Báo cáo khoa học: Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation

M I N I R E V I E W

Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation Lindsey A. Allan and Paul R. Clarke

Biomedical Research Institute, School of Medicine, College of Medicine, Dentistry and Nursing, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland, UK

Keywords apoptosis; caspase; mitosis; phosphorylation; protein kinase

Correspondence P. R. Clarke, Biomedical Research Institute, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK Fax: +44 (0) 1382 669993 Tel: +44 (0) 1382 425580 E-mail: p.r.clarke@dundee.ac.uk

(Received 23 March 2009, revised 6 July 2009, accepted 13 August 2009)

doi:10.1111/j.1742-4658.2009.07330.x

Cell death by the process of apoptosis plays important roles in development, tissue homeostasis, diseases and drug responses. The cysteine aspartyl prote- ase caspase-9 plays a central role in the mitochondrial or intrinsic apoptotic pathway that is engaged in response to many apoptotic stimuli. Caspase-9 is activated in a large multimeric complex, the apoptosome, which is formed with apoptotic peptidase activating factor 1 (Apaf-1) in response to the release of cytochrome c from mitochondria. Once activated, caspase-9 cleaves and activates the effector caspases 3 and 7 to bring about apoptosis. This pathway is tightly regulated at multiple steps, including apoptosome formation and caspase-9 activation. Recent work has shown that caspase-9 is the direct target for regulatory phosphorylation by multiple protein kinas- es activated in response to extracellular growth ⁄ survival factors, osmotic stress or during mitosis. Here, we review these advances and discuss the possible roles of caspase-9 phosphorylation in the regulation of apoptosis during development and in pathological states, including cancer.

Introduction

apoptotic machinery via this pathway, stimulating the release of cytochrome c from mitochondria [4–6]. In the cytosol, cytochrome c promotes the oligo- merization of apoptotic peptidase activating factor 1 (Apaf-1), leading to recruitment and activation of cas- pase-9 in a large complex called the apoptosome [2,7,8]. Once activated, caspase-9 cleaves and activates downstream effector caspases 3 and 7, which target key regulatory and structural proteins for proteolysis to effect cell death [1] (Fig. 1).

Abbreviations Apaf-1, apoptotic peptidase activating factor 1; AraC, cytosine arabinoside; Bad, Bcl-2 associated death promoter; Bcl-2, B-cell lymphoma 2; BH3, a Bcl-2 homology domain 3; Bid, BH3-interacting domain death agonist; CARD, caspase recruitment domain; CDK, cyclin-dependent kinase; CK2, casein kinase 2; ERK, extracellular signal-regulated kinase; GSKb, glycogen synthase kinase 3b; MAPK, mitogen-activated protein kinase; MEFs, mouse embryonic fibroblasts; MEK, mitogen-activated protein kinase kinase; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; siRNA, small interfering RNA; TPA, 12-O-tetradecanoylphorbol-13-acetate.

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Apoptosis is a controlled form of cell death that plays fundamental roles during embryonic development and in the maintenance of tissues. Defects in the regulation of apoptosis have been associated with the pathogene- sis of disease states such as neurodegeneration and cancer. Caspases, a family of cysteine proteases that cleave proteins at specific aspartyl residues, are instru- mental to the execution of apoptosis. These enzymes are expressed in cells as inactive or low-activity zymo- gens that require oligomerization and ⁄ or cleavage for activation [1]. Caspase-9 is the initiator caspase associ- ated with the intrinsic or mitochondrial pathway of apoptosis [2,3]. Many pro-apoptotic signals engage the Evidence for the critical role of caspase-9 during development and in the response to many pro-apop- totic stimuli is provided by genetic knockout studies in

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Regulation of caspase-9 by phosphorylation

Survival factor signalling pathways

Cell membrane

BH3

cAMP

Mitochondrion

MEK1,2

PKA

P

Hyperosmotic stress

Cytochrome C

ERK1,2 MAPK

Apaf-1

P

" Caspase-9

PKC

P

P

Mitosis

CDK1-cyclin B

Thr125

DYRK1A

Caspase-3

Caspase-7

Apopto(cid:2)c cell death

Fig. 1. Regulation of caspase-9 by protein kinase signalling pathways. Mitochondrial outer membrane permeabilization is initiated by pro-apoptotic proteins, containing a Bcl-2 homology domain 3 (BH3), in response to a variety of stimuli. Release of cytochrome c from mito- chondria induces the formation of the apoptosome, a multimeric complex formed by Apaf-1 and procaspase-9. Activation of caspase-9 in this complex results in the cleavage and activation of caspase-3 and caspase-7, which cleave key structural and regulatory proteins to bring about apoptotic cell death. Caspase-9 activation is regulated by protein kinases, including ERK1 ⁄ 2 and PKA, which are activated by extracellular growth ⁄ survival signals. Hyperosmotic stress induces inhibitory phosphorylation of caspase-9 by PKCf, whereas, in mitosis, CDK1-cyclin B1 phosphorylates Thr125, the same inhibitory site targeted by ERK1 ⁄ 2 in the interphase. Thr125 is also phosphorylated by DYRK1A, which regulates apoptosis during development.

