From the Departments of Hematopoiesis and
¶ Experimental Pathology, Holland Laboratory, American Red Cross,
Rockville, Maryland 20855, the § Department of Cancer
Biology, Harvard School of Public Health, Boston, Massachusetts 02115, and the
Department of Anatomy and Cell Biology, George
Washington University Medical Center, Washington, D. C. 20037
Received for publication, November 6, 2002, and in revised form, February 17, 2003
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ABSTRACT |
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SHP-2, a ubiquitously expressed Src hmology 2 (SH2) domain-containing tyrosine phosphatase, plays a critical role in
the regulation of growth factor and cytokine signal transduction. Here
we report a novel function of this phosphatase in DNA damage-induced
cellular responses. Mutant embryonic fibroblast cells lacking
functional SHP-2 showed significantly decreased apoptosis in response
to DNA damage. Following cisplatin treatment, induction of p73 and its
downstream effector p21Cip1 was essentially blocked in SHP-2
mutant cells. Further investigation revealed that activation of the
nuclear tyrosine kinase c-Abl, an essential mediator in DNA damage
induction of p73, was impaired in the mutant cells, suggesting a
functional requirement of SHP-2 in c-Abl activation. Consistent with
this observation, the effect of overexpression of c-Abl kinase in SHP-2
mutant cells on sensitizing the cells to DNA damage-induced death was
abolished. Additionally, we found that in embryonic fibroblast cells
30-40% of SHP-2 was localized in the nuclei, and that a fraction of
nuclear SHP-2 was constitutively associated with c-Abl via its SH3
domain. Phosphatase activity of nuclear but not cytoplasmic SHP-2 was
significantly enhanced in response to DNA damage. These results
together suggest a novel nuclear function for SHP-2 phosphatase in the
regulation of DNA damage-induced apoptotic responses.
DNA damage induced by ionizing radiation, ultraviolet light, and
genotoxic agents triggers intracellular signaling cascades that lead to
cellular responses such as cell cycle arrest, activation of DNA repair,
and apoptotic cell death (1, 2). p53, a well known tumor suppressing
transcription factor, is induced and activated in response to DNA
damage to mediate cell death and cell cycle arrest (3-5). However,
ionizing radiation and DNA damaging agents such as cisplatin can also
induce apoptosis in a p53-independent manner (6, 7). Recent studies
have revealed that substantial components of the p53 independent
pathway are mediated by p73 (8-10), a newly identified member of the
p53 transcription factor family (11, 12). Both p53 and p73 are
pro-apoptotic proteins; overexpression of p53 and p73 leads to
cellular apoptosis through activation of the apoptotic machinery
(13). They can also transactivate downstream effectors such as
p21Cip1, MDM2, and GADD45 to regulate cell cycle progression.
Further investigations have demonstrated that p73 but not p53, is the downstream effector and target of the nuclear tyrosine kinase c-Abl
(8-10).
c-Abl, a Src homology (SH)1 2 and SH3 domain-containing tyrosine kinase, shares high homology and
similar overall structure with the Src family tyrosine kinases.
However, a unique characteristic of c-Abl kinase is that it negatively
regulates cellular growth; overexpression of c-Abl kinase induces cell
growth arrest and apoptosis (14, 15). This kinase is primarily located
in the nucleus and is normally repressed, but is activated upon DNA
damage induced by ionizing radiation or DNA damaging drugs (16-20).
c-Abl is clearly required for DNA damage-induced apoptosis, because c-Abl-deficient cells or MCF7 cells harboring dominant negative (catalytically inactive) c-Abl show defects in DNA damage-induced apoptosis (7, 21).
Insights into how c-Abl acts came from the identification of its
upstream regulatory signals and downstream targets. DNA damage activates c-Abl kinase (16-20). Activated c-Abl induces apoptosis in a
p53-independent manner, as c-Abl induces apoptosis in both p53
null cells and p53 positive cells in which p53 is inactivated by
expressed viral proteins (7, 8, 22, 23). Moreover, DNA damage induction
of p53 is intact in c-Abl-deficient cells (7, 8). Recent studies have
revealed that the c-Abl-induced p53-independent apoptotic pathway is
mediated by the p73 transcription factor (8-10). Activation of p73
requires functional c-Abl; neither induction nor tyrosyl
phosphorylation of p73 occurs in c-Abl-deficient cells (8-10). More
importantly, it has been demonstrated that mutated in ataxia
telangiectasia (ATM) kinase, the product of the gene mutated in the
human genetic disorder ataxia telangiectasia and an important
component of the DNA damage-induced cell cycle checkpoint, interacts
and phosphorylates (activates) c-Abl at serine 465 (19, 20). In
addition to regulating apoptosis, c-Abl also has an important effect on
cell cycle progression; overexpression of c-Abl blocks G1/S
transition, and cells with compromised c-Abl function, or cells lacking
c-Abl have dysregulated cell cycles (14, 24). However, even though a
critical role for c-Abl kinase in DNA damage-induced cellular responses
has been well established, the precise knowledge of the molecular components involved and their working relationship in the
c-Abl-mediated pathways remain unclear.
