Tài liệu Báo cáo khoa học: Regulation of DNA fragmentation: the role of caspases and phosphorylation doc

REVIEW ARTICLE
Regulation of DNA fragmentation: the role of caspases
and phosphorylation
Ikuko Kitazumi and Masayoshi Tsukahara
Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co. Ltd, Gunma, Japan
Introduction
Apoptosis is a crucial cellular mechanism that is
involved in inflammation, cell differentiation and cell
proliferation. As a form of cell death, it is character-
ized by distinctive morphological and biochemical
changes, including plasma membrane blebbing, phos-
phatidylserine exposure, nuclear condensation and
DNA fragmentation [1]. These cellular changes are
largely mediated by caspases, a family of cysteinyl
aspartate-specific proteases whose target proteins are
important indicators of apoptotic cell death [2].
Keywords
apoptosis; caspase; DNA fragmentation;
okadaic acid; phosphorylation
Correspondence
M. Tsukahara, Bio Process Research and
Development Laboratories, Kyowa Hakko
Kirin Co. Ltd, 100-1 Hagiwara, Takasaki,
Gunma 370-0013, Japan
Fax: 81 27 353 7400
Tel: 81 27 353 7382
E-mail: masayoshi.tsukahara@kyowa-
kirin.co.jp
(Received 10 September 2010, revised 18
November 2010, accepted 26 November
2010)

doi:10.1111/j.1742-4658.2010.07975.x
DNA fragmentation is a hallmark of apoptosis that is induced by apopto-
tic stimuli in various cell types. Apoptotic signal pathways, which eventu-
ally cause DNA fragmentation, are largely mediated by the family of
cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic
signal transduction by cleavage of apoptosis-implicated proteins and the
caspases themselves. In the process of caspase activation, reversible protein
phosphorylation plays an important role. The activation of various pro-
teins is regulated by phosphorylation and dephosphorylation, both
upstream and downstream of caspase activation. Many kinases ⁄ phosphata-
ses are involved in the control of cell survival and death, including the
mitogen-activated protein kinase signal transduction pathways. Reversible
protein phosphorylation is involved in the widespread regulation of cellular
signal transduction and apoptotic processes. Therefore, phosphatase ⁄ kinase
inhibitors are commonly used as apoptosis inducers ⁄ inhibitors. Whether
protein phosphorylation induces apoptosis depends on many factors, such
as the type of phosphorylated protein, the degree of activation and the
influence of other proteins. Phosphorylation signaling pathways are intri-
cately interrelated; it was previously shown that either induction or inhibi-
tion of phosphorylation causes cell death. Determination of the
relationship between protein and phosphorylation helps to reveal how
apoptosis is regulated. Here we discuss DNA fragmentation and protein
phosphorylation, focusing on caspase and serine ⁄ threonine protein phos-
phatase activation.
Abbreviations
AIF, apoptosis-inducing factor; CA, calyculin A; CAD, caspase-activated DNase; DFF, DNA fragmentation factor; EndoG, endonuclease G;
ERK, extracellular signal-regulated kinase; HtrA2, high temperature requirement protein A2; ICAD, inhibitor of caspase-activated DNase;
JNK, Jun NH2 terminal kinase; MAPK, mitogen-activated protein kinase; OA, okadaic acid; PARP, poly(ADP-ribose) polymerase;
PP, serine ⁄ threonine protein phosphatase; ST, staurosporine; TM, tautomycin; XIAP, X-linked inhibitor of apoptosis.
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 427

Caspases almost exist in an inactive form whose acti-
vation is widely affected by protein phosphorylation ⁄
dephosphorylation [3–5]. Kinase ⁄ phosphatase activa-
tion initiates apoptotic signal pathways; protein
phosphorylation plays important roles in the signaling
cascade that contributes to the control of cell death
and survival signal transduction [6–8]. In this review,
we will discuss the role of caspases and phosphoryla-
tion in apoptosis, with particular emphasis on the
induction of DNA fragmentation, which is one of the
most typical characteristics of apoptosis.
Regulators of DNA fragmentation
One of the terminal processes of apoptosis is DNA
degradation. During apoptosis, DNA breakage usually
occurs in at least two stages: the first is initial cleavage
at chromatin loop domains (50–300 kb) to generate
high relative molecular mass DNA fragments; the sec-
ond is cleavage of loose parts of internucleosomal
DNA (in approximate multiples of 180 bp, oligonucle-
osomal size) into low relative molecular mass DNA
fragments [9]. Nuclear morphological changes vary
according to cell type and related factors, some of
which have been prevented using gene knockouts and
treatment inhibitors [10–13].
Several nucleases have been implicated in the degra-
dation of DNA during apoptosis, two major ones
being endonuclease G (EndoG) and DNA fragmenta-
tion factor (DFF). Each nuclease has a distinct cellular
location, is regulated in different ways and causes
DNA fragmentation by a different pathway. Translo-

cation of EndoG from the mitochondria to the nucleus
leads to DNA fragmentation [12], whereas nuclear
activation of DFF caused by caspase activation leads
to characteristic low relative molecular mass oligonu-
cleosomal DNA fragmentation [14].
In addition, caspases are a key mediator of DNA
fragmentation. Caspases activate most apoptotic path-
ways through the cleavage of a wide range of cytoplas-
mic and nuclear proteins including themselves [5].
However, it is widely reported that inactivation or an
absence of caspases does not prevent DNA fragmenta-
tion [15,16]. Apoptotic DNA fragmentation thus
occurs both caspase dependently and independently.
Nucleases behavior and involvement of caspases in
DNA fragmentation are shown schematically in Fig. 1.
DFF: CAD
⁄
ICAD
DFF is composed of two subunits, a 40 kDa caspase-
activated DNase (CAD) ⁄ DFF40 and a 45 kDa inhibi-
tor of CAD (ICAD ⁄ DFF45), the complex of which is
an inactive form. During apoptosis, activated caspase-
3 induces ICAD cleavage, which releases CAD from
ICAD in an active form [17]. CAD is a DNA-specific,
double-strand-specific endonuclease, whose activity
leads to the generation of double-stranded breaks in
internucleosomal chromatin regions [18]. CAD triggers
high relative molecular mass DNA cleavage and results
in oligonucleosomal DNA ladders [19]. It lacks exonu-
clease activity and attacks only the linker regions

between nucleosomes; DNA degraded by CAD can be
detected by agarose gel electrophoresis as a character-
istic ‘DNA ladder’ [20].
ICAD is an indispensable factor in normal CAD
function. ICAD acts as a specific chaperone for
CAD during its synthesis and, after translation,
forms a heterodimer with CAD and inhibits its
DNase activity [21,22]. It has been shown that the
CAD ⁄ ICAD complex forms a heterotetramer
(CAD ⁄ ICAD with CAD ⁄ ICAD) in nonapoptotic
cells [23]. Such ICAD ⁄ CAD complexes are mainly
localized in the nucleus due to the presence of a
nuclear localization signal at the C-termini of both
ICAD and CAD [24]. During apoptosis, activation
of capsase-3 results in ICAD cleavage, which releases
CAD to form an active homodimer in the nucleus
[25]. ICAD mutant overexpression does not affect
the extent of cell death [26], suggesting that ICAD
could be involved in the induction of DNA fragmen-
tation, but is not involved in the execution phase of
DNA fragmentation.
ICAD exists as both a long (ICAD ⁄ DFF45) and a
short (ICAD-S ⁄ DFF35) form. ICAD-S is a splicing
variant of ICAD that ends at residue 268 and lacks
the C-terminal 63 residues of ICAD [27]. This short
form also dimerizes with CAD, and partially main-
tains the function of the inhibitor and chaperone
[28]. ICAD-S cannot translocate to the nucleus
because of a splice-out nuclear localization signal in
its C-terminal [24,29]. Because ICAD cleavage and

CAD activation occur in the nucleus, it is thought
that ICAD-S is the endogenous inhibitor of CAD
[14,24].
Mitochondrial DNA fragmentation-inducing
factor: EndoG, AIF
Several pro-apoptotic proteins exist in mitochondria
and are released during apoptosis. These include
apoptosis-inducing factor (AIF) and EndoG, which
are located in the mitochondrial intermembrane space
due to the presence of mitochondrial localization
signals at their N-termini [30]. They are probably
bound by their N-termini to the surface of the inner
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
428 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
mitochondrial membrane [31]. During apoptosis, both
enzymes are cleaved and simultaneously released from
mitochondria with the loss of the mitochondrial mem-
brane potential, then translocated to the nucleus,
where they have been shown to participate in nuclear
DNA fragmentation in various cell lines [32].
184
136
112
155
P
P
P
Bad
P
14-3-3

JNK
Bcl-2
Bax
Bax
Cytochrome c
14-3-3
167
P38 MAPK
185
183
Degradation
125
p53
Bax
Akt
473
Caspase-3
ERK1
70
87
69
Apaf-1
Apoptosome
Caspase-9
Cleaved
caspase-3
Endo GAIF
Dimerization
PARP
DNA fragmentation

Nucleus
Cytoplasm
CAD
ICAD-S
CAD
CAD
ICAD
Ribosome
CAD
CAD
ICAD
CAD
ICAD
Cleaved
PARP
Nuclear export
Response to DNA damage
Bax
P
P
62
184
Bcl-xL
Bax
Phosphorylated sites
Serine residues
Threonine residues
Tyrosine residues
Phosphorylation
Dephosphorylation

