Bcl-xL Blocks Activation of Related Adhesion Focal
Tyrosine Kinase/Proline-rich Tyrosine Kinase 2 and Stress-activated
Protein Kinase/c-Jun N-terminal Protein Kinase in the Cellular
Response to Methylmethane Sulfonate*
Pramod
Pandey,
Shalom
Avraham
,
Andrew
Place,
Vijay
Kumar,
Pradip
K.
Majumder,
Keding
Cheng§,
Atsuko
Nakazawa,
Satya
Saxena§, and
Surender
Kharbanda¶
From the Department of Adult Oncology, Dana-Farber Cancer
Institute, Harvard Medical School, Boston, Massachusetts 02115, the
Division of Experimental Medicine and
Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard
Institutes of Medicine, Boston, Massachusetts 02115, and the
§ Manitoba Institute of Cell Biology, University of
Manitoba, Winnipeg, Manitoba R3E 0V9, Canada
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ABSTRACT |
The stress-activated protein kinase/c-Jun
N-terminal protein kinase (JNK) is induced in response to ionizing
radiation and other DNA-damaging agents. Recent studies indicate that
activation of JNK is necessary for induction of apoptosis in response
to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the
anti-apoptotic protein Bcl-xL, but not by caspase
inhibitors CrmA and p35. By contrast, UV-induced JNK activity is
insensitive to Bcl-xL. The results demonstrate that
treatment with MMS is associated with an increase in tyrosine
phosphorylation of related adhesion focal tyrosine kinase
(RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of
JNK and that this phosphorylation is inhibited by overexpression of
Bcl-xL. Furthermore, overexpression of a dominant-negative
mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The
results indicate that inhibition of RAFTK phosphorylation by MMS in
Bcl-xL cells is attributed to an increase in tyrosine
phosphatase activity in these cells. Hence, treatment of
Bcl-xL cells with sodium vanadate, a tyrosine phosphatase
inhibitor, restores MMS-induced activation of RAFTK and JNK. These
findings indicate that RAFTK-dependent induction of JNK in
response to MMS is sensitive to Bcl-xL, but not to CrmA and
p35, by a mechanism that inhibits tyrosine phosphorylation and thereby
activation of RAFTK. Taken together, these findings support a novel
role for Bcl-xL that is independent of the caspase cascade.
 |
INTRODUCTION |
The cellular response to certain stress inducers includes cell
cycle arrest and, in certain cases, lethality. However, the intracellular signals that control the induction of these events are
mainly unclear. Whereas p53 has been implicated in promoting apoptosis
induced by ionizing radiation
(IR)1 exposure (1, 2), other
studies have demonstrated that Bcl-2 and Bcl-xL inhibit
apoptosis in response to diverse agents (3, 4). The induction of
apoptosis by diverse stimuli is associated with activation of
aspartate-specific cysteine proteases (caspases) (5) and cleavage of
poly(ADP-ribose) polymerase (PARP) (6), protein kinase C
(PKC
)
(7), and other proteins (8). Importantly, the finding that cleavage of
these proteins is blocked by overexpression of Bcl-xL (8,
9) has indicated that the Bcl-2-related family of anti-apoptotic
proteins functions upstream to the activation of caspases. More direct
evidence for involvement of interleukin-converting enzyme (ICE)-like
proteases in apoptosis comes from studies utilizing the cowpox virus
protein CrmA (10) and the baculovirus protein p35 (11), which are
direct inhibitors of at least certain members of the caspase family.
Methylmethane sulfonate (MMS) is a monofunctional alkylating agent and
is a potent inducer of cellular stress leading to chromosomal aberrations, point mutations, and cell killing (12-14). Previous studies have shown that reactivity of MMS with the ring nitrogens of
the purine bases, particularly N-7 of guanine, correlates with induction of cell killing (12). Other studies have shown that treatment
of a variety of cell types with MMS is associated with induction of JNK
and p38 MAPK (15), but the signaling mechanisms involved in this
response are unclear.
