodies were obtained from
Transduction Laboratories. Alkaline phosphatase-conjugated secondary
antibody and reagents for chemiluminescence detection were purchased
from New England Biolabs Inc. All other reagents were from Sigma.
Cell Culture and Expression of a Catalytically Inactive Mutant of
Fyn--
MDCK-Tag3 cells stably expressing a hemagglutinin
epitope-tagged version of the porcine
2A-adrenergic
receptor (
2A-AR) were cultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum (16). A
catalytically inactive mutant cDNA of mouse Fyn
(Lys296-Met296) was subcloned into a pApuro
vector (17, 18). At 50% confluence, MDCK-Tag3 cells were transfected
with the catalytically inactive mutant of Fyn plasmid DNA using a
standard calcium phosphate precipitation/glycerol shock procedure.
After selection in puromycin (1 µg/ml), single cell-derived colonies
were isolated, amplified, and screened for the expression of mutated
Fyn by immunoblotting. Three clones with high expression of the mutated
Fyn were obtained and propagated in the complete medium under a
selection pressure of 0.5 µg/ml puromycin.
Immunoprecipitation and Immunoblotting--
MDCK-Tag3 cells were
normally serum-starved in Dulbecco's modified Eagle's medium for
~24 h before agonist stimulation. Stimulated and unstimulated cells
were washed twice with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 and then lysed on ice in
Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each leupeptin and
aprotinin). The extract was clarified by centrifugation at full speed
in a microcentrifuge. The clarified lysates were incubated sequentially
(4 h for each incubation at 4 °C) with antibodies as indicated and
protein A-Sepharose. The immunoprecipitates were collected and washed
four times with Nonidet P-40 lysis buffer. For immunoblotting, whole
cell lysates or immunoprecipitates were separated by SDS-PAGE and
transferred to a PVDF membrane. The membranes were probed with various
primary antibodies as indicated and detected using the ECL system with
alkaline phosphatase-conjugated secondary antibodies according to the
manufacturer's protocol.
GST Fusion Protein Binding Assay--
Whole cell lysates were
incubated with GST fusion proteins bound to glutathione-Sepharose for
12 h at 4 °C with rotation. Beads were pelleted and washed four
times with ice-cold Nonidet P-40 lysis buffer. Bound proteins were
released by boiling in SDS-PAGE sample buffer for 4 min.
Far-Western Blotting--
Protein blots on PVDF membranes were
first subjected to 6 M guanidine hydrochloride denaturation
at 4 °C in a buffer containing 20 mM Hepes, pH 7.6, 75 mM KCl, 0.1 mM EDTA, 2.5 mM
MgCl2, 1 mM dithiothreitol, and 0.05% Nonidet
P-40 and then to renaturation at 4 °C by five dilution steps (10 min
each) to a final concentration of 0.185 M guanidine. The
PVDF membranes were blocked with 5% nonfat dry milk in
phosphate-buffered saline/Tween, incubated with GST fusion proteins in
blocking buffer overnight, probed with monoclonal anti-GST antibody,
and then developed using the appropriate secondary antibodies and the
ECL system.
Immune Complex Tyrosine Phosphatase and Kinase Assays--
Cell
lysates were prepared in lysis buffer (25 mM Tris-HCl, pH
7.5, 10 mM 2-mercaptoethanol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each leupeptin
and aprotinin, 5 mM benzamidine, and 1% Nonidet P-40). The
cell lysates were precleared with protein A-Sepharose and control IgG
from normal mouse or rabbit serum, and SHP-2 was immunoprecipitated
with monoclonal or polyclonal anti-SHP-2 antibodies. Immune complexes
were washed four times with lysis buffer and once with phosphatase
reaction buffer (25 mM Hepes, pH 7.4, 5 mM
EDTA, and 10 mM dithiothreitol). The in vitro
phosphatase activity was measured as described previously (19).
Briefly, the synthetic peptide Raytide was labeled at its tyrosine
residue using [
-32P]ATP and pp60c-Src
tyrosine kinase. The washed immune complexes were mixed with 32P-labeled Tyr-Raytide in 30 µl of phosphatase reaction
buffer and incubated at 30 °C for 5 min. The reaction was terminated by the addition of acidic charcoal mixture (0.9 M HCl, 90 mM sodium pyrophosphate, 2 mM
NaH2PO4, and 4% (w/v) Norit A). After
centrifugation in a microcentrifuge, the amount of radioactivity
present in the supernatant was determined by scintillation counting.