Degradation
Facilitatory effect
P
P
P
P
14-3-3
Bax
184
Bax
P P
P
P
P
159
121
163
184
P
P
P
Mcl-1
Bax
P
121
163
184
P
P
Mcl-1

Bax
P
184
Mcl-1
Bax
P
184
Bax
P
184
Bcl-xL
Bax
136
112
155
P
P
P
Bad
14-3-3
184
Bad
P
14
-3
-3
P
P
P
Caspase-9

P
P
364
Activation
Cleavage
465397
PP
P
Caspase-8
150
Caspase-3
P
Cleavage
Activation
P
Caspase-3
Endo GAIF
46
Apoptosis
Transcription of
Bax , Bcl-2
P
15
37
P
P
p53
Bad
Bcl-xL
Bcl-2

Caspase-9
Activation
Cleaved
caspase-3
Activation
Activation
Activation
Caspase-8
Cleavage
Activation
Akt
P
JNK
P38 MAPK
ERK1
15
37
55
P
P
P
p53
Fig. 1. Regulation of DNA fragmentation by phosphorylation of the MAPK family and mitochondrial proteins. Phosphorylated ERK prevents
the activation of caspases and the Bcl-2 family, whereas these are activated by phosphorylated JNK and p38 MAPK, leading to caspase acti-
vation. The Bcl-2 family is also directly regulated by PP2A. Activated caspase eventually cleaves and activates pro-caspase-3. Cleaved
caspase-3 translocates to the nucleus, where it cleaves substrates such as the DNA repair enzyme PARP and ICAD. Cleavage of ICAD
results in the release and activation of CAD, which induces DNA fragmentation. In contrast, EndoG and AIF are released from mitochondria
and then translocate to the nucleus where they induce DNA fragmentation in a caspase-independent manner. Whether apoptosis is induced
or not depends on the activation balance of these proteins. PP2A affects upstream and downstream signal cascades and assists in MAPK
mediation of each other. Dephosphorylations inhibited by OA are shown by green arrows.

I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 429
Despite their similar localization, they have different
functions in DNA fragmentation. Mitochondrial nucle-
ase EndoG first induces higher order chromatin cleav-
age into high relative molecular mass DNA fragments
(> 50 kb in length), followed by inter- and intra-
nucleosomal DNA cleavages, resulting in products
with many internal single-stranded nicks spaced at
nucleosomal ( 190 bp) and subnucleosomal ( 10 bp)
periodicities. Hence, DNA fragmentation generated
by EndoG is broad compared with other nucleases
[19]. Although EndoG is both a double- and a
single-stranded DNase ⁄ RNase, it preferentially attacks
single-stranded regions in the presence of additional
co-activators [33].
Unlike EndoG, AIF does not have DNase activity.
It is a mitochondrial flavoprotein that plays an essen-
tial role in oxidoreductase activity in nonapoptotic
cells [34]. AIF has been reported to trigger chromatin
condensation and induce cleavage of DNA into high
relative molecular mass fragments through other nuc-
leases, but not to cause oligonuclesomal DNA frag-
mentation [35,36]. However, other studies have shown
that inhibition of apoptotic AIF does not prevent the
appearance of high relative molecular mass DNA frag-
ments [26]; the nuclear actions of AIF therefore remain
poorly understood.
Relationship between DNA fragmenta-
tion and caspases

Caspase activation
Caspases play important roles in cell survival and
death, and widely regulate apoptotic signal pathways.
Apoptotic caspases are generally classified into two
groups: the initiator caspases (including caspase-2, -8,
-9 and -10) and the executioner caspases (consisting
of caspases-3, -6 and -7). The functional forms of
initiator caspases directly or indirectly promote acti-
vation of the executioner caspases [37,38]. Initiator
caspases not only activate executioner caspases, but
also act as their substrates. Initiator caspases are
activated by caspase-3 and initiate a feedback ampli-
fication loop that is followed by incremented caspase
activation [39]. Executioner caspase-6 and -7 play
specialized roles in apoptosis, whereas caspase-3 is well
established as the dominant executioner caspase, the
activation of which ultimately leads to cell death [40].
Two major pathways for caspase activation have
been identified: the receptor pathway and the mito-
chondrial pathway; both pathways trigger a cascade of
downstream caspases that induces DNA fragmentation
[2]. The former pathway initiates on receipt of cell sur-
face stimuli at the death receptors. These receptors,
such as tumor necrosis factor receptor and Fas, trans-
mit signals to the interior of cells, and activate initiator
caspases [41,42]. The latter pathway is induced by
various cellular stresses, including DNA damage,
and releases apoptotic mitochondrial molecules that
lead to caspase-9 activation and regulate executioner
caspases [43].

Caspases are initially synthesized as inactive zymo-
gens, and their dimerization is crucial for stabilizing
the conformation of the active site, which is cleaved
prior to activation [44,45]. Initiator caspases are mono-
meric zymogens, which are activated by dimerization
during apoptosis, whereas the executioner caspases
exist as the inactive dimers [46]. Initiator caspases form
signaling complexes that are platforms for caspase acti-
vation. Pro-caspase-9 forms a large complex as the
apoptosome, which consists of released cytochrome c
from mitochondria and oligomers of Apaf-1 [47].
Pro-caspase-8 is activated through recruitment of the
death receptor complexes [41]. Executioner caspase
dimers are activated by upstream proteolysis or auto-
proteolysis to cleave sequentially and generate active
large and small subunits that form active hetero-
tetramers [5,38].
Caspases have multiple cleavage sites at specific
aspartic acid residues; the exact cleavage location
affects caspase activity and function [37]. In the case
of caspase-9, it is activated by autolytic cleavage via
the mitochondrial pathway [47]. Caspase-9 is also
cleaved by caspase-3 at another cleavage site. How-
ever, this fragmentation does not have caspase activity.
It enhances the activation of other caspases by alleviat-
ing endogenous X-linked inhibitor of apoptosis (XIAP)
inhibition of caspases [48]. Although cleavage is a sig-
nificant change for caspase activation, the cleaved frag-
ment does not always have caspase activity. It was
previously shown that cleavage of caspase still occurs

in the presence of caspase inhibitors, but that cleavage
fragments were inactive because they bound caspase
inhibitors [49]; cleavage fragments of caspase-3 ⁄ 7
sometimes exist in living cells, but are inactive due to
the binding of XIAP [50]. Although chemical caspase
inhibitors bind to caspase fragments and inhibit their
peptide-specific activity, other proteolytic activity still
occurs [15,51]. Moreover, cleavage is not necessary to
activate caspase-8 and probably its close paralog cas-
pase-10 [46]. Pro-caspases exist in living cells and casp-
ases are indispensable for the proliferation of some cell
lines. When deciding between maintaining cells alive or
inducing apoptosis, caspase function and activation
are regulated in terms of which pathways induce cleav-
age sites and modification of fragments.
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
430 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
Involvement of caspases in DNA fragmentation
One of the direct substrates of caspases in DNA frag-
mentation is ICAD. Its caspase-mediated cleavage
causes the release and activation of CAD from the
DFF complex [17]. ICAD possesses two caspase
cleavage sites, D117 and D224. The N-terminal cleav-
age site D117 is cleaved by multiple caspases, and
this cleavage is necessary for CAD activation. Cleav-
age of the C-terminal cleavage site D224 retains CAD
inhibitory activation that is preferentially processed
by caspase-3 [26,52,53]. Caspase activation is indis-
pensable for proteolysis of the DFF complex.
Although CAD is not cleaved by caspase-3, activation

of CAD is a caspase-3-dependent process that occurs
in the nucleus [21,24]. Caspase-3 also affects the
induction of other factors involved in DNA fragmen-
tation by cleaving substrates. Poly(ADP-ribose) poly-
merase (PARP) is a major nuclear target for caspases
that is involved in many cellular functions, including
DNA repair and maintenance of genomic stability
[54]. PARP is activated in response to DNA damage,
and its activity is shown to regulate DFF40 activity
in vitro. Caspases cleave PARP and inactivate its
DNA-repairing abilities during apoptosis; hence, inhi-
bition of caspases mostly prevents PARP cleavage
and DNA fragmentation [10].
Caspases often share common substrates. Cells have
multiple cleavage mechanisms, as shown by the cleav-
age induction of ICAD and PARP in caspase-3-
deficient cells [13,55]. However, they exhibit different
levels of activity against substrates. The close relation-
ship between capsase-3 and caspase-7 is well docu-
mented. Although caspase-7 is as efficient as caspase-3
(in some cases more effective) for several substrates in
a cell-free system, caspase-3 is a major executioner cas-
pase [56]. The different localizations and substrates of
caspases contribute to functional distinctions. For
example, pro-caspases are often present in the cytosol
fraction (caspase-2, -3, -6, -7, -8 and -9) of living cells
to separate silent precursor caspases in the cytosol
from pro-apoptotic cofactors in the mitochondria and
nucleus [57,58], although caspase localization depends
on cell lines. In the case of pro-caspase-3 and -7, they

