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INTRODUCTION |
The mammalian c-Abl and Arg nonreceptor tyrosine kinases are
expressed widely in adult tissues (1-3). The N-terminal regions of
c-Abl and Arg share ~90% identity and, as found in members of the
Src family, contain tandem Src homology 3 (SH3),1 SH2, and tyrosine
kinase (SH1) domains. Following the kinase domain, the next 135 amino
acids of both proteins contain three conserved PXXP motifs
that can serve as binding sites for SH3 domains (4, 5). The C-terminal
regions of c-Abl and Arg share 29% identity and differ from other
nonreceptor tyrosine kinases by the presence of globular and
filamentous actin binding domains (6). In addition, the C-terminal
region of c-Abl differs from that of Arg by the presence of a nuclear
localization signal (7) and DNA binding sequences (8). In concert with
these structural differences, c-Abl is expressed in both the nucleus and cytoplasm, whereas Arg has been found predominantly in the cytoplasm (5, 9).
The available evidence supports a role for c-Abl and Arg in regulating
cytoskeletal dynamics. Mammalian c-abl and arg
exhibit structural conservation with genes in the sea urchin
(E-abl), fruit fly (D-abl), and
nematode (N-abl) (10, 11). D-abl is expressed in neuronal axons (12) and functions in control of the axonal
cytoskeleton (13). Other studies have demonstrated that
D-Abl interacts with the Notch transmembrane receptor to regulate axon extension (14). Mice with targeted disruption of the
c-abl gene are born runted and exhibit head and eye
abnormalities (14). Mice deficient in Arg develop normally and exhibit
behavioral abnormalities (9). Moreover, embryos deficient in both c-Abl and Arg die before 11 days postcoitus with defects in neurulation (9).
The finding that neuroepithelial cells from
c-abl
/
arg
/
mice
have an altered actin cytoskeleton has supported involvement of c-Abl
and Arg in the regulation of actin microfilaments (9). Further support
for interactions between c-Abl and the actin cytoskeleton has been
obtained from the demonstration that clustering of integrins and
thereby docking of actin stress fibers is associated with stimulation
of c-Abl activity (15).
Other insights into a functional role for c-Abl have been derived from
the findings that overexpression of c-Abl in fibroblasts induces cell
cycle arrest (16, 17). Growth suppression is dependent on the nuclear
localization sequences, an intact SH2 domain, and tyrosine kinase
activity (17). Expression of c-Abl in Schizosaccharomyces
pombe similarly induces growth arrest by a mechanism dependent on
the c-Abl kinase function (18). In mammalian cells,
c-Abl-dependent growth arrest is mediated in part by
interactions with p53 and the induction of p21 (19, 20). Nuclear c-Abl
also associates with the DNA-dependent protein kinase
complex (21, 22) and with the product of the gene mutated in ataxia
telangiectasia (23, 24). Activation of these serine/threonine kinases in the response of cells to DNA damage is associated
with induction of c-Abl activity (21, 23-26). Activation of c-Abl contributes to DNA damage-induced apoptosis by mechanisms in part dependent on p53 and its homolog p73 (27-31). In contrast to the involvement of nuclear c-Abl in DNA damage-induced signaling, there is
no known role for Arg in the cellular response to genotoxic stress.
Recent work has shown that the cytoplasmic forms of c-Abl
and Arg are activated in the response of cells to oxidative stress. Normal cellular metabolism is associated with the production of reactive oxygen species (ROS) and, as a consequence, damage to DNA and
proteins (32, 33). Cytoplasmic c-Abl is activated in response to ROS
production by a mechanism that depends on interactions with protein
kinase C
(34-36). Activation of cytoplasmic c-Abl by ROS is
associated with targeting of c-Abl to mitochondria, release of
cytochrome c, and induction of cell death (36, 37). Other
studies have shown that Arg is activated by oxidative stress and that
this response involves Arg-mediated phosphorylation of the proapoptotic
Siva-1 protein (38). The finding that ROS-induced apoptosis is
attenuated in arg
/
cells has supported a
role for Arg in the cell death response to oxidative stress (38). Thus,
the available evidence indicates that cytoplasmic c-Abl and Arg are
both functional in the oxidative stress response.