The stress-activated protein kinase (SAPK) (also known as c-Jun
N-terminal kinase (JNK)) is induced by tumor necrosis factor (TNF),
anisomycin, and interleukin-1 (16, 17). JNK is also activated by UV
light, osmotic shock, IR, and a variety of other DNA damaging agents
(18-22). Upstream regulators of JNK include GCK, MLK-3, SPRK, and
hematopoietic protein kinase-1 (HPK-1) (23-26). Other studies have
shown that JNK is activated by immediate upstream kinases such as
SEK-1/MKK4 and/or MKK7 (27, 28). The finding that certain DNA
damaging agents, but not TNF, induce JNK by a c-Abl-dependent mechanism has also supported distinct
upstream effectors to JNK activation (20, 22, 29).
Many of the signals activating JNK also induce apoptosis. Accordingly,
JNK has been implicated as a potential mediator of the apoptotic
pathway in response to IR, TNF, certain other inducers (30), and
trophic factor deprivation (31). By contrast, other studies have shown
that TNF-induced apoptosis is independent of JNK activation (32).
Therefore, the role of JNK in mediating apoptosis may be inducer- and
cell type-specific.
Activation of the related adhesion focal tyrosine kinase (RAFTK) has
been shown to be upstream to JNK in the response to TNF or UV in
certain cell types (33). RAFTK (also known as PYK2 and
Ca2+-dependent tyrosine kinase (CADTK)) is involved in
signaling upstream to the extracellular-regulated kinase (ERK) pathway
(34-36). RAFTK is a close relative of the pp125 FAK tyrosine kinase
and is activated by various extracellular signals that increase
intracellular calcium concentrations (34, 36). Moreover, RAFTK can
tyrosine phosphorylate and thereby modulate the action of ion channels.
Thus, RAFTK may function as an intermediate that links various calcium
signals with both short and long term responses in neuronal cells (34, 36).
In this study, we demonstrate that MMS-induced activation of JNK in
U-937 cells is blocked by overexpressing Bcl-xL and not CrmA or p35. By contrast, Bcl-xL had no apparent effect on
UV-induced JNK activity. We also demonstrate that, in contrast to UV,
MMS-induced activation of JNK is RAFTK-dependent and that
tyrosine phosphorylation of RAFTK is sensitive to Bcl-xL.
Moreover, treatment of U-937/Bcl-xL cells with sodium
vanadate (SV) restores MMS-induced tyrosine phosphorylation of RAFTK
and activation of JNK.
 |
MATERIALS AND METHODS |
Cell Culture and Reagents--
Human U-937/neo,
U-937/Bcl-xL, U-937/CrmA, and U-937/p35 cells (37, 38) were
grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. PC12 cells were grown in DMEM
containing 10% HI horse serum, 5% heat-inactivated-fetal bovine
serum, and antibiotics. Cells (1 × 106/flask) were
seeded 24 h before treating with 1 mM MMS (Sigma) with
or without 200 µM SV (Sigma). Cells were also treated
with 40 J/m2 UV (UV StratalinkerTM 1800, Stratagene).
Cell Proliferation Assays--
Cells were treated with 1 mM MMS with or without 200 µM SV. The initial
number of cells seeded was 1 × 105/ml. After
indicated times, the numbers of live cells was determined by trypan
blue exclusion.
Immune Complex Kinase Assays--
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 SV, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 µg/ml of leupeptin and
aprotinin) as described (21). Lysates were incubated with anti-JNK
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at
4 °C and then for 45 min after addition of protein A-Sepharose. The
immune complexes were washed three times with lysis buffer and once
with kinase buffer and resuspended in kinase buffer containing
[
-32P]ATP (6000 Ci/mmol; NEN Life Science Products)
and GST-Jun (1-102) (39). The reactions were incubated for 15 min at
30 °C and terminated by the addition of SDS sample buffer. The
proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis
and autoradiography.
