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J Biol Chem, Vol. 274, Issue 43, 30657-30663, October 22, 1999
,
From the Department of Adult Oncology, Related adhesion focal tyrosine kinase (RAFTK)
(also known as PYK2) is a cytoplasmic tyrosine kinase related to the
focal adhesion kinase (FAK) p125FAK. RAFTK is rapidly
phosphorylated on tyrosine residues in response to various stimuli,
such as tumor necrosis factor- Protein tyrosine kinases are critical components of signaling
pathways that control cellular proliferation, differentiation, and
apoptosis. Related adhesion focal tyrosine kinase
(RAFTK),1 also known as
proline-rich tyrosine kinase (PYK2), calcium-dependent tyrosine kinase, and cellular adhesion kinase RAFTK has been shown to display integrin-dependent
phosphorylation in B lymphocytes, CMK cells, and transfected COS cells (16, 17). In contrast, RAFTK phosphorylation has been found to be
independent of integrin ligation during platelet aggregation (18).
Other studies have demonstrated that RAFTK is tyrosine-phosphorylated following Tyrosine phosphorylation of RAFTK by lysophosphatidic acid or
bradykinin stimulation contributes to activation of the MAPK pathway
(11). Furthermore, tyrosine phosphorylation of RAFTK by
lysophosphatidic acid, bradykinin, or fluoroaluminate (23) leads to
binding of the SH2 domain of Src to Tyr-402 (autophosphorylation site
(1)) of RAFTK and activation of Src (11). Since overexpression of RAFTK
Y402F mutant failed to activate MAPK, these findings, taken together,
indicated that RAFTK tyrosine phosphorylation and RAFTK-mediated MAPK
activation depend on induction of c-Src kinase activity by binding of
c-Src to autophosphorylated Tyr-402 on RAFTK. However, the molecular
mechanisms that regulate the tyrosine phosphorylation of Tyr-402 of
RAFTK are presently unclear.
The present studies have addressed the involvement of a tyrosine
phosphatase, SHPTP1, in RAFTK-mediated signaling. The results demonstrate that RAFTK binds constitutively to SHPTP1.
We also show that, by contrast to PTP1B, overexpression of SHPTP1
blocks tyrosine phosphorylation of RAFTK and its subsequent interaction
with the SH2 domain of Src. Furthermore, SHPTP1 selectively inhibits
certain functions of RAFTK, such as RAFTK-mediated activation of MAPK
but not of JNK.
Cell Culture--
Human U-937 myeloid leukemia cells were grown
in RPMI 1640 supplemented with 10% heat-inactivated (HI) fetal bovine
serum (FBS), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. PC12 cells were grown in RPMI
1640 containing 10% HI horse serum, 5% HI-FBS, and antibiotics. 293T
cells were grown in Dulbecco's modified Eagle's medium containing
10% FBS and antibiotics. 293T or PC12 cells (1 × 106/100-mm culture dish) were plated 24 h before
treating with bradykinin (Sigma). Cells (1 × 106/100
mm culture dish) were plated 24 h before transfection with various
cDNAs. U-937 or PC12 cells were treated with 1 µM
bradykinin for 5 min.
Immunoprecipitation and Immunoblot
Analysis--
Immunoprecipitations were performed as described (24).
In brief, cells were washed with phosphate-buffered saline and lysed in
1 ml of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium
vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 µg/ml of leupeptin and
aprotinin) as described. Total cell lysates were subjected to
immunoprecipitation with anti-RAFTK (2), anti-SHPTP1 (Upstate
Biotechnology Inc., Lake Placid, NY), or preimmune rabbit serum (PIRS),
and the resultant protein precipitates were analyzed by immunoblotting
with anti-SHPTP1 or anti-RAFTK antibodies. To determine the
stoichiometry of interactions, lysates were also immunoprecipitated
with anti-RAFTK or anti-SHPTP1, and lysates before and after
immunoprecipitation were analyzed by immunoblotting with anti-SHPTP1 or
anti-RAFTK antibodies, respectively.
Transient Transfections--
293T cells were grown in 100-mm
cell culture dishes, and cells were seeded a day before transfections.
Cells were transiently transfected with FLAG-SHPTP1 and FLAG-RAFTK by
calcium phosphate as described (25). After 12 h of incubation at
37 °C, the medium was replaced, and the cells were incubated for
another 24-36 h. Total cell lysates were subjected to
immunoprecipitation with anti-RAFTK, anti-SHPTP1, or PIRS, and the
precipitates were analyzed by immunoblotting with anti-FLAG antibody.