SHP-2, an SH2 domain-containing tyrosine phosphatase, is ubiquitously
expressed in a variety of tissues and cell types, and has been
demonstrated to be involved in diverse signaling pathways, including
those initiated by growth factors, cytokines, and insulin (25, 26). In
most circumstances, SHP-2 plays a positive role in transducing the
signal relay from receptor tyrosine kinases, whereby its phosphatase
activity has been shown to be required (27-30), even though the
biochemical significance of its catalytic activity remains ill-defined.
The N-terminal SH2 domain (N-SH2) plays a critical role in mediating
SHP-2 function. A targeted N-terminal deletion of SHP-2 (amino acids
46-110 including the N-SH2) results in a loss-of-function mutation for
SHP-2. As a result of this mutation, homozygous mutant
(SHP-2 Although the role for SHP-2 phosphatase in the signal transduction of
growth factors and cytokines has been relatively well established,
several clues from other studies in our laboratory indicated that SHP-2
might also be involved in cell death regulation. In the present
studies, we examined cellular responses of SHP-2 Cell Lines and Reagents--
Wild type (WT) and
SHP-2 Cell Survival Assay--
WT, SHP-2 4',6'-Diamidino-2-phenylindole Staining--
Cells grown in
slide chambers were treated with various concentrations of cisplatin.
Twenty-four hours later, the cells were fixed with methanol for 15 min,
washed with distilled water, and then stained with
4',6'-diamidino-2-phenylindole (DAPI) (1 µg/ml) for 30 min. Nuclei
were visualized under a fluorescence microscope. Apoptotic cells were
identified by condensation and fragmentation of nuclei, and the
percentage of apoptotic cells was calculated as the ratio of
apoptotic cells to total cells in randomly selected fields (40, 41). A
minimum of 300 cells was counted.
Immunoprecipitation and Immunoblotting Analysis--
Cells were
lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet
P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM
EDTA, 1 mM NaF, 2 mM
Na3VO4, and 1 mM
phenylmethylsulfonyl fluoride). Whole cell lysates (500 µg) were
immunoprecipitated with 1 µg of purified antibodies as indicated.
Immunoprecipitates were washed three times with HNTG buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1% glycerol,
0.1% Triton X-100, and 1 mM
Na3VO4) and resolved by SDS-PAGE followed by
immunoblotting with the indicated antibodies (36).
Preparation of Cytosolic and Nuclear Extracts--
Cytosolic and
nuclear extracts were prepared essentially as described (42). Cells
were lysed on ice for 15 min with the buffer containing 10 mM Hepes, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM dithiothreitol, 10 mM NaF, 0.1 mM Na3VO4, and protease inhibitor
mixture (Roche Molecular Biochemicals). Nonidet P-40 was then added to
0.5%. After further incubation on ice for 5 min, samples were vortexed
for 20 s and then spun at 13,000 rpm for 30 s. Supernatant
was collected as cytosolic extracts. Nuclear pellets were washed once
with the aforementioned buffer and then suspended and sonicated in
nuclear extraction buffer (25 mM Hepes, pH 7.5, 500 mM NaCl, 1 mM dithiothreitol, 10 mM
NaF, 10% glycerol, 0.2% Nonidet P-40, 5 mM
MgCl2). Supernatant containing nuclear extracts was finally
collected after centrifugation at 13,000 rpm for 5 min as reported. For
examination of tyrosyl phosphorylation of nuclear SHP-2, nuclear
pellets were homogenized and lysed in nuclear extraction buffer for 40 min instead of being sonicated.
