Interaction of Hematopoietic Progenitor Kinase 1 and c-Abl
Tyrosine Kinase in Response to Genotoxic Stress*
Yasumasa
Ito
,
Pramod
Pandey
,
Pradeep
Sathyanarayana§,
Pin
Ling¶,
Ajay
Rana§,
Ralph
Weichselbaum
,
Tse-Hua
Tan¶,
Donald
Kufe
, and
Surender
Kharbanda
**
From the
Department of Adult Oncology, Dana-Farber
Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, § Diabetes Research Laboratory, Department of Molecular
Biology, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts 02114,
Department of Radiation and
Cellular Oncology, University of Chicago, Chicago, Illinois 60637, and
¶ Department of Immunology, Baylor College of Medicine, Houston,
Texas 77030
Received for publication, August 10, 2000, and in revised form, January 10, 2001
 |
ABSTRACT |
The c-Abl protein tyrosine kinase is activated by
certain DNA-damaging agents and regulates induction of the
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The
hematopoietic progenitor kinase 1 (HPK1) has also been shown to act
upstream to the SAPK/JNK signaling pathway. We report here that
exposure of hematopoietic Jurkat T cells to genotoxic agents is
associated with activation of HPK1. The results demonstrate that
exposure of Jurkat cells to DNA-damaging agents is associated with
translocation of active c-Abl from nuclei to cytoplasm and binding of
c-Abl to HPK1. Our findings also demonstrate that c-Abl phosphorylates HPK1 in cytoplasm and stimulates HPK1 activity. The functional significance of the c-Abl-HPK1 interaction is supported by the demonstration that this complex regulates SAPK/JNK activation. Overexpression of c-Abl(K-R) inhibits HPK1-induced activation of
SAPK/JNK. Conversely, the dominant negative mutant of HPK1 blocks
c-Abl-mediated induction of SAPK/JNK. These findings indicate that
activation of HPK1 and formation of HPK1/c-Abl complexes are
functionally important in the stress response of hematopoietic cells to genotoxic agents.
 |
INTRODUCTION |
The cellular response to ionizing radiation
(IR)1 and other genotoxic
agents includes cell cycle arrest, activation of DNA repair, and
apoptosis or programmed cell death. However, the intracellular signals
that control these events are largely unclear. The available evidence
supports a role for the c-Abl protein tyrosine kinase in the induction
of apoptosis (1, 2). Transient transfection studies with wild-type but
not kinase-inactive c-Abl have demonstrated induction of an apoptotic
response (2). Also, cells that stably express the dominant negative
c-Abl(K-R) mutant exhibit resistance to induction of apoptosis by IR
and other DNA-damaging agents (2, 3). Similar results have been
obtained in Abl
/
fibroblasts (2, 3). The
apoptosis-resistant phenotype is more pronounced in cells expressing
c-Abl(K-R) than in c-Abl null cells. In addition, a proapoptotic role
for c-Abl is supported by c-Abl-dependent induction of the
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in
the response to genotoxic stress (4-6).
The SAPK/JNK signaling cascade plays a critical role in the responses
stimulated by DNA damage, heat shock, interleukin 1, tumor necrosis
factor
, and Fas (4-15). SAPK is phosphorylated and activated by
immediate upstream mitogen-activated protein kinase kinases (MAPKKs),
MAPKK4 (MKK4)/SEK1 (8, 16) and MKK7 (17). These MAPKKs are
activated, in turn, by the upstream MAPKK kinases including
MAPKK/extracellular signal-regulated kinase kinase kinase 1 (MEKK-1)
(18), mixed lineage kinase 3 (MLK-3) (19), transforming growth factor
-activated kinase 1 (TAK1) (20), tumor progression locus 2 (Tpl-2)
(16), mitogen-activated protein kinase upstream kinase (21), and
apoptosis signal-regulating kinase 1 (22). Furthermore, several
Ste20-related protein kinases that activate SAPK through MAPKK kinases
have been identified as MAPKK kinase kinases, including hematopoietic
progenitor kinase 1 (HPK1) (23, 24), germinal center kinase (25, 26),
HPK1/germinal center kinase-like kinase/Nck-interacting kinase (27,
28), germinal center kinase-like kinase (29), and kinase homologous to
Ste20/Sps1/germinal center kinase-related kinase (30, 31).
HPK1, a 97-kDa serine/threonine kinase, is restricted to hematopoietic
tissues in adults (23, 24). Studies have shown that HPK1 interacts with
MEKK-1 (23), MLK-3 (24), and TAK1 (32), which, in turn, can activate
MKK4/SEK1 and thereby result in activation of the SAPK signaling
pathway. Previous studies have demonstrated that four proline-rich
motifs in HPK1 are potential binding sites for SH3 domain-containing
proteins. HPK1 interacts with the SH2/SH3 domain-containing adaptor
proteins Crk and CrkL (33). Using yeast two-hybrid analysis, HPK1 has
also been shown to associate with the c-Abl SH3 domain (24). The
demonstration that Abl
/
cells exhibit a
defective SAPK response in response to certain DNA-damaging agents has
provided support for c-Abl as an upstream effector in the SAPK pathway
(5, 6) and has raised the possibility of a functional interaction
between c-Abl and HPK1.
The present studies demonstrate that exposure of Jurkat cells to IR is
associated with activation of HPK1. Similar results were obtained with
another genotoxic agent,
1-
-D- arabinofuranosylcytosine (ara-C). The results
also demonstrate that activated HPK1 forms a complex with cytoplasmic
c-Abl in the cellular response to genotoxic agents. The functional
significance of the c-Abl/HPK1 interaction is supported by the finding
that HPK-1-induced activation of SAPK is inhibited by a dominant
negative c-Abl and that kinase-inactive mutants of HPK1 block
c-Abl-mediated induction of SAPK activity.
 |
MATERIALS AND METHODS |
Cell Culture and Reagents--
Human Jurkat T cells (American
Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM
L-glutamine. Human embryonic kidney 293T cells were
cultured in Dulbecco's modified Eagle's medium containing 10%
heat-inactivated fetal bovine serum and antibiotics. MCF-7/neo and
MCF-7/c-Abl(K-R) (34) cells were cultured in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum, antibiotics, and
500 µg/ml Geneticin sulfate (Life Technologies, Inc.). Cells were
seeded at a density of 1 × 106 cells/100-mm culture dish
for 24 h before treatment with 20 Gy of IR or 10 µM
ara-C (Sigma). Irradiation was performed at room temperature with a
-ray source (Cs173; Gamma Cell 1000; Atomic Energy of
Canada, Ontario, Canada) at a fixed dose rate of 0.76 Gy/min.
