Regulation of apoptosis by endoplasmic reticulum pathways

David G Breckenridge1, Marc Germain1, Jaigi P Mathai1, Mai Nguyen1 and Gordon C Shore*,1

1Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec, Canada H3G 1Y6

Apoptotic programmed cell death pathways are activatedby a diverse array of cell extrinsic and intrinsic signals,most of which are ultimately coupled to the activation ofeffector caspases. In many instances, this involves anobligate propagation through mitochondria, causingegress of critical proapoptotic regulators to the cytosol.Central to the regulation of the mitochondrial checkpointis a complex three-way interplay between members of theBCL-2 family, which are comprised of an antiapoptoticsubgroup including BCL-2 itself, and the proapoptoticBAX,BAK and BH3-domain-only subgroups. Constitu-ents of all three of these BCL-2 classes, however, alsoconverge on the endoplasmic reticulum (ER), an organellewhose critical contributions to apoptosis is only nowbecoming apparent. In addition to propagating death-inducing stress signals itself, the ER also contributes in afundamental way to Fas-mediated apoptosis and to p53-dependent pathways resulting from DNA damage andoncogene expression. Mobilization of ER calcium storescan initiate the activation of cytoplasmic death pathwaysas well as sensitize mitochondria to direct proapoptoticstimuli. Additionally, the existence of BCL-2-regulatedinitiator procaspase activation complexes at the ERmembrane has also been described. Here, we review thepotential underlying mechanisms involved in these eventsand discuss pathways for ER–mitochondrial crosstalkpertinent to a number of cell death stimuli.Oncogene (2003) 22, 8608–8618. doi:10.1038/sj.onc.1207108

Keywords: calcium; BCL-2; caspase; mitochondria

Introduction

Apoptotic stresses originating from diverse cellular lociare typically channelled into a core death pathwayinvolving proteolytic activation of the caspase family ofcysteine proteases. BCL-2 family proteins, on the otherhand, localize to the membranes of various organellesand regulate the activation of caspases. For example,several members of the BH3-only subset of the BCL-2family respond to apoptotic signals by increasing thepermeability of the outer mitochondrial membrane(OMM). This releases apoptotic intermembrane space(IMS) proteins, including cytochrome c (cyt.c), which

binds APAF-1 in the cytosol and triggers the activationof caspase-9 (Green and Reed, 1998). Membranepermeabilization is achieved by BH3-dependent oligo-merization of a second group of multidomain proapop-totic BCL-2 proteins represented by BAX and BAK, andis inhibited by antiapoptotic BCL-2 members, includingBCL-xL and BCL-2 itself. Emerging evidence suggeststhat the endoplasmic reticulum (ER) also regulatesapoptosis both by sensitizing mitochondria to a varietyof extrinsic and intrinsic death stimuli and by initiatingcell death signals of its own. Members of all three classesof the BCL-2 family localize to the ER membrane andhave been shown to influence ER homeostasis, perhapsby influencing membrane permeability. Calcium releasefrom the ER, for example, has been implicated as a keysignalling event in many apoptotic models and, depend-ing on the mode of Ca2þ release, may either directlyactivate death effectors or influence the sensitivity ofmitochondria to apoptotic transitions. Furthermore, agrowing number of ER proteins have been shown toinfluence apoptosis by either interacting with BCL-2family members or altering ER Ca2þ responses, whereasseveral ER proteins are caspase substrates that mayregulate the execution phase of apoptosis. Moreover,recent studies on how stress in the ER is coupled toapoptosis have demonstrated that the ER, like mito-chondria, can directly initiate pathways to caspaseactivation and apoptosis (Figure 1).