In Vitro Fusion Protein Binding Assay--
GST, GST-Abl SH2, and
GST-Abl SH3 fusion proteins were purified by glutathione-Sephorose 4B
beads and the immobilized proteins on beads were equilibrated in RIPA
buffer. Cell lysates (500 µg) were incubated with 2 µg of
immobilized GST fusion proteins at 4 °C for 2 h. The protein
complexes were washed three times with RIPA buffer, resolved by
SDS-PAGE, and then subjected to immunoblotting with anti-SHP-2 antibody
as reported (20, 43).
In Vitro Kinase Assays--
The c-Abl kinase assay was performed
as reported (16, 17). Briefly, whole cell lysates (500 µg) were
immunoprecipitated with anti-c-Abl antibody (2 µg).
Immunoprecipitates were washed three times with RIPA buffer, one time
with kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl2, and 0.1 mM sodium
orthovanadate), and were finally resuspended in 30 µl of kinase
buffer containing GST-Crk (5 µg), 10 µM ATP, and 5 µCi of [ In Vitro Phosphatase Assay--
Phosphatase activity of SHP-2
was measured as we and others previously described (32, 44). Briefly,
myelin basic protein was labeled with [ To define new potential functions of SHP-2 phosphatase, we
recently tested for its role in DNA damage-induced signaling by examining cell death sensitivity to DNA damage in WT and mutant embryonic fibroblast cells carrying an amino acid 46-110 deletion mutation of SHP-2 (SHP-2
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) embryos die at midgestation with multiple
developmental defects (31). Essential roles for SHP-2 in the regulation
of a variety of signal transduction pathways and cellular processes
such as cell proliferation, differentiation, adhesion, and migration
have been characterized by using this SHP-2 gene knockout mouse model and its derived mutant cell lines (31-39).
/
mutant cells to DNA damage, and showed that SHP-2 played an important role in the genotoxic stress-induced cell death signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mutant embryonic fibroblast cell lines were
derived from day 9.0-9.5 embryos through SV40 T antigen immortalization (35-39). Rescued cell lines were generated by
transduction of WT SHP-2 cDNA into SHP-2
/
cells
through a retroviral-mediated gene transfer. All cell lines were
cultured in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum. Cisplatin and anti-tubulin antibody were purchased from
Sigma. Anti-p53 (Ab-1), c-Abl (Ab-3), ATM (Ab-3), and ATR (Ab-2)
antibodies and p60c-Src enzyme were obtained from Oncogene
Science. Anti-c-Abl (K-12), p73 (H-79), p21Cip1 (F-5), and
SHP-2 (C-18) antibodies were supplied by Santa Cruz Biotechnology, Inc.
Anti-phospho-p53Ser-15 and anti-phospho-Ser/Thr-Gln
motif antibodies were purchased from Cell Signaling Technology.
Anti-phosphotyrosine (Tyr(P)) antibody (4G10) was obtained from
Upstate Biotechnology.
/
, and
rescued SHP-2
/
cells were seeded in 96-well plates
(8000 cells/well). Cells were treated with freshly prepared cisplatin
in 0.9% saline solution 24 h later. Forty-eight hours after
treatment, the media was decanted, and the plates were submerged in
0.4% crystal violet in 50% methanol for 30 min, and then rinsed with
water. The plates were dried and the bound dye was solubilized by
incubation at 37 °C for 1 h with 200 µl of 0.5% SDS in 50% ethanol. The plates were read at 595 nm, and the cell survival rate was
then determined by comparing the OD value of the treated to the
untreated control cells (37).
-32P]ATP. The reaction was incubated at room
temperature for 30 min. Proteins in the whole reaction system were
resolved by SDS-PAGE, and phosphorylated GST-Crk was visualized by
autoradiography. For the JNK kinase assay, cell lysates (500 µg) were
incubated with 5 µg of immobilized GST-c-Jun protein at 4 °C for
3 h. After the beads were washed three times with RIPA buffer and
once with kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM
-glycerophosphate, 5 mM NaF, 0.1 mM
Na3VO4, and 2 mM dithiothreitol),
10 µl of kinase buffer containing 10 µCi of
[
-32P]ATP was then added into each reaction system.
The reaction was incubated at 30 °C for 20 min, and phosphorylation
of GST-c-Jun was examined by SDS-PAGE and autoradiography.