293T cells were transiently transfected with wild-type FLAG-RAFTK,
kinase-dead FLAG-RAFTK (RAFTK K457M), or an autophosphorylation dead
mutant of RAFTK (RAFTK Y402F) with or without wild-type FLAG-SHPTP1
(SHPTP1) or phosphatase-dead mutant of FLAG-SHPTP1 (SHPTP1 C-S). Cells
were also separately transfected with HA-PTP1B (26) and FLAG-RAFTK as a
negative control. Lysates were subjected to immunoprecipitation with
anti-RAFTK and analyzed by immunoblotting with anti-Tyr(P) (4G10,
Upstate Biotechnology). Anti-RAFTK immunoprecipitates were also
analyzed by immunoblotting with anti-RAFTK antibody. Anti-FLAG immunoprecipitates were also analyzed by immunoblotting with
anti-SHPTP1. PC12 cells were transiently transfected with wild-type
SHPTP1 or SHPTP1 C453S mutant using LipofectAMINE (Life Technologies, Inc.). After 48 h of transfection, cells were treated with 1 µM bradykinin and harvested after 5 min. Total cell
lysates were subjected to immunoprecipitation with anti-RAFTK and
analyzed by immunoblotting with anti-Tyr(P). The autoradiograms were
scanned, and the percentage inhibition in bradykinin-induced tyrosine
phosphorylation of RAFTK was expressed as the mean ± S.D of three
independent experiments.
Dephosphorylation of RAFTK in Vitro--
293T cells were grown
in 100-mm cell culture dishes, and cells were seeded a day before
transfections. Cells were transiently transfected with FLAG-RAFTK by
calcium phosphate. Cell lysates were prepared in Nonidet P-40 lysis
buffer without phosphatase inhibitors. Total cell lysates were
subjected to immunoprecipitation with anti-RAFTK antibody, and
precipitated proteins were sedimented with protein A-Sepharose beads.
The resultant immune complexes were incubated in an assay buffer
containing [ RAFTK Activity Assays--
293T cells were transiently
transfected with FLAG-RAFTK by calcium phosphate. Total cell lysates
were subjected to immunoprecipitation with anti-RAFTK, and the
resultant immune complexes were incubated with or without purified
SHPTP1 as described. After incubation, beads containing
dephosphorylated RAFTK were washed three times with wash buffer (20 mM HEPES, pH 7.4) and incubated in a kinase buffer (20 mM HEPES, pH 7.4, 10 mM MnCl2, 10 mM MgCl2) containing [ c-Jun Kinase Assays--
293T cells were transiently transfected
with various amounts of RAFTK and pEBG-SAPK with or without SHPTP1 or
SHPTP1 C-S. After 48 h, total cell lysates were prepared and
incubated with 5 µg of immobilized GST for 30 min at 4 °C. The
protein complexes were washed with lysis buffer and incubated in kinase
buffer (20 mM HEPES, pH 7.4, 10 mM
MgCl2) containing [ MAPK Assays--
293T cells were transiently transfected with
various amounts of FLAG-RAFTK with HA-MAPK with or without wild-type
SHPTP1 or SHPTP1 C453S mutant. Total cell lysates were then subjected
to immunoprecipitation with anti-HA antibodies for 2 h at 4 °C.
The protein complexes were incubated in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2)
containing [ c-Src/RAFTK Binding Assays in Vivo--
PC12 cells were
transiently cotransfected with FLAG-RAFTK and wild-type SHPTP1 or
SHPTP1 C453S mutant. Following transfections, cells were treated with 1 µM bradykinin for 5 min. Total cell lysates were
subjected to immunoprecipitation with anti-c-Src antibody and analyzed
by immunoblotting with anti-FLAG. The protein bands were scanned by
densitometer, and signal intensities were plotted and expressed as
arbitrary values ± S.D from three independent experiments.
GST-Src SH2 Domain Fusion Protein Binding Assays--
Lysates
from transfected 293T cells were incubated with affinity purified
GST-c-Src-SH2 domain (Upstate Biotechnology) fusion protein linked to
glutathione-Sepharose beads as described (30). The resulting protein
complexes were washed three times with lysis buffer containing 0.1%
detergent and boiled for 5 min in SDS sample buffer. The complexes were
then separated by SDS-PAGE and subjected to immunoblot analysis with
anti-Tyr(P) or anti-RAFTK antibodies.
Src SH2 Domain Binding Assays in Vitro--
293T cells were
transiently transfected with FLAG-RAFTK. Cell lysates were prepared and
subjected to immunoprecipitation with anti-RAFTK. The resulting immune
complexes were incubated in an assay buffer containing
[ U-937 cell lysates were subjected to immunoprecipitation with
anti-RAFTK antibody and analyzed by immunoblotting with anti-SHPTP1. The results demonstrated reactivity with a 70-kDa protein (Fig. 1A). In the reciprocal
experiment, analysis of anti-SHPTP1 immunoprecipitates with anti-RAFTK
confirmed constitutive interaction between these two proteins (Fig.
1B). Since treatment with bradykinin induces activation of
RAFTK (1), we investigated whether bradykinin affects the interaction
between RAFTK and SHPTP1. The results demonstrate that treatment of
PC12 cells with bradykinin is associated with little, if any, increase
in the association of RAFTK with SHPTP1 (Fig. 1C). The
association between RAFTK and SHPTP1 was further analyzed by
overexpression of wild-type FLAG-SHPTP1 and FLAG-RAFTK in human
embryonic kidney 293T cells that do not express RAFTK (20). Whole cell
lysates were subjected to immunoprecipitation with anti-RAFTK and
analyzed by immunoblotting with anti-FLAG antibody. Incubation of total
cell lysates with preimmune rabbit serum was used as a negative
control. Anti-SHPTP1 immunoprecipitates and total cell lysate were used
as positive controls. Reactivity at 70 kDa with anti-FLAG immunoblot in
anti-RAFTK immunoprecipitates confirmed association of these two
proteins (Fig. 1D).
