c-Abl is required for staurosporine-induced caspase activity.

Abstract

Caspases are the intracellular molecular machinery responsible for apoptotic cell death. The regulation of these critical proteolytic enzymes is known to occur on multiple levels. While their expression as inactive precursors exhibits a primary level of control, other types of regulation such as post-translational modifications also play a role. Nuclear c-Abl, a nonreceptor tyrosine kinase, plays a role in the regulation of apoptosis in response to DNA damage. The function of cytoplasmic c-Abl in cell death is not fully understood. Here, we report c-Abl dependent caspase-3 and caspase-8 activity in response to staurosporine. Despite the presence and apparent activation of the mitochondrial-dependent apoptotic pathway and cellular demise, we find no caspase-3 activity in cells lacking the Abl gene (Abl(-/-)). These findings demonstrate a novel tyrosine kinase dependent regulation of caspase-mediated cell death.

Full text

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Proc. West. Pharmacol. Soc. 48: 110-117 (2005)

c-Abl Is Required For Staurosporine-induced Caspase Activity
Brian C. Oxhorn1, Amy R. Sanguinetti2, Cynthia Corley Mastick2, and Iain L. O. Buxton*,1
110
1Departments of Pharmacology MS 318 and Biochemistry2 MS 330, University of Nevada School of Medicine,
Reno, Nevada 89557
*Email: buxton@med.unr.edu

ABSTRACT
Caspases are the intracellular molecular machinery
responsible for apoptotic cell death. The regulation of
these critical proteolytic enzymes is known to occur
on multiple levels. While their expression as inactive
precursors exhibits a primary level of control, other
types of regulation such as post-translational
modifications also play a role. Nuclear c-Abl, a non-
receptor tyrosine kinase, plays a role in the regulation
of apoptosis in response to DNA damage. The
function of cytoplasmic c-Abl in cell death is not fully
understood. Here, we report c-Abl dependent
caspase-3 and caspase-8 activity in response to
staurosporine. Despite the presence and apparent
activation of the mitochondrial-dependent apoptotic
pathway and cellular demise, we find no caspase-3
activity in cells lacking the Abl gene (Abl-/-). These
findings demonstrate a novel tyrosine kinase
dependent regulation of caspase-mediated cell death.
INTRODUCTION
Programmed cell death (PCD) is a regulated process
whereby specialized intracellular pathways are
activated with the effect of producing the orderly
destruction of the cell. Frequently termed apoptosis,
this process ensures the efficient degradation and
removal of superfluous, damaged, or infected cells
thereby avoiding injury to surrounding cells [1]. This
differs from necrotic cell death in that inflammatory
responses are notably absent since apoptotic cells
are engulfed by phagocytes prior to the release of
toxic intracellular material. While apoptosis is a
natural process crucial
development of diverse organisms, many human
pathological conditions
dysregulated apoptosis. Neurological syndromes
such as Alzheimer’s and Parkinson’s disease are
associated with an unscheduled initiation of death
pathways, while many neoplastic disorders can be
linked to the inactivation of death-activating proteins
or the initiation of death-suppressor proteins [2]. In
either scenario, an understanding of the biochemical
events that occur at various points along apoptotic
pathways is crucial to the development of therapies
aimed at enhancing or preventing PCD.
for the growth and
may be attributed to