-32P]ATP upon
phosphorylation by p60c-Src kinase. Whole cell lysates,
cytosolic, or nuclear extracts (250 µg) of WT and SHP-2 mutant cells
treated with cisplatin (25 µM, 2 h) were
immunoprecipitated with anti-SHP-2 antibody. Immunoprecipitates were
then mixed with the 32P-labeled myelin basic protein in the
phosphatase assay buffer for 30 min. The free 32P released
into the supernatant was measured after trichloroacetic acid
precipitation. SHP-2 phosphatase activities in WT and mutant cells
following cisplatin treatment were determined by the extent of myelin
basic protein dephosphorylation in vitro.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
). Cell survival assays
demonstrated that mutant cells lacking functional SHP-2 were much more
resistant to the DNA damaging chemotherapeutic drug cisplatin than
their WT counterparts (Fig. 1A). This appears to be
attributed to their differential apoptotic responses to DNA damage, as
chromatin DAPI staining showed that the percentage of cells with
typical morphological changes associated with apoptosis, including
nuclear blebbing and heterochromatin aggregation, was significantly
decreased in SHP-2
/
mutant cells following DNA damage
(Fig. 1B). The DNA fragmentation assay, a characteristic
analysis for apoptotic cells, also showed that significantly reduced
DNA fragmentation was observed in SHP-2 mutant cells treated with
cisplatin (data not shown). Moreover, the percentage of hypodiploid
cells with sub-G1 DNA content, known to be apoptotic, was
significantly decreased in SHP-2
/
cells following
either cisplatin treatment (Fig. 1C) or
-irradiation (Fig. 1D). Notably, re-introduction of functional WT SHP-2
into mutant cells (rescued cell line) partially restored their
sensitivity to DNA damage (Fig. 1), suggesting that the decrease in
apoptosis in mutant cells resulted from loss-of-function of SHP-2
rather than gain-of-function of the truncated SHP-2 expressed in the mutant cells.
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Fig. 1.
DNA damage-induced apoptosis is significantly
decreased in SHP-2 /
mutant cells. A, WT,
SHP-2
/
, and rescued fibroblast cells
(Rescued) were seeded into 96-well plates and treated with
cisplatin at the indicated concentrations 24 h later. Cell
survival rates relative to the untreated cells were measured 48 h
following the treatment by the crystal violet staining assay as
described under "Experimental Procedures." B,
exponentially growing WT, SHP-2
/
, and rescued cells
were treated with cisplatin at the indicated concentrations for 24 h. The cells were fixed, stained with (DAPI) and analyzed for
morphological characteristics associated with apoptosis. Percentage of
apoptotic cells following cisplatin treatment was determined as
described under "Experimental Procedures." C,
exponentially growing cells were treated as above. The cells were
harvested, fixed with ethanol, and stained with propidium iodide.
Cellular DNA content was monitored by FACScan (BD Biosciences).
Percentage of the cells with sub-G1 DNA content was
determined with the MODFIT LT program (Verity Software House, Topsham,
ME). D, WT, SHP-2
/
, and rescued cells were
-irradiated at the indicated dosages and a percentage of the
sub-G1 phase cells was determined 24 h later. Three to
four independent experiments were performed, similar results were
obtained. Results shown are the mean ± S.E. of triplicates from
one experiment.
Previous studies have demonstrated that p53 tumor suppressor and its
homologue p73 are major mediators in cellular death responses to DNA
damage (45, 46). The observation that DNA damage gave rise to different
cell death sensitivities between WT and SHP-2/
mutant
cells prompted us to examine p53 and p73 induction in these cells
following DNA damage. p53 induction in both WT and mutant cells was not
evident (see Fig. 8A), presumably because of immortalization
of the cell lines with SV40 large T antigen that blocks p53 function
(47-49). However, as shown in Fig. 2, p73 expression was dramatically up-regulated in WT cells exposed to
cisplatin treatment, consistent with previous reports (8). Surprisingly, p73 induction in SHP-2
/
mutant cells
was completely blocked. Prolonged incubation and increased dosages of
cisplatin did not increase the expression of p73 in these mutant cells
(data not shown). As p73 can transactivate the downstream effector
p21Cip1, the induction of p21Cip1 was next analyzed.
Consistent with the expression of p73, p21Cip1 induction was
also diminished in SHP-2 mutant cells (Fig. 2). Notably, p73 and
p21Cip1 induction was partially restored in the rescued cell
line, further confirming a role for SHP-2 tyrosine phosphatase in DNA
damage-induced intracellular responses.
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To examine potential involvement of SHP-2 tyrosine phosphatase in DNA
damage-triggered signal transduction, tyrosyl phosphorylation and
catalytic activity of SHP-2 following cisplatin treatment was assessed.