To evaluate the stoichiometry of the interaction between RAFTK and
SHPTP1, we subjected U-937 cell lysates to immunoprecipitation with
anti-RAFTK, and we analyzed the supernatants before and after immunoprecipitation by immunoblotting with anti-SHPTP1. Signal intensities from lysates before and after anti-RAFTK
immunoprecipitation were compared by laser densitometric scanning. The
results demonstrate that approximately 60% (average of three
independent experiments) of RAFTK is associated with SHPTP1 (Fig.
2A). Similar results were
obtained in a reciprocal experiment in which the cell lysates were
subjected to immunoprecipitation with anti-SHPTP1, and we analyzed the
supernatants before and after immunoprecipitation by immunoblotting
with anti-RAFTK (Fig. 2B).
Divisions of
Experimental Medicine and Hematology/Oncology,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
, changes in osmolarity, elevation in
intracellular calcium concentration, lysophosphatidic acid, and
bradykinin. Overexpression of RAFTK induces activation of c-Jun
amino-terminal kinase (also known as stress-activated protein kinase),
mitogen-activated protein kinase (MAPK), and p38 MAPK. The present
studies demonstrate that RAFTK binds constitutively to the protein
tyrosine phosphatase SHPTP1. In contrast to PTP1B, overexpression of
wild-type SHPTP1 blocks tyrosine phosphorylation of RAFTK. The results
further demonstrate that RAFTK is a direct substrate of SHPTP1 in
vitro. Moreover, treatment of PC12 cells with bradykinin is
associated with inhibition in tyrosine phosphorylation of RAFTK in the
presence of SHPTP1. Furthermore, in contrast to the phosphatase-dead
SHPTP1 C453S mutant, overexpression of wild-type SHPTP1 blocks
interaction of RAFTK with the SH2-domain of c-Src and inhibits
RAFTK-mediated MAPK activation. Significantly, cotransfection of RAFTK
with SHPTP1 did not inhibit RAFTK-mediated c-Jun amino-terminal kinase
activation. Taken together, these findings suggest that SHPTP1 plays a
negative role in PYK2/RAFTK signaling by dephosphorylating RAFTK.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
is a recently described cytoplasmic tyrosine kinase that is homologous to the focal
adhesion kinase (FAK) (1-4). RAFTK, 116-kDa kinase, is selectively
expressed in hematopoietic cells and neurons and is distinct from FAK
(2, 5). RAFTK lacks a transmembrane domain and, similar to FAK, does
not have any SH2 or SH3 domains (2). FAK and RAFTK share 45% overall
sequence identity and 60% identity in the catalytic domain. Certain
key tyrosine residues are conserved between RAFTK and FAK that function
as c-Src and Grb2 SH2 domain binding sites (1, 2, 4, 6-13). Moreover,
studies have shown that, in rat hippocampal slices and cortical
synaptosomes, RAFTK and FAK are regulated differentially by pathways
involving calcium and protein kinase C (14). Recent studies have
described identification of an another isoform of Pyk2, Pyk2-H, that is implicated in chemokine and antigen receptor signaling (15).
1 integrin or B cell antigen receptor-mediated
stimulation in both transformed and normal B cells (16, 19). More
recent work has shown that RAFTK is activated by intracellular calcium, treatment with tumor necrosis factor-, ultraviolet light, or hyperosmolarity (1, 11, 20). RAFTK is involved in calcium-stimulated regulation of ion channels (1). RAFTK also regulates stress-induced JNK
and p38 MAPK activation in PC12 cells (11, 20-22).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP for 30 min at room temperature.
Following the autophosphorylation reactions, the beads were extensively
washed to remove excess ATP, and immunopurified autophosphorylated
RAFTK was then incubated with buffer or column purified SHPTP1 for
another 30 min. The reactions were terminated by the addition of SDS
sample buffer and analyzed by 7.5% SDS-PAGE and autoradiography.
Anti-RAFTK immunoprecipitates were also incubated with purified SHPTP1
in the presence of cold ATP, and the reaction products were analyzed by
immunoblotting with anti-Tyr(P) antibody.
-32P]ATP
and 2 µg of poly(Glu-Tyr) (4:1) (Sigma) (1) as a substrate for 15 min
at 30 °C. The reactions were terminated by the addition of SDS
sample buffer and analyzed by SDS-PAGE and autoradiography.
-32P]ATP for 15 min at
30 °C using GST-Jun-(1-102) as substrate as described (27, 28).
Kinase reactions were terminated by addition of SDS-PAGE sample buffer,
and phosphorylated proteins were analyzed by SDS-PAGE and autoradiography.
-32P]ATP for 15 min at 30 °C using
myelin basic protein (MBP) as substrate (29). Kinase reactions were
then stopped by addition of SDS-PAGE sample buffer and analyzed by
SDS-PAGE and autoradiography. 293T cells were also transfected with 2 µg of c-Raf-1 (provided by J. Auruch, MGH, Boston) with HA-MAPK with
or without wild-type SHPTP1 or SHPTP1 C453S mutant. Total cell lysates
were then subjected to immunoprecipitation with anti-HA antibodies, and
protein complexes were incubated in kinase buffer containing
[-32P]ATP and MBP as described. After 15 min, kinase
reactions were then stopped by addition of SDS-PAGE sample buffer and
analyzed by SDS-PAGE and autoradiography. Total lysates were also
separately analyzed by immunoblotting with anti-Raf, anti-HA, or
anti-SHPTP1 antibodies.