[16] (Fig. 1). Human caspase-9 was first reported to be phosphorylated in 1998 [19], and it has subsequently been shown to be targeted by multiple protein kinases that respond to extracellular signals, cellular stresses or are activated during the cell cycle (Fig. 2A). Here, we review the phosphorylation of caspase-9 and we discuss the potential developmental and pathological relevance of this mechanism of regulation. the mitochondrial through mice. Caspase-9 null mice die perinatally, exhibiting enlarged, deformed brains associated with decreased apoptosis during brain development [9,10]. This is lar- gely reminiscent of defects observed in caspase-3 [11] and Apaf-1 [12,13] null mice. In addition, caspase-9 to a lesser extent, null embryonic stem cells and, mouse embryonic fibroblasts (MEFs) and thymocytes, are resistant to a wide variety of stimuli that induce apoptosis pathway [9,10,14]. The intrinsic apoptotic pathway is

Thr125: an integration point for multiple signalling pathways

Inhibition of caspase-9 activation by phosphorylation at Thr125

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A major inhibitory phosphorylation site in human cas- pase-9 is Thr125, which forms part of a Thr-Pro motif that is targeted by multiple proline-directed kinases in response to different stimuli and during specific phases of the cell-division cycle [20–23]. The Thr125 site is conserved in many (but not all) mammals, while Thr-Pro or Ser-Pro sites are also located in this region of caspase-9 from zebrafish and clawed frog (Fig. 2B). Phosphorylation of Thr125, or its mutation to the acidic residue Glu, which mimics constitutive phos- regulated at multiple stages to ensure that apoptosis does not occur inappropriately. Cytochrome c release from mitochon- dria is tightly controlled by B-cell lymphoma 2 (Bcl-2) family proteins that control the permeabilization of the outer mitochondrial membrane [15]. Downstream of cytochrome c release, apoptosome function may be influenced by the levels of expression of caspase-9 and Apaf-1, which are decreased in several tumour types [16]. The enzymatic activity of caspase-9 is inhibited directly by X-linked inhibitor of apoptosis (XIAP) [17], which can also mediate caspase-9 ubiquitination and degradation by the proteasome [18]. In addition, regulation of the apoptosome through protein phos- phorylation can provide acute control of the pathway

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Regulation of caspase-9 by phosphorylation

A

107 T T

125 T T

144 S S

T T

T T S

B Human Monkey Dog Mouse Rat Zebrafish Frog

99 KPTLENL PVVLRPE-----IRKPEVLRPE PRPVDIGSGGFGDVGALE LRG-- S KPTLENL PVVLRPE-----IRKPEVLRPE PRPVDIGSGGCGDVGAPE LRG-- S KLTPGKLAPVVLGPA-----ELQPQVVRPSVPRPTDNGSGRFSDVCVQEISKG-- S QPAVGNL PVVLGPEELWPARLKPEVLRPE PRPVDIGSGGAHDVCVPGKIRG-- S QPALGNL PVVLGPEELWPTRLRPEVLTPE PRPVDIGSGRAHDVCTPGKIER-- S DKPNIASPRLVPLRPE-------SLPVHKTY P-PSET-------VVRPTRPRR-- LQPIPTTP PV-LKP--------LPKAEPAEYP------------AREIR RKGTL

S

S

195

196

SS S

S

183 S S S S S S S

153 Y Y Y Y Y Y Y

Human Monkey Dog Mouse Rat Zebrafish Frog

NADLA ILSMEPCGHCLIINNVNFCRESGLRTRTG NIDCEKLRRRF LHFM 200 NADLA VLSMEPCGHCLIINNVNFCRESGLSTRTG SIDCEKLQRRF LLHFM 200 NGDLA ALNADPCGYCLIINNVNFCPESRLTARGG NIDCEKLQRRFCLLRFT 230 HADMA TLDSDPCGHCLIINNVNFCPSSGLGTRTG NLDRDKLEHRFRWLRFM 238 HADMA TLDSDPCGHCLIINNVNFCPSSGLSTRIG HVDCEKLQHRFCWLRFM 238 DSIQC KMDASPCGVCLIINNINFEKASELNDRKG NIDCDKLEKRFKALNFE 211 DKDKD PMSSDPIGFCLIINNMNFHECTGLSTRTG DIDRDKLANRMR FHFE 183