ER stress-induced pathways

The ER is the first stop on the secretory pathwaywherein chaperone-assisted polypeptide folding andmodification ensures that proteins obtain their matureconformation. When the capacity of the ER to foldproperly proteins is compromised or overwhelmed, ahighly conserved unfolded protein response (UPR)signal transduction pathway is activated. The UPRhalts general protein synthesis while upregulating ERresident chaperones and other regulatory components ofthe secretory pathway (Travers et al., 2000), giving thecell a chance to correct the environment within the ER(Patil and Walter, 2001). However, if the damage is toostrong and homeostasis cannot be restored, the mam-malian UPR ultimately initiates apoptosis. This switch,from metabolic arrest, which provides an opportunityfor repair of the ER folding capacity, to cell death,which eliminates an overly damaged cell, is analogous to*Correspondence: Gordon C Shore; E-mail: gordon.shore.mcgill.ca

Oncogene (2003) 22, 8608–8618& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00

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the p53 response to genotoxic stress. In contrast tothe p53 switching mechanisms, however, theanalogous processes relating to the ER remain poorlyunderstood.

ER stress: the survival response

In mammals, three ER transmembrane proteins, Ire1,ATF6, and PERK, respond to the accumulation ofunfolded proteins in the lumen (see Sidrauski et al.,1998; Kaufman, 1999; Patil and Walter, 2001 forexcellent reviews). Ire1 and PERK are normally keptin an inactive state through an association between theirN-terminal lumenal domains and the chaperone BiP.Under conditions of ER stress, BiP dissociates (to bindunfolded proteins) and Ire1 and PERK undergo homo-oligomerization, stimulating trans-autophosphorylationwithin their serine/threonine kinase domains. Ire1 alsocontains a C-terminal endonuclease domain that excisesa short sequence from the mRNA of the X-Box bindingprotein (XBP-1), generating an active bZIP transcrip-tion factor that stimulates transcription of ER chaper-one genes (Ma and Hendershot, 2001; Shen et al., 2001;Yoshida et al., 2001; Calfon et al., 2002). PERK, on theother hand, phosphorylates the translation initiationfactor eIF2a, which halts translation and prevents thecontinual accumulation of newly synthesized proteinsinto the ER when protein folding conditions arecompromised (Harding et al., 1999). Upregulation ofER chaperone genes is also mediated by a second,perhaps redundant, pathway involving ATF6. In thiscase, ATF6 undergoes proteolytic cleavage at the ER,

which releases its active bZIP transcription factordomain to the nucleus (Yoshida et al., 1998; Ye et al.,2000).

ER stress: the death response

Experimentally, ER stress is induced by pharmacologi-cal agents that inhibit N-linked glycosylation (tunica-mycin, TN), block ER to Golgi transport (brefeldin A,BFA), impair disulfide bond formation (dithiothreitol,DTT), or disrupt ER Ca2þ stores (thapsigargin, TG, aninhibitor of the sarcoplasmic/endoplasmic reticulumCa2þ ATPase (SERCA) pumps, or A-23187, a Ca2þ

ionophore). All of these agents eventually induceapoptosis within 20–48 h depending on the cell type,suggesting that if the damage to the ER is too great, or ifbalance is not restored within a certain window of time,an apoptotic response is elicited (Patil and Walter,2001). The mechanism by which ER stress is coupled toactivation of caspases was for the most part a mysteryuntil caspase-12 was characterized by Nakagawa andYuan (2000). Caspase-12 is ubiquitously expressed and,like all caspases, is synthesized as an inactive proenzymeconsisting of a regulatory prodomain and two catalyticp20 and p10 subunits (Van de Craen et al., 1997;Nakagawa and Yuan, 2000). However, unlike othercaspases, caspase-12 is remarkably specific to insultsthat elicit ER stress and is not proteolytically activatedby other death stimuli (Nakagawa et al., 2000).Accordingly, caspase-12-null mice and cells are partiallyresistant to apoptosis induced by ER stress but not byother apoptotic stimuli (Nakagawa et al., 2000).