-32P]ATP for 30 min at room temperature. Following
the autophosphorylation reactions, the beads were extensively washed to
remove excess ATP and then incubated with buffer or column purified
SHPTP1 for 30 min. Following dephosphorylation reactions, RAFTK protein
was eluted from the protein A-Sepharose beads in 0.5% SDS elution buffer. Reaction mixtures were then diluted with lysis buffer to a
final concentration of 0.1% SDS and then incubated with GST-Src-SH2 domain fusion protein for 45 min. Following extensive washing, the
beads were boiled in SDS sample buffer and analyzed by 7.5% SDS-PAGE
and autoradiography.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Association of RAFTK and SHPTP1.
A, lysates from U-937 cells were subjected to
immunoprecipitation with anti-RAFTK or preimmune rabbit serum
(PIRS). Immunoprecipitates were analyzed by immunoblotting
(IB) with anti-SHPTP1. B, U-937 cell lysates were
immunoprecipitated with PIRS, anti-RAFTK, or anti-SHPTP1, and the
resulting immunoprecipitates were then analyzed by immunoblotting with
anti-RAFTK. C, PC12 cells were treated with 1 µM bradykinin (Brad.) and harvested at the
indicated times. Total cell lysates were subjected to
immunoprecipitation with anti-SHPTP1, and the immunoprecipitates were
analyzed by immunoblotting with anti-RAFTK. D, 293T cells
were transiently transfected with FLAG-RAFTK and FLAG-SHPTP1. Total
cell lysates were then subjected to immunoprecipitation with anti-RAFTK
and analyzed by immunoblotting with anti-FLAG. PIRS and anti-SHPTP1
immunoprecipitates were also analyzed by immunoblotting with
anti-FLAG.
View larger version (11K):
[in a new window]
Fig. 2.
Stoichiometry of the interaction between
RAFTK and SHPTP1. A, U-937 cell lysates were subjected
to immunoprecipitation with anti-RAFTK. Lysates from B
(before immunodepletion) and A (after immunodepletion) were
separated by SDS-PAGE and analyzed by immunoblotting (IB)
with anti-SHPTP1 antibody. B, total cell lysates were
subjected to immunoprecipitation with anti-SHPTP1. Lysates from
B (before immunodepletion) and A (after
immunodepletion) were separated by SDS-PAGE and analyzed by
immunoblotting with anti-RAFTK antibody.
The increase in tyrosine phosphorylation of RAFTK in response to
various inducers has suggested that RAFTK is activated by phosphorylation on tyrosine (1, 3, 20, 23). As demonstrated previously,
overexpression of RAFTK in 293T cells leads to approximately a
12-15-fold induction of its tyrosine phosphorylation (Fig.
3A). RAFTK autophosphorylates
itself on Tyr-402 (11). As expected, overexpression of RAFTK Y402F or
dominant negative RAFTK K-M mutants in 293T cells did not increase
their level of tyrosine phosphorylation (Fig. 3A). The
ability of SHPTP1 to bind to RAFTK suggests that this protein might be
a physiological target for SHPTP1. To address this issue, we
cotransfected 293T cells with wild-type RAFTK and SHPTP1 or
phosphatase-dead SHPTP1 (SHPTP1 C453S) (31) and analyzed anti-RAFTK
immunoprecipitates by immunoblotting with anti-phosphotyrosine. Importantly, in contrast to SHPTP1 C453S mutant, the level of tyrosine-phosphorylated RAFTK is substantially reduced in 293T cells
transfected with wild-type SHPTP1 (Fig. 3B, top panel). The
blots were then stripped and reprobed with anti-RAFTK antibodies to
ensure that the immunoprecipitates contained equal levels of RAFTK
(Fig. 3B, middle panel). Total cell lysates were also
analyzed by immunoblotting with anti-SHPTP1 (Fig. 3B, bottom
panel). Since overexpression of SHPTP1 can nonspecifically
dephosphorylate all potential targets within the cell, 293T cells were
transfected with RAFTK with or without SHPTP1, and total cell lysates
were analyzed by immunoblotting with anti-Tyr(P). As a control, RAFTK Y402F mutant was separately transfected in 293T cells. The results demonstrate that many other phosphotyrosyl proteins are not affected by
SHPTP1 expression, indicating that this phosphatase does not indiscriminately dephosphorylate all potential targets within the cell
(Fig. 3C, upper panel). This effect was without any
difference in the RAFTK protein levels (Fig. 3C, lower
panel). Moreover, to confirm specificity of SHPTP1, a non-SH2
domain containing tyrosine phosphatase, PTP1B, was overexpressed with
RAFTK, and anti-RAFTK immunoprecipitates were analyzed by
immunoblotting with anti-Tyr(P). The results demonstrate that, by
contrast to SHPTP1, overexpression of PTP1B failed to block tyrosine
phosphorylation of RAFTK (Fig. 3D). Taken together, these
findings indicated that activation of RAFTK by autophosphorylation on
Tyr-402 is specifically regulated by SHPTP1 in vivo.