Immunoblot Analysis--
Cell lysates were separated by
electrophoresis in SDS-polyacrylamide gels and then transferred to
nitrocellulose paper. Immunoblot analyses were performed using
anti-Bcl-x (40), anti-PKC
(Santa Cruz) or anti-Tyr(P) (4G10, Upstate
Biotechnology, Lake Placid, NY) antibodies. The antigen-antibody
complexes were visualized by chemiluminescence (ECL, Amersham Pharmacia
Biotech). Preparation of lysates and immunoblotting for PARP were
performed as described using the C-2-10 anti-PARP monoclonal antibody
(41). After washing with phosphate-buffered saline/Tween, the membranes
were incubated with horseradish peroxidase-conjugated anti-mouse IgG
(Amersham Pharmacia Biotech) for anti-PARP antibody.
Immunoprecipitations--
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 SV, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 µg/ml leupeptin and aprotinin)
as described (42). Lysates were incubated with anti-RAFTK antibody (43)
for 1 h at 4 °C and then for 45 min after addition of protein
A-Sepharose. The immune complexes were analyzed by immunoblotting with
anti-Tyr(P) (4G10, Upstate Biotechnology) or anti-RAFTK antibodies.
Transient Transfections--
PC12 cells were transiently
transfected with vector or Flag-RAFTK (K-M) with pEBG-SAPK using
LipofectAMINE (Life Technologies, Inc.). After transfection, cells were
treated with MMS, and total cell lysates were subjected to incubation
with GST protein adsorbed on GSH beads. The protein precipitates were
analyzed by GST-Jun immune complex kinase assays as described above.
Total cell lysates were also analyzed by immunoblotting with
anti-GST-SAPK.
Measurment of Total PTPase Activity--
U-937 or
U-937/Bcl-xL cells were treated with 200 µM
SV and harvested after 3 h. Cells were lysed in phosphate-free 50 mM Tris-HCl, pH 7.5, buffer using a sonicator. The PTPase
activity in whole cell lysate was measured by a nonradioactive
photometric enzyme immunoassay kit (Boehringer Mannheim). The reactions
were performed directly in the wells of a microtiter plate with
biotin-labeled synthetic tyrosine-phosphorylated peptide substrate (100 mM) bound to the streptavidin matrix. Following the
addition of vanadate (500 µM) to quench the reactions,
the fractions of unmetabolized substrate in the reactions were
determined immunochemically using peroxidase conjugated
anti-phosphotyrosine antibodies. The development of color was monitored
at 405 nm in a microtiter plate reader.
 |
RESULTS AND DISCUSSION |
To determine the involvement of Bcl-xL in MMS- and
UV-induced apoptosis, we assessed cleavage of the 116-kDa PARP protein to an 85-kDa fragment in response to these agents. As expected, MMS-treatment of U-937 cells resulted in PARP cleavage (Fig.
1A). Similar findings were
obtained when U-937 cells were treated with UV, and there was no
detectable cleavage of PARP when U-937/Bcl-xL cells were
treated with MMS or UV (Fig. 1A). Previous studies have
shown that PKC
undergoes caspase-3-mediated proteolytic cleavage in
an apoptotic pathway induced in response to diverse forms of stress
(7, 44). To further determine the activation of caspase-3 by MMS or UV,
cell lysates from MMS- or UV-treated U-937 and U-937/Bcl-xL
cells were analyzed by immunoblotting with PKC
. Similar to PARP
cleavage, the results demonstrate that MMS or UV-treatment of U-937
cells results in PKC
cleavage (Fig. 1B). Moreover, there
was no detectable cleavage of PKC
when U-937/Bcl-xL cells were treated with MMS or UV (Fig. 1B). We also
compared the proliferation of U-937 and U-937/Bcl-xL cells in the
presence of varying concentrations of MMS. At lower MMS concentrations (25-50 µM), there was little if any effect of MMS on
proliferation of either cell type. At higher concentrations of MMS (1 mM), in contrast to U-937/Bcl-xL, approximately
50% of U-937 cells were dead (based on trypan blue exclusion) by
12-16 h. Moreover, more than 75% U-937 cells were dead when treated
with 1 mM MMS for 24-36 h. These findings indicate that
overexpression of Bcl-xL significantly blocks MMS-induced