are mostly localized in the cytosol [59], whereas
CAD ⁄ ICAD is activated in the nucleus [14]. During
apoptosis, both caspases are activated and caspase-3,
but not caspase-7, translocates from the cytosol into
the nucleus [59], subsequently cleaving ICAD. Active
caspase-7 has been shown to be located in the nucleus
[55]; ICAD can also be cleaved by caspase-7, but at a
lower level of efficiency [13,56]. Distributional differ-
ences of caspases according to species and cell type
contribute to the conflicting reports as to whether
caspases are dependent on DNA fragmentation.
Caspase-independent DNA fragmentation
Mitochondrial proteins EndoG and AIF cause caspase-
independent DNA fragmentation. AIF has a direct
effect on nuclei, triggering high relative molecular mass
DNA fragmentation in a caspase-independent manner
[35]. The release of mitochondrial DNase EndoG is
dependent on Bcl-2 family proteins, which normally
require active caspases for their activation [12].
Even though the release process is often regulated by
caspases, activities of both EndoG and AIF are then
caspase-independent [60].
Additionally, high temperature requirement protein
A2 (HtrA2) ⁄ Omi, the pro-apoptotic mitochondrial ser-
ine protease, causes caspase-independent cell death
when it is released from mitochondria during apoptosis
[31,61]. After cell damage, HtrA2 accumulates in the
nucleus and activates the transcription factor p73,
which activates pro-apoptotic genes such as bax [62].
Pro-apoptotic activity of HtrA2 results from both its

serine protease activity and its ability to act as an
inhibitor of apoptosis antagonist, which enhances cas-
pase activation [63]. The release of HtrA2 ⁄ Omi from
mitochondria into the cytosol and pro-apoptotic activ-
ity via XIAP inhibition is closely related to caspase
activity; HtrA2 ⁄ Omi activity contributes to the pro-
gression of caspase-independent cell death in mito-
chondria [64]. Although activation of these proteins is
highly dependent on caspase activation, DNA frag-
mentation has been shown to occur during caspase
inhibition [15,16]. In the case of cell death stimulation
that does not activate caspases, alternative pathways
induce caspase activity, resulting in DNA fragmen-
tation.
DNA fragmentation resulting from
phosphorylation-induced apoptotic
pathways
Cell signal transduction is regulated by the biochemical
modification of proteins that alters their conformation,
stabilization, reaction to substrates and function.
Reversible protein phosphorylation and dephosphory-
lation at serine and threonine residues can modulate
cell survival through positively or negatively changing
protein stability, transcriptional activity and apoptotic
ability [7,8]. Caspases play central roles in apoptotic
pathways, which induce DNA fragmentation [2,21].
Phosphorylation regulates many caspase activity-
induced signal pathways; phosphorylation is also
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 431

directly involved in the change in active form of cas-
pases and DNA fragmentation-induced factors [5,11].
The induction of DNA fragmentation is closely linked
to the phosphorylation of proteins such as mitochon-
drial proteins, caspases, transcriptional factors and
nuclear proteins.
Phosphorylation of MAPK signaling pathways
Many protein kinases are associated with cell survival
and death; a key pathway in apoptosis is the mitogen-
activated protein kinase (MAPK) signaling pathways.
These pathways promote activation and nuclear trans-
location of transcription factors that modify gene
expression through phosphorylation-dependent sub-
strate activation [65].
MAPK pathways consist of three major kinases:
the activation of p38 MAPK, the extracellular signal-
regulated kinases (ERK) and Jun NH2 terminal kinases
(JNK) [66]. JNK and p38 MAPK activation triggers
apoptosis in response to many types of cellular stress,
including DNA damage [67]. These two pathways
share several upstream regulators and are simulta-
neously activated. p38 MAPK isoforms a, b, c and d
have been identified and may have both overlapping
and specific functions depending on the cellular con-
text and ⁄ or stimuli [7]. ERK translocates to the
nucleus and phosphorylates a variety of substrates that
promote cell proliferation. Activated ERK-1 inhibits
the induction of mitochondrial permeability transition,
thus blocking mitochondrial apoptotic pathways
[68,69]. ERK pathway activity is suppressed by

JNK ⁄ p38 kinases during apoptosis [70]; this represents
an example of cross-talk or cross-signaling in which
one signaling pathway is regulated by another
[66,69,71].
Additionally, phosphatases, which have an effect
opposite to kinases, play important roles in the down-
regulation of MAPK activity. Especially, the ser-
ine ⁄ threonine protein phosphatase (PP) is a key
regulator of cellular protein dephosphorylation. PP
can be classified as type 1 (PP1) or type 2 (PP2), and
PP2A regulates both cell survival and apoptotic cellu-
lar reactions [72]. PP2A has been shown to dephos-
phorylate p38 MAPK, thereby impairing its activity,
and its inhibition results in the induction of apoptosis
via caspase activation, for example [16,71].
Phosphorylation of mitochondrial apoptotic
proteins
The antiapoptotic Bcl-2 family members are important
regulators of cell survival in their control of mitochon-
drial pathways. These proteins both prevent and
induce entry into the apoptotic cell death cascade, for
example by activating caspases [73]. The family is
divided into three subfamilies: antiapoptotic proteins
(Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1), pro-apoptotic
proteins (Bax, Bak and Bok) and BH3-only proteins
(Bad, Bid, Bik, Blk, Hrk, BNIP3 and BimL). Bcl-2
family proteins mostly mediate the activity of other
proteins in the same family [74]. Antiapoptotic Bcl-2
family members bind to pro-apoptotic family mem-
bers, interrupting cell death signals [75], but with very

different effects depending on the binding proteins.
For instance, the apoptotic effects of Bax on mito-
chondria are inhibited by heterodimerization with
Bcl-xL, which maintains Bax in the cytoplasm; con-
versely, Bad shows the apoptotic effects on binding to
Mcl-1 and Bcl-xL at the mitochondrial outer mem-
brane, where Bad causes degradation of antiapoptotic
proteins and cell death [76,77].
A major antiapoptotic Bcl-2 protein, Mcl-1, modu-
lates pro-apoptotic Bcl-2 family proteins through its
phosphorylation. JNK and ERK mediate phosphoryla-
tion of Mcl-1 at Ser121 and especially at Thr163,
which stabilizes it to prolong its half-life [78]. Phos-
phorylation at Ser64 enhances the antiapoptotic
activity of Mcl-1 through increased binding to pro-
apoptotic proteins such as Bak [79]. Conversely,
Ser159 phosphorylation of Mcl-1 enhances its degrada-
tion through the ubiquitin proteasome pathway and
induces apoptosis [80]. Phosphorylation of Bcl-xL and
Bcl-2 regulates their functions negatively and posi-
tively, respectively. Phosphorylation of Bcl-xL at Ser62
disables the ability of Bcl-xL to bind Bax [81]. Bcl-2
has several phosphorylated sites, including Thr69 and
Ser87, and its degradation is promoted through
dephosphorylation of these sites. Ser70 is the major
physiological phosphorylation site for the survival
function of Bcl-2 [82].
The pro-apoptotic Bcl-2 proteins relocate to the sur-
face of mitochondria during apoptosis. They induce
the permeabilization of the mitochondrial membrane

with the release of cytochrome c and the formation of
the apoptosome [38]. The major pro-apoptotic protein
Bax exists mainly in the cytosol or loosely attaches to
mitochondria in an inactive form. Inactivated Bax is
phosphorylated at Ser184 by the physiological Bax
kinase Akt, and heterodimerizes with antiapoptotic
Bcl-2 family members such as Bcl-xL [83]. Activation
of Bax by dephosphorylation results in translocation
from the cytosol to mitochondria, where it forms large
oligomers. This translocation is inhibited by ERK-1
[69,84]. Bax dimerization leads to the formation of a
pore or channel in the mitochondrial outer membrane,
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
432 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
enabling multiple mitochondrial proteins to be released
into the cytosol with cytotoxic activities [85].
The activated BH3-only protein Bad is also localized
mostly in the cytosol in normal cells, and is phosphor-
ylated at Ser112, Ser136 and Ser155 in an ERK-1-
dependent manner [69]. Dephosphorylation of Ser136,
which is regulated by dephosphorylation of Ser112, is
a key action in mediating apoptosis. After dephosphor-
ylation of both Ser112 and Ser136, Bad is dephospho-
rylated at Ser155, which allows translocation to
mitochondria and the binding of Bcl-xL [86], and
increases the release of cytochrome c from mitochon-
dria into the cytosol through inactivation with Bcl-xL
and Bcl-2 [69]. Inactivated Bax and Bad bind 14-3-3,
the phosphoserine ⁄ threonine binding proteins in the
cytosol. 14-3-3 prevents Bax and Bad dissociation from

translocating to the mitochondria by a conformation
change, and 14-3-3 binding leads to protection of Bad
phosphorylation at Ser112, Ser136 and Ser155 [86].
The dissociation of Bax and Bad from 14-3-3 is pro-
moted by JNK via phosphorylation of 14-3-3 at
Ser184; this reduces the affinity of 14-3-3 for Bax and
Bad and translocation of Bax and Bad to mitochon-
dria independently of caspase activation [77,87].
Phosphorylation of caspases
Phosphorylation of caspases switches the cellular apop-
totic signal on and off. Caspase activation is under the
direct control of kinases and phosphatases, and the
indirect control of phosphorylation through the regula-
tion of other apoptotic proteins. Furthermore, many
kinases and phosphatases are cleaved by activated
caspases. The initiator caspase-9 has several sites that
are phosphorylated by multiple protein kinases [88],
including the major phosphorylation site Thr125.
The direct phosphorylation of this site by ERK, but
not JNK or p38 MAPK/MAKP, suppresses the pro-
cessing of caspase-9 [89]. Caspase-9 dephosphorylation
and, as a consequence, its activation are involved in
regulating the activity of an isoform of PP1, PP1a [3].
Similarly, activation of caspase-8 and -3 is regulated
through their phosphorylation and dephosphorylation.
Phosphorylation of caspase-8 at Tyr397 and Tyr465 by
Lyn, a nonreceptor tyrosine kinase of the Src family,
renders it resistant to activational cleavage, thus inhib-
iting apoptosis [90]. In addition to these sites, phos-
phorylation of caspase-8b at Tyr380 by Src suppresses