Mitochondrion

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Figure 1 ER stress signalling pathways. See text for details

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Caspase-12 is localized at the cytosolic face of the ER,placing it in a position to respond to ER stress as aproximal signalling molecule (Nakagawa and Yuan,2000). However, the mechanism of caspase-12 activationis unclear. In mouse glial cells undergoing ER stresscaused by oxygen and glucose deprivation, caspase-12was cleaved by calpain. In vitro, m-calpain cleavedcaspase-12 at T132 and K158, which releases theprodomain from the catalytic subunits and increasesenzymatic activity (Nakagawa and Yuan, 2000). It hasalso been suggested that caspase-12 activation is linkedto Ire1 signalling. The cytosolic tail of Ire1 can recruitTRAF2 (Urano et al., 2000) and when overexpressed,TRAF2 can interact with caspase-12 and weaklyinduce its oligomerization and cleavage (Yoneda et al.,2001). Moreover, Ire1 induces apoptosis whenoverexpressed (Wang et al., 1998), which could be dueto caspase-12 activation. The CARD in caspase-12’sprodomain might mediate homotypic interactionwith other CARD containing proteins, allowing itsrecruitment into such an activation complex (analogousto caspase-9 recruitment to Apaf-1), or mediate caspase-12 oligomerization and autoactivation at theER. Overexpression of full-length caspase-12 inducesits oligomerization and self-cleavage between the p20and p10 subunits at D318 (Nakagawa et al., 2000; Raoet al., 2001; Fujita et al., 2002), but only caspase-12lacking its prodomain induces apoptosis. Therefore,caspase-12 activation likely involves both calpain-dependent removal of the prodomain and self-cleavageat D318.

Ire1 signalling might play a role in coupling the UPRsurvival response with an apoptotic cascade, and thefate of the cell would be determined by an interplaybetween these opposing signals. This scheme is reminis-cent of TNF death receptor signalling in which NFkBsurvival pathways and a caspase-8 death pathway aresimultaneously activated (Baud and Karin, 2001). Atpresent, however, an Ire1/TRAF2/caspase-12 complexremains speculative: interactions between the endogen-ous proteins have not been reported and Ire1 activationand caspase-12 cleavage occur with different kineticsfollowing ER stress (Harding et al., 2000). Thedependence of caspase-12 activation on Ire1 could betested in Ire1-deficient cells, and the dependence of Ire1-mediated apoptosis on caspase-12 could be tested incaspase-12-deficient cells. Interestingly, Zong et al., 2003found that caspase-12 processing is abolished in BAX,BAK-1-cells, and caspase-12 cleavage could be inducedby the expression of ER-targeted BAK. Of note,procaspase-12 has also been reported to be cleaved bydownstream executioner caspases, such as caspase-7, butthis event likely represents a downstream amplificationloop (Rao et al., 2001). It raises the possibility, however,that caspase-12 might also amplify the activity of otherinitiator caspases.

Following its activation at the ER, caspase-12 maydirectly process downstream caspases in the cytosol ortarget other as yet unidentified substrates that influencethe progression of apoptosis. Two groups recentlyreported that caspase-12 can directly cleave procas-

pase-9 in vitro, leading to caspase-9-dependent activa-tion of caspase-3 (Morishima et al., 2002; Rao et al.,2002). Inhibition of caspase-12 by expression of MAGE-3, a protein that binds the p10 subunit and blockscatalytic activity, prevents TG- and TN-induced proces-sing of caspase-9 and -3 (Morishima et al., 2002). Inaddition, ER stress-induced processing of procaspase-9can occur in the absence of cyt.c release and in APAF-1-null fibroblasts (Rao et al., 2002). These results arguethat caspase-12 can directly trigger caspase-9 activationand apoptosis independent of the mitochondrial cyt.c/Apaf-1 pathway, at least in certain cell types. Manystudies, however, implicate the involvement of mito-chondria in ER stress-induced apoptosis (below), whichmight represent a redundant pathway to caspaseactivation or, alternatively, might provide a pathwayfor accumulating Smac/Diablo and HtrA in the cytosol,where their inhibition of IAPs supports optimal activa-tion of caspases.