cell death. Moreover, overexpression of Bcl-xL also blocked
MMS- and UV-induced internucleosomal DNA fragmentation, a hallmark of
apoptosis (data not shown). Taken together, these findings indicated
that overexpression of Bcl-xL is associated with inhibition
of apoptosis in the response to MMS and UV. Whereas JNK activation has
been associated with induction of apoptosis, we assayed anti-JNK
immunoprecipitates for phosphorylation of GST-Jun. The results
demonstrate increased JNK activity and no change in JNK protein levels
in wild type cells treated with MMS or UV (Fig. 1, C and
D, and data not shown). Importantly, MMS-induced JNK
activity was completely inhibited in cells overexpressing Bcl-xL (Fig. 1C). By contrast,
Bcl-xL had no detectable effect on UV-induced JNK activity
(Fig. 1D). To rule out a clonal effect, three additional,
independently selected clones of U-937/Bcl-xL cells (clones
2, 9, and 10) that express high levels of this protein (data not shown)
were treated with MMS. Analysis of anti-JNK immunoprecipitates from
these clones also demonstrated inhibition of JNK activation by
Bcl-xL (Fig. 1E). Thus, whereas both MMS- and
UV-induced apoptosis is inhibited in U-937/Bcl-xL cells,
only MMS-induced JNK activity is blocked by Bcl-xL. These
findings suggest that Bcl-xL functions upstream to the
activation of JNK by MMS, but not UV.

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Fig. 1.
A and B, effects of
Bcl-xL on MMS- or UV-induced proteolytic cleavage of PARP
and PKC . U-937 or U-937/Bcl-xL cells were treated with 1 mM MMS for 6 h or 40 J/m2 for 3 h.
Total cell lysates were analyzed by immunoblotting with anti-PARP
(A) or anti-PKC (B) antibodies. C
and D, activation of JNK by MMS (C) and UV
(D) in U-937 cells expressing Bcl-xL. U-937 or
U-937/Bcl-xL (clone 6) cells were treated with 1 mM MMS for 1 h (C) or 40 J/m2
UV for 15 min (D). Total cell lysates were
immunoprecipitated with anti-JNK antibody, and in vitro
immune complex kinase reactions were performed using GST-Jun fusion
protein as substrate. The proteins were separated by 10%
SDS-polyacrylamide gel electrophoresis, stained with Coomassie Blue,
and analyzed by autoradiography. E, different clones of
U-937/Bcl-xL cells were treated with 1 mM MMS
for 1 h. mms-treated U-937 cells were used as control. In
vitro immune complex kinase assays in anti-JNK immunoprecipitates
were performed as described above.
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Previous studies have shown that Bcl-xL functions upstream
to activation of caspase 3 (7, 44). Because MMS- and UV-induced PARP
cleavage is inhibited in U-937/Bcl-xL cells, we asked
whether this event is also sensitive to CrmA or p35. There was no
detectable cleavage of PARP in MMS-treated U-937/CrmA or U-937/p35
cells (Fig. 2A). Similar
results were obtained when cell lysates were analyzed for cleavage of
PKC
(data not shown). However, the finding that p35, but not CrmA,
blocks UV-induced cleavage of PARP (Fig. 2B) indicated that
UV and MMS activate caspases by different mechanisms. Analysis of
U-937/CrmA and U-937/p35 cells for MMS- or UV-induced JNK activity
demonstrated that, in contrast to Bcl-xL, overexpression of
CrmA or p35 in U-937 cells has no detectable effect on activation of
JNK in response to these agents (Fig. 2C). The inhibition of JNK by MMS in U-937/Bcl-xL, but not in U-937/CrmA or
U-937/p35 cells, suggested that activation of JNK by MMS is upstream to caspase activation. To further assess this issue, U-937 cells treated
with MMS for various times were analyzed for activation of JNK and
cleavage of procaspase-3. The results demonstrate that whereas JNK is
activated by MMS at 1 h, cleavage of procaspase-3 is detected only
after 4-6 h (Fig. 2D). Taken together, these findings
indicate that in U-937 cells treated with MMS, Bcl-xL functions upstream of JNK activation by a mechanism either upstream from or independent of caspase inhibition. In this context, previous studies have shown that caspases act both upstream and downstream to
JNK activation depending upon cell type and inducer (45-47).