302 307 310

C

S S S S S

Human Monkey Dog Mouse Rat Zebrafish Frog

S S VAST PEDE PG NPEPDATP 318 S S VAST PEDE SG NPEPDATP 318 S S S VASA PEDR PG D EPDAVP 348 VACT SQGR LD D EPDAVP 356 T S S S S VAFT SQDKAFD D EPDAVP 356 VSPDDIQPCIGGIDDEMDAIP 329 VTSETPPLSPTSTSLQSDATP 301

Fig. 2. Caspase-9 is phosphorylated at multiple sites. (A) The diagram shows a linear representation of procaspase-9 showing the position of phosphorylation sites and their respective protein kinases. Phosphorylation sites conserved between human and mouse caspase-9 are shown in red, and nonconserved sites are shown in black. Sites of cleavage within the linker region between the large and small subunit domains that are targeted during autoprocessing (Casp-9) and by caspase-3 (Casp-3) are indicated. (B) Alignment of primary sequences of caspase-9 from vertebrates showing conservation of phosphorylated residues (in red; numbers correspond to the human sequence). Sequences are shown that correspond to the region of residues 99–200 in the human (Homo sapiens; NCBI reference sequence NP_001220.2), monkey (Macaca mulatta; XP_001082859.1; 93% identical to human caspase-9 over the whole sequence), dog (Canis lupus familiaris; XP_852163.1; 77.5%), mouse (Mus musculus; NP_056548.2; 78.6%), rat (Rattus norvegicus; NP_113820.1; 78.1%), zebrafish (Danio rerio; NP_001007405.1; 51.1%) and clawed frog (Xenopus laevis; NP_001079035; 51%). (C) Alignment of capase-9 sequences corre- sponding to residues 298-318 of human caspase-9. Confirmed sites of phosphorylation in mouse and human caspase-9 are underlined and the conservation of these sites in other mammalian caspase-9 sequences is highlighted in red. Potential phosphorylation sites (not highlighted) are also present in this region of caspase-9 from zebrafish and clawed frog.

(Thr125Ala) extracts depleted of endogenous

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phorylation at the site, inhibits caspase-9 processing induced by constitutively active Apaf-1 [21]. In cyto- solic caspase-9 and reconstituted with in vitro-translated caspase-9, phosphorylation of Thr125 blocks the ability of cytochrome c to induce caspase-3 activity [21]. In cells, replacement of endogenous caspase-9 with a nonphosphorylatable mutant sensitizes the cells to apoptosis induced by microtubule poisons that arrest cells in mitosis [20]. Thus, phosphorylation of Thr125 inhibits caspase-9 activation, compromises its ability to activate its downstream targets and plays

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Regulation of caspase-9 by phosphorylation

a critical role in the restraint of apoptosis in response to certain stimuli.

staurosporine. Both in cell extracts and in interphase mammalian cells stimulated with epidermal growth factor or the phorbol ester 12-O-tetradecanoylphorbol- 13-acetate (TPA), inhibitors of mitogen-activated pro- tein kinase kinase 1 (MEK1) and mitogen-activated protein kinase kinase 2 (MEK2), which prevent the activation of their substrates (ERK1 and ERK2), strongly inhibit the phosphorylation of Thr125 [21]. In vitro, Thr125 is directly phosphorylated by purified ERK2, which interacts with caspase-9 in cells through a docking domain situated in the N-terminal CARD of caspase-9 (Fig. 2A). The interaction of ERK2 with cas- pase-9 and the phosphorylation of Thr125 requires Arg10 in caspase-9, which is likely to form an interac- tion with Asp160 within a 157TTCD160 motif that is present in ERK2, but not in c-Jun N-terminal kinase or p38a [22].

Exactly how phosphorylation of Thr125 inhibits caspase-9 activation is not certain because this process is not understood fully at the molecular level. At pres- ent, structural data are not available for the region encompassing Thr125, which comprises a putative flexible linker region between the caspase recruitment domain (CARD) (through which caspase-9 binds Apaf- 1) and the large subunit of the processed enzyme [24] (Fig. 2A). Phosphorylation at Thr125 might inhibit a conformational change associated with the activation of caspase-9 bound to Apaf-1, although such a conforma- tional change is still contentious [25,26]. Caspase-9 recruitment to Apaf-1 is unaffected by Thr125 phos- phorylation [21], so the phosphorylated protein has the potential to act in a dominant negative manner, com- promising the activation of unphosphorylated caspase-9 molecules recruited to the same apoptosome platform [8]. This might occur either through effectively lowering the concentration of caspase-9 monomers capable of dimer formation on the apoptosome, or through the for- mation of heterodimers between phosphorylated and unphosphorylated caspase-9 molecules that cannot be activated.