caspase-8 activity and function [91]. Moreover, p38
MAPK can directly phosphorylate and inhibit the
activities of caspase-8 at Ser364 and caspase-3 at
Ser150 [4]. After phosphorylation of Tyr310, caspase-8
is dephosphorylated at both Tyr397 and Tyr465 by the
Src-homology domain 2-containing tyrosine phospha-
tase-1, which allows its cleavage and activation [90],
and caspase-3 at threonine residues by PP2A interac-
tion [51] initiates apoptosis. Conversely, kinases
involved in the phosphorylation of caspases are regu-
lated by cleaved caspases [8]. Caspases, kinases and
phosphatases are regulated by each other and control
cell survival.
Phosphorylation of intranuclear protein
Core nucleosomal histone H2AX is phosphorylated at
sites of DNA double-stranded breaks in DNA-injured
cells. H2AX is a member of the histone H2A family,
which differs from other species by containing a
Ser139 phosphorylation site in the C-terminal tail.
Phosphorylation of H2AX on Ser139 is a key event in
the repair of DNA damage and the induction of DNA
degradation leading to cell death; therefore, the phos-
phorylated form of H2AX (cH2AX) is a sensitive
marker for DNA double-stranded breaks [92,93].
It has been reported that the last residue at C-termi-
nal Tyr142 is phosphorylated under normal conditions,
preventing recruitment of DNA repair factors to phos-
phorylated Ser139 [94]. The phosphorylation site
Ser139 is directly phosphorylated by JNK and p38b
MAPK [11,95]. cH2AX associates not only with DNA

damage repair factors [96], but also with DNA degra-
dation-induced factors at damaged DNA sites. cH2AX
mediates the caspase-3 downstream target CAD [95],
and also interacts with AIF to promote DNA degrada-
tion [36]. H2AX regulates both caspase-dependent and
-independent DNA fragmentation during apoptosis.
Phosphorylation of H2AX is also regulated indirectly
via the p53 tumor suppressor. Once activated, p53 acts
as a transcription factor, eliciting the transcription of
genes that induce cell cycle arrest or programmed cell
death through interaction with a large number of other
signal transduction pathways [97]. Thr55 phosphoryla-
tion is required for p53 nuclear export, and inhibition
of this phosphorylation restores the nuclear localiza-
tion of p53, and sensitizes it to DNA damage [98].
Phosphorylated p53 suppresses cH2AX accumulation,
leading to higher DNA damage and activation of
p53 ⁄ p21, which in turn further inhibits H2AX [99].
Effects of phosphatase inhibitors on
DNA fragmentation
Phosphatase and kinase inhibitors are commonly used
to induce apoptosis. Despite their conflicting effects on
protein phosphorylation, both inhibitors can equally
cause DNA fragmentation [26,51]. PP1⁄ 2A inhibitor
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 433
okadaic acid (OA) and protein kinase inhibitor stauro-
sporine (ST) are typical inhibitors that promote cell
death. Both inhibitors increase MAPK-involved cell
death signaling leading to caspase activation [100,101].

OA is a component of diarrhetic shellfish poisoning
toxin [102]. It is a potent inhibitor of PP1 and PP2A
that increases the tyrosine phosphorylation and inacti-
vation of PP2A [68] with 100-fold greater selectivity
for PP2A over PP1 [103]. OA induces various cellular
reactions that can either induce or prevent apoptosis
through phosphorylation modulating (Fig. 1). Inhibi-
tion of PP upsets the balance between serine ⁄ threonine
phosphorylation and dephosphorylation of various
proteins, leading to altered signal transduction and
gene expression. The following section focuses on the
effects of OA on apoptosis.
Apoptotic effect of OA
The inhibition of PP positively regulates apoptosis by
activating pro-apoptotic factors and inactivating antia-
poptotic factors. Many PP dephosphorylation signals
are involved in the induction of DNA fragmentation,
such as activation of the caspase cascade and MAPK
family. Treatment with OA has been shown to alter
mitochondrial membrane permeability due to the
release of cytochrome c and AIF, and to enhance
apoptosis in HeLa cells [16], primary cultures of nor-
mal human foreskin keratinocytes [100] and Jurkat
T leukemia cells [104]. OA affects antiapoptotic Bcl-2
family members that are involved in mitochondrial
apoptotic pathways. PP2A plays a role in the dephos-
phorylation of Bcl-xL at Ser62 in response to oxidative
stress, and treatment with OA has been shown to
enhance phosphorylated Bcl-xL, leading to diminished
Bcl-xL ⁄ Bax interaction in human retinal pigment epi-

thelial ARPE-19 cells [105] and the human cervical
carcinoma cell line KB-3 [81]. OA also induces the
phosphorylation and degradation of Mcl-1 in periph-
eral blood neutrophils [51] and at Thr163 and other
sites in the Burkitt lymphoma subline BL41-3 [106].
Repression of antiapoptotic proteins by OA treatment
activates the caspase cascade in T leukemia cells via a
mitochondrial feedback amplification loop [104].
PP1 and PP2A are involved in p53-dependent cell
death pathways through the direct dephosphorylation
of p53. p53 functions in the nucleus to regulate pro-
apoptotic genes, whereas cytoplasmic p53 directly acti-
vates pro-apoptotic Bcl-2 proteins such as Bax [107].
Inhibition of PP1 by OA enhances the phosphorylation
of p53 at Ser15 and Ser37, decreases the expression
of bcl-2 and increases the expression of bax in human
laryngeal epithelial cells and human lung fibroblast
WI-38 cells [108,109]. PP2A dephosphorylation of p53
at Ser15 and Ser37 is inhibited by OA in the human
acute lymphoblastic leukemia cell line MOLT4 and
JB6 mouse skin epidermal cell line Cl41 [110,111].
Phosphorylation of p53 at these residues is important
for transcriptional activity. PP2A inhibition also
enhances the phosphorylation of p53 at Ser46, and
apoptotic signaling such as caspase activation in the
normal human lymphoblast cell type GM02814 [112].
Phosphorylated p53 regulates H2AX [99], thus PP
indirectly mediates the accumulation of cH2AX. Addi-
tionally, because PP2A directly dephosphorylates
cH2AX, OA treatment increases cH2AX in human

myeloid leukemia K562 cells [113]. Thus, OA effects
range from upstream of apoptotic signal pathways to
downstream proteins.
Antiapoptotic effect of OA
Although treatment with OA induces apoptosis, OA
also protects cells against other apoptotic signals.
PP2A can activate Bad via two different routes, direct
dephosphorylation of Ser112 and negative regulation
of the ERK pathway via p38 MAPK, both of which
lead to impaired phosphorylation of Ser112 [70]. After
dephosphorylation of Ser112, Ser136 becomes suscepti-
ble to multiple phosphatases. PP2A dephosphorylates
Bad mainly on Ser112, as well as on Ser136 and
Ser155 [6]. Treatment with OA was shown to
phosphorylate Bad at Ser112 and Bcl-2 at Ser70, and
activate ERK, thereby preventing tumor necrosis
factora ⁄ cycloheximide-induced JNK activation, cyto-
chrome c release and caspase activation in rat epithelial
IEC-6 cells [68].
Apoptotic activation of Bad results from 14-3-3 dis-
sociation after dephosphorylation of Ser112 and
Ser136, and sequential dephosphorylation of Ser155 by
PP2A. Activated Bad binds to Bcl-XL to prevent
antiapoptotic activation in both the interleukin-
3-dependent murine prolymphocytic cell line FL5.12
and the mouse embryonic fibroblast cell line NIH 3T3
[86]. Dephosphorylated Bax is directly increased by
PP2A, and indirectly through inhibition of Akt phos-
phorylation on Ser473 by p38a MAPK-mediated PP2A.
OA increases phosphorylation of Bax, then inhibits

disruption of the Bcl-2⁄ Bax complex, which leads to
cytochrome c release in the human epithelial cell line
A549 and mouse cardiomyocyte cell line [84,114]. OA-
induced Bcl-2 phosphorylation induces its antiapoptotic
function to prevent formation of the Bcl-2 ⁄ p53 complex
in association with apoptotic cell death [115].
Additionally, OA induces the direct inhibition of
capsase-9 to increase phosphorylated caspase-9 in the
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
434 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
murine T cell line TS1ab [3]. Dephosphorylation of cas-
pase-9 by PP1a is required for cytochrome c-induced
activation and subsequent caspase-3 activation.
Balance between apoptotic and antiapoptotic
effects of OA
There are many conflicting findings concerning the
effects of OA on apoptosis. It has been reported that
OA cytotoxicity is chiefly cell type-dependent and con-
centration-dependent [116]. Because low concentrations
of OA inhibit PP2A and high concentrations of OA
inhibit PP1 [108], the effects of OA on apoptosis
appear to depend on inhibition of PP type.
In addition to OA, several other phosphatase inhibi-
tors are often used, which differ in their sensitivity to
PP1 and PP2A. Calyculin A (CA) has nearly equiva-
lent inhibitory activities against PP1 and PP2A. Tauto-
mycin (TM) has PP1 selectivity approximately 10 times
higher than PP2A. In contrast, OA has 100-fold
greater selectivity for PP2A than PP1 [117]. Fostriecin
is a highly selective inhibitor of PP2A enzymes and