In a simplified model of apoptosis, two distinct
signaling stages, initiation and execution, can be said
to occur. Initiation can be accomplished by ligands
acting on receptors at the plasma membrane [3] or
from release of apoptogenic substances from
intracellular organelles such as mitochondria [4] or
nuclei. Regardless of their origin, apoptotic signals
ultimately activate aspartic acid-directed cysteinyl
proteases termed caspases [5], which mark the
execution phase. There are currently 14 known
members of the caspase family, which are the
enzymes responsible for the proteolytic activity
characteristic of PCD [6]. Caspase activity provides
the molecular basis for apoptotic morphology, which
includes chromatin condensation,
fragmentation, and membrane
Caspases can be classified as initiators containing
long NH2-terminal pro-domains or as effectors with
short pro-domains. The effector caspases mediate
cell disassembly while the initiator caspases transmit
the “death” signal by proteolytically activating effector
caspases.
nucleosomal
blebbing [7].
Our understanding of the precise mechanisms by
which these enzymes are regulated remains
incomplete, however numerous processes have been
described. Representing the principal and perhaps
most important level of their regulation, caspases are
synthesized as inactive precursors (zymogens). In
response to proapoptotic signals, caspases become
activated either autocatalytically or by proteolysis via
upstream caspases in a cascade-like fashion.
Activation involves cleavage of the enzymes into
large and small subunits followed by re-association of
the subunits to form heterodimers and tetramers [8;9].
A second, highly significant, means of regulation
involves direct inhibition of certain caspases by a
family of proteins termed inhibitors of apoptosis
(IAP’s) [10]. Other modes of regulation have also
been reported. Post-translational modifications have
been shown to alter the cleavage or proteolytic
activity of caspases. Cardone, et al. (1998) describe
an protein kinase B/Akt-mediated regulation of
caspase-9 by direct phosphorylation thereby inhibiting
its proteolytic activation [11]. In addition, caspase-3
can be regulated by S-nitrosylation of the catalytic
cysteine residues [12].

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The tyrosine kinase c-Abl is a ubiquitously expressed
non-receptor tyrosine kinase that shuttles between
the nucleus and the cytoplasm. Activation of nuclear
c-Abl has been shown to occur in response to DNA
damaging agents including ionizing radiation and
cisplatin [13;14]. Activation of c-Abl in the nucleus
leads to activation of the p53 homologue, p73 and
subsequent induction of apoptosis [15-17]. In
addition, c-Abl has a proapoptotic role in response to
NF-κB regulated signaling pathways for cell survival.
Nuclear c-Abl phosphorylates IκBα on tyrosine 305
thus increasing IκBα stability which blocks NF-κB
transcriptional activity [18]. In addition to its function
in the nucleus, the cytoplasmic pool of c-Abl has also
been shown to induce apoptosis in response to
cellular stress. Oxidative stress activates c-Abl in the
cytoplasm and this activation is required for apoptosis
[19]. The mechanism by which cells undergo c-Abl
dependent apoptosis in response to cellular stress
remains unknown.
In the present study, we address the question of the
relationship between c-Abl and caspase-dependent
apoptotic pathways. As a model system, we utilized
mouse embryonic fibroblasts (MEF) established from
c-Abl knockout mice (Abl-/-) and a companion set of
Abl-/- cells reconstituted with c-Abl by stable
transfection with the gene encoding c-Abl (Abl+) [20].
The expression level of c-Abl in the reconstituted cell
line has been shown to be equivalent to endogenous
c-Abl expression. Cell death is initiated by exposing
MEF cultures to staurosporine, a frequently used
agent for examining
Staurosporine, a microbial alkaloid, is a broad-