The present studies demonstrate that c-Abl forms heterodimers with Arg
in the response to oxidative stress. The functional significance of
these findings is supported by the demonstration that both c-Abl and
Arg are required for ROS-induced apoptosis.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
293 cells, MCF-7, MCF-7/c-Abl(K-R) (27), and
MCF-7/Arg(K-R) (38) cells, and mouse embryo fibroblasts (MEFs;
wild-type, c-abl
/
, and
arg
/
) were grown in Dulbecco's modified
Eagle's medium supplemented with 10% heat-inactivated fetal bovine
serum, 2 mM L-glutamine, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Transient transfections were
performed with LipofectAMINE (Invitrogen). Cells were treated with
H2O2 (Sigma), menadione (Sigma), 20 ng/ml tumor
necrosis factor-
(TNF-
; Promega, Madison, WI), 10 µg/ml cycloheximide (Sigma), or 0.1 µM Taxol
(paclitaxel; Sigma).
Vectors--
FLAG-tagged c-Abl, Arg, and their truncated
variants were expressed by cloning into the pcDNA3.1-based FLAG
vector. His/Exp-tagged constructs were prepared by cloning into
pcDNA4HisMAX (Invitrogen). Myc-tagged constructs were prepared by
cloning into the pCMV-Myc vector (Clontech).
Retroviruses expressing FLAG-c-Abl(K-R) or FLAG-Arg(K-R) were prepared
by cloning into a MSCV-based retroviral vector (MSCVpuro; Clontech).
Immunoprecipitation and Immunoblot Analysis--
Cell lysates
were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5; 1 mM phenylmethylsulfonyl fluoride; 1 mM
dithiothreitol; 10 mM sodium fluoride; and 10 µg/ml
aprotinin, leupeptin, and pepstatin A) containing 1% Nonidet P-40.
Soluble protein was subjected to immunoprecipitation with anti-Myc
(Santa Cruz), anti-FLAG (M5, Sigma), anti-c-Abl (24-11, Santa Cruz) or
anti-Exp (Invitrogen). Immunoblot analysis was performed with anti-Myc,
anti-FLAG, anti-Arg (38), anti-c-Abl, anti-Exp-horseradish peroxidase
or anti-Tyr(P) (4G10, Upstate Biotechnology). The antigen-antibody
complexes were visualized by chemiluminescence (ECL, Amersham
Biosciences).
Binding Assays--
Glutathione S-transferase (GST)
fusion proteins were prepared as described (28, 38). Cell lysates were
incubated with 5 µg of GST or GST fusion proteins conjugated to
Sepharose 4B beads for 2 h at 4 °C. The adsorbates were washed
with lysis buffer and then subjected to immunoblotting with anti-FLAG.
An aliquot of the total lysate (2% v/v) was included as a control. For
direct binding assays, purified GST fusion proteins were incubated with in vitro translated 35S-labeled proteins. The
adsorbates were analyzed by SDS-PAGE and autoradiography.
Surface Plasmon Resonance--
Recombinant GST-c-Abl was coupled
to a carboxymethyl dextran sensor chip CM5 in the presence of
EDC, ethanolamine HCl, and N-hydroxysuccinimide.
Binding assays were performed in a Biocore 1000 (BIAcore AB, Uppsala,
Sweden). Purified recombinant Arg (9) diluted in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20) was applied to the chip at a flow rate
of 10 µl/min at 35 °C. BIAevaluation software 3.0 and the 1:1
Langmuir binding model were used to assess binding kinetics.
Kinase Assays--
Lysates from
c-abl
/
cells expressing FLAG-Arg were
subjected to immunoprecipitation with anti-FLAG. The protein complexes
were washed, normalized by immunoblotting, heat inactivated at 80 °C for 20 min, and then resuspended in kinase buffer (20 mM
HEPES, pH 7.5, 75 mM KCl, 10 mM
MgCl2, 10 mM MnCl2) containing 2.5 µCi of [
-32P]ATP and kinase-active Abl (New England
Biolabs) for 30 min at 30 °C. The reaction products were analyzed by
SDS-PAGE and autoradiography.