The relevance of caspase-12 signalling has beensomewhat clouded by the fact that a human orthologremains elusive Fischer et al. reported that the humancaspase-12 gene has acquired deleterious mutations thatprevent the expression of a functional protein. In spiteof this discovery, two reports showed that antibodiesagainst murine caspase-12 detect an appropriately sizedprotein in human cells that is processed following ERstress (Nakagawa et al., 2000; Rao et al., 2001). Proofthat a functional human caspase-12 protein does indeedexist, therefore, must await purification of the endogen-ous enzyme.

ER stress-induced death: contribution of mitochondria

Several lines of evidence suggest that mitochondria arean important component of the ER stress-inducedapoptotic pathway. First, ER stress agents causemitochondrial release of cyt.c and loss of mitochondrialtransmembrane potential (Hacki et al., 2000; Boya et al.,2002); second, BCL-2/BCL-XL inhibit ER stress-in-duced apoptosis (McCormick et al., 1997; Hacki et al.,2000; McCullough et al., 2001); and third, Bax�/�,Bak�/� MEFs are resistant to TG-, TN-, and BFA-induced apoptosis (Wei et al., 2001). While the lattertwo findings could be due to potential roles of BCL-2family members at the ER (see below), Kroemer andcoworkers confirmed the involvement of mitochondriaby showing that the cytomegalovirus-encoded mito-chondrial inhibitor of apoptosis (vMIA) potentlyinhibited ER stress apoptosis (Boya et al., 2002).Signalling between the ER and mitochondria presum-ably involves the activation of one or more BH3-onlyproteins. BAD is a candidate, since TG or A23187 havebeen shown to induce its Ca2þ /calcineurin-dependentdephosphorylation and activation (Wang et al., 1999).Increased [Ca2þ ]c has also been observed followingother conditions of ER stress (Carlberg et al., 1996) and,therefore, BAD activation may be a conserved compo-nent of the pathway. Additionally, the UPR might

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transcriptionally upregulate other BH3-only proteins,analogous to the response of BH3-only genes to p53-mediated stress responses. A recent report documentingthe upregulation of PHMA following ER stress suggeststhis is the case (Reimertz et al., 2003).

ER stress-induced cyt.c release is apparently depen-dent on the c-Abl tyrosine kinase, because c-Abl�/�mouse embryonic fibroblasts are resistant to A23187-,BFA-, and TN-induced cyt.c release and apoptosis (Itoet al., 2001). c-Abl redistributes from the ER to themitochondria within several hours of TG application,which parallels an increase in its kinase activity. Themechanism by which c-Abl exerts its action at this site isunclear, but it may function in concert with JNKkinases, which are recruited and activated by Ire1 duringER stress (Urano et al., 2000), and are essential formediating cyt.c release in other cell death pathways(Tournier et al., 2000). UPR upregulation of CHOP/GADD153, a nuclear transcription factor that repressesthe Bcl-2 promoter (McCullough et al., 2001), maysensitize mitochondria to the proapoptotic effects ofBH3-only proteins by decreasing the cellular levels ofBCL-2 protein. Consistent with this, tunicamycin-induced apoptosis is impaired in CHOP�/� MEFs,and CHOP�/� mice injected with tunicamycin showdecreased apoptosis in the renal tubular epithelium(Zinszner et al., 1998).

Therefore, stress in the ER unleashes a mitochondrial-dependent apoptotic pathway and a caspase-12, mito-chondrial-independent pathway (Figure 1). These twoarms of the ER stress response apparently operateindependent of one another, since zVAD-fmk, whicheffectively inhibits caspase-12 in vitro (Nakagawa andYuan, 2000), has no effect on ER stress-induced cyt.crelease (Hacki et al., 2000), and caspase-12�/� MEFsonly partially resist apoptosis (Nakagawa et al., 2000).This duality in signalling may ensure complete andefficient caspase activation in physiological settings.