Isolation of the Cytosolic Fraction--
Cytosolic fractions
were prepared as described previously (35). Cells were washed twice
with phosphate-buffered saline and then suspended in ice-cold buffer
(20 mM HEPES, pH 7.5, 1.5 mM MgCl2,
10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, and 10 µg/ml leupeptin and aprotinin) containing 250 mM sucrose. The cells were disrupted by five strokes in a
Dounce homogenizer. After centrifugation of the lysate at 10,000 × g for 5 min at 4 °C, the supernatant fraction was
centrifuged at 105,000 × g for 30 min at 4 °C. The
resulting supernatant was used as the soluble cytosolic fraction.
Isolation of the Nuclear Fraction--
Nuclear proteins were
isolated as described previously (36). In brief, cells were washed
three times with phosphate-buffered saline and resuspended in 4 cell
volumes of hypotonic lysis buffer (10 mM HEPES, pH 7.5, 2 mM MgCl2, 10 mM KCl, 10 µg/ml
leupeptin, and 10 µg/ml aprotinin). After incubation on ice for 30 min to allow swelling, the cells were disrupted in a Dounce homogenizer (15-20 strokes). The homogenate was layered on a cushion of 1 M sucrose in hypotonic solution and subjected to
centrifugation for 10 min. The nuclei were then suspended in lysis
buffer containing 0.5% Nonidet P-40. After incubation at 4 °C for
30 min, the suspension was centrifuged at 12,000 × g,
and the supernatant was used as the nuclear fraction.
Immunoprecipitation and Immunoblot Analysis--
Total cell
lysates were prepared as described in lysis buffer containing 1%
Nonidet P-40 (37). Equal amounts of total, cytosolic, or nuclear
proteins were subjected to immunoprecipitation with anti-c-Abl (K-12;
Santa Cruz Biotechnology) or anti-HPK1 (32). Immune complexes were
recovered by incubation with protein A-Sepharose for 1 h at
4 °C, washed three times with lysis buffer, separated by SDS-PAGE,
and then transferred to nitrocellulose filters. After blocking with 5%
dried milk in phosphate-buffered saline-Tween, the filters were
incubated with anti-HPK1, anti-c-Abl (Ab-3; Oncogene Research
Products), anti-Flag M2 (Sigma), anti-P-Tyr (4G10; Upstate Biotechnology), anti-Lamin A (Santa Cruz Biotechnology), anti-
-actin (Santa Cruz Biotechnology), or anti-SAPK (Santa Cruz Biotechnology). The filters were analyzed by ECL (Amersham Pharmacia Biotech).
Plasmids and Peptides--
The pSR
-c-Abl wild-type and
pSR
-c-Abl(K-290R) have been described previously (6, 34).
HA-c-Abl was provided by Dr. Jean Y. J. Wang (University of
California, San Diego, CA); GST-Jun(1-102) as described (38);
pEBG-SAPK, pEBG-SEK1 as described (6); pCIneo-Flag-HPK1 wild-type,
pCIneo-Flag-HPK1(M46), GST-HPK1KD, GST-HPK1CD as described (23). The
plasmid GST-Crk(120-225) was provided by Dr. Stephan Feller (Bavarian
Julius-Maximilians University, Wurzburg Germany). The peptides
PR1 (H2N-PELPPAIPRR-COOH), PR2 (H2N-PPPLPPKPK-COOH), PR3
(H2N-PPPNSPRPGPPP-COOH), and PR4 (H2N-KPPLLPPKKE-COOH) were prepared as
described previously (33).
Fusion Protein Binding Assays and Peptide Competition
Assays--
GST and GST-Abl SH3 (39) were purified by affinity
chromatography using glutathione-Sepharose beads and equilibrated in lysis buffer. Cell lysates were incubated with 5 µg of immobilized GST or GST-c-Abl SH3 for 2 h at 4 °C. The resulting protein
complexes were washed three times with lysis buffer and boiled for 5 min in SDS sample buffer. The complexes were then separated by SDS-PAGE and subjected to immunoblot analysis with anti-HPK1. GST-c-Abl SH3
fusion protein was incubated with PR2 (33), PR3, or PR4. The fusion
protein-peptide mixtures were incubated separately with cell lysates
for 30 min at room temperature. After washing, bound proteins were
analyzed by immunoblotting.
c-Abl and HPK1 Kinase Assays--
Cell lysates were subjected to
immunoprecipitation with anti-HPK1 or anti-c-Abl as described
previously (35). The protein complexes were washed and incubated in
kinase buffer (20 mM HEPES, pH 7.4, and 10 mM
MgCl2) containing 2.5 µCi of [
-32P]ATP
and either GST-Crk(120-225) (40) or myelin basic protein (MBP; Sigma)
as substrates for 15 min at 30 °C. The reaction products were
analyzed by SDS-PAGE and autoradiography.
c-Jun Kinase Assays--
293T cells were transfected with
pEBG-SAPK, pEBG-SEK-1, Flag-HPK1, Flag-HPK1(M46), and c-Abl or
c-Abl(K-R). After 12 h of incubation at 37 °C, the medium was
replaced, and the cells were incubated for another 24 h. Cell
lysates were prepared as described, and 200-250 µg of soluble
proteins were incubated with 5 µg of immobilized GST for 30 min at
4 °C. The protein complexes were washed with lysis buffer and then
incubated in kinase buffer containing [
-32P]ATP and
GST-c-Jun(2-100) (38) for 15 min at 30 °C. Reactions were
terminated by the addition of SDS-PAGE sample buffer and boiling.
Phosphorylated proteins were resolved by SDS-PAGE and analyzed by
autoradiography. Cell lysates were also subjected to immunoblotting
with anti-GST (Santa Cruz Biotechnology).
Transient Transfections and Immunoprecipitations--
293T cells
were cotransfected by the calcium phosphate method with HA-c-Abl and
Flag-HPK1. After incubation for 36 h, the cells were lysed in
lysis buffer containing 1% Nonidet P-40 and then subjected to
immunoprecipitation with anti-HA (Boehringer Mannheim), and the
immunoprecipitates were analyzed by immunoblotting with anti-Flag. 293T
cells were also transiently transfected by the calcium phosphate method
with Flag-HPK1 or Flag-HPK1(M46) in the presence of c-Abl or
c-Abl(K-R). After incubation for 36 h, the cells were lysed in
lysis buffer containing 1% Nonidet P-40 and then subjected to HPK1
kinase assay or immunoblot analysis with anti-Flag. MCF-7/neo or
MCF-7/c-Abl(K-R) cells were transiently transfected with Flag-HPK1 by
LipofectAMINE (Life Technologies, Inc.). Total cell lysates were
subjected to immunoprecipitation with anti-Flag and then subjected to
immunoblot analysis with anti-P-Tyr. Autoradiograms were scanned by
laser densitometry, and the intensity of the signals was quantitated
with the ImageQuant program (Molecular Dynamics, Sunnyvale, CA).