Apoptosis Assays--
The DNA content was assessed by staining
ethanol-fixed and citrate buffer-permeabilized cells with propidium
iodide and monitoring by a fluorescence-activated cell sorter (BD
Biosciences). The numbers of cells with sub-G1 DNA
were determined with a MODFIT LT program.
 |
RESULTS |
ROS Induce c-Abl·Arg Heterodimers--
To determine whether
c-Abl associates with Arg in vivo, lysates from MCF-7 cells
were subjected to immunoprecipitation with anti-c-Abl or, as a control,
mouse IgG. Immunoblot analysis of the precipitates with anti-Arg
demonstrated a low level of c-Abl·Arg complexes (Fig.
1A). Significantly, treatment
of the cells with 40 and 160 µM
H2O2 was associated with detectable increases
in c-Abl·Arg complexes (Fig. 1A). Exposure to 640 µM H2O2, however, resulted in an
association between c-Abl and Arg which was comparable with that found
in control cells (Fig. 1A). Based on the total amount of Arg
in lysates subjected to immunoprecipitation, less than 1% of the Arg
protein was complexed with c-Abl in control cells. Treatment with 40 µM H2O2 increased the formation
of c-Abl·Arg complexes 3.2-fold such that ~3% of the Arg protein
was complexed with c-Abl. Although total cellular levels of Arg and
c-Abl were similar in these cells, the fraction of c-Abl complexed to
Arg was also ~3% in response to H2O2.
Similar findings were obtained in MEFs treated with
H2O2 (Fig. 1B). The association
between c-Abl and Arg was increased after exposure to 10, 40, and 160 µM H2O2, whereas treatment with
640 µM H2O2 had little effect
(Fig. 1B). To determine whether c-Abl and Arg interact in
response to other inducers of oxidative stress, wild-type MEFs were
treated with menadione, a redox-cycling agent that increases ROS
generation (39). Like H2O2, treatment with 25 µM menadione increased the formation of c-Abl·Arg
complexes, whereas exposure to higher concentrations had less of an
effect (Fig. 1C, left). Similar results were
obtained when cells were treated with 20 ng/ml TNF-
to induce an
endogenous oxidative stress response (40, 41) (Fig. 1C,
right). As a control, anti-c-Abl immunoprecipitates from
c-abl
/
, and arg
/
MEFs showed no detectable signals when probed with anti-Arg (Fig. 1D, left). As additional controls, anti-c-Abl
reacted specifically with c-Abl, whereas anti-Arg reacted specifically
with Arg in immunoblot analyses of arg
/
and
c-abl
/
MEFs (Fig. 1D,
right).

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Fig. 1.
Oxidative stress induces binding of c-Abl and
Arg. A and B, MCF-7 cells (A) and
MEFs (B) were treated with the indicated concentrations of
H2O2 for 3 h. Anti-c-Abl
immunoprecipitates (IP) were analyzed by immunoblotting
(IB) with anti-Arg or anti-c-Abl. Immunoprecipitations with
IgG were included as controls. Lysates not subjected to
immunoprecipitation were analyzed by immunoblotting with anti-Arg and
anti-c-Abl. C, MEFs were treated with the indicated
concentrations of menadione for 1.5 h (left panel) or
20 ng/ml TNF- for 1 h (right panel). Anti-c-Abl
immunoprecipitates were analyzed by immunoblotting with anti-Arg and
anti-c-Abl. D, anti-c-Abl immunoprecipitates from wild-type,
c-abl / , and arg /
MEFs were analyzed by immunoblotting with anti-Arg and anti-c-Abl
(left panel). Lysates from wild-type (WT),
arg / , and c-abl /
MEFs were subjected to immunoblot analysis with anti-c-Abl or anti-Arg
(right panel).
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To extend these findings, 293 cells were transfected to express
Myc-tagged c-Abl (Myc-c-Abl) and FLAG-tagged Arg (FLAG-Arg). Immunoblot
analysis of anti-Myc immunoprecipitates with anti-FLAG demonstrated
detection of complexes containing c-Abl and Arg (Fig. 2A). In the reciprocal
experiment, immunoblot analysis of anti-FLAG immunoprecipitates with
anti-Myc provided further support for the association of c-Abl and Arg
in cells (Fig. 2B). To define the kinetics of the
interaction between c-Abl and Arg, the parameters for binding of Arg
were determined using GST-c-Abl immobilized to the sensor chip in a
BIAcore. Arg bound to c-Abl with a dissociation constant
(Kd) of 0.05 µM (Fig.