As shown in Fig. 3A, in WT
fibroblast cells, tyrosyl phosphorylation of SHP-2 was significantly
increased shortly after cisplatin treatment. However, phosphorylation
of the truncated form of SHP-2 (SHP-2) expressed in
SHP-2
/
mutant cells was unchanged at all time points
examined (Fig. 3A). Subsequent in vitro
phosphatase assays demonstrated that catalytic activity of SHP-2 was
doubled in the WT cells following cisplatin treatment (Fig.
3B). By contrast, although SHP-2
in the
untreated mutant cells had 2 times higher basal activity as we
previously reported (32), its phosphatase activity remained unchanged
after DNA damage (Fig. 3B). Together, these results suggest
that SHP-2 tyrosine phosphatase is involved in DNA damage-induced signaling and that the truncated form of SHP-2 expressed in the mutant
cells might not be functioning in response to DNA damage.
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Previous studies have clearly demonstrated that induction and
stabilization of p73 but not p53 in response to DNA damage is completely dependent on nuclear c-Abl kinase activity (8-10). The
blockade of p73 induction in SHP-2/
mutant cells
indicated that c-Abl activation following cisplatin treatment in these
cells might be impaired. To test this hypothesis, tyrosyl
phosphorylation response of c-Abl kinase was first examined to
determine its activation status. As shown in Fig.
4A, c-Abl became
phosphorylated in WT cells 2-3 h after DNA damage. In contrast, phosphorylation of c-Abl in SHP-2 mutant cells was significantly reduced. Several c-Abl antibodies from various manufacturers were used
for this experiment, producing similar results. We also checked longer
time points following genotoxic insult (4, 8, and 24 h) in which
no obvious phosphorylation of c-Abl was observed in mutant cells (data
not shown). Subsequent examination of the tyrosyl phosphorylation
response of baseline p73 through anti-p73 immunoprecipitation followed
by anti-phosphotyrosine immunoblotting revealed that its
phosphorylation in WT cells was rapidly induced after DNA damage as
previously reported (9, 10). Interestingly, this response in
SHP-2
/
mutant cells was undetectable (Fig.
4B). As tyrosyl phosphorylation of p73 in response to DNA
damage is fully attributed to c-Abl kinase activity (9, 10), these
results further suggested that c-Abl activation in mutant cells was
compromised. Subsequently, this notion was supported by in
vitro c-Abl kinase assays. Using GST-Crk as the substrate, we
showed that in response to cisplatin-induced DNA damage, activation of
c-Abl kinase was barely detectable in the SHP-2 mutant cells, but was
partially restored in the rescued cell line following reintroduction of
WT SHP-2 (Fig. 4C), confirming a requirement of SHP-2 for
c-Abl activation.
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Interestingly, activation of JNK kinase, known to be implicated in
mediating stress-induced apoptosis (50), was dramatically enhanced in
SHP-2/
mutant cells (Fig. 4D), suggesting
that decreased sensitivity of SHP-2
/
cells to DNA
damage-induced apoptosis was not related to JNK activity. This result
additionally indicates that SHP-2 tyrosine phosphatase negatively
regulates DNA damage-induced JNK kinase activation.
c-Abl kinase has been previously demonstrated to negatively regulate
cellular growth. Overexpression of WT c-Abl leads to cell growth arrest
and sensitizes cellular apoptotic responses to DNA damage, whereas
interference of endogenous c-Abl function by overexpression of kinase
inactive c-Abl (c-Abl K/R) reduces DNA damage-induced cell death
(14-16). To further confirm the functional requirement of SHP-2 for
c-Abl activation in response to DNA damage, we transfected WT and c-Abl
K/R into WT and SHP-2/
mutant cells and assessed the
effects of WT c-Abl and c-Abl K/R in these cells. As shown in Fig.
5, the sensitivity of WT cells to
cisplatin was enhanced by transfection of the WT c-Abl as compared to
the pSR vector control, whereas expression of the c-Abl K/R mutant in
WT cells significantly reduced cell death. Interestingly, although the
transfected WT c-Abl was equally overexpressed in SHP-2
/
mutant cells, the effect of overexpression of
c-Abl in these mutant cells was abrogated, suggesting that the c-Abl
kinase did not function in mutant cells without WT SHP-2. Because
endogenous c-Abl in SHP-2
/
cells was disabled as
described above (Fig. 4), the dominant negative effect of catalytically
inactive c-Abl K/R in mutant cells was not observed. Together, these
results further confirm a requirement of SHP-2 for c-Abl activation and
thereby cell death responses to DNA damage.
|
Co-immunoprecipitation experiments showed that SHP-2 protein was
detected in the anti-c-Abl immunocomplex, suggesting direct or indirect
interactions between SHP-2 and c-Abl. Interestingly, their association
was constitutive, being independent on DNA damage treatment (Fig.