In Vitro Phosphorylation of HPK1--
Recombinant c-Abl protein
was incubated with GST-HPK1-KD or GST-HPK1-CD fusion proteins in the
presence of [
-32P]ATP for 30 min at 30 °C.
Phosphorylation of the reaction products was assessed by SDS-PAGE and
autoradiography. 293T cells were transiently transfected with
pCIneo-Flag-HPK1. Cell lysates were subjected to immunoprecipitation
with anti-Flag, and the precipitates were incubated with recombinant
purified c-Abl or kinase-inactive c-Abl(K-R) in the presence of
[
-32P]ATP for 30 min at 30 °C. Phosphorylation of
the reaction products was assessed by SDS-PAGE and autoradiography.
 |
RESULTS |
Genotoxic Stress Induces the Interaction of c-Abl and HPK1 in
Jurkat Cells--
To assess whether c-Abl and HPK1 associate in cells,
lysates from human Jurkat T cells were subjected to immunoprecipitation with anti-HPK1, and the protein precipitates were analyzed by immunoblotting with anti-c-Abl. Immunoblot analysis of precipitates using a control antibody or preimmune rabbit serum demonstrated little,
if any, detection of c-Abl (Fig.
1a; data not shown). However,
a similar analysis of anti-HPK1 immunoprecipitates demonstrated the
coprecipitation of HPK1 and c-Abl (Fig. 1a). To assess
interactions between c-Abl and HPK1 in response to genotoxic agents,
Jurkat cells were treated with 10 µM ara-C and harvested
at 3 h. Analysis of anti-HPK1 immunoprecipitates by immunoblotting
with anti-c-Abl demonstrated induction of HPK1-c-Abl complexes (Fig.
1b). Whereas ara-C incorporates into DNA and inhibits DNA
replication (41), IR induces single- and double-strand DNA breaks. The
finding that exposure of Jurkat cells to IR is also associated with
increased binding of c-Abl and HPK1 indicated that this response is
induced by diverse types of genotoxic stress (Fig. 1b).

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Fig. 1.
c-Abl associates with HPK1. a,
total cell lysates from Jurkat cells were subjected to
immunoprecipitation with anti-c-Abl, anti-HPK1, or preimmune rabbit
serum (PIRS). The protein precipitates were separated by
SDS-PAGE and transferred to nitrocellulose filters. The filters were
analyzed by immunoblotting with anti-c-Abl antibody. b,
Jurkat cells were treated with 10 µM ara-C or exposed to
20 Gy of IR and harvested after 3 h. Cell lysates (approximately
150 µg of total protein) were subjected to immunoprecipitation with
anti-HPK1 and analyzed by immunoblotting with anti-c-Abl (top
panel). As a control, anti-HPK1 immunoprecipitates were analyzed
by immunoblotting with anti-HPK1 (bottom panel). Total cell
lysates (10 µg of total protein; +ve) were also analyzed
by immunoblotting with anti-c-Abl or anti-HPK1.
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To further determine the interaction of c-Abl with HPK1, we transiently
overexpressed Flag-HPK1 with HA-c-Abl in 293T cells and analyzed
anti-HA immunoprecipitates with anti-Flag. Reactivity of anti-Flag with
a 100-kDa protein supported the coprecipitation of HPK1 with c-Abl
(Fig. 2a). In the reciprocal
experiment, anti-Flag immunoprecipitates were subjected to immunoblot
analysis with anti-HA. The results confirmed the detection of complexes
containing HPK1 and c-Abl (data not shown). Lysates from transfected
293T cells were also subjected to immunoprecipitation with anti-HA. Analysis of protein precipitates by immunoblotting with anti-HA demonstrated equal levels of c-Abl (Fig. 2a). Taken
together, these findings indicate that c-Abl associates with HPK1 in
cells. To assess whether interaction between c-Abl and HPK1 is induced by genotoxic stress under conditions overexpressing c-Abl and HPK1,
36 h after the transfection with Flag-HPK1 and HA-c-Abl, cells
were treated with 10 µM ara-C for 3 h. Analysis of
anti-HA immunoprecipitates by immunoblotting with anti-Flag
demonstrated a significant induction of HPK1-c-Abl complex in response
to ara-C (Fig. 2b). Four proline-rich sequences (PR1-PR4)
are present in the C-terminal region of HPK1 (33). One of these
proline-rich sequences (SGPPPNSPRPGPPPS; aa 430-444) displays homology
with motifs located in the C-terminal domains of 3BP1, 3BP2, and ST5 that bind c-Abl SH3. To determine whether the c-Abl SH3 domain binds to
HPK1, lysates from irradiated Jurkat cells were incubated with GST or
GST-c-Abl SH3, and the resulting precipitates were analyzed by
immunoblotting with anti-HPK1. The results demonstrate that in contrast
to GST, HPK1 was detectable in the adsorbates to GST-c-Abl SH3 (Fig.
2c). Because the HPK1 proline-rich motif PR3 (but not PR1,
PR2, or PR4) matches the c-Abl SH3-binding consensus motif
(PXXXXPXPP), we examined the ability of HPK1
proline-rich peptides to block the formation of c-Abl/HPK1 complexes.
The results demonstrate that the PR3 proline-rich peptide efficiently
blocks the interaction of HPK1 with c-Abl, whereas PR2 and PR4 had at best a marginal effect on c-Abl/HPK1 complex (Fig. 2d).
These findings collectively indicate that the interaction between HPK1 and c-Abl likely involves c-Abl-SH3 and HPK1-Pro. It is possible that
the coprecipitation of HPK1 and c-Abl is due to interactions of each
kinase with other molecules.

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Fig. 2.
c-Abl associates with HPK1 via its SH3
domain. a, 293T cells were transiently transfected with
Flag-HPK1 in the presence of HA-c-Abl or empty vector. Lysates were
subjected to immunoprecipitation with anti-HA, and the precipitates
were analyzed by immunoblotting with anti-Flag (top panel).