inhibits PP2A at 10 000–40 000 times lower concentra-
tion than that required for PP1 inhibition [103]. The
apoptotic effects of OA and fostriecin (PP1 < PP2A)
and CA (PP1 = PP2A) were observed; however, TM
(PP1 > PP2A) did not exhibit any pro-apoptotic
effects in the interleukin-3-dependent murine pro-B cell
line [6], the endothelium-derived permanent human cell
line EA.hy926 [70] or Jurkat cells [104]. Inhibition of
PP2A equivalent to PP1 (PP1 = PP2A) or better than
PP1 (PP1 < PP2A) (OA, fostriecin, CA) induces
apoptosis; on the other hand, inhibition of PP1 rather
than PP2A (PP1 > PP2A) (TM) fails to induce apop-
tosis. It is possible that apoptosis is induced when PP1
has greater activation than PP2. Additionally, inhibi-
tion of PP1 by CA or TM prevents Fas-mediated
apoptosis, whereas inhibition of PP2A by OA protects
Jurkat cells from anisomycin [118]. The effects of OA
on apoptosis therefore depend on the kind of inducer,
as well as inhibition of PP type and cell type.
The effects of OA on cellular signaling are also
affected by intrinsic regulation. PP2A is a downstream
target of p38 MAPK, whose activity regulates the sub-
cellular localization of PP2A [70,114]; meanwhile,
PP2A dephosphorylates p38d MAPK [100,119]. p38
MAPK acts to limit the phosphorylation of JNK
through increased activation of PP2A [71]; thus the
MAPK family regulates its members via PP2A. PP2A
affects not only upstream but also downstream pro-
teins for apoptotic signaling. OA-induced activity of
the MAPK family mediates the downregulation of var-

ious phosphorylations, such as those of mitochondrial
proteins, caspases and MAPK themselves. p38 MAPK
binds and regulates caspase-3, forming a complex that
is predominantly observed in the nucleus during Fas-
induced apoptosis of the human hepatoma cell line
Bel-7402, for example [120]. Furthermore, cells have
multiple apoptosis-induced mechanisms, as shown by
induction of OA-induced DNA fragmentation by
caspase-dependent and -independent pathways [15].
Despite the same substrate, the effects of OA vary
between phosphorylation sites. The inhibition of PP2A
in Fas-engaged neutrophils led to an increased phos-
phorylation of caspase-3 at Ser150, which inhibited its
activity and thereby delayed the apoptotic process
[119]. On the other hand, treatment with OA caused
phosphorylation of caspase-3 at the threonine residue,
and degradation of pro-caspase-3 activated caspase-3
via the inhibition of PP in HeLa cells [51]. PP1 and
PP2A have a large number of substrates, and whether
OA treatment induces apoptosis appears to depend on
the overall balance of the above activities.
Comparing phosphatase and kinase inhibitors
The protein phosphatase inhibitor OA and the protein
kinase inhibitor, the broad spectrum inhibitor of pro-
tein kinase ST for example, often exert opposing
effects on protein modification by modulating one sub-
strate of different reactions. For example, OA
increases phosphorylation of both ERK and Bad in
BL41-3 cells [106]. In contrast, treatment with ST
causes Bad dephosphorylation and alters mitochon-

drial membrane permeabilization in intact NIH 3T3
cells [86] and human hepatoma HepG2 cells [121].
Phosphorylation of ERK1 ⁄ 2, upstream of Bad, is simi-
larly degraded by ST in rat primary hepatocytes [101].
Interestingly, phosphatase and kinase inhibitors act
on identical cell death pathways and eventually induc-
tion of DNA fragmentation [26,104]. Both OA and ST
induce phosphorylation and activation of JNK and
p38 MAPK, which are involved in the increase of
release of cytochrome c into the cytoplasm and caspase
activation [16,71,122]. ST rapidly increased p53 cyto-
plasmic accumulation, which activated Bax in the
mouse cerebellar neural stem cell line C17.2 [123].
Treatment with OA increased levels of phosphorylated
p53 at Ser15 (at least one phosphorylated site), which
binds to microtubules and cannot be efficiently translo-
cated into the nucleus; this resulted in inhibition of its
transcriptional activity [124].
However, these inhibitors are essentially different,
although they lead in part to induce similar reactions.
Both ST and OA phosphorylate the same substrate
but at different phosphorylation sites. Stimulation with
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 435
ST induces JNK- and p38 MAPK-mediated phosphor-
ylation of Bax at Thr167, leading to its activation in
HepG2 cells [122]. On the other hand, treatment with
OA increases Akt-mediated phosphorylation of Bax at
Ser184, which is important in the cytosolic retention of
Bax [83,84]. The opposite reactions regulate functional

properties of cell death pathway-involved protein in
different ways. Because of the effect on upstream and
downstream proteins as well as the target proteins, we
therefore have to consider the combinations of apopto-
sis inducer ⁄ inhibitor, detection method and target
proteins.
Conclusion
Many signal-transducing proteins have multiple phos-
phorylation sites, each of which induces different
downstream signaling reactions through a close rela-
tionship between protein modification sites and confor-
mations. Cellular kinases ⁄ phosphatases affect a wide
variety of phosphorylation sites on one protein.
Following phosphorylation ⁄ dephosphorylation, succes-
sive changes depend on the kinases ⁄ phosphatases
involved and the effect of upstream proteins. Even
with the same outcome, a wide range of signaling
transduction factors are involved. For example, OA
and ST similarly cause DNA fragmentation, but have
conflicting effects on phosphorylation. Moreover,
cellular signaling pathways mediate each other via phos-
phorylation. It is difficult to determine which protein is
required for a signaling pathway. Therefore, it must be
noted that signal transductions interact with each other,
and that signal inducers ⁄ inhibitors affect more than just
their target proteins.
References
1 Taatjes DJ, Sobel BE & Budd RC (2008) Morphologi-
cal and cytochemical determination of cell death by
apoptosis. Histochem Cell Biol Rev 129, 33–43.

2 Li J & Yuan J (2008) Caspases in apoptosis and
beyond. Oncogene Rev 27, 6194–6206.
3 Dessauge F, Cayla X, Albar JP, Fleischer A, Ghadiri
A, Duhamel M & Rebollo A (2006) Identification of
PP1alpha as a caspase-9 regulator in IL-2 deprivation-
induced apoptosis. J Immunol 177, 2441–2451.
4 Alvarado-Kristensson M, Melander F, Leandersson K,
Ro
¨
nnstrand L, Wernstedt C & Andersson T (2004)
p38-MAPK signals survival by phosphorylation of
caspase-8 and caspase-3 in human neutrophils. J Exp
Med 199, 449–458.
5 Duncan JS, Turowec JP, Vilk G, Li SS, Gloor GB &
Litchfield DW (2010) Regulation of cell proliferation
and survival: convergence of protein kinases and cas-
pases. Biochim Biophys Acta Rev 1804, 505–510.
6 Chiang CW, Yan L & Yang E (2008) Phosphatases
and regulation of cell death. Methods Enzymol 446,
237–257.
7 Wagner EF & Nebreda AR (2009) Signal integration
by JNK and p38 MAPK pathways in cancer develop-
ment. Nat Rev Cancer Rev 9, 537–549.
8 Kurokawa M & Kornbluth S (2009) Caspases and
kinases in a death grip. Cell Rev 138, 838–854.
9 Samejima K, Tone S & Earnshaw WC (2001)
CAD ⁄ DFF40 nuclease is dispensable for high molecular
weight DNA cleavage and stage I chromatin conden-
sation in apoptosis. J Biol Chem 276, 45427–45432.
10 West JD, Ji C & Marnett LJ (2005) Modulation of

DNA fragmentation factor 40 nuclease activity by
poly(ADP-ribose) polymerase-1. J Biol Chem 280,
15141–15147.
11 Lu C, Shi Y, Wang Z, Song Z, Zhu M, Cai Q & Chen
T (2008) Serum starvation induces H2AX phosphory-
lation to regulate apoptosis via p38 MAPK pathway.
FEBS Lett 582, 2703–2708.
12 Li LY, Luo X & Wang X (2001) Endonuclease G is
an apoptotic DNase when released from mitochondria.
Nature 412, 95–99.
13 Wolf BB, Schuler M, Echeverri F & Green DR (1999)
Caspase-3 is the primary activator of apoptotic DNA
fragmentation via DNA fragmentation factor-
45 ⁄ inhibitor of caspase-activated DNase inactivation.
J Biol Chem 274, 30651–30656.
14 Chen D, Stetler RA, Cao G, Pei W, O’Horo C, Yin
XM & Chen J (2000) Characterization of the rat DNA
fragmentation factor 35 ⁄ inhibitor of caspase-activated
DNase (short form). The endogenous inhibitor of
caspase-dependent DNA fragmentation in neuronal
apoptosis. J Biol Chem 275, 38508–38517.
15 Kitazumi I, Maseki Y, Nomura Y, Shimanuki A,
Sugita Y & Tsukahara M (2010) Okadaic acid induces
DNA fragmentation via caspase-3-dependent and
caspase-3-independent pathways in Chinese hamster
ovary (CHO)-K1 cells. FEBS J 277, 404–412.
16 Jayaraj R, Gupta N & Rao PV (2009) Multiple signal
transduction pathways in okadaic acid induced apop-
tosis in HeLa cells. Toxicology 256, 118–127.
17 Enari M, Sakahira H, Yokoyama H, Okawa K,