MEK1 ⁄ 2 inhibitors that reduce caspase-9 phosphor- ylation at Thr125, including PD98059, PD184352, UO126 and PD0325901 [21,23], do not distinguish between ERK1 and ERK2. Indeed, it is likely that ERK1, as well as ERK2, can phosphorylate caspase-9 at Thr125, because the substrate specificity of these kinases is indistinguishable. MEK1 ⁄ 2 inhibitors also target MEK5 (with some differences in potency) thereby blocking activation of the downstream MAPK ERK5 [27]. However, ERK5 does not appear to play any role in the phosphorylation of caspase-9 at Thr125 under these conditions [21,28]. Phosphorylation by the mitogen-activated protein kinase extracellular signal-regulated kinase

Phosphorylation by cyclin-dependent kinase 1-cyclin B1

strategy, we demonstrated that Thr125 was first identified as an inhibitory phosphory- lation-site caspase-9 using a cell-free system made from cytosolic extracts of human HeLa cells in which the intrinsic apoptotic pathway was activated by addition of cytochrome c [21]. In this system, caspase-9 activa- tion was inhibited by okadaic acid (an inhibitor of type 1 and type 2A serine-threonine phosphatases) that not only inhibits the dephosphorylation of caspase-9 but also increases the activity of protein kinases that target caspase-9 such as extracellular signal-regulated kinase 1 (ERK1) and extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinases (MAPKs) by inducing their phosphorylation in the extracts. Isolation of caspase-9 from the extracts, and analysis of a tryptic digest by mass spectrometry, iden- tified Thr125 as the major phosphorylation site, with Thr107 as an additional minor site also induced by okadaic acid (Fig. 2A). An antibody that specifically recognizes caspase-9 when phosphorylated at Thr125 was raised and has been used subsequently to study the regulation of Thr125 phosphorylation [21].

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Phosphorylation of Thr125 in cell extracts was inhib- ited by the broad-specificity protein kinase inhibitor Analysis of the phosphorylation of Thr125 during the cell cycle revealed that this inhibitory site was phos- phorylated in both G1 and S phases, when ERK1 and ERK2 are activated following release from a thymidine arrest, and in mitosis, when ERK1 and ERK2 are not active [20]. Phosphorylation of Thr125 in mitotic cells is mediated by the major mitotic cyclin-dependent kinase (CDK), CDK1-cyclin B1, which forms a stable complex with caspase-9 during the G2 phase and mito- sis. CDK1-cyclin B1-mediated Thr125 phosphorylation was also observed when cells were arrested in mitosis with the microtubule poisons nocodazole or taxol (Fig. 3). These drugs can also induce apoptosis in a caspase-9-dependent manner, because death is pre- vented in cells depleted of caspase-9 [20] and in cas- pase-9 null MEFs [14,29]. Using an RNA interference and rescue cells expressing a nonphosphorylatable caspase-9, in which Thr125 was mutated to Ala, were more susceptible to apoptosis induced by microtubule poisons [20]. Immu-

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Regulation of caspase-9 by phosphorylation

A

plays a critical role in determining the sensitivity of these cells to cell killing by microtubule poisons.

threshold could restrain apoptosis

B

signals generated during active

The demonstration that phosphorylation of caspase- 9 at Thr125 exhibits temporal regulation during the cell division cycle established a direct link between the cell cycle machinery and the intrinsic pathway of apoptosis [20,30]. These findings raised the possibility that the inhibitory phosphorylation of caspase-9 sets a variable threshold for the activation of downstream caspases and subsequent apoptosis during the cell cycle. This in response to intrinsic signals generated during specific stages of the cell cycle. For example, the inhibitory phosphorylation of caspase-9 by the ERK1 ⁄ 2 MAPK could provide protection against potentially pro-apop- cell growth totic Increased induced following mitogenic stimulation. phosphorylation of caspase-9 by CDK1-cyclin B1 would raise the threshold for apoptosis to be triggered during mitosis. This might be necessary to protect mitotic cells against the loss of extracellular survival factor signalling when dividing cells round up or because intracellular apoptotic signals are generated during the major reorganization of the cell that facili- tates chromosome segregation [30].