As a positive control (+ve), total cell lysate was also
analyzed by immunoblotting with anti-Flag. As controls, anti-HA and
anti-Flag immunoprecipitates were analyzed by immunoblotting with
anti-HA (middle panel) and anti-Flag (bottom
panel), respectively. IgH, immunoglobulin heavy chain.
b, 293T cells were transiently transfected with Flag-HPK1
and HA-c-Abl. Thirty-six h after the transfection, cells were treated
with 10 µM ara-C for 3 h. Total cell lysates were
subjected to immunoprecipitation with anti-HA. The precipitates and
lysate were analyzed by immunoblotting with anti-Flag (top
panel) or anti-HA (middle panel). As a control,
anti-Flag immunoprecipitates were also analyzed by immunoblotting with
anti-Flag (bottom panel). c, Jurkat cell lysates
(150 µg of total protein) were incubated with GST or GST-c-Abl SH3
fusion proteins. The protein adsorbates and lysate (10 µg of total
protein; +ve) were analyzed by immunoblotting with
anti-HPK1. d, 293T cells were transiently transfected with
Flag-HPK1, and total lysates were divided in three equal portions.
GST-c-Abl SH3 fusion protein was incubated with PR2 (32), PR3, or PR4.
The fusion protein-peptide mixtures were incubated separately with
lysates for 1 h at 4 °C. After washing, bound proteins were
analyzed by immunoblotting with anti-Flag.
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|
HPK1 is localized primarily in the cytoplasm (23). To define the
subcellular localization of the interaction between c-Abl and HPK1, we
subjected nuclear and cytoplasmic lysates from control and
ara-C-treated cells to immunoprecipitation with anti-HPK1. The
immunoprecipitates were then analyzed by immunoblotting with anti-c-Abl. Signal intensities from the anti-c-Abl immunoblotting experiments (n = 3) were analyzed by densitometric
scanning. Immunoblot analysis of the immunoprecipitates from control
and ara-C-treated cells demonstrated little if any reactivity with
anti-c-Abl in the nuclear fraction (Fig.
3a). Formation of HPK1/c-Abl
complexes was significantly increased in the cytoplasm but not in the
nucleus of ara-C-treated cells (Fig. 3a). Studies have shown
that although c-Abl contains three nuclear localization signals, it is
not localized exclusively to the nucleus (42, 43). c-Abl contains a
functional nuclear export signal, and the subcellular localization of
c-Abl is determined by a balance of nuclear import and export (44). To
assess whether c-Abl translocates to the cytoplasm in response to
genotoxic stress, Jurkat cells were treated with ara-C for different
intervals of time. Nuclear and cytoplasmic fractions were isolated and
analyzed by immunoblotting with anti-c-Abl. As controls, nuclear and
cytoplasmic fractions were also analyzed by immunoblotting with
anti-Lamin A and anti-
-actin, respectively. The results demonstrate
that treatment of Jurkat cells with ara-C was associated with
significant decreases in nuclear c-Abl levels (Fig. 3b, left
panel). Moreover, levels of cytoplasmic c-Abl were increased in
response to ara-C (Fig. 3b, right panel). Translocation of
c-Abl from nucleus in the response to genotoxic stress may initiate
formation of complexes with multiple molecules in cytoplasm. Indeed,
densitometric scanning of autoradiograms and quantitative analysis
demonstrate that the formation of c-Abl complexes with HPK1 in
cytoplasm is significantly less than the translocation of c-Abl from
the nucleus to the cytoplasm. These findings support a model in which
c-Abl is activated in the nucleus in response to genotoxic stress,
translocates to the cytoplasm, and thereby interacts with HPK1.

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Fig. 3.
Kinase activities of c-Abl and HPK1 are
required for their optimal interaction. a, Jurkat cells were
treated with 10 µM ara-C for 6 h. Nuclear and
cytoplasmic fractions were subjected to immunoprecipitation with
anti-HPK1. The precipitates were analyzed by immunoblotting with
anti-c-Abl. Signal intensities from the anti-c-Abl immunoblotting
experiments were analyzed by densitometric scanning. The data represent
the fold increase in signal intensities compared with untreated
controls. The results are expressed as the mean ± S.D. from three
independent experiments. b, Jurkat cells were treated with
10 µM ara-C for the indicated times. Nuclear (left
panels) and cytoplasmic (right panels) fractions were
isolated and analyzed by immunoblotting with anti-c-Abl (top
panels), anti-Lamin A (bottom left panel), or
anti- -actin (bottom right panel) antibodies.
c, 293T cells were transiently cotransfected with c-Abl and
Flag-HPK1 or kinase-inactive mutant Flag-HPK1(M46). After treatment of
cells with ara-C, anti-c-Abl immunoprecipitates were analyzed by
immunoblotting with anti-Flag (top panel). As a control,
anti-c-Abl immunoprecipitates were analyzed by immunoblotting with
anti-c-Abl (middle panel). Total cell lysates were also
analyzed by immunoblotting with anti-Flag (bottom panel).
d, 293T cells were transiently cotransfected with Flag-HPK1
and c-Abl or dominant negative mutant c-Abl(K-R). After treatment of
cells with ara-C, anti-c-Abl immunoprecipitates were analyzed by
immunoblotting with anti-Flag (top panel). As a control,
anti-c-Abl immunoprecipitates were analyzed by immunoblotting with
anti-c-Abl (middle panel). Total cell lysates were also
analyzed by immunoblotting with anti-Flag (bottom
panel).
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To determine whether the kinase function of HPK1 is necessary for the
interaction with c-Abl, we transiently cotransfected Flag-HPK1 or a
kinase-inactive Flag-HPK1(M46) mutant with c-Abl in 293T cells. After
treatment with ara-C, anti-c-Abl immunoprecipitates were analyzed by
immunoblotting with anti-Flag. The results demonstrate that in contrast
to HPK1(M46), overexpression of wild-type HPK1 is associated with an
increase in binding with c-Abl (Fig. 3c). Because c-Abl is
also activated by ara-C and IR (11), we asked whether the association
of HPK1 with c-Abl is also dependent on the c-Abl kinase function. To
address this issue, 293T cells were transiently transfected with
Flag-HPK1 and c-Abl or dominant negative c-Abl(K-R) mutant and then
treated with ara-C. Anti-c-Abl immunoprecipitates were analyzed by
immunoblotting with anti-Flag. The results demonstrate that the
interaction between HPK1 and c-Abl is significantly increased in cells
overexpressing wild-type c-Abl (Fig. 3d). Taken together, these findings suggest that the kinase function of c-Abl and HPK1 may
be necessary for their interaction. However, our data do not rule out
the possibility that the loss of interaction between c-Abl and HPK1
might also be due to improper folding of these mutants. Additional
studies using purified recombinant c-Abl and HPK1 proteins are required
to delineate this issue.