Iwamatsu A & Nagata S (1998) A caspase-activated
DNase that degrades DNA during apoptosis, and its
inhibitor ICAD. Nature 391, 43–50.
18 Hanus J, Kalinowska-Herok M & Widlak P (2008)
The major apoptotic endonuclease DFF40 ⁄ CAD is a
deoxyribose-specific and double-strand-specific enzyme.
Apoptosis 13, 377–382.
19 Widlak P, Li LY, Wang X & Garrard WT (2001)
Action of recombinant human apoptotic endonuclease
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
436 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
G on naked DNA and chromatin substrates. J Biol
Chem 276, 48404–48409.
20 Widlak P & Garrard WT (2006) Unique features of
the apoptotic endonuclease DFF40 ⁄ CAD relative to
micrococcal nuclease as a structural probe for chroma-
tin. Biochem Cell Biol Rev 84, 405–410.
21 Cao G, Pei W, Lan J, Stetler RA, Luo Y, Nagayama
T, Graham SH, Yin XM, Simon RP & Chen J (2001)
Caspase-activated DNase ⁄ DNA fragmentation factor
40 mediates apoptotic DNA fragmentation in transient
cerebral ischemia and in neuronal cultures. J Neurosci
21, 4678–4690.
22 Sakahira H, Enari M & Nagata S (1999) Functional
differences of two forms of the inhibitor of caspase-
activated DNase, ICAD-L, and ICAD-S. J Biol Chem
274, 15740–15744.
23 Lechardeur D, Dougaparsad S, Nemes C & Lukacs
GL (2005) Oligomerization state of the DNA fragmen-
tation factor in normal and apoptotic cells. J Biol

Chem 280, 40216–40225.
24 Lechardeur D, Drzymala L, Sharma M, Zylka D,
Kinach R, Pacia J, Hicks C, Usmani N, Rommens JM
& Lukacs GL (2000) Determinants of the nuclear
localization of the heterodimeric DNA fragmentation
factor (ICAD ⁄ CAD). J Cell Biol 150, 321–334.
25 Woo EJ, Kim YG, Kim MS, Han WD, Shin S, Robin-
son H, Park SY & Oh BH (2004) Structural mecha-
nism for inactivation and activation of CAD ⁄ DFF40
in the apoptotic pathway. Mol Cell 14, 531–539.
26 Yuste VJ, Sa
´
nchez-Lo
´
pez I, Sole
´
C, Moubarak RS,
Bayascas JR, Dolcet X, Encinas M, Susin SA & Com-
ella JX (2005) The contribution of apoptosis-inducing
factor, caspase-activated DNase, and inhibitor of
caspase-activated DNase to the nuclear phenotype and
DNA degradation during apoptosis. J Biol Chem 280,
35670–35683.
27 Gu J, Dong RP, Zhang C, McLaughlin DF, Wu MX
& Schlossman SF (1999) Functional interaction of
DFF35 and DFF45 with caspase-activated DNA frag-
mentation nuclease DFF40. J Biol Chem 274, 20759–
20762.
28 Scholz SR, Korn C, Gimadutdinow O, Knoblauch M,
Pingoud A & Meiss G (2002) The effect of ICAD-S on

the formation and intracellular distribution of a nucle-
olytically active caspase-activated DNase. Nucleic
Acids Res 30, 3045–3051.
29 Samejima K & Earnshaw WC (2000) Differential
localization of ICAD-L and ICAD-S in cells due to
removal of a C-terminal NLS from ICAD-L by alter-
native splicing. Exp Cell Res 255, 314–320.
30 Var
ˇ
echa M, Amrichova
´
J, Zimmermann M, Ulman V,
Luka
´
s
ˇ
ova
´
E & Kozubek M (2007) Bioinformatic and
image analyses of the cellular localization of the apop-
totic proteins endonuclease G, AIF, and AMID during
apoptosis in human cells. Apoptosis 12, 1155–1171.
31 Uren RT, Dewson G, Bonzon C, Lithgow T, New-
meyer DD & Kluck RM (2005) Mitochondrial release
of pro-apoptotic proteins: electrostatic interactions can
hold cytochrome c but not Smac ⁄ DIABLO to mito-
chondrial membranes.
J Biol Chem 280, 2266–2274.
32 Bajt ML, Cover C, Lemasters JJ & Jaeschke H (2006)
Nuclear translocation of endonuclease G and apopto-

sis-inducing factor during acetaminophen-induced liver
cell injury. Toxicol Sci 94, 217–225.
33 Kalinowska M, Garncarz W, Pietrowska M, Garrard
WT & Widlak P (2005) Regulation of the human
apoptotic DNase ⁄ RNase endonuclease G: involvement
of Hsp70 and ATP. Apoptosis 10, 821–830.
34 Miramar MD, Costantini P, Ravagnan L, Saraiva
LM, Haouzi D, Brothers G, Penninger JM, Peleato
ML, Kroemer G & Susin SA (2001) NADH oxidase
activity of mitochondrial apoptosis-inducing factor.
J Biol Chem 276, 16391–16398.
35 Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow
BE, Brothers GM, Mangion J, Jacotot E, Costantini
P, Loeffler M et al. (1999) Molecular characterization
of mitochondrial apoptosis-inducing factor. Nature
397, 441–446.
36 Artus C, Boujrad H, Bouharrour A, Brunelle MN,
Hoos S, Yuste VJ, Lenormand P, Rousselle JC,
Namane A, England P et al. (2010) AIF promotes
chromatinolysis and caspase-independent programmed
necrosis by interacting with histone H2AX. EMBO J
29, 1585–1599.
37 Riedl SJ & Shi Y (2004) Molecular mechanisms of
caspase regulation during apoptosis. Nat Rev Mol Cell
Biol Rev 5, 897–907.
38 Riedl SJ & Salvesen GS (2007) The apoptosome: sig-
nalling platform of cell death. Nat Rev Mol Cell Biol
Rev 8, 405–413.
39 Guerrero AD, Chen M & Wang J (2008) Delineation of
the caspase-9 signaling cascade. Apoptosis 13, 177–186.

40 Slee EA, Adrain C & Martin SJ (2001) Executioner
caspase-3, -6, and -7 perform distinct, non-redundant
roles during the demolition phase of apoptosis. J Biol
Chem 276, 7320–7326.
41 Micheau O & Tschopp J (2003) Induction of TNF
receptor I-mediated apoptosis via two sequential
signaling complexes. Cell 114, 181–190.
42 Wilson NS, Dixit V & Ashkenazi A (2009) Death
receptor signal transducers: nodes of coordination in
immune signaling networks. Nat Immunol Rev 10,
348–355.
43 Saelens X, Festjens N, Vande Walle L, van Gurp M,
van Loo G & Vandenabeele P (2004) Toxic proteins
released from mitochondria in cell death. Oncogene
Rev 23, 2861–2874.
44 Donepudi M & Gru
¨
tter MG (2002) Structure and
zymogen activation of caspases. Biophys Chem Rev
101–102, 145–153.
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 437
45 Piana S, Sulpizi M & Rothlisberger U (2003) Struc-
ture-based thermodynamic analysis of caspases reveals
key residues for dimerization and activity. Biochemistry
42, 8720–8728.
46 Boatright KM, Renatus M, Scott FL, Sperandio S,
Shin H, Pedersen IM, Ricci JE, Edris WA, Sutherlin
DP, Green DR et al. (2003) A unified model for apical
caspase activation. Mol Cell 11, 529–541.

47 Malladi S, Challa-Malladi M, Fearnhead HO & Brat-
ton SB (2009) The Apaf-1 procaspase-9 apoptosome
complex functions as a proteolytic-based molecular
timer. EMBO J 28, 1916–1925.
48 Denault JB, Eckelman BP, Shin H, Pop C & Salvesen
GS (2007) Caspase 3 attenuates XIAP (X-linked inhib-
itor of apoptosis protein)-mediated inhibition of
caspase 9. Biochem J 405, 11–19.
49 Egger L, Schneider J, Rheˆ me C, Tapernoux M, Ha
¨
cki
J & Borner C (2003) Serine proteases mediate
apoptosis-like cell death and phagocytosis under
caspase-inhibiting conditions. Cell Death Differ 10,
1188–1203.
50 Paulsen M, Ussat S, Jakob M, Scherer G, Lepenies I,
Schu
¨
tze S, Kabelitz D & Adam-Klages S (2008) Inter-
action with XIAP prevents full caspase-3 ⁄ -7 activation
in proliferating human T lymphocytes. Eur J Immunol
38, 1979–1987.
51 Park HY, Song MG, Lee JS, Kim JW, Jin JO, Park
JI, Chang YC & Kwak JY (2007) Apoptosis of human
neutrophils induced by protein phosphatase 1 ⁄ 2A inhi-
bition is caspase-independent and serine protease-
dependent. J Cell Physiol 212, 450–462.
52 Tang D & Kidd VJ (1998) Cleavage of DFF-45 ⁄ ICAD
by multiple caspases is essential for its function during
apoptosis. J Biol Chem 273, 28549–28552.