The phosphatase activity was evaluated by the extent of Tyr-Raytide
dephosphorylation in vitro. For measuring the activities of
Src family kinases, cell lysates prepared in Nonidet P-40 lysis buffer
were precleared with protein A-Sepharose and control IgG from normal
rabbit serum, and the lysates were immunoprecipitated with a polyclonal
antibody (SRC-2) that recognizes the C-terminal sequence of the Src
family members Src, Fyn, and Yes. Immunoprecipitates were washed four times with Nonidet P-40 lysis buffer and once with phosphate-buffered saline and resuspended in 15 µl of a buffer solution containing 40 mM Hepes, pH 7.0, 2% (v/v) glycerol, and 0.02% Nonidet
P-40. Kinase reactions were initiated by the addition of 100 µM ATP, 25 mM MgCl2, 5 mM MnCl, 50 µM
Na3VO4, and 10 µCi of
[
-32P]ATP in the presence of 300 µM Src
family kinase-specific peptide (KVEKIGEGTYGVVKK) in a total volume of
35 µl. After incubation at 30 °C for 10 min, peptide
phosphorylation was stopped by the addition of 15 µl of 50% acetic
acid, and the reaction mixture was then applied onto P-81
phosphocellulose filter paper. Papers were washed four times with
0.75% phosphoric acid and washed once with acetone, dried, and then
counted in a scintillation counter.
 |
RESULTS |
Specific Activation of SHP-2 (but Not SHP-1) by the
2A-AR and LPA Receptor--
To determine whether SHP-2
or SHP-1 can be activated by the Gi protein-coupled
2A-AR or LPA receptor, we measured the enzymatic activities of the tyrosine phosphatases following the stimulation of
MDCK-Tag3 cells with the receptor's agonist. MDCK-Tag3 cells express
the endogenous LPA receptor and porcine
2A-AR (16). Lysates from control or agonist-treated cells were immunoprecipitated with monoclonal anti-SHP-2 or anti-SHP-1 antibody and subjected to
immune complex tyrosine phosphatase assay. The phosphatase activity was
measured using 32P-labeled Tyr-Raytide as a substrate. As
shown in Fig. 1, both UK14304 (an agonist
of the
2A-AR) and LPA markedly stimulated the enzymatic
activity of SHP-2 with similar kinetics. The stimulatory effects
occurred fast (<1 min), reached a maximal level (4-5-fold increase)
at 3 min, and declined after 10 min. Similar results were observed when
the cell lysates were immunoprecipitated with polyclonal anti-SHP-2
antibody. The UK14304-stimulated phosphatase activity of SHP-2 was
blocked by the selective
2A-AR antagonist rauwolscine
(data not shown). In contrast, we did not detect any change of
phosphatase activity in SHP-1 immunoprecipitates by stimulation of the
cells with UK14304 and LPA under these conditions (Fig. 1), although
the cells express about equivalent amounts of SHP-1 and SHP-2.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Specific activation of SHP-2 (but not SHP-1)
by the 2A-AR and LPA
receptor. MDCK-Tag3 cells were stimulated with either 500 nM UK14304 or 10 µM LPA for the indicated
time periods. Cell lysates were immunoprecipitated with monoclonal
anti-SHP-2 or anti-SHP-1 antibodies, and the immunoprecipitates were
then subjected to in vitro immune complex phosphatase assay
using 32P-labeled Tyr-Raytide as a substrate. The
phosphatase activity was evaluated by the extent of
32Pi release from 32P-labeled
Tyr-Raytide. Data are the average of duplicate values and are
representative of three independent experiments.
|
|
Since the catalytic activity of SHP-2 can be stimulated by tyrosine
phosphorylation of the phosphatase (8, 20), we determined the tyrosine
phosphorylation of SHP-2. MDCK-Tag3 cells were stimulated with UK14304
or LPA for 3 min, and the SHP-2 immunoprecipitates were subjected to
immunoblot analysis with a mixture of monoclonal antibodies to
phosphotyrosine (5:1 (v/v) PY20/4G10). As shown in Fig.
2A, both agonists stimulated
the tyrosine phosphorylation of SHP-2, which migrates at 70 kDa.