spectrum inhibitor of kinases that induces apoptosis
in nearly every mammalian model examined [21].
Here we demonstrate a c-Abl dependent activation of
both caspase-3 and caspase-8 in response to
staurosporine. In addition, c-Abl appears to act on
both enzymes following zymogen cleavage.
apoptotic pathways.
Methods
Cell Line: Abl-/- and Abl+ mouse fibroblasts were kindly
provided by Dr. Jean Y. Wang (UCSD, CA) [22;23]. All
cells are cultured on plastic in complete Dulbecco’s
modified essential media (CDMEM) with 10% fetal bovine
serum (FBS), 10x (763 U/ml:100 U/ml) penicillin-
streptomycin (P/S), and 0.5µg/ml amphotericin B.
Colorimetric Caspase Activity: Apoptosis is induced by the
addition of staurosporine 50nM in CDMEM for 4 (caspase-
8 and 9) or 5 (caspase-3) hours. Where indicated, cells
are pretreated with either β-methyl-cyclodextrin 5mM or
filipin III 1µg/ml in RAK for 20 minutes @ 37°C prior to the
addition of staurosporine. Soluble cellular lysates from
staurosporine treated cells are incubated with the substrate
IETD-p-nitroaniline, LEHD-p-nitroaniline,
nitroaniline in the presence or absence of the reversible
aldehyde inhibitor DEVD-CHO or irreversible ketone
inhibitors IETD-FMK or LEDH-FMK in 96-well plates at
37°C. A405 readings are obtained at various times. Rates
of enzymatic cleavage of p-nitroaniline are calculated from
the slopes of the best-fit lines using linear regression
analysis and normalized for total protein content as
determined by the BCA method. Recombinant enzymes
are employed as positive controls.
or DEVD-p-
Cytochrome C Release: Near-confluent monolayers are
exposed to staurosporine as for caspase activity assays.
All adherent and detached cells are enzymatically removed
using trypsin/EDTA, pelleted by centrifugation 600xg for 10
minutes at 4°C. Cell pellets are washed in ice-cold
phosphate buffered saline (PBS) containing (in mM): NaCl
(118), KCl (4.7), KH2PO4 (0.6), NaH2PO4 (0.6) and
centrifuged at 600xg for 6 minutes at 4°C. The cells are
permeabilized for 5 minutes on ice in a lysis buffer pH 7.4
containing (in mM): Sucrose (250), NaCl (137mM), KCl
(70mM), Na2HPO4 (4.2), KH2PO4 (1.4), leupeptin (1),
AEBSF (1), and digitonin 700µg/ml. The cytosolic
compartment is retained
centrifugation at 7200xg for 5 minutes at 4°C. Standard
electrophoresis sample buffer (4x) is added to a final
concentration of 1x. The resultant mitochondria-containing
pellet is suspended in 1x sample buffer. The proteins are
denatured by boiling and separated by 15% SDS-Page.
Western analysis is completed as described below.
in the supernatant by
Western Blot Analysis: Near-confluent monolayers are
exposed to staurosporine (where indicated) as for caspase
activity assays. Cells are scraped into a buffer (SOL)
containing (in mM): Tris pH 7.2 (10), NaCl (150), Octyl
glucoside (60), NaF (5), EDTA (10), EGTA (10), Leupeptin
(1), AEBSF (1), and 1% Triton X-100. Soluble material is
extracted by centrifugation at 7200xg for 4 minutes at 4°C.
Equal amounts of protein as determined by the BCA
method are diluted in standard sample buffer, denatured by
boiling, & separated by 12% SDS-Page (caspases) or 8%
SDS-Page (c-Abl). The proteins are transferred to PVDF
membranes, blocked overnight in 0.5% gelatin in 0.05%
Tween-20 in tris-base saline (TNT), and incubated with
primary antibodies (anti-caspase-3, anti-caspase-8, or anti-
c-Abl). Detection is achieved by subsequent incubation
with alkaline phosphatase (AP)-conjugated secondary
antibodies and exposure using 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium (BCIP/NBT).
Materials: Dulbecco’s modified Eagle’s medium (DMEM)
and fetal bovine serum are purchased from Atlanta
Biological (Atlanta, GA). Trypsin (1:250) is purchased from
GibcoBRL (Grand Island, NY). Anti-caspase-3 (L-18
clone) is purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Anti-caspase-8 (IgY), colorimetric caspase
substrates, recombinant caspase enzymes and caspase