Regulation of apoptosis by ER Ca2þ signals

The release of Ca2þ from the ER is primarily achievedby two well-characterized types of channels, the inositol1,4,5-triphosphate receptor (IP3R) and the Ryanodinereceptor (RyR) families (Pozzan et al., 1994; Berridgeet al., 2000). Also, other Ca2þ release channels likelyexist (Cavalli et al., 2002). Release of calcium from theER has been observed in many forms of apoptosis, andregulated oscillations of Ca2þ levels by IP3R-mediatedspikes may directly regulate the cell death machinery.Jurkat cells deficient in type 1 IP3R (IP3R1) do notincrease [Ca2þ ]c in response to dexamethasone, ionizingradiation, and T-cell receptor or Fas stimulation, andresist subsequent apoptosis (Jayaraman and Marks,1997). Moreover, targeted disruption of all three IP3Risoforms in the chick DT40 B-cell line blocked Ca2þ

mobilization and apoptosis by B-cell receptor cross-linking, and the degree of resistance increased with thenumber of IP3R genes deleted, suggesting that all threeisoforms can contribute to apoptotic signalling (Suga-

wara et al., 1997). How IP3R activation is coupled tosuch diverse death signals, however, remains to bedetermined.

The way in which Ca2þ signals engage cell deathpathways may largely be decided by the spatio-temporalpattern and intensity of ER Ca2þ release. IP3R/RyR-dependent global increases in [Ca2þ ]c can influenceapoptosis by several mechanisms. For example, Ca2þ -dependent activation of calpains has been implicated inactivating caspases in several forms of apoptosis (Wang,2000), including ER stress- (Nakagawa and Yuan,2000), B-cell receptor- (Ruiz-Vela et al., 1999, 2001),and radiation-induced apoptosis (Waterhouse et al.,1998). In addition, calpain cleavage of BAX has beenreported to increase its proapoptotic activity (Woodet al., 1998), and in some cases calpain may cooperatewith caspases in the execution phase of apoptosis (Woodand Newcomb, 1999). As discussed above, Ca2þ signalscan lead to activation of BAD, and Ca2þ-dependentactivation of the transcription factor MEF2 leads toupregulation of Nur77/TR3 (Youn et al., 1999), whichcan bind to mitochondria and induce cyt.c release (Liet al., 2000). In contrast, privileged transport of Ca2þ

between juxtaposed ER and mitochondrial membranesmay sensitize mitochondria to the effects of proapopto-tic BCL-2 family members (Hajnoczky et al., 2000),perhaps through Ca2þ -induced opening of the mito-chondrial permeability transition pore. For an in depthdescription of the mechanisms by which ER Ca2þ

signals can influence apoptosis, the reader is referredto chapter XXX in this issue.

BCL-2 family proteins and the ER: antiapoptoticmembers

Although the function of BCL-2 proteins is bestcharacterized at the mitochondrion, these proteins alsolocalize to other intracellular membranes such as theER/nuclear envelope. Studies employing BCL-2 selec-tively targeted to the ER with the transmembranesequence of cytochrome b5 (b5-BCL-2) have demon-strated that BCL-2 has antiapoptotic activity at thislocation. For example, b5-BCL-2 prevents apoptosisinduced by ER stress agents, ceramide, Myc, ionizingradiation, or BAX overexpression (Hacki et al., 2000;Annis et al., 2001; Rudner et al., 2001; Wang et al.,2001). Annis et al. observed that b5-BCL-2 conferredprotection against apoptotic stimuli that caused anobvious mitochondrial depolarization prior to therelease of cyt.c (Myc, C2 ceramide), but not againststimuli that induced cyt.c release in the absence of largemitochondrial depolarizations (etoposide). Wild-type(wt) BCL-2, in contrast, could inhibit both types ofstimuli. The fact that ER-localized BCL-2 can onlyinhibit certain apoptotic pathways suggests that itsantiapoptotic effect at the ER may not be the result ofsimple sequestration of endogenous proapoptotic BCL-2 proteins, in which case its membrane location wouldbe irrelevant. This assumes, however, that the various

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pathways tested are indeed coupled to proapoptoticBCL-2 binding partners.