UK14034 and LPA also promoted the co-immunoprecipitation of SHP-2 with
three tyrosine-phosphorylated proteins with apparent molecular masses
of ~175, 125, and 115 kDa, respectively (data not shown). To verify
the tyrosine phosphorylation of SHP-2 and to determine the dynamic
effect of UK14304, we performed an alternative experiment. Cell lysates
were immunoprecipitated with antibody PY20 and analyzed by
immunoblotting with a polyclonal anti-SHP-2 antibody. Unexpectedly, two
SHP-2 protein bands (~68 and 70 kDa) were detected by the anti-SHP-2
antibody (Fig. 2B). The upper 70-kDa protein band represents
the tyrosyl-phosphorylated form of SHP-2 since tyrosine-phosphorylated
SHP-2 migrates at 70 kDa as shown in Fig. 2A. It has been
shown that phosphorylation of SHP-2 increases its apparent molecular
size to ~70 kDa in response to growth factors and cytokines (8,
20-22). The phosphorylated form of SHP-2 (70 kDa) was not detected in
the whole cell lysates. We estimate that <1% of SHP-2 protein was
phosphorylated by UK14304 in MDCK-Tag3 cells. The lower 68-kDa protein
band represents the non-tyrosine-phosphorylated form of SHP-2, and this
may be complexed via the interaction of its SH2 domains with
phosphotyrosine proteins including the tyrosine-phosphorylated SHP-2.
The intensity of the phosphorylated and non-phosphorylated SHP-2 (70- and 68-kDa bands) was increased in a time-dependent manner,
with a maximum at 3 min following the stimulation with UK14304. The
stimulatory effects of UK14304 on SHP-2 tyrosine phosphorylation and
SHP-2 association with tyrosine-phosphorylated proteins were abolished by PP1, a selective Src family kinase inhibitor (Fig. 2B).
PP1 interacts specifically with Src family kinase and is a competitive inhibitor of ATP. PP1 selectively inhibits Lck, Fyn, and Hck as compared with other tyrosine kinases such as ZAP-70, JAK2, and the EGF
receptor (23). In addition, the adaptor protein Grb2, which mediates
signals to Ras through the nucleotide exchange factor Sos, was found
co-immunoprecipitated with SHP-2 in response to the UK14304 treatment.
The kinetics of the Grb2 association with SHP-2 was similar to that of
the SHP-2 tyrosine phosphorylation evoked by UK14304. The
agonist-induced SHP-2 association with Grb2 was also abolished by the
Src family kinase inhibitor PP1 (Fig. 2C). In contrast, we
could not detect the tyrosine phosphorylation of SHP-1 and its
association with Grb2 in SHP-1 immunoprecipitates either before or
after UK14304 or LPA treatment (data not shown). Therefore, although
SHP-1 shares a high degree of homology with SHP-2, only SHP-2 was
specifically activated by the
2A-AR and LPA receptor in
MDCK-Tag3 cells.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
Tyrosine phosphorylation of SHP-2 and its
association with Grb2. A, MDCK-Tag3 cells were either
left unstimulated ( ) or stimulated with 500 nM UK14304
(UK) or 10 µM LPA for 3 min. Lysates were
prepared and immunoprecipitated (IP) with polyclonal
anti-SHP-2 antibody. Immune complexes were subjected to immunoblotting
(IB) with a mixture of monoclonal antibodies to
phosphotyrosine (5:1 (v/v) PY20/4G10) (upper panel). The
same blot was stripped and reprobed with monoclonal anti-SHP-2 antibody
(lower panel). B, MDCK-Tag3 cells were pretreated
with or without 10 µM PP1 for 30 min as indicated and
then were either left unstimulated ( ) or stimulated with 500 nM UK14304 for the indicated time periods. Cell lysates
were immunoprecipitated with control IgG from normal mouse serum or
monoclonal anti-phosphotyrosine antibody PY20. The immunoprecipitates
and whole cell lysates from UK14304-treated (3 min) MDCK-Tag3 cells
were subjected to immunoblotting with a polyclonal antibody to SHP-2.