2C). These findings demonstrate that c-Abl binds to Arg in
the response to oxidative stress and that the interaction is
direct.

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Fig. 2.
A and B, 293 cells were
transfected to express Myc-c-Abl and FLAG-Arg. Lysates were subjected
to immunoprecipitation (IP) with anti-Myc (A) or
anti-FLAG (B). The immunoprecipitates were analyzed by
immunoblotting (IB) with anti-FLAG or anti-Myc.
C, a sensor chip was conjugated with GST-c-Abl. Arg was
injected over the chip at concentrations ranging from 20 to 80 nM. Raw binding data were analyzed by BIAevaluation
software 3.0 and fit to a 1.1 Languir binding model.
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c-Abl SH3 Interacts with the Arg C Terminus--
To define the
interaction between c-Abl and Arg, lysates from cells expressing
FLAG-Arg were incubated with GST or GST fusion proteins containing the
c-Abl SH3 or SH2 domains. The results show that FLAG-Arg associates
with GST-c-Abl SH3 and not GST-c-Abl SH2 (Fig.
3A). To assess whether the
interaction between c-Abl SH3 and Arg is direct, the GST fusion
proteins were incubated with in vitro translated
35S-FLAG-Arg. The finding that GST-c-Abl SH3 associates
with 35S-FLAG-Arg supported a direct interaction (Fig.
3B). Moreover, the finding that there is no detectable
binding of 35S-FLAG-Arg with the c-Abl SH2 domain supported
specificity of the c-Abl SH3-Arg interaction (Fig. 3B).
Other studies were performed with 35S-labeled Arg(1-501)
or Arg(532-1182) to define regions of Arg responsible for binding to
c-Abl SH3. The results demonstrate that GST-c-Abl SH3 binds to
Arg(532-1182) and not Arg(1-501) (Fig. 3C). Arg(532-1182)
contains six proline-rich sequences that could function as binding
sites for the c-Abl SH3 domain (Fig. 3D). To define the Arg
site(s) responsible for the c-Abl SH3 interaction, FLAG-Arg proteins
mutated at each of the PXXP sequences were incubated with
GST-c-Abl SH3. Analysis of the adsorbates with anti-FLAG demonstrated
that binding of c-Abl SH3 is decreased, but not completely abrogated,
with the Arg(P570A/P573A) mutant (Fig. 3D). By contrast, the
other Arg mutants had little if any effect on c-Abl SH3 binding (Fig.
3D). These findings demonstrate that the c-Abl SH3 domain interacts, at least in part, with the Arg proline-rich site at amino
acids 567-576.

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Fig. 3.
Direct interaction between the c-Abl SH3
domain and Arg in vitro. A, lysates
from 293 cells expressing FLAG-Arg were incubated with GST or the
indicated GST fusion proteins. The adsorbates were analyzed by
immunoblotting (IB) with anti-FLAG. Lysate was included as a
control. B, GST or the indicated GST fusion proteins were
incubated with in vitro translated 35S-labeled
FLAG-Arg. The adsorbates were analyzed by SDS-PAGE and autoradiography.
C, GST or GST-c-Abl SH3 were incubated with
35S-labeled FLAG-Arg(1-501) (left panel) or
35S-labeled Arg(532-1182) (right panel). The
adsorbates were analyzed by SDS-PAGE and autoradiography. D,
proline-rich sequences in Arg(532-1182) are listed with their
respective mutants. Lysates from 293 cells expressing wild-type
FLAG-Arg or the indicated mutants were incubated with GST-c-Abl SH3.
The adsorbates were analyzed by immunoblotting with anti-FLAG
(left panel). Lysates not incubated with GST-c-Abl SH3 were
analyzed with anti-FLAG as controls for expression of the Arg proteins
(right panel).