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Fig. 2.
A, effects of CrmA or p35 on MMS- or
UV-induced proteolytic cleavage of PARP. U-937/p35 or U-937/CrmA cells
were treated with 1 mM MMS for 6 h or 40 J/m2 for 3 h. Total cell lysates were analyzed by
immunoblotting with anti-PARP antibody. B and C,
U-937/p35 or U-937/CrmA cells were treated with 1 mM MMS
for 2 h (B) or 40 J/m2 UV for 15 min
(C). Total cell lysates were immunoprecipitated with
anti-JNK antibody, and in vitro immune complex kinase
reactions were performed using GST-Jun fusion protein as substrate. The
proteins were separated by 10% SDS-polyacrylamide gel electrophoresis,
stained with Coomassie Blue, and analyzed by autoradiography.
D, U-937 cells were treated with 1 mM MMS for
the indicated times. Total cell lysates were subjected to
immunoprecipitation with anti-JNK and in vitro immune
complex kinase assays were performed as described (top
panel). Total cell lysates were also analyzed by immunoblotting
with anti-caspase-3 antibody (bottom panel).
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Previous studies have shown that activation of JNK by TNF, MMS, or UV
is induced by c-Abl-independent mechanisms (20, 22, 29). In this
context, other studies have shown that the RAFTK tyrosine kinase plays
a key role as an upstream regulator of the JNK pathway in response to
UV in PC12 cells (33). RAFTK is activated by phosphorylation on
tyrosine (33). Treatment of U-937 cells with MMS, but not UV, resulted
in increased tyrosine phosphorylation of RAFTK (Fig.
3A). There was also no
detectable tyrosine phosphorylation of RAFTK following treatment with
agents, such as cis-platinum and IR, that activate c-Abl (data not
shown). Whereas Bcl-xL blocks MMS-induced JNK activation
and MMS induces tyrosine phosphorylation of RAFTK, we asked whether
Bcl-xL functions upstream to RAFTK. The results demonstrate
that Bcl-xL blocks MMS-induced tyrosine phosphorylation of
RAFTK (Fig. 3B). By contrast, inhibition of caspases by
overexpressing CrmA or p35 had no effect on RAFTK tyrosine
phosphorylation (data not shown). RAFTK is also expressed in PC12
neuroblastoma cells (34, 43). To determine the role of RAFTK in
MMS-induced activation of JNK in PC12 cells, total lysates from control
and MMS-treated PC12 cells were subjected to immunoprecipitation with
anti-RAFTK, and the protein precipitates were analyzed by
immunoblotting with anti-Tyr(P). The results demonstrate that, similar
to U-937 cells, treatment of PC12 cells with MMS is associated with
increases in tyrosine phosphorylation of RAFTK (Fig.
4A). Furthermore, JNK
activation is also increased in response to MMS in these cells (Fig.
4B). To further confirm a direct role for RAFTK in
MMS-induced activation of JNK, PC12 cells were transiently transfected
with a dominant negative mutant of RAFTK (RAFTK K-M), treated with MMS,
and assayed for activation of JNK. As a control, PC12 cells expressing
the empty vector were also treated with MMS. The results demonstrate
that treatment of PC12 cells expressing RAFTK K-M mutant, but not the
empty vector, with MMS is associated with a significant inhibition of
JNK activity (Fig. 4C). Other studies have shown that the
protein-tyrosine kinase c-Src functions upstream to JNK in response to
MMS (48). Because c-Src is activated by a RAFTK-dependent
mechanism (35), these results together indicate that RAFTK acts as an
upstream activator of the JNK signaling pathway in the cellular
response to MMS.

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Fig. 3.
Activation of RAFTK by MMS.