C, MDCK-Tag3 cells were pretreated with or without 10 µM PP1 for 30 min as indicated. The cells were then
unstimulated ( ) or stimulated with 500 nM UK14304 for the
indicated time periods. Lysates were immunoprecipitated with polyclonal
anti-SHP-2 antibody. Immune complexes were subjected to immunoblotting
with monoclonal anti-Grb2 antibody (upper panel). The same
blot was stripped and reprobed with monoclonal anti-SHP-2 antibody
(lower panel).
|
|
Grb2 Binds Directly via Its SH2 Domain to SHP-2--
The SH2
domain of Grb2 is predicted to bind to the consensus sequence
pYXNX (24), of which there are two in
SHP-2, namely at tyrosine 304 (Y304INA) and at
tyrosine 542 (Y542TNI). To analyze the
interaction between SHP-2 and Grb2, we first performed an in
vitro binding assay as shown in Fig.
3A. The
glutathione-agarose-bound GST fusion proteins containing the SH2 or SH3
domain of Grb2 were incubated with lysates of UK14304-stimulated
MDCK-Tag3 cells and washed extensively, and the beads were resolved by
SDS-PAGE. Immunoblot analysis with a monoclonal anti-SHP-2 antibody
showed that SHP-2 coprecipitated only with a GST fusion protein
containing the SH2 domain of Grb2 (Fig. 3A). The next set of
in vitro binding assays and far-Western analyses was carried
out to determine whether Grb2 interacts directly with SHP-2 and to
resolve the issue of whether SHP-2 must be tyrosine-phosphorylated to
affect this association. MDCK-Tag3 cells were stimulated with LPA and
UK14304 to promote the tyrosine phosphorylation of SHP-2, and the SHP-2
immunoprecipitates were incubated with a GST fusion protein containing
the SH2 domain of Grb2. As shown in Fig. 3B, LPA and UK14304
markedly increased the association of SHP-2 with the GST-Grb2 SH2
fusion protein. However, the results using GST fusion protein may also
imply that Grb2 binds indirectly to SHP-2 through an association with
proteins bound to SHP-2. We therefore used far-Western blots to resolve this issue. SHP-2 immunoprecipitates were transferred to PVDF membranes, denatured in 6 M guanidine HCl, and then
gradually renatured. The membranes were subsequently incubated with the GST fusion protein containing the SH2 domain of Grb2. Proteins on the
blot that interacted directly with the GST fusion protein were
visualized using anti-GST antibodies. As shown in Fig. 3C, the GST-Grb2 SH2 fusion protein could bind directly to a 70-kDa protein
band migrating with the tyrosine-phosphorylated form of SHP-2, and the
binding was markedly increased upon UK14304 treatment. These results
demonstrate that Grb2 binds directly via its SH2 domain to SHP-2
following stimulation with UK14304 and LPA.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 3.
Grb2 interacts directly via its SH2 domain
with SHP-2. A, MDCK-Tag3 cells were stimulated with 500 nM UK14304 for 3 min, and the cell lysates were incubated
with GST or the GST fusion protein containing the SH2
(GST-SH2) or SH3 (GST-SH3) domain of Grb2
overnight at 4 °C. Beads were pelleted, washed, resolved by
SDS-PAGE, and immunoblotted with monoclonal anti-SHP-2 antibody.
B, MDCK-Tag3 cells were either left unstimulated ( ) or
stimulated with 500 nM UK14304 (UK) or 10 µM LPA for 3 min. Lysates were immunoprecipitated
(IP) with control IgG from normal rabbit serum or a
polyclonal antibody to SHP-2. The immunoprecipitates were washed and
incubated with the GST-Grb2 SH2 fusion protein (Grb2 SH2).
Beads were pelleted, washed, resolved by SDS-PAGE, and immunoblotted
(IB) with monoclonal anti-GST antibody (upper
panel). The same blot was stripped and reprobed with a monoclonal
antibody to SHP-2 (panel b). C, cells were left
unstimulated ( ) or stimulated with 500 nM UK14304 for 3 min, and the lysates were immunoprecipitated with polyclonal anti-SHP-2
antibody. The SHP-2 immunoprecipitates were transferred to a PVDF
membrane, denatured in 6 M guanidine HCl, and then
gradually renatured. The membranes were subsequently incubated with the
GST fusion protein containing the SH2 domain of Grb2. Proteins on the
blot that interacted directly with the GST fusion protein were
visualized using anti-GST antibody (upper panel). The same
blot was stripped and reprobed with monoclonal anti-SHP-2 antibody
(lower panel).