Fig. 3. Phosphorylation of caspase-9 at Thr125 restrains apoptosis during mitosis. (A) Phosphorylation of caspase-9 during mitosis by CDK1-cyclin B1 inhibits apoptosis. We propose that CDK1-cyclin B1 also induces an upstream apoptotic signal, either directly or indi- rectly through the induction of mitosis. In the case of a normal mitosis, inactivation of CDK1-cyclin B1 by the rapid degradation of cyclin B1 after the mitotic checkpoint is satisfied causes exit from mitosis, inactivation of the apoptotic signal and dephosphorylation of caspase-9. (B) During a prolonged mitotic arrest caused by spon- taneous defects in spindle assembly or in response to anti-mitotic drugs, we propose that an upstream apoptotic signal accumulates while caspase-9 is slowly dephosphorylated. When the upstream signal overcomes the threshold set by caspase-9 phosphorylation, the caspase cascade is initiated and causes apoptosis. Whether caspase activation is initiated in the mitotic state or after exit from mitosis would depend on the relative rates of caspase-9 dephos- phorylation and the slow degradation of cyclin B1, which results in mitotic slippage when it drops below a threshold required to maintain mitosis.

the spindle assembly checkpoint

subsequently following exit

The phosphorylation of caspase-9 Thr125 plays a particularly important role in cells treated with micro- tubule poisons [31]. There is increasing evidence that the apoptotic response of cells to such drugs requires a protracted delay in mitosis [32]. For example, cell killing by antimitotic drugs, such as nocodazole [33], taxol [34] and a mitotic kinesin inhibitor [35], requires the activity of to maintain the mitotic state. During an extended mito- tic arrest, cyclin B1 is degraded slowly [32] and this is associated with a gradual net dephosphorylation of caspase-9 (Allan & Clarke, unpublished data). When the level of cyclin B1 falls below the threshold required to maintain the mitotic state, a cell exits mitosis prematurely by a process called mitotic slip- page [32]. In these circumstances, caspase-9 dephos- phorylation would also occur, presumably resulting in initiation of apoptosis from a G1-like state if an upstream apoptotic signal is still present. Thus, the propensity of a cell to initiate apoptosis during mito- sis, or from mitosis, would depend upon the relative threshold of apop- tosis set by caspase-9 phosphorylation compared with the threshold for mitotic exit determined by cyclin B levels [31]. That survival

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nostaining for histone H3 Ser10 phosphorylation, a commonly used mitotic marker, together with active caspase-3, showed that caspase activation was initiated during mitosis but peaked much earlier in the absence of Thr125 phosphorylation. This work demonstrated that phosphorylation of caspase-9 at Thr125 restrains apoptosis during mitotic arrest in human cells and in response to microtubule poisons requires restraint of caspase-9 activity by Thr125 phos- phorylation indicates that a prolonged mitotic arrest

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Regulation of caspase-9 by phosphorylation

caspase-9 at Thr125 by DYRK1A plays a role in its anti-apoptotic activity [40].

stress. to hyperosmotic

engenders an as-yet undefined pro-apoptotic signal (Fig. 3B). Interestingly, recent work has demonstrated that cells exhibit considerable variation in their apop- totic response to such conditions [36,37] and this is influenced by the level of cyclin B1 [37]. Thus, the thresholds for initiation of apoptosis and exit from mitosis are likely to differ between cell types. The vari- ation in responses of individual cells of a certain type suggests a stochastic element in the process, perhaps in the relative rates of caspase-9 dephosphorylation and cyclin B1 degradation.

Phosphorylation of Thr125 by DYRK1A and the role of p38a MAPK

DYRK1A also plays a role in the phosphorylation of Thr125 of caspase-9 in human and mouse cells Interestingly, responding although acute chemical inhibition of the stress-acti- vated MAPK p38a has only a small inhibitory effect on Thr125 phosphorylation induced under these condi- tions, the response is completely ablated in p38a) ⁄ ) MEFs [28]. These results indicate an unexpected func- tional interaction between p38a and DYRK1A in the response to hyperosmotic stress. Although p38a might itself phosphorylate Thr125, it is unlikely to interact with caspase-9 through the same docking mechanism as ERK2 [22] and may therefore control the phosphor- ylation of caspase-9 indirectly in conjuction with DYRK1A [28].

Additional sites of phosphorylation in caspase-9

Ser196 and protein kinase B

In addition to its phosphorylation catalysed by ERK1 ⁄ 2 and CDK1-cyclin B1, Thr125 of human cas- pase-9 displays a basal level of phosphorylation in cells that persists when both of these pathways are inhibited chemically. The residual phosphorylation is inhibited when the dual-specificity tyrosine-phosphorylation reg- ulated protein kinase DYRK1A is ablated using small interfering RNA (siRNA) or is inhibited chemically by harmine [23]. DYRK1A is implicated in Down syn- drome (trisomy 21), being encoded by a gene in the Down Critical Region on chromosome 21. Consistent with an ability to phosphorylate Thr125 of caspase-9 directly, the mature DYRK1A enzyme exhibits ser- ine ⁄ threonine kinase activity towards other substrates with a minimal sequence specificity (Ser ⁄ Thr-Pro) like that of MAPKs and CDKs to which it is distantly related [38,39]. DYRK1A also interacts with caspase-9, and co-expression experiments showed that DYRK1A inhibited the cleavage of wild-type caspase-9 associated with its activation but did not inhibit the cleavage of the Thr125Ala nonphosphorylatable mutant [23,40].