A, U-937 cells were treated with 1 mM MMS for 30 min or 40 J/m2 UV for 15 min. Total cell lysates were
immunoprecipitated with anti-RAFTK antibody. The proteins were
separated by 7.5% SDS-polyacrylamide gel electrophoresis, transferred
to nitrocellulose, and analyzed by immunoblotting with anti-Tyr(P)
(top panel) or anti-RAFTK (bottom panel)
antibodies. B, U-937/Bcl-xL cells were treated
with 1 mM MMS for 30 min. Anti-RAFTK
immunoprecipitates were analyzed by immunoblotting with
anti-Tyr(P).
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Fig. 4.
RAFTK-dependent activation of JNK
by MMS. A, PC12 cells were treated with 1 mM MMS for 30 min. Total cell lysates were subjected to
immunoprecipitation with anti-RAFTK and analyzed by immunoblotting with
anti-Tyr(P) antibody. B, total cell lysates from control and
MMS-treated PC12 cells were subjected to immunoprecipitation with
anti-JNK antibody. The protein precipitates were analyzed by in
vitro immune complex kinase assays as described. C,
PC12 cells were transiently transfected with vector or Flag-RAFTK K-M.
The cells were also cotransfected with pEBG-SAPK. Fourty-eight hours
after transfection, cells were treated with 1 mM MMS and
harvested after 30 min. Cell lysates were incubated with GST, and the
protein precipitates were analyzed by in vitro immune
complex kinase assays (top panel). GST-protein precipitates
were also analyzed by immunoblotting with anti-GST-SAPK
(middle panel). The fold activation in JNK activity is
expressed as the mean ± S.D. of three independent experiments
(bottom panel).
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Tyrosine phosphorylation of proteins has been implicated as playing
critical roles in regulating cell death and survival. Previous studies
have shown that treatment of certain cell types with tyrosine
phosphatase inhibitor, SV, is associated with the regulation of
apoptosis (49, 50). Because the results of the present study
demonstrate that overexpression of Bcl-xL inhibits tyrosine
phosphorylation of RAFTK, we sought to determine whether a tyrosine
phosphatase is involved in this regulation. To compare the overall
tyrosine phosphatase activity in U-937 and U-937/Bcl-xL cells, we measured the background tyrosine phosphorylation of proteins
in these cell types and total tyrosine phosphatase activity. U-937 and
U-937/Bcl-xL cells treated with SV were also assayed for
total tyrosine phosphatase activity. The results demonstrate that
overexpression of Bcl-xL in U-937 cells is associated with an increase (approximately 3-4-fold) in total tyrosine phosphatase activity, and as expected, treatment with SV inhibited this activity to
basal levels (Fig. 5A). In
concert with the increase in phosphatase activity, background tyrosine
phosphorylation of proteins in U-937/Bcl-xL cells is
significantly lower than that in U-937 cells (Fig. 5B). To
determine the role of a tyrosine phosphatase in the regulation of RAFTK
activity by MMS, cells were treated with SV with or without MMS, and
anti-RAFTK immunoprecipitates were analyzed by immunoblotting with
anti-Tyr(P). The results demonstrate that treatment with SV increases
MMS-induced tyrosine phosphorylation of RAFTK in U-937 cells (Fig.
5C). Importantly, treatment of U-937/Bcl-xL cells with SV restores tyrosine phosphorylation of RAFTK in response to
MMS (Fig. 5D). Taken together, these findings indicate that MMS-induced tyrosine phosphorylation and activation of RAFTK is regulated by a tyrosine phosphatase in U-937 cells. The 4-fold higher
basal activity of tyrosine phosphatases in U-937/Bcl-xL cells contributes to inhibition of MMS-induced tyrosine phosphorylation of RAFTK.

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Fig. 5.
Pretreatment of cells with SV potentiates
MMS-induced increases in tyrosine phosphorylation of RAFTK.