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To assess binding in vivo, lysates from 293 cells expressing
FLAG-Arg(1-501) or FLAG-Arg(532-1182) were subjected to
immunoprecipitation with anti-c-Abl. Immunoblot analysis of the
precipitates with anti-c-FLAG demonstrated that c-Abl associates with
Arg(1-501) (Fig. 4A,
left) and Arg(532-1182) (Fig. 4A,
right). In the reciprocal analysis, coexpression of Arg and
c-Abl(1-486) or c-Abl(487-1130) demonstrated that Arg associates with
the c-Abl N- and C-terminal regions (Fig. 4B). In studies
performed with 293 cells expressing Exp-c-Abl(1-486) and
Arg(532-1182), immunoblot analysis of anti-Exp immunoprecipitates with
anti-Arg also demonstrated association of the c-Abl N-terminal and the
Arg C-terminal regions (Fig. 4C). By contrast, when
anti-FLAG immunoprecipitates from 293 cells expressing FLAG-Arg(1-501)
and Exp-c-Abl(1-486) were subjected to immunoblotting with anti-Exp,
there was no detectable interaction between these N-terminal regions of
c-Abl and Arg (data not shown). Moreover, in similar experiments
performed on 293 cells expressing FLAG-Arg(532-1182) and
Exp-c-Abl(487-1130), there was no apparent association of these c-Abl
and Arg C-terminal regions (data not shown). Taken together with the
in vitro binding data, the results demonstrate that the
c-Abl SH3 domain interacts with the Arg C-terminal region and that the
c-Abl C-terminal region interacts with the Arg N-terminal region.

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Fig. 4.
Binding of the c-Abl N-terminal and Arg
C-terminal regions in vivo. A,
anti-c-Abl immunoprecipitates (IP) from 293 cells expressing
FLAG-Arg(1-501) (left panel) or FLAG-Arg(532-1182)
(right panel) were analyzed by immunoblotting
(IB) with anti-FLAG or anti-c-Abl. B, lysates
from 293 cells expressing FLAG-Arg and His/Exp-c-Abl(1-486)
(left panel) or His/Exp-Arg and FLAG-c-Abl(487-1130)
(right panel) were subjected to immunoprecipitation with
anti-FLAG. The immunoprecipitates were analyzed by immunoblotting with
anti-Exp and anti-FLAG. C, lysates from 293 cells expressing
His/Exp-c-Abl(1-486) and FLAG-Arg(532-1182) were subjected to
immunoprecipitation with anti-c-Exp. The immunoprecipitates were
analyzed by immunoblotting with anti-Arg and anti-c-Abl.
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The Arg SH3 Domain Binds Directly to the c-Abl C Terminus--
To
determine whether the Arg SH3 domain binds to c-Abl, GST-Arg SH3 was
incubated with in vitro translated 35S-labeled
FLAG-c-Abl. The results demonstrate that Arg SH3 binds directly to
c-Abl (Fig. 5A). To extend
this finding, we generated a c-Abl C-terminal (amino acids 487-1130)
fragment. The results demonstrate that GST-Arg SH3 binds to
35S-labeled c-Abl(487-1130) (Fig. 5B). These
findings indicate that, in addition to binding of c-Abl SH3 to the Arg
C terminus, a direct interaction between Arg SH3 and the c-Abl
C-terminal region contributes to the formation of c-Abl·Arg complexes
(Fig. 5C).

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Fig. 5.
Direct binding of the Arg SH3 domain to the
c-Abl C terminus in vitro. A, GST or
the indicated GST fusion proteins were incubated with
35S-labeled FLAG-Arg. The adsorbates were analyzed by
SDS-PAGE and autoradiography. B, GST or GST-Arg SH3 were
incubated with 35S-labeled FLAG-c-Abl(487-1130). The
adsorbates were analyzed by SDS-PAGE and autoradiography. C,
schematic representation of the interactions that contribute to the
formation of c-Abl·Arg heterodimers.
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c-Abl-mediated Phosphorylation of Arg--
To determine whether
Arg is a substrate for c-Abl, in vitro
translated/heat-inactivated Arg was incubated with a 45-kDa
kinase-active Abl and [
-32P]ATP. Analysis of the
reaction products demonstrated that Abl phosphorylates the 145-kDa Arg
protein (Fig. 6A). To assess
tyrosine phosphorylation of Arg in vivo, a kinase-inactive
FLAG-Arg(K-R) mutant was expressed in 293 cells. Immunoblot analysis of
anti-FLAG immunoprecipitates with anti-Tyr(P) demonstrated that
ectopically expressed Arg is constitutively phosphorylated on tyrosine
(Fig. 6B). To confirm that Arg is phosphorylated by c-Abl in
cells, c-abl
/
and
c-abl+ cells were infected with a retrovirus
expressing FLAG-Arg(K-R). Immunoblot analysis of anti-FLAG
immunoprecipitates with anti-Tyr(P) showed that tyrosine
phosphorylation of FLAG-Arg(K-R) is substantially higher in
c-abl+ compared with
c-abl
/
cells (Fig. 6C). These
findings indicate that Arg is phosphorylated, at least in large part,
by a c-Abl-dependent mechanism.