6A). Extension of cisplatin
treatment to 4, 8, 12, and 24 h did not increase their physical
association (data not shown). In addition, SHP-2 was also detected in
the immunocomplex pulled down with anti-ATM antibody. Likewise, their
association was found to be constitutive. By contrast, the truncated
form of SHP-2 expressed in mutant cells was dramatically reduced in these immunocomplexes. To determine whether the association between c-Abl and SHP-2 is direct, in vitro GST fusion protein
binding assays were performed. As shown in Fig. 6B, the
GST-SH3 domain but not the GST-SH2 domain of c-Abl pulled down WT
SHP-2, suggesting a direct interaction between these two signaling
proteins, with their interaction being mediated by the c-Abl SH3
domain. By contrast, binding of the truncated form of SHP-2 expressed
in the mutant cells with GST-Abl SH3 was greatly reduced.
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Because c-Abl is localized both in the nucleus and the cytoplasm of fibroblast cells, we wanted to determine which fraction of c-Abl associated with SHP-2. Cytosolic and nuclear extracts from WT fibroblast cells were prepared and examined for c-Abl/SHP-2 association by immunoprecipitation and immunoblotting. As shown in Fig. 6C, c-Abl is predominantly localized in the nucleus, and nuclear c-Abl associates with SHP-2 independent of DNA damage stimulation (Fig. 6A). Additionally, this result indicated that a fraction of SHP-2 was localized in the nucleus. Indeed, subsequent reprobing of stripped blots with anti-SHP-2 antibody showed that although SHP-2 phosphatase was mainly localized to the cytoplasm, a substantial amount of SHP-2 was also detected in nuclear extracts. Statistical analysis of the relatively quantitative results from multiple experiments showed that in embryonic fibroblast cells, 36 ± 7% of SHP-2 is localized in the nucleus. It is noteworthy that this observation appears not to be because of significant contamination of nuclear extracts by the cytosol, because tubulin, a cytosolic structural protein, was only detected in the cytosolic but not nuclear lysates. Moreover, anti-SHP-2 immunostaining of WT embryonic fibroblast cells (Fig. 6D) also showed significant signals in nuclei. By comparison, only the cytoplasm was stained positive by anti-tubulin antibody.
Because SHP-2 is localized both in the cytoplasm and the nucleus, we
next examined phosphatase activity of SHP-2 in cytosolic and nuclear
extracts from cisplatin-treated WT cells to determine which fraction of
SHP-2 participates in DNA damage signaling. In response to DNA damage,
tyrosyl phosphorylation and phosphatase activity of the nuclear
fraction of SHP-2 were enhanced, while catalytic activity of cytosolic
SHP-2 remained unchanged (Fig. 7). This
result suggests that nuclear but not cytosolic SHP-2 is involved in DNA
damage signaling, further confirming the nuclear function of SHP-2 in
DNA damage-induced cellular response.
|
We next attempted to dissect the potential mechanism by which SHP-2
regulates DNA damage-induced c-Abl activation, and examined c-Abl
upstream activators. To date, ATM kinase and possibly the ATM-related
kinase (ATR) have been demonstrated to be responsible for the
activation of c-Abl after DNA damage (19, 20, 51). Therefore, we wanted
to determine whether SHP-2 might modulate c-Abl activation through its
upstream ATM/ATR kinases. To examine ATM/ATR kinase activity, the
phosphorylation status of p53 on serine 15, a putative in
vivo substrate of ATM/ATR kinases (2, 52-54), was analyzed using
a specific anti-phospho-p53Ser-15 antibody. Phosphorylation
of p53 on serine 15 was dramatically induced upon cisplatin treatment
(Fig. 8A). Interestingly, p53 phosphorylation response in WT and SHP-2/
mutant
cells was not significantly altered. Furthermore, as the ATM/ATR
substrate proteins contain specific Ser/Thr-Gln motifs that are
phosphorylating sites of these kinases (51, 52, 55), the activation
status of ATM/ATR kinases was also determined by examining the
phosphorylation status of ATM/ATR substrate proteins in whole cell
lysates using a specific anti-phospho-Ser/Thr-Gln antibody.