53 Sakahira H, Enari M & Nagata S (1998) Cleavage of
CAD inhibitor in CAD activation and DNA degrada-
tion during apoptosis. Nature 391, 96–99.
54 Agarwal A, Mahfouz RZ, Sharma RK, Sarkar O,
Mangrola D & Mathur PP (2009) Potential biological
role of poly (ADP-ribose) polymerase (PARP) in male
gametes. Reprod Biol Endocrinol Rev 7, 143–162.
55 Germain M, Affer EB, D’Amours D, Dixit VM, Salve-
sen GS & Poirier GG (1999) Cleavage of automodified
poly(ADP-ribose) polymerase during apoptosis.
Evidence for involvement of caspase-7. J Biol Chem
274, 28379–28384.
56 Walsh JG, Cullen SP, Sheridan C, Lu
¨
thi AU, Gerner
C & Martin SJ (2008) Executioner caspase-3 and cas-
pase-7 are functionally distinct proteases. Proc Natl
Acad Sci USA 105, 12815–12819.
57 Milisav I, Nipic
ˇ
D&S
ˇ
uput D (2009) The riddle of
mitochondrial caspase-3 from liver. Apoptosis 14,
1070–1075.
58 van Loo G, Saelens X, Matthijssens F, Schotte P,
Beyaert R, Declercq W & Vandenabeele P (2002)
Caspases are not localized in mitochondria during life
or death. Cell Death Differ
9, 1207–1211.

59 Kamada S, Kikkawa U, Tsujumoto Y & Hunter T
(2005) Nuclear translocation of caspase-3 is dependent
on its proteolytic activation and recognition of a sub-
strate-like protein(s). J Biol Chem 280, 857–860.
60 Arnoult D, Gaume B, Karbowski M, Sharpe JC,
Cecconi F & Youle RJ (2003) Mitochondrial release of
AIF and EndoG requires caspase activation down-
stream of Bax ⁄ Bak-mediated permeabilization. EMBO
J 22, 4385–4399.
61 Suzuki Y, Takahashi-Niki K, Akagi T, Hashikawa T
& Takahashi R (2004) Mitochondrial protease Omi ⁄
HtrA2 enhances caspase activation through multiple
pathways. Cell Death Differ 11, 208–216.
62 Marabese M, Mazzoletti M, Vikhanskaya F & Brog-
gini M (2008) HtrA2 enhances the apoptotic functions
of p73 on bax. Cell Death Differ 15, 849–858.
63 Verhagen AM, Silke J, Ekert PG, Pakusch M, Kauf-
mann H, Connolly LM, Day CL, Tikoo A, Burke R,
Wrobel C et al. (2002) HtrA2 promotes cell death
through its serine protease activity and its ability to
antagonize inhibitor of apoptosis proteins. J Biol
Chem 277, 445–454.
64 Blink E, Maianski NA, Alnemri ES, Zervos AS, Roos
D & Kuijpers TW (2004) Intramitochondrial serine
protease activity of Omi ⁄ HtrA2 is required for cas-
pase-independent cell death of human neutrophils. Cell
Death Differ 11, 937–939.
65 Krishna M & Narang H (2008) The complexity of
mitogen-activated protein kinases (MAPKs) made sim-
ple. Cell Mol Life Sci Rev 65, 3525–3544.

66 Junttila MR, Li SP & Westermarck J (2008) Phospha-
tase-mediated crosstalk between MAPK signaling
pathways in the regulation of cell survival. FASEB J
Rev 22, 954–965.
67 Wada T & Penninger JM (2004) Mitogen-activated
protein kinases in apoptosis regulation. Oncogene Rev
23, 2838–2849.
68 Ray RM, Bhattacharya S & Johnson LR (2005) Pro-
tein phosphatase 2A regulates apoptosis in intestinal
epithelial cells. J Biol Chem 280, 31091–31100.
69 Pucci B, Indelicato M, Paradisi V, Reali V, Pellegrini
L, Aventaggiato M, Karpinich NO, Fini M, Russo
MA, Farber JL et al. (2009) ERK-1 MAP kinase pre-
vents TNF-induced apoptosis through Bad phospho-
rylation and inhibition of Bax translocation in HeLa
Cells. J Cell Biochem 108, 1166–1174.
70 Grethe S & Po
¨
rn-Ares MI (2006) p38 MAPK regulates
phosphorylation of Bad via PP2A-dependent suppres-
sion of the MEK1 ⁄ 2-ERK1 ⁄ 2 survival pathway in
TNF-a induced endothelial apoptosis. Cell Signal 18,
531–540.
71 Avdi NJ, Malcolm KC, Nick JA & Worthen GS
(2002) A role for protein phosphatase-2A in p38
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
438 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
mitogen-activated protein kinase-mediated regulation
of the c-Jun NH
2

-terminal kinase pathway in human
neutrophils. J Biol Chem 277, 40687–40696.
72 Eichhorn PJ, Creyghton MP & Bernards R (2009) Pro-
tein phosphatase 2A regulatory subunits and cancer.
Biochim Biophys Acta Rev 1795, 1–15.
73 Lomonosova E & Chinnadurai G (2009) BH3-only
proteins in apoptosis and beyond: an overview. Onco-
gene Rev 27, S2–S19.
74 Skommer J, Wlodkowic D & Deptala A (2007) Larger
than life: mitochondria and the Bcl-2 family. Leuk Res
Rev 31, 277–286.
75 Petros AM, Olejniczak ET & Fesik SW (2004) Struc-
tural biology of the Bcl-2 family of proteins. Biochim
Biophys Acta Rev 1644, 83–94.
76 Dewson G & Kluck RM (2009) Mechanisms by which
Bak and Bax permeabilise mitochondria during apop-
tosis. J Cell Sci Rev 122, 2801–2808.
77 Sunayama J, Tsuruta F, Masuyama N & Gotoh Y
(2005) JNK antagonizes Akt-mediated survival signals
by phosphorylating 14-3-3. J Cell Biol 170, 295–304.
78 Kodama Y, Taura K, Miura K, Schnabl B, Osawa Y
& Brenner DA (2009) Antiapoptotic effect of c-Jun
N-terminal Kinase-1 through Mcl-1 stabilization in
TNF-induced hepatocyte apoptosis. Gastroenterology
136, 1423–1434.
79 Kobayashi S, Lee SH, Meng XW, Mott JL, Bronk SF,
Werneburg NW, Craig RW, Kaufmann SH & Gores
GJ (2007) Serine 64 phosphorylation enhances the
antiapoptotic function of Mcl-1. J Biol Chem 282,
18407–18417.

80 Maurer U, Charvet C, Wagman AS, Dejardin E &
Green DR (2006) Glycogen synthase kinase-3 regulates
mitochondrial outer membrane permeabilization and
apoptosis by destabilization of MCL-1. Mol Cell 21,
749–760.
81 Upreti M, Galitovskaya EN, Chu R, Tackett AJ, Terr-
ano DT, Granell S & Chambers TC (2008) Identifica-
tion of the major phosphorylation site in Bcl-xL
induced by microtubule inhibitors and analysis of its
functional significance. J Biol Chem 283, 35517–35525.
82 Deng X, Gao F, Flagg T & May WS Jr (2004) Mono-
and multisite phosphorylation enhances Bcl2’s anti-
apoptotic function and inhibition of cell cycle entry
functions. Proc Natl Acad Sci USA 101, 153–158.
83 Gardai SJ, Hildeman DA, Frankel SK, Whitlock BB,
Frasch SC, Borregaard N, Marrack P, Bratton DL &
Henson PM (2004) Phosphorylation of Bax Ser184 by
Akt regulates its activity and apoptosis in neutrophils.
J Biol Chem 279, 21085–21095.
84 Xin M & Deng X (2006) Protein phosphatase 2A
enhances the proapoptotic function of Bax through
dephosphorylation. J Biol Chem 281, 18859–18867.
85 Dejean LM, Martinez-Caballero S, Guo L, Hughes C,
Teijido O, Ducret T, Ichas F, Korsmeyer SJ, Antons-
son B, Jonas EA et al. (2005) Oligomeric Bax is a
component of the putative cytochrome c release chan-
nel MAC, mitochondrial apoptosis-induced channel.
Mol Biol Cell 16, 2424–2432.
86 Chiang CW, Kanies C, Kim KW, Fang WB, Park-
hurst C, Xie M, Henry T & Yang E (2003) Protein

phosphatase 2A dephosphorylation of phosphoserine
112 plays the gatekeeper role for BAD-mediated apop-
tosis. Mol Cell Biol 23, 6350–6362.
87 Tsuruta F, Sunayama J, Mori Y, Hattori S, Shimizu
S, Tsujimoto Y, Yoshioka K, Masuyama N & Gotoh
Y (2004) JNK promotes Bax translocation to mito-
chondria through phosphorylation of 14-3-3 proteins.
EMBO J 23, 1889–1899.
88 Allan LA & Clarke PR (2009) Apoptosis and auto-
phagy: regulation of caspase-9 by phosphorylation.
FEBS J Rev 276, 6063–6073.
89 Martin MC, Allan LA, Mancini EJ & Clarke PR
(2008) The docking interaction of caspase-9 with
ERK2 provides a mechanism for the selective inhibi-
tory phosphorylation of caspase-9 at threonine 125.
J Biol Chem 283, 3854–3865.
90 Jia SH, Parodo J, Kapus A, Rotstein OD & Marshall
JC (2008) Dynamic regulation of neutrophil survival
through tyrosine phosphorylation or dephosphoryla-
tion of caspase-8. J Biol Chem 283, 5402–5413.
91 Cursi S, Rufini A, Stagni V, Condo
`
I, Matafora V,
Bachi A, Bonifazi AP, Coppola L, Superti-Furga G,
Testi R et al. (2006) Src kinase phosphorylates cas-
pase-8 on Tyr380: a novel mechanism of apoptosis
suppression. EMBO J 25, 1895–1905.
92 Dickey JS, Redon CE, Nakamura AJ, Baird BJ, Sedel-
nikova OA & Bonner WM (2009) H2AX: functional
roles and potential applications. Chromosoma Rev 118,