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24 487296 120 144168
0
20
40
60
80
100
Abl -/-
Abl +
Time [Hour]
Viable Cells
[%]
inhibitors are purchased from Calbiochem (San Diego,
CA). Anti-cytochrome C (Ab-2 clone) and anti-c-Abl
antibodies are purchased from Oncogene (Boston, MA).
Alkaline phosphatase-conjugated secondary antibodies are
purchased from Promega (Madison, WI). All other
materials and chemicals are purchased from Sigma (St.
Louis, MO).
Figure 1. Staurosporine induces cell death independent of c-Abl
expression. Equal numbers of cells were allowed to establish in
culture overnight. Cells were
staurosporine 50 nM for 5 hrs in CDMEM. The cells were washed
once in warm PBS to remove residual staurosporine and returned
to culture in fresh CDMEM. Cells were enzymatically removed at
the time points indicated and tallied by a Coulter® Z1 particle
counter. Data is plotted as the percentage of cells remaining in
culture relative to starting numbers. “Viable cells” are those cells
remaining adherent to the culture dish. Data are mean ± SEM, n
= 3. A non-linear regression analysis employing the equation
describing single exponential decay is performed to generate
curves and to compare the rates of cell death.
transiently exposed to
RESULTS
Staurosporine induces cell death in cultured
MEF’s
To examine the role of c-Abl in mediating apoptotic
biochemical pathways, we utilized fibroblasts derived
from c-Abl knockout mice (Abl-/-). These same Abl-/-
cells stably transfected with wild type c-Abl using a
retroviral vector served as control cells (Abl+). The
Abl+ cells were expanded from a population of cells,
and not from a single transfected cell, avoiding
possible clonal variations [24]. Equal numbers of Abl-
/- and Abl+ cells are inoculated into growth conditions
(Dulbecco’s Modified Eagle’s Medium with 10% fetal
bovine serum: CDMEM) and allowed to plate
overnight on plastic in a 37°C humidified incubator
95% O2, 5% CO2. Cells are transiently exposed to
staurosporine (50 nM x 5 hrs) followed by the addition
of fresh CDMEM and then returned to the incubator.
The remaining viable cells are counted every 24
hours for 5 days. Both cell types undergo cell
shrinking, blebbing, and detachment from the culture
dishes as determined by bright-field microscopy,
consistent with classically described changes in cell
morphology associated
Staurosporine induces a rapid loss in cell number in
cultures independent of c-Abl expression (Figure 1).
The insult is likely terminal since neither cell type is
able to proliferate in culture following drug exposure
and data can be best-fit with an equation describing
exponential decay in the population. In addition, the
rate of cell death (Figure 1) is significantly more rapid
in the Abl+ cells (decay rate: Abl+, K = 0.032 ± 0.0062;
Abl-/-, K = 0.010 ± 0.0027; P = 0.0023).
with apoptosis.
Figure 2. Expression of c-Abl is required for staurosporine-
induced caspase-3 activity. Staurosporine (50 nM x 5 hrs) treated
cells are lysed in a CHAPS-containing buffer. Clarified
supernatants are incubated with DEVD-p-nitroaniline (200 µM) in
the presence and absence of DEVD-CHO (0.1 µM). Specific
activity is determined as described in METHODS. Values are
normalized for total protein content determined by the BCA
method. DMSO = white bars. Staurosporine = gray bars. Data
are mean ± SEM, n = 3.
c-Abl is required for staurosporine-induced
caspase-3 activity
Cellular disassembly in the setting of apoptosis is
mediated by effector caspases such as caspase-3.
We sought to compare the activity of staurosporine-
induced caspase-3 in our model. Abl-/- and Abl+ cells
are grown on plastic to near-confluence and exposed
to 50 nM staurosporine for 5 hrs. Compared to
control conditions, cells expressing c-Abl show
significant caspase-3 activity in response to
Abl-/-
Abl+
0.0
0.1
0.2
0.3
0.4
0.5
DMSO
STS
n=3
DEVDase Activity
[pmol/min/µg protein]