|
|
Activation of SHP-2 Is Sensitive to Pertussis Toxin and Requires
Src Family Tyrosine Kinase--
Pretreatment of the cells with the
selective Src family kinase inhibitor PP1 abolished the UK14304-induced
tyrosine phosphorylation of SHP-2 and its association with
tyrosine-phosphorylated proteins and Grb2 as shown in Fig. 2
(B and C). This suggests an involvement of a Src
family kinase in the activation of SHP-2. Pertussis toxin-sensitive activation of Src family kinase by UK14304 and LPA has been reported (25, 26). To measure the UK14304-stimulated activity of Src family
kinase, cell lysates were immunoprecipitated with polyclonal antibody
SRC-2, which recognizes the common C-terminal sequence of the family
members Src, Fyn, and Yes. The immunoprecipitable kinase activity was
determined by its ability to phosphorylate a Src kinase-specific
substrate peptide (KVEKIGEGTYGVVKK) (27). We found that the Src family
kinase activity was increased as early as 30 s after the addition
of UK14304, peaked at 2 min (2.3-fold; 56,270 ± 6200 cpm with
UK14304 versus 24,465 ± 2800 cpm without UK14304), and
returned to basal levels by 5 min. We next examined the effects of
pertussis toxin and the selective Src family kinase inhibitor PP1 on
the activation of SHP-2 by UK14304 and LPA. As shown in Fig.
4, UK14304 and LPA markedly increased the
activity of SHP-2, and the increase in activity was completely
suppressed by pertussis toxin and PP1 pretreatment of the cells. PP1
also markedly inhibited the basal activity of SHP-2. These results indicate that UK14304- and LPA-induced activation of SHP-2 in MDCK-Tag3
cells is mediated by a pertussis toxin-sensitive G protein and may
require the activity of Src family kinase.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Pertussis toxin and PP1 block the
agonist-stimulated activity of SHP-2. MDCK-Tag3 cells were
pretreated with 625 ng/ml pertussis toxin (PTX) overnight or
with 10 µM PP1, a specific Src family kinase inhibitor,
for 30 min as indicated, and the cells were then stimulated with 500 nM UK14304 or 10 µM LPA for 3 min. Lysates
were immunoprecipitated with monoclonal anti-SHP-2 antibody, and the
immunoprecipitates were then subjected to in vitro immune
complex phosphatase assay using 32P-labeled Tyr-Raytide as
a substrate. The phosphatase activity was evaluated by the extent of
32Pi release from 32P-labeled
Tyr-Raytide. Data are the average of duplicate values and are
representative of three independent experiments. PP1 also suppressed
the basal activity of SHP-2.
|
|
Physical Interaction of SHP-2 with Fyn Kinase--
Since the
UK14304- or LPA-stimulated activation of SHP-2 was inhibited by the
selective Src family kinase inhibitor PP1, and the activation of Src
family kinase was prior to that of SHP-2 in MDCK-Tag3 cells, we
addressed the question as to whether SHP-2 could interact directly or
indirectly with one of the members of the Src kinase family. Cell
lysates were immunoprecipitated with specific polyclonal antibodies
against Src, Fyn, Lck, Yes, and Lyn, which are present in MDCK-Tag3
cells (data not shown), and the immunoprecipitates were then
immunoblotted with a monoclonal anti-SHP-2 antibody. As shown in Fig.
5A, among the members of the
Src family, only Fyn was specifically co-immunoprecipitated with SHP-2.
Two SHP-2 protein bands of ~68 and 70 kDa, representing non-tyrosine-phosphorylated and tyrosine-phosphorylated SHP-2, respectively, were detected in the Fyn immune complex. Comparing the
amount of SHP-2 present in total cell lysates versus the
amount coprecipitated with Fyn, we found that <1% of
non-phosphorylated SHP-2 (68 kDa) was recovered in the Fyn
immunoprecipitates and that the phosphorylated SHP-2 (70 kDa)
associated with Fyn accounted for only <0.5% of total SHP-2. The
association of both forms of SHP-2 with Fyn was increased following 3 min of stimulation with UK14304 (Fig. 5A) and LPA (data not
shown). The UK14304-promoted association of SHP-2 with Fyn was blocked
by pretreatment of the cells with the Src family kinase inhibitor PP1.