A, U-937 or U-937/Bcl-xL cells were treated with
200 µM SV for 3 h. Total tyrosine phosphatase
activity was measured as described in text. B, total lysates
from U-937 or U-937/Bcl-xL cells were analyzed by
immunoblotting with anti-Tyr(P) antibody. C, U-937 cells
were treated with MMS with or without SV. Total cell lysates were
subjected to immunoprecipitation with anti-RAFTK and analyzed by
immunoblotting with anti-Tyr(P). D, U-937/Bcl-xL
cells were treated with MMS with or without SV and anti-RAFTK
immunoprecipitates were analyzed by immunoblotting with anti-Tyr(P)
antibody.
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To determine whether treatment of cells with SV affects MMS-induced JNK
activity, U-937 and U-937/Bcl-xL cells were treated with SV
with or without MMS, and anti-JNK immunoprecipitates were analyzed by
immune complex JNK kinase assays. The results demonstrate that
treatment with SV significantly increases MMS-induced JNK activity in
U-937 cells (Fig. 6A). More
importantly, treatment of U-937/Bcl-xL cells with SV
restored JNK activation in response to MMS (Fig. 6B). In
this context, a recent study has suggested that activation of JNK by
MMS and UV is mediated by distinct upstream modulators and that the
MMS-response may be regulated by a tyrosine phosphatase (15). Moreover,
activation of RAFTK by MMS is regulated by a tyrosine phosphatase and
functions upstream to the activation of caspases. Taken together, the
findings of our study indicate that MMS induces tyrosine
phosphorylation of RAFTK and contributes directly to the activation of
JNK.

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Fig. 6.
Pretreatment of cells with SV restores
MMS-induced activation of JNK and cell death. A, U-937
cells were treated with MMS with or without SV. Total cell lysates were
subjected to immunoprecipitation with anti-JNK and analyzed by in
vitro immune complex kinase assays. B, U-937/Bcl-xL
cells were treated with MMS with or without SV, and anti-JNK
immunoprecipitates were analyzed by in vitro immune complex
kinase assays. C, U-937 and U-937/Bcl-xL cells
were treated with 1 mM MMS with or without 200 µM SV. The initial number of cells seeded was 1 × 105/ml. After 12 h of treatment, the number of dead
cells were determined by trypan blue exclusion. The results expressed
as percentage of dead cells (mean ± S.D.) of three independent
experiments.
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To determine whether inhibition of phosphatase activity by SV also
affects MMS-induced cell death, U-937 and U-937/Bcl-xL cells were treated with SV with or without MMS, and cell death was
assessed by trypan blue exclusion. The results demonstrate that
treatment with SV increases MMS-induced cell death in U-937 cells (not
shown). More importantly, SV treatment of U-937/Bcl-xL cells, which are resistant to MMS, was associated with approximately a
30% increase in cell death in response to MMS (Fig. 6C).
Taken together, these findings suggest that inhibition of phosphatase activity by SV potentiates cell death in response to MMS.
Control of apoptosis and progression of cell cycle are closely linked
processes, acting to preserve homeostasis and developmental morphogenesis. Proteins that regulate apoptosis, such as Bcl-2, have
also been implicated in control of the cell (51-53). In this context,
previous studies have shown that the hypoxia-induced genes involved in
cell cycle regulation are p53, p21, and Bcl-2 (51). Moreover, it has been shown that overexpression of Bcl-2 in
breast cancer cells is associated with prolongation of the cell cycle,
particularly at the G1/S boundary (54). By contrast, overexpression of Bcl-xL in U-937 cells is associated with
more rapid growth kinetics as compared with parent cells. Thus, Bcl-2 and Bcl-xL may function differently in control of cell
cycle progression. Nevertheless, the role of Bcl-xL in
regulating the cell cycle, which is different from its anti-apoptotic
function, could contribute the levels and/or activation of phosphatases
observed in these cells.