To assess in part the functional significance of the interaction of
c-Abl and HPK1, we incubated purified recombinant c-Abl with
GST-HPK1-KD (HPK1 kinase domain; aa 1-291) or GST-HPK1-CD (C-terminal
domain; aa 292-833) (Fig. 4a)
fusion proteins in the presence of [
-32P]ATP. Analysis
of the reaction products demonstrated little, if any, phosphorylation
of either GST-HPK1-KD or GST-HPK1-CD (Fig. 4b, left panel).
The unavailability of full-length GST-HPK1 protein precluded us from
using it as a substrate. The potential HPK1 binding sequence for
the c-Abl SH3 domain (SGPPPNSPRPGPPPS; aa 430-444) is present in the
HPK1-CD, whereas the c-Abl phosphorylation site (YXXP) (39)
in HPK1 (Y232QPP; aa 232-235) is localized in HPK1-KD. To
determine whether full-length HPK1 acts as a substrate for c-Abl, 293T
cells were transiently transfected with full-length Flag-HPK1. Cell
lysates were subjected to immunoprecipitation with anti-Flag, and the precipitates were incubated with recombinant c-Abl in the presence of
[
-32P]ATP. As control, anti-Flag immunoprecipitates
were incubated separately with recombinant c-Abl(K-R) protein in the
presence of [
-32P]ATP. Analysis of the reaction
products demonstrated c-Abl-mediated phosphorylation of full-length
HPK1 (Fig. 4b, right panel). As a control, anti-Flag
immunoprecipitates were analyzed separately by immunoblotting with
anti-Flag. The results demonstrate equal expression of Flag-HPK1 in
multiple transfections (data not shown). To confirm c-Abl-mediated
tyrosine phosphorylation of HPK1, 293T cells were cotransfected with
Flag-HPK1 with increasing amounts of c-Abl. Lysates were subjected to
immunoprecipitation with anti-Flag and analyzed by immunoblotting with
anti-P-Tyr. Signal intensities from anti-P-Tyr immunoblotting
experiments were analyzed by densitometric scanning. The results from
quantitation of signal intensities demonstrate 3.2 ± 0.4-fold
induction in tyrosine phosphorylation of HPK1 by c-Abl (Fig.
4c).

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Fig. 4.
c-Abl phosphorylates HPK1 in
vitro. a, a schematic diagram displays various
HPK1 constructs, including wild-type HPK1, the kinase domain
(HPK1-KD; amino acids 1-291) and the C-terminal domain
(HPK1-CD; amino acids 292-833). b, left
panel, GST-HPK1-KD (KD) and GST-HPK1-CD (CD)
fusion proteins were incubated separately with purified c-Abl in the
presence of [ -32P]ATP. As a control, GST-Crk(120-225)
fusion protein was incubated separately with purified c-Abl in the
presence of [ -32P]ATP. After kinase reactions, the
products were analyzed by SDS-PAGE and autoradiography (left
panel). The GST-HPK1-KD and GST-HPK1-CD proteins were visualized
by Coommassie Blue staining (data not shown). 293T cells were
transiently transfected with Flag-HPK1 wild-type. Lysates were
subjected to immunoprecipitation with anti-Flag, and the precipitates
were incubated with recombinant c-Abl or recombinant c-Abl(K-R)
proteins in the presence of [ -32P]ATP. As a control,
GST-Crk(120-225) fusion protein was incubated with purified c-Abl in
the presence of [ -32P]ATP. The phosphorylated products
were analyzed by SDS-PAGE and autoradiography (right panel).
c, 293T cells were transfected with Flag-HPK1 (1 µg) and
increasing concentrations (0, 2, 4, and 8 µg) of c-Abl. Cell lysates
were subjected to immunoprecipitation with anti-Flag, and the
precipitates were analyzed by immunoblotting with anti-P-Tyr (top
panel) or anti-Flag (bottom panel).
|
|
Because c-Abl phosphorylates HPK1 in vitro, we asked
whether HPK1 is tyrosine-phosphorylated in the cellular response to
genotoxic stress. Jurkat cells were treated with ara-C or exposed to
IR. Total cell lysates were subjected to immunoprecipitation with anti-HPK1, and the precipitates were analyzed by immunoblotting with
anti-P-Tyr. As a control, anti-HPK1 immunoprecipitates were also
analyzed by immunoblotting with anti-HPK1. The results demonstrate a
2-3-fold induction in tyrosine phosphorylation of HPK1 in response to
genotoxic agents (Fig. 5a;
data not shown). Exposure of cells to genotoxic agents is associated
with activation of c-Abl (4-6). We therefore investigated whether
genotoxic stress affects c-Abl-mediated tyrosine phosphorylation of
HPK1. MCF-7 cells expressing neo cassette (MCF-7/neo) and MCF-7 cells
expressing c-Abl(K-R) (MCF-7/c-Abl(K-R)) were transiently transfected
with Flag-HPK1. After transfection, cells were exposed to IR or treated
with ara-C and harvested after 3 h. Cytoplasmic lysates were
subjected to immunoprecipitation with anti-Flag antibody, and the
protein precipitates were analyzed by immunoblotting with anti-P-Tyr.
As a control, anti-Flag immunoprecipitates were also analyzed by
immunoblotting with anti-Flag. Exposure of MCF-7/neo cells to IR was
associated with increases (3 ± 0.5-fold; Fig. 5b, bottom
panel) in tyrosine phosphorylation of HPK1 (Fig. 5b).
Moreover, IR had no detectable effect on tyrosine phosphorylation of
HPK1 in MCF-7/c-Abl(K-R) cells (Fig. 5b). Similar results
were obtained when MCF-7/neo or MCF-7/c-Abl(K-R) cells were treated with ara-C (Fig. 5b). Because MCF-7/c-Abl(K-R) cells
stably overexpress c-Abl(K-R), and because of the very minimal basal
activity of c-Abl(K-R), the background level of tyrosine
phosphorylation of c-Abl(K-R) is high compared with the endogenous
levels seen in MCF-7/neo. Collectively, these findings demonstrate that
genotoxic stress induces tyrosine phosphorylation of HPK1 by a
c-Abl-dependent mechanism.