683–692.
93 Cook PJ, Ju BG, Telese F, Wang X, Glass CK &
Rosenfeld MG (2009) Tyrosine dephosphorylation of
H2AX modulates apoptosis and survival decisions.
Nature 458, 591–596.
94 Solier S & Pommier Y (2009) The apoptotic ring: a
novel entity with phosphorylated histones H2AX and
H2B and activated DNA damage response kinases.
Cell Cycle 8, 1853–1859.
95 Lu C, Zhu F, Cho YY, Tang F, Zykova T, Ma WY,
Bode AM & Dong Z (2006) Cell apoptosis: require-
ment of H2AX in DNA ladder formation, but not for
the activation of caspase-3. Mol Cell 23, 121–132.
96 Paull TT, Rogakou EP, Yamazaki V, Kirchgessner
CU, Gellert M & Bonner WM (2000) A critical role
for histone H2AX in recruitment of repair factors
to nuclear foci after DNA damage. Curr Biol 10 ,
886–895.
97 Harris SL & Levine AJ (2005) The p53 pathway: posi-
tive and negative feedback loops. Oncogene Rev 24,
2899–2908.
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 439
98 Cai X & Liu X (2008) Inhibition of Thr-55 phosphory-
lation restores p53 nuclear localization and sensitizes
cancer cells to DNA damage. Proc Natl Acad Sci USA
105, 16958–16963.
99 Gabai VL, Sherman MY & Yaglom JA (2010) HSP72
depletion suppresses cH2AX activation by genotoxic
stresses via p53 ⁄ p21 signaling. Oncogene 29, 1952–

1962.
100 Kraft CA, Efimova T & Eckert RL (2007) Activation
of PKCd and p38d MAPK during okadaic acid depen-
dent keratinocyte apoptosis. Arch Dermatol Res 299,
71–83.
101 Wang Y, Zhang B, Peng X, Perpetua M &
Harbrecht BG (2008) Bcl-xL prevents staurosporine-
induced hepatocyte apoptosis by restoring protein
kinase B ⁄ mitogen-activated protein kinase activity
and mitochondria integrity. J Cell Physiol 215,
676–683.
102 Vale C & Botana LM (2008) Marine toxins and the
cytoskeleton: okadaic acid and dinophysistoxins. FEBS
J Rev 275, 6060–6066.
103 Walsh AH, Cheng A & Honkanen RE (1997) Fostrie-
cin, an antitumor antibiotic with inhibitory activity
against serine ⁄ threonine protein phosphatases types 1
(PP1) and 2A (PP2A), is highly selective for PP2A.
FEBS Lett 416, 230–234.
104 Boudreau RT, Conrad DM & Hoskin DW (2007)
Apoptosis induced by protein phosphatase 2A (PP2A)
inhibition in T leukemia cells is negatively regulated by
PP2A-associated p38 mitogen-activated protein kinase.
Cell Signal 19, 139–151.
105 Antony R, Lukiw WJ & Bazan NG (2010) Neuropro-
tectin D1 induces dephosphorylation of BCL-X
L
in a
PP2A-dependent manner during oxidative stress and
promotes retinal pigment epithelial cell survival. J Biol

Chem 285, 18301–18308.
106 Domina AM, Vrana JA, Gregory MA, Hann SR &
Craig RW (2004) MCL1 is phosphorylated in
the PEST region and stabilized upon ERK activa-
tion in viable cells, and at additional sites with
cytotoxic okadaic acid or taxol. Oncogene 23, 5301–
5315.
107 Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM,
Newmeyer DD, Schuler M & Green DR (2004) Direct
activation of Bax by p53 mediates mitochondrial mem-
brane permeabilization and apoptosis. Science 303,
1010–1014.
108 Li DC, Liu JP, Schmid PC, Schlosser R, Feng H, Liu
WB, Yan Q, Gong L, Sun SM, Deng M et al. (2006)
Protein serine ⁄ threonine phosphatase-1 dephosphory-
lates p53 at Ser-15 and Ser-37 to modulate its tran-
scriptional and apoptotic activities. Oncogene 25,
3006–3022.
109 Haneda M, Kojima E, Nishikimi A, Hasegawa T,
Nakashima I & Isobe K (2004) Protein phosphatase 1,
but not protein phosphatase 2A, dephosphorylates
DNA-damaging stress-induced phospho-serine 15 of
p53. FEBS Lett 567, 171–174.
110 Dohoney KM, Guillerm C, Whiteford C, Elbi C,
Lambert PF, Hager GL & Brady JN (2004)
Phosphorylation of p53 at serine 37 is important for
transcriptional activity and regulation in response to
DNA damage. Oncogene 23, 49–57.
111 Qin J, Chen HG, Yan Q, Deng M, Liu J, Doerge S,
Ma W, Dong Z & Li DW (2008) Protein phosphatase-

2A is a target of epigallocatechin-3-gallate and modu-
lates p53-Bak apoptotic pathway. Cancer Res 68,
4150–4162.
112 Mi J, Bolesta E, Brautigan DL & Larner JM (2009)
PP2A regulates ionizing radiation-induced apoptosis
through Ser46 phosphorylation of p53. Mol Cancer
Ther 8, 135–140.
113 Chowdhury D, Keogh MC, Ishii H, Peterson CL,
Buratowski S & Lieberman J (2005) c-H2AX
dephosphorylation by protein phosphatase 2A
facilitates DNA double-strand break repair. Mol Cell
20, 801–809.
114 Zuluaga S, Alvarez-Barrientos A, Gutie
´
rrez-Uzquiza
A, Benito M, Nebreda AR & Porras A (2007) Nega-
tive regulation of Akt activity by p38a MAP kinase in
cardiomyocytes involves membrane localization of
PP2A through interaction with caveolin-1. Cell Signal
19, 62–74.
115 Deng X, Gao F & May WS (2009) Protein phospha-
tase 2A inactivates Bcl2’s antiapoptotic function by
dephosphorylation and up-regulation of Bcl2-p53 bind-
ing. Blood 113, 422–428.
116 Souid-Mensi G, Moukha S, Mobio TA, Maaroufi K
& Creppy EE (2008) The cytotoxicity and genotoxicity
of okadaic acid are cell-line dependent. Toxicon 51,
1338–1344.
117 Hemmings BA, Favre B & Turowski P (1997) Differ-
ential inhibition and posttranslational modification of

protein phosphatase 1 and 2A in MCF7 cells treated
with calyculin-A, okadaic acid, and tautomycin. J Biol
Chem 272, 13856–13863.
118 Chatfield K & Eastman A (2004) Inhibitors of protein
phosphatases 1 and 2A differentially prevent intrinsic
and extrinsic apoptosis pathways. Biochem Biophys
Res Commun 323, 1313–1320.
119 Alvarado-Kristensson M & Andersson T (2005)
Protein phosphatase 2A regulates apoptosis in
neutrophils by dephosphorylating both p38 MAPK
and its substrate caspase 3. J Biol Chem 280, 6238–
6244.
120 Wang Y, Sun L, Xia C, Ye L & Wang B (2009)
P38MAPK regulates caspase-3 by binding to caspase-3
in nucleus of human hepatoma Bel-7402 cells during
anti-Fas antibody- and actinomycin D-induced apop-
tosis. Biomed Pharmacother 63, 343–350.
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
440 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
121 Roy SS, Madesh M, Davies E, Antonsson B, Danial
N & Hajno
´
czky G (2009) Bad targets the permeability
transition pore independent of Bax or Bak to switch
between Ca2+-dependent cell survival and death. Mol
Cell 33, 377–388.
122 Kim BJ, Ryu SW & Song BJ (2006) JNK- and p38
kinase-mediated phosphorylation of Bax leads to its
activation and mitochondrial translocation and to
apoptosis of human hepatoma HepG2 cells. J Biol

Chem 281, 21256–21265.
123 Geng Y, Walls KC, Ghosh AP, Akhtar RS, Klocke BJ
& Roth KA (2010) Cytoplasmic p53 and activated
Bax regulate p53-dependent, transcription-independent
neural precursor cell apoptosis. J Histochem Cytochem
58, 265–275.
124 Be
´
ghin A, Matera EL, Brunet-Manquat S &
Dumontet C (2008) Expression of Arl2 is associ-
ated with p53 localization and chemosensitivity
in a breast cancer cell line. Cell Cycle 7, 3074–
3082.
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 441