The PP1 treatment also blocked the basal interaction of SHP-2 with Fyn
(Fig. 5A). Blotting of the same membrane with specific
antibodies to each member of the Src kinase family revealed that a
comparable amount of Fyn (Fig. 5A, lower panel)
and Src, Yes, Lck, and Lyn (data not shown) was immunoprecipitated.
These results indicate that the specific interaction between SHP-2 and
Fyn requires the activity of Src family kinase. To characterize the
interaction between SHP-2 and Fyn, we performed an in vitro
binding assay using GST or the GST fusion protein containing the SH2 or
SH3 domain of Fyn. As shown in Fig. 5B, two SHP-2 protein
bands (68 and 70 kDa) coprecipitated with the GST-Fyn SH2 fusion
protein, and the coprecipitation was increased upon UK14304 treatment,
whereas the GST fusion protein containing the SH3 domain of Fyn
coprecipitated only the lower 68-kDa band of SHP-2 (the
non-phosphorylated form). The association of the non-phosphorylated
form of SHP-2 with the GST-Fyn SH3 fusion protein was also increased by
UK14304.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 5.
Physical interaction of SHP-2 with Fyn
kinase. A, MDCK-Tag3 cells were pretreated with or
without 10 µM PP1 for 30 min and then either left
unstimulated ( ) or stimulated (+) with 500 nM UK14304
(UK). Lysates were immunoprecipitated (IP) with a
polyclonal antibody to Src, Fyn, Yes, Lck, or Lyn as indicated. The
immunoprecipitates were subjected to immunoblotting (IB)
with monoclonal anti-SHP-2 antibody. B, MDCK-Tag3 cells were
either left unstimulated ( ) or stimulated (+) with 500 nM
UK14304 for 3 min. Lysates were prepared and incubated with GST or the
GST fusion protein containing the SH2 (GST-SH2) or SH3
(GST-SH3) domain of Fyn. Beads were pelleted and washed, and
the released proteins were subjected to immunoblotting with a
monoclonal antibody to SHP-2 (left panel). The left
panel was overexposed, and the agonist-promoted association of
SHP-2 with the Fyn SH3 domain is shown in the right
panel.
|
|
Fyn Kinase-directed Activation of SHP-2--
The physical and
specific interaction between SHP-2 and Fyn suggests that the activation
of SHP-2 evoked by UK14304 and LPA may be directly mediated by Fyn
kinase in MDCK-Tag3 cells. To examine the role of Fyn kinase in the
activation of SHP-2, we transfected MDCK-Tag3 cells with a
catalytically inactive mutant cDNA of mouse Fyn
(Lys296-Met296) and prepared cell clones
stably overexpressing this inactive Fyn mutant, Fyn(K
)
(Fig. 6A). Immune complex
kinase assay reconfirmed that the exogenously expressed
Fyn(K
) mutant was totally inactive. Overexpression of the
Fyn(K
) mutant did not affect the expression of other
members of the Src kinase family in MDCK-Tag3 cells (data not shown).
As shown in Fig. 6 (B and C), overexpression of
the Fyn(K
) mutant markedly inhibited the UK14304-induced
tyrosine phosphorylation of SHP-2, SHP-2 association with Grb2, and the
increase in SHP-2 enzymatic activity evoked by UK14304 and LPA. These
results demonstrate that Fyn kinase plays a direct role in mediating
the activation of SHP-2 by these Gi protein-coupled
receptors in MDCK-Tag3 cells. Similar results were observed in the
other two cell clones expressing the Fyn(K
) mutant (data
not shown).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
Fyn kinase-directed activation of SHP-2.
A, lysates from vector- or Fyn(K )
mutant-transfected MDCK-Tag3 cells were subjected to immunoblotting
with monoclonal anti-Fyn antibody. B, MDCK-Tag3 cells stably
expressing vector or the Fyn(K ) mutant were either left
unstimulated ( ) or stimulated with 500 nM UK14304
(UK) for 3 min. Lysates were immunoprecipitated
(IP) with polyclonal anti-SHP-2 antibody, and the
immunoprecipitates were subjected to immunoblotting (IB)
with a mixture of monoclonal anti-phosphotyrosine antibodies (5:1 (v/v)
PY20/4G10) (upper panel) or monoclonal anti-Grb2 antibody
(center panel). The same blot was stripped and reprobed with
monoclonal SHP-2 antibody (lower panel). C,
MDCK-Tag3 cells stably expressing vector or the Fyn(K )
mutant were stimulated with 500 nM UK14304 or 10 µM LPA for 3 min. Lysates were immunoprecipitated with
monoclonal anti-SHP-2 antibody, and the immunoprecipitates were then
subjected to in vitro immune complex phosphatase assay using
32P-labeled Tyr-Raytide as a substrate. The phosphatase
activity was evaluated by the extent of 32Pi
release from 32P-labeled Tyr-Raytide. Data are presented as
-fold increase over unstimulated controls and represent the means ± S.E. of three separate experiments.