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Cardone et al. [19] reported that caspase-9 was phos- phorylated at Ser196 by protein kinase B (PKB), also known as Akt (Fig. 2A). Indeed, this was the first such report of caspase-9 phosphorylation. Cytosolic extracts made from cells with elevated PKB activity as a result of transfection with an activated mutant of the small GTPase Ras failed to support caspase activation fol- lowing the addition of cytochrome c. This inhibition was lost if cells were incubated with an inhibitor of phosphoinositide 3-kinase, the upstream activator of PKB, before the preparation of cytosolic extracts. Furthermore, apoptosis induced by transfection of wild-type caspase-9 was inhibited by the co-expression of active PKB. This effect was partially lost when caspase-9 Ser196Ala, a mutant that cannot be phos- phorylated at Ser196, was used. However, inhibition of caspase-3-like activity was observed in MDCK cells, a canine kidney cell line, even though the Ser196 phos- phorylation site is absent in dog caspase-9 (Fig. 2B). The site is not conserved in monkey, mouse or rat, and PKB fails to phosphorylate murine caspase-9 in vitro [41]. Thus, a direct role for PKB in caspase-9 regulation remains unclear. Additional targets through which PKB mediates its anti-apoptotic effects are known, for exam- ple, Bcl-2-associated death promoter (Bad) and glyco- gen synthase kinase 3b (GSK3b) [42]. However, none is able to explain the ability of PKB to inhibit apoptosis downstream of cytochrome c release, suggesting that PKB targets as-yet unidentified substrates to compro- mise caspase activation. DYRK1A is a predominantly nuclear kinase and appears to phosphorylate caspase-9 in the nucleus, because caspase-9 it preferentially phosphorylates tagged with a nuclear localization signal compared to that tagged with a nuclear export signal [23]. This is interesting because, although caspase-9 is considered to be predominantly cytoplasmic, it has been observed in the nucleus [23], and its phosphorylation there by DYRK1A suggests that there might be specific regula- tion of nuclear caspase-9. Alternatively, caspase-9 might be phosphorylated in the nucleus before relocat- ing to the cytoplasm, providing a mechanism by which nuclear DYRK1A might regulate the activity of cas- pase-9 in the cytoplasm. DRYK1A has recently been shown to play an important role in the control of apoptosis during retinal cell development in the mouse and it has been suggested that phosphorylation of

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Regulation of caspase-9 by phosphorylation

Phosphorylation of Ser144 by protein kinase C zeta

this mutant prevented apoptosis

processing was blocked either by an inhibitor of c-Abl kinase activity or in cells expressing a nonphosphory- latable mutant of caspase-9, Tyr153Phe. Furthermore, expression of in response to AraC or ionizing radiation [45]. However, it is not clear why there would be a requirement for phosphorylation for caspase-9 activation in response to a specific stimulus and not others. One possible explanation for the effect of the Tyr153Phe mutation is that it simply inhibits the enzyme, creating a domi- nant mutant that is not only incapable of initiating the apoptotic programme but also has a dominant nega- tive effect on the endogenous wild-type caspase-9, like the effect of an active-site mutant, Cys287Ala [2,46]. The general role of Tyr153 phosphorylation in the regulation of caspase-9 activation therefore remains to be confirmed.

Phosphorylation in the linker region between the large and small subunit domains of caspase-9

An atypical isoform of protein kinase C (PKC), PKCf, phosphorylates human caspase-9 at another site, Ser144, in cytosolic extracts treated with okadaic acid [43]. Mutation of caspase-9 to prevent Ser144 phos- phorylation, or inhibition of PKCf, promotes caspase- 3 activation in this system, and phosphorylation of Ser144 is sufficient to inhibit caspase-9 activation. In human cells, Ser144 is phosphorylated in response to hyperosmotic stress, which rapidly activates PKCf, but is not phosphorylated in response to growth factors, phorbol ester or other cellular stresses. In addition, the association of PKCf with caspase-9 found in non- stressed cells is disrupted by hyperosmolarity. How- ever, phosphorylation of caspase-9 at Ser144 is not required for the dissociation of PKCf, indicating that other mechanisms regulate their dynamic interaction [43]. Phosphorylation of human caspase-9 by PKCf provides a potential mechanism (in addition to the DYRK1A-dependent and p38a-dependent phosphory- lation of Thr125) to restain apoptosis and promote cell survival in response to hyperosmotic stress.