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Fig. 5.
c-Abl-mediated tyrosine phosphorylation of
HPK1 in response to genotoxic stress. a, Jurkat cells were
treated with 20 Gy of ionizing radiation and harvested after different
times. Total cell lysates were subjected to immunoprecipitation with
anti-HPK1, and the precipitates were analyzed by immunoblotting with
anti-P-Tyr (top panel) or anti-HPK1 (bottom
panel). b, MCF-7/neo and MCF-7/c-Abl(K-R) cells were
transiently transfected with Flag-HPK1 and treated with 10 µM ara-C for 3 h or exposed to 20 Gy of IR and
harvested after 3 h. Total cell lysates were subjected to
immunoprecipitation with anti-Flag and analyzed by immunoblotting with
anti-P-Tyr (top panel) or anti-Flag (middle
panel). The bottom panel depicts the fold increase in
tyrosine phosphorylation expressed as the mean + S.D. from
three independent experiments.
|
|
c-Abl Activates HPK1 in Vitro and in the Response to Genotoxic
Stress--
To further assess the functional significance of the
interaction between c-Abl and HPK1, we investigated whether c-Abl
affects HPK1 activity. 293T cells were cotransfected with Flag-HPK1 and empty vector or c-Abl. Anti-Flag immunoprecipitates were assayed for
HPK1 kinase activity using MBP as a substrate. Analysis of the reaction
products by autoradiography demonstrated that overexpression of c-Abl
is associated with an increase (~3-fold) in the kinase activity of
HPK1 (Fig. 6a). Because HPK1
is a serine/threonine kinase, we next assessed whether HPK1 activates
c-Abl. To address this issue, 293T cells were transfected with HA-c-Abl
and HPK1 or empty vector, and anti-HA immunoprecipitates were assayed
for phosphorylation of GST-Crk(120-225) (40). The results demonstrate that coexpression of HPK1 and c-Abl had no detectable effect on c-Abl
kinase activity (Fig. 6b).

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Fig. 6.
c-Abl activates HPK1. a, 293T
cells were transfected with Flag-HPK1 (1 µg) in the presence of
different concentrations (0, 1, 2, and 5 µg) of c-Abl or empty
vector. Total cell lysates were subjected to immunoprecipitation with
anti-Flag and assayed for phosphorylation of MBP in the presence of
[ -32P]ATP. The reaction products were separated by
SDS-PAGE and analyzed by autoradiography (top panel). The
fold increase in MBP phosphorylation is described as the mean of three
independent experiments. As a control, total lysates were also analyzed
by immunoblotting with anti-Flag (middle panel) or
anti-c-Abl (bottom panel). b, 293T cells were
transiently cotransfected with HA-c-Abl (1 µg) in the presence of
increasing concentrations (0, 1, 2, and 5 µg) of HPK1 or empty
vector. After transfection, anti-HA immunoprecipitates were assayed for
phosphorylation of GST-Crk(120-225) in the presence of
[ -32P]ATP. The reaction products were separated by
SDS-PAGE and analyzed by autoradiography (top panel). As a
control, total lysates were also analyzed by immunoblotting with
anti-Flag (middle panel) or anti-HA (bottom
panel).
|
|
To determine whether HPK1 is activated in the response to genotoxic
stress, lysates from Jurkat cells treated with ara-C were subjected to
immunoprecipitation with anti-HPK1. The immunoprecipitates were assayed
for phosphorylation of MBP. The results demonstrate an increase (5 ± 1.1-fold) in phosphorylation of MBP by HPK1 in ara-C-treated cells
as compared with control cells (Fig.
7a). Similar results were
obtained when Jurkat cells were exposed to IR (Fig. 7b). To
define the subcellular localization of HPK1 activation, we assayed
nuclear and cytoplasmic lysates from control and ara-C-treated cells
for HPK1 activity. The results demonstrate increased activation of HPK1
in cytoplasmic but not nuclear lysates of the ara-C-treated cells (Fig.
7c). Similar results were obtained when Jurkat cells were
exposed to IR (data not shown). We next determined the role of c-Abl in
the regulation of HPK1 activity in response to genotoxic agents.
Studies have shown that HPK1 is expressed in Jurkat cells and other
cell types that are predominantly hematopoietic. Due to the extremely
low transfection efficiency of Jurkat cells, we transiently transfected
293T cells with Flag-HPK1 and empty vector, c-Abl or c-Abl(K-R). After
transfections, cells were treated with 10 µM ara-C and
harvested after 3 h. Anti-Flag immunoprecipitates were analyzed
for phosphorylation of MBP. As controls, anti-Flag immunoprecipitates
and total cell lysates were analyzed separately by immunoblotting with
anti-Flag and anti-c-Abl, respectively. The results demonstrate
increased phosphorylation of MBP in cells overexpressing wild-type
c-Abl but not Abl(K-R) (Fig. 7d). Moreover, to demonstrate
whether the kinase function of HPK1 is necessary for HPK1 activation in
response to genotoxic stress, we transiently overexpressed empty
vector, Flag-HPK1, or Flag-HPK1(M46) and treated with ara-C.
Anti-Flag immunoprecipitates were analyzed for phosphorylation of MBP.
The results demonstrate increased phosphorylation of MBP in cells
overexpressing wild-type HPK1 but not HPK1(M46) (Fig. 7e).
To further determine activation of HPK1 in response to IR, we
transiently overexpressed Flag-HPK1 in 293T cells and then exposed
cells to IR. Anti-Flag immunoprecipitates were analyzed for
phosphorylation of MBP. The results demonstrate that
exposure of cells to IR is associated with an increase in activation of HPK1 (Fig. 7f).

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Fig. 7.
Genotoxic agents induced activation of HPK1.
a, Jurkat cells were treated with 10 µM ara-C
and harvested at 3 h. Total cell lysates were subjected to
immunoprecipitation with anti-HPK1. The immunoprecipitates were
analyzed in immune complex kinase assays using MBP as a substrate
(top panel). As a control, total cell lysates were analyzed
by immunoblotting with anti-HPK1 (bottom panel). The fold
increase in MBP phosphorylation is described as the mean of two
independent experiments. b, Jurkat cells were exposed to 20 Gy of IR and harvested after 3 h. Total cell lysates were
subjected to immunoprecipitation with anti-HPK1 and assayed as
described above. c, Jurkat cells were treated with 10 µM ara-C and harvested after 3 h. Nuclear
(left panels) and cytoplasmic (right panels)
lysates were subjected to immunoprecipitation with anti-HPK1. The
immunoprecipitates were analyzed in immune complex kinase assay using
MBP as a substrate (top panels). As a control, total cell
lysates were analyzed by immunoblotting with anti-HPK1 (bottom
panels). d, 293T cells were transiently transfected
with empty vector, c-Abl, or c-Abl(K-R) in the presence of Flag-HPK1.