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Fig. 6.
c-Abl phosphorylates Arg. A,
in vitro translated FLAG-Arg was immunoprecipitated
(IP) with anti-FLAG and heat inactivated (HI) at
80 °C for 20 min. The immunoprecipitates were incubated with Abl and
[ -32P]ATP. The reaction products were analyzed by
SDS-PAGE and autoradiography (upper panel). The
immunoprecipitates were also subjected to immunoblotting
(IB) with anti-FLAG (lower panel). B,
anti-FLAG immunoprecipitates from 293 cells expressing FLAG-Arg(K-R)
were analyzed by immunoblotting with anti-Tyr(P) or anti-FLAG.
C, Anti-FLAG immunoprecipitates from
c-abl / and c-abl+
MEFs infected with a retrovirus expressing FLAG-Arg(K-R) were analyzed
by immunoblotting with anti-Tyr(P) or anti-FLAG.
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c-Abl and Arg Are Required for ROS-induced Apoptosis--
To
assess involvement of c-Abl and Arg in the response of cells to ROS,
cells were studied for H2O2-induced apoptosis.
Compared with MCF-7 cells expressing the empty vector, treatment of
MCF-7/c-Abl(K-R) cells with 250 µM
H2O2 resulted in an attenuated apoptotic
response (Fig. 7A). Similar
findings were obtained in MCF-7 cells stably expressing Arg(K-R) (Fig.
7A). In studies of MEFs, c-abl
/
cells exhibited little if any apoptosis in response to treatment with
40 or 250 µM H2O2 (Fig.
7B). The finding that stable expression of c-Abl in the
c-abl
/
cells reconstitutes the apoptotic
response to ROS demonstrates dependence on c-Abl (Fig. 7B).
The arg
/
cells were also less sensitive to
ROS-induced apoptosis compared with arg+ cells
(Fig. 7B). The results further demonstrate that compared with MCF-7 cells expressing the empty vector, menadione-induced apoptosis is attenuated in MCF-7/c-Abl(K-R) and MCF/Arg(K-R) cells (Fig. 7C). Similar results were obtained when these cells
were treated with TNF-
(Fig. 7C). By contrast, apoptosis
induced by stabilization of microtubules with paclitaxel was unaffected
by a expression of c-Abl(K-R) or Arg(K-R) (Fig. 7C). These
findings collectively support a model in which both c-Abl and Arg are
required for the apoptotic response to oxidative stress.

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Fig. 7.
c-Abl and Arg regulate the apoptotic response
to oxidative stress. A, MCF-7 cells stably expressing
empty vector, c-Abl(K-R), or Arg(K-R) were left untreated (solid
bars) or treated with 250 µM
H2O2 (open bars) for 24 h.
B, c-abl / ,
c-abl+, arg / , and
arg+ MEFs were left untreated (solid
bars) or treated with 40 µM (hatched
bars) or 250 µM (open bars)
H2O2 for 24 h. The percentage of cells
with sub-G1 DNA was determined by flow cytometry.
The results are expressed as the mean ± S. D. of three
experiments. C, MCF-7 cells stably expressing the empty
vector (open bars), c-Abl(K-R) (hatched bars),
and Arg(K-R) (solid bars) were treated with 25 µM menadione for 24 h, 20 ng/ml TNF- + 10 µg/ml
cycloheximide for 36 h, or 0.1 µM paclitaxel (Taxol)
for 36 h. Control cells (C) were left untreated. The
percentage of cells with sub-G1 DNA is expressed as the
mean ± S.D. of three experiments.