|
Whereas the present findings demonstrate that overexpression of
wild-type SHPTP1 is associated with inhibition of autophosphorylation of RAFTK, we asked whether RAFTK is a direct target of SHPTP1 in
vitro. To address this issue, we immunopurified RAFTK from 293T
cells transiently overexpressing RAFTK and incubated with purified
recombinant phosphatase-active SHPTP1 protein in the presence of cold
or [32-P]ATP. RAFTK autophosphorylation was determined
in these reactions either by immunoblotting with anti-Tyr(P) or by
SDS-PAGE and autoradiography, respectively. The results demonstrate
that autophosphorylation of RAFTK is inhibited in the presence of
SHPTP1 in vitro (Fig. 4,
A and B). Since purified SHPTP1 dephosphorylates
RAFTK autophosphorylation site Tyr-402 in vitro, we asked
whether dephosphorylation of RAFTK affects its kinase function. To
address this, we first incubated purified SHPTP1 with immunopurified
RAFTK in the presence of ATP. After washing, RAFTK activity was
measured in a kinase reaction containing [32-P]ATP and
poly(Glu-Tyr) (4:1) as a substrate (1). The results demonstrate little,
if any, change in RAFTK-mediated phosphorylation of poly(Glu-Tyr) in
the presence or absence of SHPTP1 (Fig. 4C). Taken together,
these findings indicate the following: (i) RAFTK is a direct substrate
of SHPTP1, and (ii) dephosphorylation of RAFTK by SHPTP1 does not
inhibits its kinase activity.
|
To assess the functional significance of the interaction of RAFTK with
SHPTP1 in vivo, we asked whether SHPTP1 regulates tyrosine phosphorylation of RAFTK induced in response to bradykinin. To address
this issue, PC12 cells were transfected with wild-type or C453S mutant
of SHPTP1 and then treated with bradykinin. Total cell lysates were
subjected to immunoprecipitation with anti-RAFTK and analyzed by
immunoblotting with anti-Tyr(P). Three independent experiments were
performed, and signal intensities were determined by densitometric
analysis. The results demonstrate that treatment of PC12 cells with
bradykinin is associated with induction of tyrosine phosphorylation of
RAFTK (Fig. 5A) as well as
induction of MAPK activity (Fig. 5B). Importantly, by
contrast to SHPTP1 C453S mutant, analysis of anti-RAFTK
immunoprecipitates with anti-Tyr(P) in cells overexpressing wild-type
SHPTP1 demonstrated over 3-fold inhibition in tyrosine phosphorylation
of RAFTK (Fig. 5C, left and right panels).
Collectively, these findings support SHPTP1-mediated dephosphorylation
of RAFTK in vitro as well as in response to bradykinin.
|
Few insights are available regarding the functional role of RAFTK.
Recent studies have shown that RAFTK regulates stress-induced c-Jun
amino-terminal protein kinase (JNK) activation (11, 20). As
demonstrated previously, the finding that overexpression of RAFTK in
293T cells induces phosphorylation of GST-Jun (20) supported
RAFTK-mediated activation of JNK in these cells (Fig. 6A). The activation of JNK was
dependent on expression of the RAFTK protein (Fig. 6A, upper
panel). Increased activation of JNK in these assays was without
any change in JNK protein levels (Fig. 6A, lower panel).
Since RAFTK is activated by certain agents such as tumor necrosis
factor- or UV light and contributes to activation of JNK (11,
20-22), we asked whether SHPTP1 is involved in regulation of this
stress pathway. To address this issue, we cotransfected pEBG-SAPK and
RAFTK with SHPTP1 or SHPTP1 C453S mutant and assayed
glutathione-Sepharose protein precipitates for phosphorylation of
GST-Jun fusion protein. The results demonstrate that cotransfection of
pEBG-SAPK with SHPTP1 or SHPTP1 C435S mutant was associated with
similar levels of RAFTK-mediated JNK activation (Fig. 6B).
Taken together, these findings indicated that, although SHPTP1
regulates tyrosine phosphorylation of RAFTK, this effect is independent
of RAFTK-dependent activation of JNK.
|
Recent studies have demonstrated that RAFTK also regulates MAPK
activation (1, 11). Since MAPK is activated in response to stimuli that
induce tyrosine phosphorylation of RAFTK, we examined the possibility
that overexpression of SHPTP1 could influence RAFTK-mediated MAPK
activation. To address this issue, we cotransfected HA-MAPK and
FLAG-RAFTK with FLAG-SHPTP1 or FLAG-SHPTP1 C453S mutant and analyzed
anti-HA immunoprecipitates for phosphorylation of MBP. As reported
previously (11), overexpression of RAFTK in 293T cells induced the
phosphorylation of MBP by a mechanism dependent on the level of RAFTK
expression (Fig. 7A). More
importantly, in contrast to the SHPTP1 C453S mutant, cotransfection of
RAFTK with SHPTP1 significantly blocked RAFTK-dependent
activation of MAPK (Fig. 7B). The inhibition of
RAFTK-induced activation of MAPK was without any change in RAFTK or
MAPK protein levels (Fig. 7B). These findings indicated that
SHPTP1 plays a negative role in RAFTK signal transduction by acting on
signaling molecules that regulate RAFTK-mediated MAPK activation.