|
|
 |
DISCUSSION |
In this study, we found that SHP-2 (but not SHP-1) was
specifically activated following the stimulation of the
2A-AR and LPA receptor, which lack intrinsic tyrosine
kinase activity in MDCK-Tag3 cells. We demonstrated for the first time
that the activation of SHP-2 by these Gi protein-coupled
receptors was mediated by Fyn kinase specifically associated with
SHP-2.
It has been shown that the catalytic activity of SHP-2 is critical for
its ability to modulate certain cellular responses to agonist. The
phosphatase activity of SHP-2 is required for
-thrombin-induced
early gene transcription and DNA synthesis, fibroblast growth
factor-induced Xenopus development, EGF-induced cell cycle
progression, and, in some cell types, PDGF-induced mitogenesis (12, 15,
28). In addition, the catalytic activity of SHP-2 is also required for
the MAP kinase activation in response to EGF, insulin, insulin-like
growth factor-1, and fibroblast growth factor (11-14). Our findings
that SHP-2 (but not SHP-1) is specifically activated and
tyrosine-phosphorylated by UK14304 (an agonist of the
2A-AR) and by LPA indicate that SHP-2 is involved in
signaling of the Gi protein-coupled receptors. SHP-2 is
tyrosine-phosphorylated in response to EGF, PDGF, and an agonist
(
-thrombin) of a G protein-coupled receptor as well as ligands for
cytokine receptors that activate Janus tyrosine kinases JAK1 and JAK2,
such as interleukin-2 and -3 and the granulocyte-macrophage
colony-stimulating factor (7, 8, 15, 20, 29). Recently, Ali et
al. (30) reported that angiotensin II stimulated the activity of
SHP-2 and SHP-2 tyrosine phosphorylation through the Gq
protein-coupled receptor AT1. The activity of SHP-2 has
been reported to increase upon its tyrosine phosphorylation (8),
whereas others found no difference in activity upon tyrosine
phosphorylation of SHP-2 (7). Our data show that the tyrosine
phosphorylation of SHP-2 is associated with its increased phosphatase
activity in response to UK14304 and LPA in MDCK-Tag3 cells.
Tyrosine phosphorylation of SHP-1 by the Src family member Lck has been
reported in T lymphocytes (31). SHP-2 is constitutively tyrosine-phosphorylated in cells transformed by v-Src (7), suggesting a
potential involvement of Src family kinase in the activation of SHP-2.
In MDCK-Tag3 cells, Src family kinase was activated rapidly in response
to UK14304 and peaked at 2 min, which preceded the maximal activation
of SHP-2. PP1 (23), a newly developed selective inhibitor of Src family
kinase with a preferential effect on Lck, Fyn, and Hck, blocked the
UK14304- and LPA-induced activation of SHP-2, SHP-2 tyrosine
phosphorylation, and SHP-2 association with Grb2 and
tyrosine-phosphorylated proteins in MDCK-Tag3 cells. Similar inhibitory
effects on the agonist-stimulated activation of SHP-2 were obtained by
overexpressing a catalytically inactive mutant of Fyn in MDCK-Tag3
cells. Thus, the activation of SHP-2 by the Gi
protein-coupled receptors is mediated by Fyn kinase in MDCK-Tag3 cells.
Among the Src family members (Src, Fyn, Lck, Yes, and Lyn) present in
MDCK-Tag3 cells, only Fyn was physically and specifically associated
with SHP-2, and the interaction between them was increased in response
to UK14304 and LPA. The interaction between Fyn and SHP-2 is dependent
on the Src family kinase activity since the selective inhibitor of Src
family kinase (PP1) blocked the basal and UK14304-induced association
of SHP-2 with Fyn. As evidenced by the in vitro pull-down
experiments, the GST-Fyn SH2 domain fusion protein can associate with
both the phosphorylated form (70 kDa) and the non-phosphorylated form
(68 kDa) of SHP-2, and the association is increased upon UK14304
stimulation. SHP-2 contains several potential tyrosine phosphorylation
sites, such as Y304INA,
Y542TNI, and Y580ENV.