After treatment of cells with ara-C for 3 h, anti-Flag
immunoprecipitates were assayed for phosphorylation of MBP (top
panel). As a control, anti-Flag immunoprecipitates were analyzed
by immunoblotting with anti-Flag (middle panel). Total cell
lysates were also analyzed by immunoblotting with anti-c-Abl
(bottom panel). e, 293T cells were transiently
transfected with empty vector, Flag-HPK1, or Flag-HPK1(M46). After
treatment of cells with ara-C, anti-Flag immunoprecipitates were
assayed for phosphorylation of MBP (top panel). As a
control, total cell lysates were analyzed by immunoblotting with
anti-Flag (bottom panel). f, 293T cells were
transiently transfected with Flag-HPK1. After exposure of cells to IR
for different time intervals, anti-Flag immunoprecipitates were assayed
for phosphorylation of MBP (top panel). As a control, total
cell lysates were analyzed separately by immunoblotting with anti-Flag
(bottom panel).
|
|
c-Abl and HPK1 Synergistically Activate SAPK/JNK--
Treatment of
cells with diverse genotoxic agents activates SAPK (5, 7-15). We next
asked whether SAPK is activated in Jurkat cells in the response to
genotoxic stress. To assess SAPK activation, Jurkat cells were either
treated with 10 µM ara-C or exposed to 20 Gy of IR and
harvested after different times. Total cell lysates were subjected to
immunoprecipitation with anti-SAPK and assayed for phosphorylation of
GST-c-Jun. In concert with previous findings, treatment of Jurkat cells
with ara-C or IR was also associated with activation of SAPK (Fig.
8, a and b).
Studies have shown that both HPK1 and c-Abl are upstream activators of
SAPK (5, 6, 23, 24, 33, 45-47). To assess whether c-Abl and HPK1 cooperate in the activation of SAPK, 293T cells were transiently cotransfected with pEBG-SAPK and SEK1 in the presence and absence of
HPK1 and/or c-Abl. Lysates were incubated with glutathione beads, and
the precipitates were assayed for GST-c-Jun phosphorylation. Transient
expression of c-Abl induced a 4-5-fold increase in SAPK activity as
compared with empty vector. Overexpression of HPK1 with SEK1 and SAPK
was associated with 13-15-fold activation of SAPK. Moreover, HPK1 and
c-Abl together induced SAPK activity 18-20-fold over basal level (Fig.
8c). These findings suggest that c-Abl interacts with HPK1
to activate SAPK. To determine whether a dominant negative mutant of
c-Abl (c-Abl(K-R)) affects HPK1-induced SAPK activation, 293T cells
were transiently transfected with HPK1 and c-Abl(K-R). Cells were also
cotransfected with SEK1 and pEBG-SAPK. Lysates were subjected to
protein precipitation with glutathione beads, and the precipitates were
assayed for phosphorylation of GST-c-Jun. The results demonstrate that
c-Abl(K-R) significantly inhibits HPK1-induced SAPK activation (Fig.
8c). Conversely, overexpression of HPK1(M46) also inhibited
c-Abl-induced activation of SAPK (Fig. 8c). Taken together,
these findings indicated that c-Abl and HPK1 synergize for activation
of SAPK.

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Fig. 8.
c-Abl and HPK1 cooperate to synergistically
activate JNK. a and b, Jurkat cells were either
treated with 10 µM ara-C (a) or exposed to 20 Gy of IR (b) and harvested after the indicated times. Total
cell lysates were subjected to immunoprecipitation with anti-SAPK. The
immunoprecipitates were analyzed in immune complex kinase assay using
GST-c-Jun as a substrate (top panels). As control, total
cell lysates were analyzed by immunoblotting with anti-SAPK
(bottom panels). c, 293T cells were transiently
transfected in duplicate with the indicated cDNAs. Total cell
lysates were subjected to protein precipitation with glutathione beads.
The adsorbates were assayed in immune complex kinase assay using
GST-c-Jun as a substrate (top panel). As a control, total
lysates were analyzed separately by immunoblotting with anti-GST
(middle panel). The bottom panel depicts the fold
increase in GST-c-Jun phosphorylation expressed as the mean ± S.D
of two independent experiments performed in duplicate.
|
|
Studies have shown that MLK-3 acts as a substrate for HPK1 and that
HPK1-induced SAPK activation is inhibited by overexpression of a
dominant negative mutant of MLK-3 (24). Our recent studies have shown
that c-Abl phosphorylates and activates MEKK-1 in nuclei (48).
Moreover, c-Abl-induced activation of SAPK is inhibited by
overexpression of a dominant negative mutant of MEKK-1 (48). MEKK-1
stimulates SEK1/MKK4, which in turn activates SAPK (8, 18, 49). The
finding that MEKK-1(K-R) fails to completely block c-Abl-induced SAPK
activation further indicates that c-Abl also stimulates the SAPK/JNK
pathway by MEKK-1-independent mechanisms (48). To determine whether
c-Abl and HPK1 function upstream to MLK-3, 293T cells were transiently
cotransfected with c-Abl or HPK1 and MLK-3(K-R). Cells were also
cotransfected with SEK1 and pEBG-SAPK. Total cell lysates were
subjected to protein precipitation with glutathione beads and assayed
for GST-c-Jun phosphorylation. The results demonstrate that
transfection of MLK-3(K-R) also inhibits c-Abl-induced activation of
SAPK (data not shown). The results further demonstrate that
overexpression of MLK-3(K-R) blocks HPK-1-induced activation of SAPK
(data not shown). Taken together, these findings indicate that the
c-Abl/HPK1 complex functions upstream to MLK-3 and induces SAPK
activation in the response to genotoxic stress, at least in Jurkat cells.
 |
DISCUSSION |
Eukaryotic cells respond to DNA damage with cell cycle arrest,
activation of DNA repair, and, in the event of irreparable damage, the
induction of apoptosis. The signals that determine cell fate, that is,
repair of DNA damage and survival or activation of cell death
mechanisms, remain unclear. The c-Abl tyrosine kinase is activated in
the cellular response to certain DNA-damaging agents (5, 6, 14, 47, 50,
51). Previous studies have also demonstrated that c-Abl functions
upstream to activation of the SAPK/JNK pathway in the response of cells
to genotoxic stress (5, 6, 14, 47, 51). The exposure of diverse types
of mammalian cells to genotoxic agents is associated with SAPK
activation (5-7, 11, 48, 52-54). Other studies have demonstrated that
activation of SAPK in the DNA damage response is associated with the
induction of apoptosis (7, 17, 52, 53). Whereas c-Abl has also been
linked to DNA damage-induced apoptosis, the precise role for c-Abl as
an upstream effector of the SAPK pathway has been controversial. In
this context, other work has indicated that c-Abl is not required for
the activation of SAPK by genotoxic agents (51). The discrepancy
between findings may be related to the demonstration that c-Abl is
necessary for activation of SAPK in proliferating cells but not
growth-arrested cells (1). As further support for c-Abl involvement,
recent work has shown that c-Abl directly activates MEKK-1, an upstream
effector in the SEK1
SAPK cascade, in the DNA damage response (48).