Previous studies have shown that c-Raf-1 acts upstream to the MAPK
pathway (32-34). Therefore, it is possible that the inhibitory effect
of SHPTP1 on RAFTK-mediated activation of MAPK, demonstrated in the
present study, could be a result of SHPTP1-mediated dephosphorylation of other targets downstream of RAFTK, such as c-Raf-1. To address this
issue, 293T cells were transiently transfected with HA-MAPK with
c-Raf-1 and wild-type or C453S mutant of SHPTP1. Following transfections, total cell lysates were subjected to immunoprecipitation with anti-HA, and in vitro immune complex kinase assays were
performed using MBP as a substrate. The results demonstrate that
overexpression of wild-type SHPTP1 has little, if any, effect on
c-Raf-1-mediated activation of MAPK (Fig. 7C). Taken
together, these findings suggested that RAFTK-dependent
activation of MAPK is inhibited by SHPTP1-mediated dephosphorylation of
RAFTK.
|
Previous studies have demonstrated that RAFTK tyrosine phosphorylation
and RAFTK-mediated MAPK activation depend on c-Src kinase activity
stimulated by binding to autophosphorylated Tyr-402 on RAFTK (11).
Therefore, we next examined the role of SHPTP1 in regulating the
interaction of RAFTK with c-Src. To address this issue, RAFTK protein
was immunopurified by transiently overexpressing FLAG-RAFTK in 293T
cells. Immunopurified RAFTK was incubated in a kinase buffer containing
[-32P]ATP at 30 °C for 30 min. Following extensive
washing, autophosphorylated RAFTK protein was incubated with purified
SHPTP1 protein for an additional 30 min. The resulting dephosphorylated
RAFTK protein was eluted from the beads and subjected to incubation
with GST-c-Src-SH2 domain fusion protein. Following fusion
protein-binding reactions, the bound proteins were analyzed by
autoradiography. The results demonstrate that incubation of SHPTP1 with
RAFTK significantly inhibits the interaction of c-Src-SH2 domain with
RAFTK (Fig. 8A). In
vitro binding experiments using a GST fusion protein containing the SH2 domain of c-Src were also performed in lysates overexpressing wild-type RAFTK with or without wild-type or C453S SHPTP1. Lysate from
overexpressing RAFTK Y402F mutant was separately used as control. GST
fusion protein containing the SH2 domain of Src was associated with
binding to tyrosine-phosphorylated RAFTK but not to the RAFTK Y402F
mutant (Fig. 8B). In contrast to SHPTP1 C453S mutant,
cotransfection of RAFTK with wild-type SHPTP1 completely blocked RAFTK
interaction with the SH2 domain of c-Src (Fig. 8B). To
address further this issue in vivo, PC12 cells were
transiently transfected with FLAG-RAFTK and wild-type SHPTP1 or SHPTP1
C453S mutant. Cells were treated with bradykinin, and total cell
lysates were subjected to immunoprecipitation with anti-c-Src antibody. The precipitates were then analyzed by immunoblotting with anti-FLAG. The results demonstrate that, in concert with the in vitro
findings, overexpression of SHPTP1 significantly inhibits
bradykinin-induced interaction of c-Src with RAFTK (Fig.
8C).
|
Recent studies have shown that autophosphorylation of RAFTK on Tyr-402 leads to binding of the SH2 domain of c-Src and contributes to activation of MAPK (11). Since SHPTP1 also associates with RAFTK, inhibition of RAFTK-mediated MAPK activation in the presence of SHPTP1 might be due to competition of SHPTP1 and c-Src for binding to RAFTK. To address this, 293T cells were cotransfected with wild-type SHPTP1 and FLAG-RAFTK or FLAG-RAFTK Y402F mutant, and anti-RAFTK immunoprecipitates were analyzed by immunoblotting with anti-SHPTP1. The results demonstrate that, similar to wild-type RAFTK, RAFTK Y402F mutant also interacts with SHPTP1 (Fig. 8D). Taken together, these findings indicated that SHPTP1 inhibits recruitment of c-Src to RAFTK by blocking the interaction of the SH2-domain of c-Src and not by competing for binding to RAFTK.
Our results suggest that the tyrosine phosphatase SHPTP1 regulates
autophosphorylation of RAFTK on Tyr-402, thereby inhibiting the
interaction of the SH2 domain of c-Src with RAFTK. In this context,
recent studies have shown that overexpression of a tyrosine kinase Csk
(negative regulator of Src; see Ref. 35) inhibits RAFTK-mediated MAPK
activation and further indicate the role of Src in mediating RAFTK
response. The present results indicate that SHPTP1 plays an important
role in RAFTK-mediated MAPK activation. Our findings also demonstrate
that SHPTP1-mediated dephosphorylation of RAFTK on Tyr-402 is not
associated with inhibition of the kinase function of RAFTK. Other
studies have shown that kinase activity of RAFTK is necessary for
RAFTK-mediated induction of JNK (11). In concert with these findings,
the results of the present study demonstrate that SHPTP1 had no effect
on RAFTK-mediated JNK activation. Other studies have shown that
overexpression of RAFTK in rat and mouse fibroblasts leads to apoptotic
cell death (36). The amino-terminal domain and tyrosine kinase activity
of RAFTK is required for efficient induction of apoptotic death (36).