Phosphorylation of ATM/ATR substrates in both WT and SHP-2
/
cells treated with cisplatin was comparable
(Fig. 8B). Taken together, these results suggest that
ATM/ATR kinases were equally activated by cisplatin-induced DNA damage
in WT and mutant cells. Therefore, the SHP-2 mutation
appears to block c-Abl activation through a mechanism(s) other than
ATM/ATR kinases.
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DISCUSSION |
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In this report, we defined a novel nuclear function of SHP-2 tyrosine phosphatase distinct from its critical cytoplasmic role in the regulation of growth factor and cytokine signal transduction. We provided evidence that SHP-2 phosphatase is involved in the DNA damage-triggered signaling pathway whereby its tyrosyl phosphorylation and catalytic activity become significantly increased upon DNA damage treatment. Mutant cells lacking functional SHP-2 demonstrated markedly decreased sensitivities to DNA damage-induced apoptosis. Activation of c-Abl tyrosine kinase and induction of p73 and its effector p21Cip1 were essentially blocked by the SHP-2 mutation. We also show that a substantial amount of SHP-2 protein is localized in the nucleus, and that SHP-2 constitutively associates with nuclear c-Abl via its SH3 domain. These results represent the first demonstration of a connection between SHP-2 phosphatase and c-Abl kinase and consequently the c-Abl-mediated DNA damage response.
It appears that impaired activation of c-Abl kinase in
SHP-2/
mutant cells accounts for their decreased
apoptotic response to DNA damage. p73 induction and hence the induction
of its downstream effector p21Cip1 were essentially blocked in
SHP-2
/
cells. Although p53 in WT and SHP-2 mutant
fibroblast cells was not significantly induced following DNA damage
presumably because of SV40 T antigen immortalization (47-49), in the
embryonic stem cell system no significant change in p53 induction was
observed between WT and SHP-2
/
mutant embryonic stem
cells exposed to DNA damage.2
Therefore, it is conceivable that p73 but not p53 induction is blocked
by the loss-of-function mutation of SHP-2. Because p73 (but
not p53) induction and tyrosyl phosphorylation are fully attributed to
c-Abl kinase activity (8-10), the complete blockade of the induction
of p73 and its downstream effector p21Cip1 together with the
abolished phosphorylation response of baseline p73 in
SHP-2
/
mutant cells suggests that DNA damage-induced
c-Abl activation is impaired in the mutant cells. Consistent with this
notion, c-Abl phosphorylation analyses and in vitro kinase
assays (Fig. 4) showed that c-Abl kinase activation in mutant cells
lacking functional SHP-2 phosphatase was significantly reduced.
Moreover, this conclusion is supported by c-Abl transfection data, in
which the effects of overexpressed WT and dominant negative c-Abl on DNA damage-induced cell death were abrogated in
SHP-2
/
mutant cells (Fig. 5).
The underlying mechanism(s) by which SHP-2 promotes c-Abl activation
and c-Abl-mediated DNA damage signaling pathways remains to be
determined. The tyrosine phosphatase activity of SHP-2 plays a positive
role in a number of signaling pathways induced by growth factors and
cytokines. However, a detailed biochemical basis for its positive
function is ill-defined. Likewise, it is unclear how SHP-2 promotes
c-Abl activation in response to DNA damage. It is unlikely that SHP-2
regulates c-Abl kinase through its upstream activators, as ATM/ATR
kinases appeared to be equally activated by DNA damage in both WT and
SHP-2/
mutant cells (Fig. 8). In addition to ATM/ATR
kinases, c-Abl activity can be regulated by several other mechanisms.
It is especially important to emphasize that the SH3 domain of c-Abl
negatively regulates its kinase activity through an intramolecular
interaction between SH3 and the linker region between its SH2 and
kinase domains, either deletion or point mutations within the SH3
domain result in constitutive tyrosine kinase activity and oncogenic
activation (56-61). c-Abl kinase can be also regulated by its
inhibitors (62-64). In our studies, because both in vivo
immunoprecipitation and in vitro binding assays showed that
SHP-2 phosphatase constitutively associated with c-Abl kinase via its
SH3 domain (Fig. 6), and a potential motif for c-Abl SH3
binding (a proline-rich consensus sequence
XPXXXXPXXP (43, 65, 66)) has been
identified in SHP-2 (428WPDHGVPSEP436), it is
possible that SHP-2 phosphatase facilitates c-Abl activation by
directly acting on c-Abl or c-Abl-associated inhibitors. It is
interesting to note that SHP-2 phosphatase also binds to the SH3 domain
of c-Src kinase and activates Src by a non-enzymatic mechanism (67).