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staurosporine (Figure 2). In stark contrast, the Abl-/-
cells do not respond to the apoptotic stimulus with
increased caspase-3 activity. The lack of activity in
the knockout cells could easily be attributed to the
lack of caspase-3 protein expression. However,
Western blot analysis indicates both cell lines express
pro-caspase-3 in relatively equal levels (Figure 3). In
addition, upon treatment with staurosporine, similar
levels of the 17kDa active fragment are also present
and independent of c-Abl expression (Figure 3). To
confirm the state of c-Abl protein expression, lysates
from quiescent Abl-/- and Abl+ cells were examined by
Western blot analysis. Our results verified the
presence of c-Abl only in the reconstituted cell line
and that the expression levels are equivalent to those
seen in control fibroblasts (data not shown). These
data suggest c-Abl acts on the caspase cascade by
altering the activity of caspase-3 after the inactive
zymogen has been cleaved.

Figure 3. Caspase-3 expression and staurosporine-induced
caspase-3 cleavage is independent of c-Abl expression. Abl-/-
and Abl+ cells are treated and prepared as described in
METHODS. DMSO 0.055% or staurosporine (STS) 50nM is
added to cultures for 5 hours. Under control conditions, pro-
caspase-3 (32 kDa) is present in both cell lines. Active caspase-3
(17 kDa fragment) is apparent in both cell lines following
staurosporine treatment. CON = positive control Jurkat T cell
lysate. Data are representative of Western blots repeated in
triplicate.
c-Abl does not effect cytochrome C release or
caspase-9 activity
We find cleaved caspase-3 in both Abl-/- and Abl+
cells. In the setting of programmed cell death, this
biochemical step is usually accomplished by initiator
caspases such as caspase 9 or caspase 8. In most
cell models, staurosporine activates the effector
caspases via the mitochondrial release of cytochrome
C with subsequent cleavage and activation of
caspase-9. We questioned whether this pathway
remained intact in our cells. An increase in cytosolic
cytochrome C translocation is evident in both Abl-/-
and Abl+ cells in response to a 2-hr staurosporine
treatment (Figure 4A). Densitometric analysis
indicates a significant 1.509 +/- 0.131 fold increase in
cytosolic cytochrome C content in the knockout cell
line (P < 0.05 versus control) while the Abl+ cells have
a 1.431 +/- 0.098 fold increase (Figure 4B). As
expected, both Abl-/- and Abl+ cells show caspase-9
activity following a 4-hr exposure to staurosporine
(Figure 5).
Figure 4. Cytosolic translocation of cytochrome C is c-Abl
independent. A) Cytosolic and mitochondrial fractions obtained
from cells treated with staurosporine (50 nM for 2 hrs) or DMSO
(0.055% for 2 hrs) are separated by electrophoresis on a 15%
SDS gels. Cytochrome C (15 kDa) is detected in both
mitochondrial and cytosolic fractions. Data are representative of
Western blots repeated in triplicate. B) Densitometric analysis
indicates significant 1.509 +/- 0.131 and 1.431 +/- 0.098 fold
increases in the ratio of cytosolic/mitochondrial cytochrome C
content in Abl-/- and Abl+ cells in response to staurosporine
compared to control (Mean ± SEM, n=3).
c-Abl is required for staurosporine-induced
caspase-8 activity
While not the principal pathway mediating xyz, the
activation of caspase-8 by staurosporine has been
demonstrated and suggested to be a post-
Pro
Caspase-3
CON
DMSO
Abl-/-
Abl+
Active
Caspase-3
ST
Abl-/- Abl+ CON
17kDa
32kDa
Mito.
Cytoch. C
Cytosol.
Cytochr. C
17kD
17kD
DMSO
DMSO
STS STS
Abl-/- Abl+
A
ControlAbl-/-
Abl+
0.0
0.5
1.0
1.5
2.0
1.509+/-0.131
1.431+/-0.098
DMSO
STS
n=3
Fold Increase
Cytosolic Cytochrome c
B

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Figure 5. Expression of c-Abl is not required for staurosporine-
induced caspase-9 activity. Staurosporine (50 nM x 4 hrs) treated
cells (gray bars) or controls (DMSO = white bars) are lysed in a
CHAPS-containing buffer. Clarified supernatants are incubated
with LEHD-p-nitroaniline (200µM) in the presence and absence of
LEHD-FMK (1 µM). Specific activity is determined as described
in METHODS. Values normalized for total protein content
determined by the BCA method are mean ± SEM, n = 3.