CrmA is a member of the serpin family that inhibits ICE by forming an
active site-directed complex (10). CrmA expression blocks apoptosis
induced by TNF or activation of Fas (55). Other studies have shown that
p35 inhibits the proteolytic activity of Ced-3, ICE, CPP32, Ich-1, and
Ich-2 but not granzyme B (11). The present studies demonstrate that in
contrast to UV, MMS-induced cleavage of PARP, PKC
, and
internucleosomal DNA fragmentation is blocked by overexpression of CrmA
(Fig. 2 and data not shown). These findings suggest that MMS induces
apoptosis by a mechanism that is distinct, at least in part, from that
induced by UV. Recent work has shown that whereas induction of
apoptosis by TNF and Fas is sensitive to CrmA, DNA damage-induced
apoptosis is dependent on activation of caspases that are CrmA
insensitive (56). Thus, a CrmA-sensitive caspase is necessary for
induction of apoptosis by only certain stress inducers that include
TNF, Fas, and MMS.
The activation of a CrmA-sensitive or -insensitive caspase appears
nonetheless to be independent of JNK activation, because neither CrmA
nor p35 affects the induction of this kinase by diverse classes of
inducers. Other studies have shown that a caspase-mediated cleavage of
MEKK-1, a kinase that acts upstream to activation of JNK (57), is
necessary for anoikis (46, 58). The present findings demonstrate that
MMS, but not UV, induces JNK activity in U-937 cells by a
RAFTK-dependent mechanism. Previous work has shown that
whereas IR-, cis-platinum- and ara-C-induced activation of JNK is
c-Abl-dependent, MMS and UV induce JNK by c-Abl-independent mechanisms (16, 18, 25). The present finding that MMS-induced, but not
UV-induced, JNK involves activation of RAFTK provides further support
for distinct signaling pathways in the responses to diverse stress
causing agents. We also show that Bcl-xL blocks MMS-induced
tyrosine phosphorylation of RAFTK and activation of JNK. The effects of
Bcl-xL on a RAFTK
JNK pathway are selective for
MMS-induced signals because Bcl-xL had no effect on
UV-induced JNK activity. MMS is a monofunctional alkylating agent that
alkylates DNA and damages membrane proteins (14). By contrast, the UV response is initiated in the cytoplasm by a
Ha-Ras-dependent mechanism (16, 59). Thus, sensitivity of
JNK activation to Bcl-xL could be dependent on subcellular
localization of the stress signal and whether RAFTK is involved as an
upstream effectors. Although Bcl-xL blocks activation of
caspases, the effects of Bcl-xL on the RAFTK
JNK
pathway are independent of p35 expression and thereby caspase activity.
Recent studies have shown that overexpression of RAFTK is associated
with induction of apoptosis in 293 cells (60). Moreover, the finding
that inhibition of JNK significantly blocks ionizing radiation- or
TNF-induced apoptosis has supported a direct role for JNK in apoptosis
(30). The present findings demonstrate that in contrast to CrmA or p35,
Bcl-xL blocks MMS-induced activation of RAFTK, JNK, and
induction of apoptosis. Thus, the RAFTK
JNK pathway is upstream to
caspase activation in the cascade of MMS-induced apoptosis.
 |
ACKNOWLEDGEMENTS |
We thank Drs. John Kyriakis, Joseph Avruch,
Ajay Rana, Jim Woodgett, and Leonard Zon for providing various JNK
cDNA constructs and anti-GST SAPK antibody and Dr. Hawa Avraham for
critical reading of the manuscript. We thank Rebecca Farber for
excellent technical assistance.
 |
FOOTNOTES |
*
This investigation was supported by United States Public
Health Service Grants CA75216 (to S. K.) (awarded by the National Cancer Institute, Department of Health and Human Services) and HL55445
(to S. A.) and by a Heart and Stroke Foundation of Canada grant (to
S. S.).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 Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2938; Fax: 617-632-2934; E-mail: surender_kharbanda{at}dfci.harvard.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
IR, ionizing
radiation;
SV, sodium vanadate;
SAPK, stress-activated protein kinase;
JNK, c-Jun N-terminal protein kinase;
PARP, poly(ADP-ribose)
polymerase;
proline-rich tyrosine kinase, RAFTK, related adhesion focal
tyrosine kinase;
MMS, methylmethane sulfonate;
PKC
, protein kinase C
;
TNF, tumor necrosis factor;
GST, glutathione
S-transferase;
ICE, interleukin-converting enzyme;
PYK2, proline-rich tyrosine kinase.
 |
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