Furthermore, apoptosis induced by RAFTK is suppressed by
overexpression of catalytically active c-Src (36) probably by
activating the MAPK pathway. The present findings that RAFTK binds
constitutively to SHPTP1 supports the existence of distinct RAFTK
pools. Moreover, the finding that SHPTP1 down-regulates RAFTK-induced
MAPK activation supports a role for SHPTP1 as a negative regulator of
the RAFTK 
MAPK cascade and regulates RAFTK-mediated apoptosis.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. John Kyriakis, Joseph Avruch, and Leonard Zon for providing various SAPK, c-Raf-1, and HA-MAPK cDNA constructs and anti-GST-SAPK antibody; Drs. Dehua Pei and Christopher Walsh for SHPTP1 and SHPTP1 C453S cDNAs; Dr. Jonathan Chernoff for providing PTP1B in mammalian expression vector. We also thank Dr. Hawa Avraham for critical reading of the manuscript. We thank Rebecca Farber, Atsuko Nakazawa, and Andrew Place for excellent technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by United States Public Health Service Grant CA75216 (to S. K.) awarded by the National Cancer Institute, Department of Health and Human Services, and by National Institutes of Health Grant HL55445 (to S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Adult Oncology, Dana-farber Cancer Inst., Harvard Medical School, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2938; fax: 617-632-2934; E-mail: surender_kharbanda@dfci.harvard.edu.
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ABBREVIATIONS |
|---|
The abbreviations used are: RAFTK, related adhesion focal tyrosine kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun amino-terminal protein kinase; Pyk2, proline-rich tyrosine kinase 2; SHPTP1, Src homology 2 domain containing protein tyrosine phosphatase; FAK, focal adhesion kinase; PTP1B, protein tyrosine phosphatase 1B; MBP, myelin basic protein; SAPK, stress-activated protein kinase; GST, glutathione S-transferase; PIRS, preimmune rabbit serum; PAGE, polyacrylamide gel electrophoresis; FBS, fetal bovine serum; HI, heat-inactivated; HA, hemagglutinin.
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REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
| 1. | Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musachhio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745[CrossRef][Medline] [Order article via Infotrieve] |
| 2. |
Avraham, S.,
London, R.,
Fu, Y.,
Ota, S.,
Hiregowdara, D.,
Li, J.,
Jiang, S.,
Pasztor, L. M.,
White, R. A.,
Groopman, J. E.,
and Avraham, H.
(1995)
J. Biol. Chem.
270,
27742-27751 |
| 3. |
Yu, H.,
Li, X.,
Marchetto, G. S.,
Dy, R.,
Hunter, D.,
Calvo, B.,
Dawson, T. L.,
Wilm, M.,
Anderegg, R. J.,
Graves, L. M.,
and Earp, H. S.
(1996)
J. Biol. Chem.
271,
29993-29998 |
| 4. |
Sasaki, H.,
Nagura, K.,
Ishino, M.,
Tobioka, H.,
Kotani, K.,
and Sasaki, T.
(1995)
J. Biol. Chem.
270,
21206-21219 |
| 5. |
Salgia, R.,
Avraham, S.,
Pisick, E.,
Li, J.-L.,
Raja, S.,
Greenfield, E. A.,
Sattler, M.,
Avraham, H.,
and Griffin, J. D.
(1996)
J. Biol. Chem.
271,
31222-31226 |
| 6. |
Kharbanda, S.,
Saleem, A.,
Yuan, Y.,
Emoto, Y.,
Prasad, K.,
and Kufe, D.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
6132-6136 |
| 7. |
Schaller, M. D.,
Borgman, C. A.,
and Parsons, J. T.
(1993)
Mol. Cell. Biol.
13,
785-791 |
| 8. |
Cobb, B. S.,
Schaller, M. D.,
Leu, T. H.,
and Parsons, J. T.
(1994)
Mol. Cell. Biol.
14,
147-155 |
| 9. |
Schaller, M. D.,
Hildebrand, J. D.,
Shannon, J. D.,
Fox, J. W.,
Vines, R. R.,
and Parsons, J. T.
(1994)
Mol. Cell. Biol.
14,
1680-1688 |
| 10. | Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786-791[Medline] [Order article via Infotrieve] |
| 11. | Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S. A., and Schlessinger, J. (1996) Nature 383, 547-550[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Zheng, C.,
Xing, Z.,
Bian, Z. C.,
Guo, C.,
Akbay, A.,
Warner, L.,
and Guan, J.-L.
(1998)
J. Biol. Chem.
273,
2384-2389 |
| 13. |
Kharbanda, S.,
Saleem, A.,
Shafman, T.,
Emoto, Y.,
Taneja, N.,
Rubin, E.,
Weichselbaum, R.,
Woodgett, J.,
Avruch, J.,
Kyriakis, J.,
and Kufe, D.