The present findings extend the role of c-Abl in the activation of SAPK
signaling by demonstrating that c-Abl interacts with HPK1 in
transducing signals to SEK1 and SAPK.
HPK1 is a mammalian Ste20/PAK-like serine/threonine kinase that is
primarily found in hematopoietic cells (23, 24). Whereas little is
known about the signals responsible for activation of HPK1, studies
have shown that HPK1 activity is induced in cells treated with
transforming growth factor
(55). Notably, HPK1 functions as an
upstream effector of transforming growth factor
-induced activation
of SAPK (55). Other studies have demonstrated that HPK1 is
phosphorylated by the epidermal growth factor receptor (56). Epidermal
growth factor stimulation induces the binding of HPK1 with the Grb2
adaptor protein and recruitment of these complexes to the
autophosphorylated epidermal growth factor receptor (56). HPK1 also
associates with the Crk and CrkL adaptor proteins in signaling that
results in activation of the SAPK pathway (33). The present results
demonstrate that HPK1 is activated in the response of cells to
DNA-damaging agents. In this context, IR exposure is associated with
the accumulation of single- and double-strand DNA breaks (57). By
contrast, ara-C is a nucleoside analog that incorporates into DNA and
causes arrest of DNA replication by functioning as a relative chain
terminator (41, 58-60). The finding that both IR and ara-C activate
HPK1 indicates that this response is induced by diverse types of DNA
damage. In addition, the findings that IR and ara-C induce the
activation of SAPK (5-7, 11, 48, 52-54) suggest that HPK1 could
contribute to SAPK-mediated signals induced by these genotoxic agents.
Indeed, expression of a kinase-inactive HPK1 mutant partially abrogated
IR- and ara-C-induced SAPK activation.
The present findings provide further support for an interaction between
c-Abl and HPK1 in the DNA damage response. The results demonstrate that
genotoxic stress induces the association of c-Abl and HPK1. In
vitro studies indicate that the c-Abl SH3 domain interacts
directly with a proline-rich motif in the HPK1 C-terminal region.
Moreover, studies in cells cotransfected to express c-Abl and HPK1
demonstrate that c-Abl phosphorylates HPK1. The finding that HPK1 is
phosphorylated on tyrosine in IR- or ara-C-treated cells expressing
wild-type c-Abl, but not in cells expressing c-Abl(K-R), further
indicates that HPK1 is phosphorylated by a c-Abl-dependent
mechanism in the DNA damage response. The functional significance of
the c-Abl-HPK1 interaction is supported by the finding that c-Abl
activates HPK1. Conversely, the results indicate that HPK1 has no
apparent effect on c-Abl activity. These findings support a model in
which HPK1 is a downstream effector of the c-Abl response to genotoxic
stress. The recent demonstration that c-Abl is also activated in cells
exposed to hydrogen peroxide has supported a role for c-Abl in the
response to diverse types of stress (61). The available evidence,
however, indicates that the interaction between c-Abl and HPK1 is
induced by genotoxic stress and not by oxidative stress (data not shown).
Previous work has shown that nuclear c-Abl functions as an upstream
effector of the MEKK1
SEK1
SAPK pathway in the response of cells to
DNA damage in nonhematopoietic cells (48). However, the incomplete
abrogation of DNA damage-induced SAPK activation by the kinase-inactive
MEKK1(K-R) mutant indicates that c-Abl can also stimulate the SAPK
pathway by a MEKK1-independent mechanism (48). HPK1 is predominantly a
cytoplasmic kinase, and DNA damage-induced formation of c-Abl/HPK1
complexes is detectable in the cytoplasm and not in the nucleus. In
concert with these findings, the results demonstrate that in response
to DNA damage, nuclear c-Abl translocates to the cytoplasm. Therefore,
whereas nuclear c-Abl interacts with MEKK-1 (48), after translocation
to the cytoplasm, c-Abl associates with HPK1. Coexpression of c-Abl and
HPK1 was associated with a synergistic effect on SAPK
activation, and that this activation of SAPK is sensitive to
MLK-3. Moreover, expression of either c-Abl(K-R) or HPK1(M46) blocked
DNA damage-induced SAPK activation. Thus, the kinase functions of both
cytoplasmic c-Abl and HPK1 are required in the induction of SAPK
activity, at least in hematopoietic cells. These findings support a
model in which c-Abl mediates activation of HPK1 and confers an
additional signal that is also necessary for SAPK activation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Leonard Zon, John Kyriakis,
Joseph Avruch, Charles Sawyer, Stephen Feller, Ruibao Ren, Jean Y. J.
Wang, Dennis Templeton, Melanie Cobb, Hawa Avraham, Bruce Meyer, and
Jim Woodgett for providing necessary reagents. We also thank Kamal
Chauhan for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported by United States Public Health
Service Grants CA75216 (to S. K.) and CA 55241 and CA 29431 (to
D. K.) awarded by the National Cancer Institute, Department of Health and Human Services and by Grants AI 8738649 and AI 42532 (to T.-H. T.)
awarded by the National Institute of Allergy and Infectious Diseases,
Department of Health and Human Services.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: ILEX Oncology Inc., 20 Overland St., Boston, MA, 02215. Tel.: 617-717-1605: Fax: 617-262-7184;
E-mail; skharbanda{at}ilexonc.com.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M007294200
 |
ABBREVIATIONS |
The abbreviations used are:
IR, ionizing
radiation;
SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal
kinase;
HPK, hematopoietic progenitor kinase;
MAPKK, mitogen-activated
protein kinase kinase;
MEKK, MAPKK/extracellular signal-regulated
kinase kinase kinase;
MLK, mixed lineage kinase;
ara-C, 1-
-D-arabinofuranosylcytosine;
PAGE, polyacrylamide gel
electrophoresis;
GST, glutathione S-transferase;
PR, proline-rich sequence;
MBP, myelin basic protein;
aa, amino acid(s);
SEK1, SAP kinase/extracellular signal-regulated kinase kinase1;
HA, hemagglutinin.
 |
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