Figure 6. Expression of c-Abl is required for staurosporine-
induced caspase-8 activity. Staurosporine (50 nM x 4 hrs) treated
cells are lysed in a CHAPS-containing buffer. Where indicated,
cells were pretreated with β-methyl-cyclodextrin (5 mM) or filipin
III (1 µg/ml) for 20 min prior to staurosporine exposure. Clarified
supernatants are incubated with IETD-p-nitroaniline (200 µM) in
the presence and absence of IETD-FMK (1 µM). Specific activity
is determined as described in METHODS. Values are normalized
for total protein content determined by the BCA method. DMSO =
white bars. Staurosporine = light gray bars. Staurosporine/β-
methyl-cyclodextrin = dark gray bars. Staurosporine/filipin III =
black bars. Data are mean ± SEM, n = 3.
mitochondrial event. Stepczynska, et al, (2001)
demonstrated a staurosporine-induced activation of
caspase-8 and caspase-3 even in the presence of a
catalytically dead caspase-9 mutant [25]. We chose
to examine this alternate pathway using the Abl-/- and
Abl+ model.
Figure 7. Caspase-8 expression and staurosporine-induced
caspase-8 cleavage is independent of c-Abl expression. Abl-/-
and Abl+ cells are treated and prepared as described in
METHODS. Cells are exposed to DMSO 0.055% or
staurosporine (50 nM) for 4 hrs. Under control conditions
(DMSO), pro-caspase-8 (57 kDa) is present in both cell lines.
Staurosporine (STS) treatment is associated with the appearance
of an active caspase-8 fragment (18kDa fragment) and the
diminished presence of 57 kDa pro-caspase-8. CON = positive
control Jurkat T cell lysate. Data are representative of Western
blots repeated in triplicate.
After a 4-hr exposure to staurosporine, Abl+ cells
show a robust activation of caspase-8 relative to
control (Figure 6). Under similar conditions, Abl-/-
cells possess virtually no caspase-8 activity following
stimulation. Since the processing and activation of
caspase-8 is known to occur at the membrane in
response to signaling via death receptors [26], we
asked whether this biochemical pathway involved
microdomains. The sequestering of cholesterol
disrupts signaling pathways that depend upon intact
microdomain structure [27;28]. Cells were pretreated
with cholesterol-altering agents prior to staurosporine
exposure. As demonstrated in figure 6, both β-
methyl-cyclodextrin (5 mM) and filipin III (1 µg/ml)
attenuate staurosporine-induced caspase-8 activity.
Filipin III reduces apparent activity by approximately
47% while β-methyl-cyclodextrin decreases activity to
levels seen in vehicle control. The sequestering of
cholesterol has no effect on caspase-3 and caspase-
9 activity or on the release of cytochrome C from the
mitochondria (data not shown). Similar to the results
shown for caspase-3, Western analysis indicates
comparable expression of caspase-8 in quiescent
Abl-/- and Abl+ cells (Figure 7). In addition,
staurosporine-induced zymogen cleavage is apparent
in both cell types as evidenced by the appearance of
an 18kDa active fragment as well as the diminished
intensity of full-length pro-caspase-8 (Figure 7).
Abl-/-
Abl+
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
DMSO
STS
LEHDase Activity
[pmol/min/µg]
Abl-/-
Abl+
0.000
0.025
0.050
0.075
0.100
0.125
0.150
DMSO
STS
STS/Cyclodextrin
STS/Fillipin III
n=3
IETDase Activity
[pmol/min/µg protein]
Pro-
Caspase-8
Active
Caspase-8
57kDa
18kDa
DMSO STS
Abl-/- Abl+ Abl-/- Abl+
CON