Y542TNI and Y580ENV are
potential binding sites for the Src and Fyn SH2 domains (32). It has
been shown that tyrosine 542 is the major in vivo site of
tyrosine phosphorylation on SHP-2 (20, 33). Fyn could bind directly via
its SH2 domain to tyrosine-phosphorylated SHP-2. Fyn could also
possibly bind directly via its SH2 domain to an intermediary molecule
that can be tyrosine-phosphorylated by a Src family kinase and that
then interacts with the SH2 domains of SHP-2. Indeed, we observed that
UK14034 and LPA promoted the co-immunoprecipitation of SHP-2 with three
tyrosine-phosphorylated proteins with apparent molecular masses of
~175, 125, and 115 kDa, respectively, in MDCK-Tag3 cells. The 115-kDa
protein band was identified as SHPS-1 (SHP
substrate-1) by a specific antibody (data not
shown). SHPS-1 is a transmembrane glycoprotein that undergoes tyrosine
phosphorylation and binds directly to the SH2 domains of SHP-2 in
response to various agonists (34). Takeda et al. (35)
recently reported that LPA-induced tyrosine phosphorylation of SHPS-1
and its association with SHP-2 are mediated by a Src family kinase in
Chinese hamster ovary cells. In addition, using far-Western blotting,
we found that the SH2 domains of SHP-2 cannot bind directly to Fyn
(data not shown).
The adaptor protein Grb2 functions to couple signals to Ras by
recruiting the nucleotide exchange factor Sos to the plasma membrane
(36). It has been reported that PDGF promotes interaction of the SHP-2
SH2 domains with the tyrosine-phosphorylated PDGF receptor, SHP-2
tyrosine phosphorylation, and concomitant binding of the SHP-2
phosphotyrosine (Tyr542) to the Grb2 SH2 domain, thereby
leading to activation of the Ras/MAP kinase pathway (9, 10). SHP-2 is
also tyrosine-phosphorylated and binds to the SH2 domain of Grb2 in
response to interleukin-3 and the granulocyte-macrophage
colony-stimulating factor (20). We found that UK14304 and LPA induced
association of Grb2 with SHP-2 in MDCK-Tag3 cells. Consistent with the
previous reports, the association is mediated directly by the SH2
domain of Grb2 and the tyrosine phosphorylation of SHP-2 as evidenced
by in vitro pull-down and far-Western blot experiments.
Stimulation of the cells with UK14304 and LPA may result in the
formation of SHP-2/Grb2·Sos complexes, which in turn may lead to
Ras/MAP kinase activation. However, recent studies have indicated a
more complicated role for SHP-2 than serving as a binding protein for
Grb2 (34, 37). SHP-2 may dephosphorylate its physiological substrates
such as the SIRP family of transmembrane proteins including SHPS-1 to exert its positive role in the cell signaling pathway. Recruitment of
Grb2·Sos complexes to the plasma membrane requires tyrosine phosphorylation of Grb2-binding sites on membrane-associated scaffolds. Tyrosine phosphorylation of several proteins (such as the adaptor protein Shc, focal adhesion kinase, the EGF receptor, and
p185neu) that contain potential Grb2-binding sites has been
reported following stimulation of Gi protein-coupled
receptors (38, 39). The data presented herein raise the possibility
that Grb2 may also function to target SHP-2 to the membrane, where
SHP-2 can bind to and dephosphorylate its physiological substrates
associated with the membrane.
Src family kinase has been demonstrated to play a key role in signal
pathways mediated by Gi protein-coupled receptors (38, 39).
We found that SHP-2 (but not SHP-1) was selectively activated upon the
stimulation of the
2A-AR and LPA receptor in MDCK-Tag3 cells and that the activation of SHP-2 by the Gi
protein-coupled receptors was directly mediated by Fyn kinase through
its specific physical interaction with SHP-2. Thus, we have
demonstrated that SHP-2 is a specific target regulated by Fyn in the
activity of the Gi protein-coupled receptors in MDCK cells.