Multiple sites phosphorylated by protein kinase A

In murine cells, casein kinase 2 (CK2) can phosphory- late caspase-9 at Ser348 (which corresponds to Ser310 in humans; Fig. 2C), protecting it from cleavage at Asp353 by caspase-8 in a cytochrome c-independent pathway of caspase-9 activation initiated by tumour necrosis factor-a [47]. A related mechanism of phos- phorylation by CK2 inhibits the cleavage and activa- tion of the pro-apoptotic Bcl-2 homology domain 3 (BH3)-only domain protein, BH3-interacting domain death agonist (Bid) [48]. Global analyses of phosphor- ylated proteins by mass spectrometry have identified a cluster of residues in this region of caspase-9 that are phosphorylated in the murine protein (Thr345, Ser348 – the CK2 site – and Ser350; [49]) and the human pro- tein (Ser302, Ser307 and Ser310; [50]). Whether CK2 or other kinases are responsible for the phosphoryla- tion of human caspase-9 within this region remains to be determined.

Protein kinase A (PKA), also called cAMP-dependent protein kinase, directly phosphorylates caspase-9 at in vitro [44] three sites (Ser99, Ser183 and Ser195) (Fig. 2). However, these sites are dispensable for the ability of PKA to inhibit caspase activity downstream of cytochrome c release in cytosolic extracts. PKA blocks recruitment of caspase-9 to the apoptosome, perhaps by compromising Apaf-1 oligomerization, because caspase-9 processing by a truncated form of Apaf-1, which is thought to be constitutively oligomer- is unaffected by PKA. Interestingly, PKA can ized, also directly phosphorylate Apaf-1 in vitro [44] (Fig. 1) but further studies are required to determine what effect this might have on apoptosome activity.

Potential biological and pathological roles of caspase-9 phosphorylation

Tyr153, c-Abl and the response to DNA damage Development

the regulation of

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Much of the intrinsic apoptotic pathway that has been identified operates at the level of controlling cytochrome c release from mitochon- dria in response to permeabilization of the outer mitochondrial membrane. Whether or not a cell can survive cytochrome c release, even when caspase acti- vation is inhibited, is still controversial, but the pres- ence of additional cells in the brains of mice lacking The tyrosine kinase c-Abl is involved in the apoptotic response to DNA damage and has been found to inter- act with caspase-9 in cells [45]. In contrast to the inhibitory effects of phosphorylation of caspase-9 at Thr125 and Ser144, phosphorylation of Tyr153 by c-Abl (Fig. 2) has been reported to be required for caspase activation downstream of cytochrome c release in response to cytosine arabinoside (AraC), an inhibi- tor of DNA replication. AraC-induced caspase-9 auto-

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Regulation of caspase-9 by phosphorylation

In part, postmitochondrial

stress. Apaf-1, caspase-9 or caspase-3 suggests that such cells survive despite mitochondrial permeabilization [51]. regulation may reflect the role of caspase-dependent positive-feed- back mechanisms that promote cytochrome c release and are dampened down by inhibition of caspase activation [52,53].

to large changes in extracellular osmolarity. Abnormal osmotic stress can result in apoptosis in a number of cell types, whereas caspase-9) ⁄ ) MEFs and thymocytes are resistant to apoptosis induced by sorbitol [10], sug- gesting that caspase-9 determines cell survival follow- ing osmotic Inhibitory phosphorylation of caspase-9 at Thr125 (under the control of p38a and DYRK1A [28]) and at Ser144 by PKCf (human cas- pase-9 [43]) in response to hyperosmotic stress might therefore be important in the acute adaptive response to hyperosmolarity that provides an opportunity for transiently stressed cells to recover. This hypothesis remains to be tested directly in vivo.

Tumorigenesis and responses to chemotherapy

In cultured cells arrested in mitosis, it has been dem- onstrated that inhibitory phosphorylation of caspase-9 at Thr125 plays a critical role in the restraint of cas- pase-3 activation [20]. This indicates that regulation of the pathway at this level can indeed determine cell fate. In vivo, phosphorylation of caspase-9 is likely to be under control of growth ⁄ survival factor signalling as well as being responsive to cellular stresses and mitosis in order to provide spatio-temporal control of apopto- sis during development. Although such roles may not be revealed by knockout of the caspase-9 gene, they might be shown by its replacement with a nonphos- phorylatable mutant.

basal Increased inhibition.

caspase-9 at Thr125 in retinal

the disease [58]. In cells,

Abnormal phosphorylation and inhibition of cas- pase-9 may cause developmental defects. For instance, increased phosphorylation of Thr125 by DYRK1A might be involved in Down syndrome. In a mouse model for Down syndrome in which DYRK1A is overexpressed, normal caspase-9-dependent apoptosis is suppressed in the retina. Indeed, DYRK1A phos- phorylates cells, although it remains to be demonstrated whether this is the mechanism by which DYRK1A suppresses apopto- sis [40]. However, this work suggests that inhibition of apoptosis by DYRK1A contributes to the develop- mental abnormalities, and possibly also the patholo- gies, associated with Down syndrome [54].