(1995)
J. Biol. Chem.
270,
18871-18874 |
| 14. |
Siciliano, J. C.,
Toutant, M.,
Derkinderen, P.,
Sasaki, T.,
and Girault, J.-A.
(1996)
J. Biol. Chem.
271,
28942-28946 |
| 15. |
Dikic, I.,
Dikic, I.,
and Schlessinger, J.
(1998)
J. Biol. Chem.
273,
14301-14308 |
| 16. |
Li, J.,
Avraham, H.,
Rogers, R. A.,
Raja, S.,
and Avraham, S.
(1996)
Blood
88,
417-428 |
| 17. |
Astier, A.,
Manié, S. N.,
Avraham, H.,
Hirai, H.,
Law, S. F.,
Zhang, Y.,
Golemis, E. A.,
Fu, Y.,
Druker, B. J.,
Haghayeghi, N.,
Freedman, A. S.,
and Avraham, S.
(1997)
J. Biol. Chem.
272,
19719-19724 |
| 18. |
Raja, S.,
Avraham, S.,
and Avraham, H.
(1997)
J. Biol. Chem.
272,
10941-10947 |
| 19. |
Astier, A.,
Avraham, H.,
Manié, S. N.,
Groopman, J.,
Canty, T.,
Avraham, S.,
and Freedman, A. S.
(1997b)
J. Biol. Chem.
272,
228-232 |
| 20. | Tokiwa, G., Dikic, I., Lev, S., and Schlessinger, J. (1996) Science 273, 792-794[Abstract] |
| 21. |
Pandey, P.,
Avraham, S.,
Place, A.,
Kumar, V.,
Majumder, P. K.,
Cheng, K.,
Nakazawa, A.,
Saxena, S.,
and Kharbanda, S.
(1999)
J. Biol. Chem.
274,
8618-8623 |
| 22. |
Pandey, P.,
Avraham, S.,
Kumar, S.,
Nakazawa, A.,
Place, A.,
Ghanem, L.,
Rana, A.,
Kumar, V.,
Majumder, P. K.,
Avraham, H.,
Davis, R. J.,
and Kharbanda, S.
(1999)
J. Biol. Chem.
274,
10140-10144 |
| 23. |
Jeschke, M.,
Standke, G. J. R.,
and Susa, M.
(1998)
J. Biol. Chem.
273,
11354-11361 |
| 24. | Kharbanda, S., Pandey, P., Jin, S., Inoue, S., Bharti, A., Yuan, Z.-M., Weichselbaum, R., Weaver, D., and Kufe, D. (1997) Nature 386, 732-735[CrossRef][Medline] [Order article via Infotrieve] |
| 25. |
Bharti, A.,
Kraeft, S.,
Gounder, M.,
Pandey, P.,
Jin, S.,
Yuan, Y.-M.,
Lees-Miller, S.,
Weichselbaum, R.,
Weaver, D.,
Chen, L.-B.,
Kufe, D.,
and Kharbanda, S.
(1998)
Mol. Cell. Biol.
18,
6719-6728 |
| 26. |
Liu, F.,
Hill, D. E.,
and Chernoff, J.
(1996)
J. Biol. Chem.
271,
31290-31295 |
| 27. | Saleem, A., Yuan, Z.-M., Kufe, D. W., and Kharbanda, S. (1995) J. Immunol. 154, 4150-4156[Abstract] |
| 28. | Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. M., Weichselbaum, R. R., and Kufe, D. W. (1995) Nature 376, 785-788[CrossRef][Medline] [Order article via Infotrieve] |
| 29. |
Kharbanda, S.,
Saleem, A.,
Emoto, Y.,
Stone, R.,
Rapp, U.,
and Kufe, D.
(1994)
J. Biol. Chem.
269,
872-878 |
| 30. |
Kumar, S.,
Pandey, P.,
Bharti, A.,
Jin, S.,
Weichselbaum, R.,
Weaver, D.,
Kufe, D.,
and Kharbanda, S.
(1998)
J. Biol. Chem.
273,
25654-25658 |
| 31. | Pei, D., Lorenz, U., Klingmüller, U., Neel, B. G., and Walsh, C. T. (1994) Biochemistry 33, 15483-15493[CrossRef][Medline] [Order article via Infotrieve] |
| 32. |
Winston, L. A.,
and Hunter, T.
(1994)
J. Biol. Chem.
270,
30837-30840 |
| 33. |
Minden, A.,
Lin, A.,
McMohan, M.,
Lange-Carter, C.,
Derijard, B.,
Davis, R. J.,
Johnson, G. L.,
and Karin, M.
(1994)
Science
266,
1719-1723 |
| 34. |
Toppmair, J.,
Bruder, J. T.,
Munoz, H.,
Lloyd, P. A.,
Kyriakis, J. M.,
Banerjee, P.,
Avruch, J.,
and Rapp, U. R.
(1994)
J. Biol. Chem.
269,
7030-7035 |
| 35. | Nada, S., Okada, M., MacAuly, A., Cooper, J. A., and Nakagawa, H. (1991) Nature 351, 69-72[CrossRef][Medline] [Order article via Infotrieve] |
| 36. |
Xiong, W.-C.,
and Parsons, J. T.
(1997)
J. Cell Biol.
139,
529-539 |
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