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|
 |
DISCUSSION |
c-Abl Interacts with Arg--
Recent findings that c-Abl and Arg
are both activated in the cellular response to oxidative stress
suggested that these related proteins may share similar functions (36,
38). The present studies were thus performed to determine whether c-Abl
and Arg interact in ROS-induced signaling. The results of
coimmunoprecipitation studies demonstrate that endogenous c-Abl and Arg
associate in the response to oxidative stress. These findings were
confirmed by showing the association of ectopically expressed c-Abl and Arg in 293 cells. The results further demonstrate that c-Abl and Arg
interact directly. The in vitro findings support direct
binding of the c-Abl SH3 domain to a proline-rich site (amino acids
567-576) in the Arg C-terminal region. By contrast, there was no
detectable binding of the c-Abl SH3 domain to the Arg N-terminal
region. In concert with these results, expression of c-Abl(1-486) and Arg(572-1182) confirmed that the c-Abl N-terminal region associates with the Arg C-terminal region in cells. In addition, binding of c-Abl
to the Arg(P570A/P573A) mutant was decreased compared with that found
for wild-type Arg. The results also demonstrate that the Arg SH3 domain
associates with the c-Abl C-terminal region. In concert with an
interaction between c-Abl and Arg, we demonstrate that Arg functions as
an in vitro substrate for c-Abl phosphorylation. Moreover,
the results show that tyrosine phosphorylation of Arg in cells
expressing c-Abl is substantially higher than that found in
c-abl
/
cells. These findings demonstrate
that c-Abl forms heterodimers with Arg in vivo by mechanisms
involving intermolecular binding of the respective SH3 domains and
C-terminal regions (Fig. 5C) and that Arg is phosphorylated
by a c-Abl-dependent mechanism.
Induction of c-Abl·Arg Heterodimers in the Oxidative Stress
Response--
Certain insights into the involvement of c-Abl in the
response of cells to oxidative stress came from the finding that ROS induce tyrosine phosphorylation and activation of protein kinase C
(34, 35). In an apparent feedback mechanism, protein kinase C
activates c-Abl, and, in turn, c-Abl phosphorylates protein kinase C
on Tyr-512 (35, 36). Activation of c-Abl is associated with targeting
to mitochondria, release of mitochondrial cytochrome c, and
induction of apoptosis (36, 37). Arg is also activated in the oxidative
stress response and contributes to the induction of apoptosis by
interacting with the proapoptotic Siva-1 protein (38). The present
results demonstrate that oxidative stress induces the formation of
c-Abl·Arg heterodimers. Moreover, the findings show that binding of
c-Abl and Arg is dependent on the concentration of
H2O2. Thus, treatment with 10 to greater than 160 µM H2O2 was associated with
increases in c-Abl·Arg heterodimers, whereas treatment with 640 µM H2O2 resulted in binding of
c-Abl and Arg at a level found in control cells. The finding that
menadione and TNF-
also induce the formation of c-Abl·Arg
heterodimers is in concert with the effects of these agents on redox
cycling and ROS generation (39, 41, 42). Thus, c-Abl and Arg form heterodimers in response to diverse agents that induce oxidative stress.
Regulation of the Apoptotic Response to Oxidative Stress by c-Abl
and Arg--
ROS have been implicated in the regulation of both
mitogenic and apoptotic signaling pathways. Mitogenic signals induced
by growth factors or activated Ras are mediated through ROS production (42, 43). Other work has indicated that ROS induce topoisomerase II-mediated cleavage of chromosomal DNA and thereby apoptosis (44). The
p66shc adaptor protein (45) and the p85 subunit
of phosphatidylinositol 3-kinase (46) have also been implicated in the
apoptotic response to oxidative stress. Moreover, p53-induced apoptosis
is mediated by ROS-dependent mechanisms (44, 47, 48). The
present results provide support for involvement of both c-Abl and Arg
in the apoptotic response to oxidative stress. Stable expression of
either kinase-inactive c-Abl(K-R) or Arg(K-R) blocked
H2O2-induced apoptosis of MCF-7 cells. In
concert with these findings, the apoptotic response of MEFs to
H2O2-induced oxidative stress was attenuated by
targeted disruption of either c-abl or arg.
Menadione- and TNF-
-induced apoptosis was also attenuated by
expression of c-Abl(K-R) or Arg(K-R). The present results also
demonstrate that, in contrast to oxidative stress, both c-Abl and Arg
are dispensable for paclitaxel-induced apoptosis. These findings thus
provide the first evidence that c-Abl and Arg form heterodimers and
that both c-Abl and Arg are required for the apoptotic response to
oxidative stress.