Because c-Abl shares high homology with c-Src kinase, it is likely that
SHP-2 promotes c-Abl activation through a similar mechanism,
i.e. SHP-2 may displace the inhibitory SH3 domain away from
the linker region, thereby co-stabilizing the active state of c-Abl
kinase. As the association between c-Abl and the mutant form of SHP-2
was diminished because of either its N-terminal truncation or its
decreased protein expression, activation of c-Abl in response to DNA
damage in SHP-2 mutant cells was compromised. Nevertheless, because
phosphatase activity of WT SHP-2 is significantly increased upon DNA
damage, it remains possible that SHP-2 might promote c-Abl activation
through inactivating c-Abl inhibitor(s). If this is the case, it would
be very interesting to determine the direct downstream substrate(s) of
SHP-2 phosphatase and the biochemical significance of its enzymatic
activity in this context.
The mutant cell lines used in this study harbor a deletion and
loss-of-function mutation of SHP-2. Although the truncated form of SHP-2 lacking N-terminal amino acids 46-110 (including the
N-SH2 domain) is expressed, the protein level is only about 25% of WT
SHP-2. Previous studies using these cell lines have suggested that it
is biologically inert (32-39). Consistent with those studies, the
mutant form of SHP-2 also appears to be non-functional in the DNA
damage-triggered signaling pathway, because its tyrosyl phosphorylation
and phosphatase activity remained unchanged following DNA damage (Fig.
3, A and B). More importantly, reintroduction of
functional SHP-2 into the mutant cells partially corrected their
biological and biochemical responses to DNA damage. Although the rescue
effects were not complete because of the low expression level of
reintroduced WT SHP-2 in the rescued cell line, the partial correction
of the defective signaling and cellular responses by WT SHP-2 clearly
suggests that the targeted mutation of SHP-2 is a hypomorphic mutation
and that the phenotype displayed by the mutant cells resulted from
loss-of-function rather than gain-of-function of the truncated form of
SHP-2 expressed in mutant cells. Regardless of whether the defective
cellular response to DNA damage in SHP-2 mutant cells results from the
N-terminal truncation of SHP-2 or the decreased expression level of the
mutant form of SHP-2, our results obtained using this mutant cell model
still support an important role for SHP-2 in DNA damage-induced cell
death responses. Additionally, it is noteworthy that because of very
early (embryonic day 8.5-10.5) embryonic lethality of
SHP-2/
mice, the number of primary embryonic
fibroblast cells that can be derived from mutant embryos at this stage
is extremely limited. For this reason, the fibroblast cell lines we
used were immortalized with SV40 T antigen. Even though p53 function in
these cell lines is blocked, p73 is fully functional, as SV40 T antigen
selectively inactivates p53 but not p73 (47-49). Indeed,
p21Cip1, a downstream effector of p73, was significantly
induced in the immortalized WT cells following DNA damage (Fig.
2A).
In summary, the biological and biochemical evidence presented in this
report suggest a previously uncharacterized nuclear function of SHP-2
tyrosine phosphatase, i.e. it plays an important role in the
DNA damage-induced apoptosis. SHP-2 is constitutively associated with
c-Abl kinase and promotes c-Abl activation in response to genotoxic
insult. These findings represent the first demonstration of a link
between SHP-2 phosphatase and c-Abl-mediated DNA damage responses, and
suggest a potential application of SHP-2 phosphatase in cancer treatment.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Bert Vogelstein, Gen-Sheng Feng, Robert G. Hawley, Mark Williams, and Michael Chase for reagents, helpful comments, and critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant R01HL68212-01A1 (to C. K. Q.).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 Hematopoiesis, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0445; Fax: 301-738-0444; E-mail: quc@usa.redcross.org.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211327200
2 L. Yuan, W. M. Yu, and C. K. Qu, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: SH2, Src homology domain 2; WT, wild-type: GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; ATR, ATM-related kinase; ATM, mutated in ataxia telangiectasia; DAPI, 4,6'-diamidino-2-phenylindole.
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