The classical MAPK pathway, comprising the Ras– Raf–MEK–ERK cascade is constitutively active in many tumour types and is associated with suppression of apoptosis in response to growth factor deprivation and chemotherapeutic drugs [57]. ERK1 ⁄ 2-mediated phosphorylation of caspase-9 is likely to contribute to this phosphorylation through overexpression of DYRK1A could also poten- tially cause resistance to apoptosis in cancer cells. immunohistochemical analyses revealed Interestingly, elevated levels of caspase-9 Thr125 phosphorylation in both early- and late-stage gastric carcinomas compared with nonmalignant surrounding tissue and normal gas- tric mucosa, suggesting that this might have a role in the development of the expression of caspase-9 Thr125Glu, a mutant that may mimic phosphorylation at Thr125, reduces hypoxia- induced cell death [59]. Hyperphosphorylation of Thr125 might therefore contribute to the survival of tumour cells in the hypoxic environment often found in solid tumours.

In addition to a role in determining cell survival during development, there is also evidence that activa- tion of the intrinsic apoptotic pathway may be restricted spatially within cells to allow intracellular remodelling without cell death. Furthermore, there are increasing amounts of data to support nonapoptotic roles for caspases in cells [55]. For instance, a role for caspase-9 in muscle cell differentiation has been reported, where knockdown of caspase-9 expression prevented the differentiation of myoblasts into myo- tubes [56]. Such functions of caspase-9 could also be controlled by post-translational mechanisms, such as inhibitory phosphorylation. Inhibition of caspase-9 through mitotic phosphoryla- tion by CDK1-cyclin B1 [20] could contribute to tumorigenesis by promoting the survival of cells that undergo an aberrant or delayed mitosis, resulting in aneuploidy. Phosphorylation of caspase-9 plays a role in determining the cell-killing activity of antimitotic drugs such as taxol [20], which is commonly used as a chemotherapeutic drug in the treatment of breast and ovarian cancer, and specific inhibition of caspase-9 phosphorylation could potentially sensitize otherwise resistant cancer cells to such drugs.

The cellular response to hyperosmotic stress

Summary

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identification of The sites of phosphorylation in caspase-9 suggest that this critical initiator of apoptosis The cellular response to hyperosmolarity is particularly important in certain cell types, such as those in the kidney medulla or oral epithelium, which are exposed

L. A. Allan & P. R. Clarke

Regulation of caspase-9 by phosphorylation

elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413.

8 Riedl SJ & Salvesen GS (2007) The apoptosome: signal- ling platform of cell death. Nat Rev Mol Cell Biol 8, 405–413.

9 Hakem R et al. (1998) Differential requirement for cas- pase 9 in apoptotic pathways in vivo. Cell 94, 339–352.

10 Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Karasuyama H, Su MS, Rakic P & Flavell RA (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325–337.

11 Kuida K, Zheng TS, Na S, Kuan C, Yang D,

Karasuyama H, Rakic P & Flavell RA (1996) Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature 384, 368–372.

is targeted by multiple protein kinase signalling path- ways that respond to extracellular stimuli, cellular stresses or are modulated during the cell division cycle. Phosphorylation of a major site, Thr125, results in the inhibition of caspase-9 activation. In human cells, phosphorylation of caspase-9 at Thr125 by CDK1- cyclin B1 plays a critical role in the restraint of apop- tosis during mitotic arrest. This site is also targeted by ERK1 ⁄ 2 in response to growth factor signalling and by DYRK1A, which plays important roles during development. Other sites in caspase-9 are targeted by additional protein kinases. In future studies, it will be important to determine the potential significance of caspase-9 phosphorylation during mammalian develop- ment and tumorigenesis in vivo.

Acknowledgements

12 Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA & Gruss P (1998) Apaf1 (CED-4 homolog) regulates pro- grammed cell death in mammalian development. Cell 94, 727–737.

13 Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A,

Hakem R, Penninger JM & Mak TW (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750.

We acknowledge Suzanne Brady, Michelle Lickrish, Malcolm McTaggart, Morag Martin and Anne Seifert for their contributions to the study of caspase-9 phos- phorylation in our laboratory. Our work on caspase-9 phosphorylation has been supported by the Royal Soci- ety, Medical Research Council, Association for Interna- tional Cancer Research and Cancer Research UK.

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