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DISCUSSION
Phosphorylation and de-phosphorylation events are
known to control numerous signaling pathways. The
importance of these protein modifications cannot be
understated in the setting of programmed cell death.
A cell’s fate; whether to survive or die may be nothing
more than the balance between survival signals and
death signals. This critical balance may shift in either
direction based on an accumulation of particular
specific signaling events such as phosphorylation.
Depending on the current state of a cell, the same
ligand can activate a proapoptotic or prosurvival
response. For example, tumor necrosis factor-α
(TNF-α) receptor activation can lead to cell death via
recruitment and activation of caspase-8 [29].
Alternately, TNF-α promotes
immunomodulatory genes and cell survival via NF-
κB-dependent transcription [30]. C-Abl mediated
phosphorylation of IκBα in the nucleus abolishes NF-
κB transcriptional activity thereby promoting the
caspase-dependent death pathway [31]. Nuclear c-
Abl has been extensively described as a mediator of
cell death in response to genotoxic stressors [32-34].
Here we describe a novel role for c-Abl in
staurosporine-induced caspase activation.
expression of
In this report, we demonstrate that staurosporine
induces caspase-3 activity as evidence by specific
DEVDase proteolysis as well as appearance of the 17
kDa active fragment assayed by Western blot.
Interestingly, caspase-3 proteolytic activity requires
expression of the non-receptor tyrosine kinase c-Abl.
We propose that the regulation of caspase-3 activity
by c-Abl occurs downstream of zymogen cleavage
since we detect equivalent appearance of the 17 kDa
active fragment in cells lacking c-Abl. Effector
caspases such as caspase-3 are proteolytically
activated in a cascade-like fashion by upstream
caspases that include caspase-8 and caspase-9
originating from divergent pathways [35].
In a simplified model, caspase-9 becomes activated
by signals acting on the mitochondria. Proapoptotic
signals act on mitochondria inducing the translocation
of cytochrome C into the cytosol whereby an
apoptosome is formed by association of cytochrome
C, Apaf-1, dATP, and procaspase-9 [36;37].
Assembly of these proteins leads to the autocatalytic
activation of caspase-9, which then proteolytically
activates downstream effector caspases. The
cytosolic translocation of cytochrome C subsequent
caspase-9 activity is independent of c-Abl expression.
The mechanism of staurosporine-induced cell death
has been traditionally accepted to be via activation of
the mitochondria-dependent pathways. A secondary
pathway has also been reported. Stepczynska, et al,
(2001) demonstrated caspase-8 processing and
caspase-3 activation in response to staurosporine
despite the expression of catalytically inactive
caspase-9 [38]. This suggests that caspase-8
mediated caspase-3 activation is independent of
caspase-9 activity. This prompted us to examine the
role of c-Abl expression in cleavage and activation of
caspase-8 in staurosporine treated cells. We find
staurosporine-induced caspase-8 activity in MEF’s is
dependent on c-Abl expression. Although proteolytic
activity is dependent on c-Abl, we find cleavage of
caspase-8 to be independent of Abl. Similar to that
seen for caspase-3, cleavage of procaspase-8
zymogen is comparable in both Abl-/- and Abl+ cells as
evidenced by the appearance of the 18 kDa active
fragment as well as reduction in amounts of full-
length zymogen.
The subcellular local where caspase-8 activation
occurs is at the plasma membrane via recruitment of
caspase-8 zymogen through associations with
proteins containing the death domain sequence in
response to ligand-bound death receptors such as
the TNF-α receptor. TNF-α mediated cell death has
been shown to be dependent on intact microdomains
and the TNF receptor co-localizes with microdomains
[39]. We hypothesize caspase-8 activation requires
intact plasma membrane microdomains. In support of
our hypothesis, disruption of microdomains by
cholesterol-altering agents attenuated staurosporine-
induced caspase-8 activity. This data further
supports the notion that caspase-8 activation occurs
at the plasma membrane and exists independently of
caspase-9 activity.
Thus we propose a model of regulation of caspase
activity involving a non-receptor tyrosine kinase. In
this model, c-Abl plays a permissive role in the
proteolytic activity of the cleaved enzymes and does
not appear to effect zymogen cleavage per se. Our
data does not clarify whether c-Abl facilitates the
formation of tetrameric caspase complexes. In
addition, the direct action of c-Abl in our apoptotic
model remains elusive. However, it is conceivable
that a c-Abl mediated phosphorylation of caspases
induces a conformational change in the tetrameric
enzyme allowing substrates access to the active
cysteine residues. At this point however, we have no
evidence that c-Abl directly phosphorylates cleaved

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zymogens, although, both caspase-8 and caspase-3
do contain the putative phosphorylation consensus
sites (YxxP) preferred by c-Abl. Alternately, a
phosphorylation event may allow for the dissociation
of an IAP family protein from the caspase thereby
permitting proteolytic activity. In either case, it is
interesting to find a tyrosine kinase-mediated
regulatory mechanism since previous reports of
kinase regulated caspase activity involved the
serine/threonine protein kinase Akt [40].
It is of interest to note that despite lacking the gene,
Abl-/- cells appear to have developed alternate
biochemical pathways to ensure cellular demise in
response to toxin exposure. Nuclear c-Abl has been
implicated in the regulation of gene expression [41]
as well as erythroid differentiation [42]. It is therefore
conceivable that c-Abl plays a significant role in the
embryonic development of apoptotic pathways. We
have taken caution to verify the expression of the
caspases discussed in this paper. However, due to
the enormous complexity of apoptotic signaling
mechanisms, to conclude that Abl-/- cells do not differ
from wild-type fibroblasts would be naive. Our data
suggest the existence of redundant pathways
resulting in cell death. We suggest, however, that c-
Abl augments caspase-mediated cell demise by
regulating the activity of both initiator and effector
caspases. This type of regulation may be present as
a fail-safe mechanism to guarantee the removal of
damaged or defective cells.
ACKNOWLEDGMENTS
This work was supported by grants from the American Heart
Association National Center and the National Institutes of Health
(NIH HL56422) and a grant from the Foundation for Research to
ILOB and an NIH pre-doctoral fellowship award (NR07379) to
BCO.
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