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. 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(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 . ARTICLE Regulation of DNA fragmentation: the role of caspases and phosphorylation Ikuko Kitazumi and Masayoshi Tsukahara Bio Process Research and Development. under the direct control of kinases and phosphatases, and the indirect control of phosphorylation through the regula- tion of other apoptotic proteins. Furthermore,