Both pro- and antiapoptotic BCL-2 proteins havepredicted pore forming properties (Antonsson andMartinou, 2000) and in theory could influence ion fluxacross cellular membranes. Not surprisingly, therefore,many studies implicate BCL-2 in regulating ER Ca2þ

homeostasis. However, the mechanism by which it doesso is controversial and may depend on the cell typetested. For example, several groups have reported thatBCL-2 increases the ER Ca2þ content and/or preventsER Ca2þ release during apoptosis (Lam et al., 1994;Distelhorst et al., 1996; He et al., 1997; Ichimiya et al.,1998; Nutt et al., 2002). In contrast, studies by Rizzutoand co-workers convincingly demonstrated that over-expression of BCL-2 reduces the steady-state level of[Ca2þ ]ER by increasing the permeability of the ERmembrane to Ca2þ (Pinton et al., 2000). Consistent withthis latter finding, there are now several examples in theliterature where manipulations that lead to decreased[Ca2þ ]ER protected cells against apoptosis, whereasmanipulations that increased [Ca2þ ]ER sensitized cellsto apoptosis (Nakamura et al., 2000, Pinton et al., 2001;Lilliehook et al., 2002). Changes in [Ca2þ ]ER affect theintensity IP3R/RyR Ca2þ spikes, which are known tosensitize mitochondria to BH3-dependent cyt.c releaseduring apoptosis (Csordas et al., 2002). It has also beenreported that BCL-2 can increase the capacity ofmitochondria to store Ca2þ (Murphy et al., 1996;Ichimiya et al., 1998; Zhu et al., 1999), presumably bypreventing the opening of the PTP, which releasesmatrix Ca2þ . Thus, by localizing to both ER andmitochondria, BCL-2 might prevent apoptotic crosstalkbetween the two compartments by lowering the amountof free [Ca2þ ]ER for IP3R/RyR release and by increasingthe tolerance of mitochondria to high Ca2þ loads.

BCL-2 family proteins and the ER: proapoptotic BAXand BAK

The proapoptotic multidomain BCL-2 family membersBAX and BAK have also been reported to localize tothe ER in some cell types (Nutt et al., 2001) and theseproteins may induce apoptosis from this location. Forexample, when overexpressed in human PC-3 cells, BAXand BAK localize to both the mitochondria and ER,and induce caspase-independent emptying of ER Ca2þ

pools concomitant with an increase in [Ca2þ ]m (Nuttet al., 2001; Pan et al., 2001). Coexpression of BCL-2/BCL-xL inhibits this Ca2þ mobilization, and aninhibitor of mitochondrial Ca2þ uptake, RU360,blocked BAX/BAK- and staurosporin-induced increasein [Ca2þ ]m, and subsequent cyt.c release and apoptosis(Nutt et al., 2001). Moreover, BAX-null DU-145 cellsresist staurosporin-induced ER Ca2þ release, mitochon-drial Ca2þ uptake and cyt.c release, all of which areovercome by re-expression of BAX (Nutt et al., 2002).Consistent with a role for these proteins at the ER, BAKand BAX interact with the cytosolic tail of the ERchaperone calnexin in yeast, and BAK-induced lethality

in S. pombe is dependent upon this interaction (Torgleret al., 1997). Moreover, b5-BCL-2 can inhibit BAX-induced apoptosis (Wang et al., 2001) as can BAXinhibitor-1, an antiapoptotic transmembrane protein ofthe ER (Xu and Reed, 1998). BAX, BAK double-nullcells, which are variant to diverse apoptosis stimuli; havereduced [Ca2þ ]ER that result in decreased Ca2þ uptakeby the mitochondria following release of Ca2þ from theER by apoptotic agents (Scorrano et al., 2003). Overexpression of SERCA corrected the Ca2þ imbalance inthese cells and fully or partially restored apoptosis inresponse to several intrinsic cell death stimuli. ThusBAX and BAK have dual roles at the ER andmitochondria and regulate Ca2þ-dependent cross linkbetween the two organelles.

BCL-2 family proteins and the ER: BH3-only members

Recent models hold that the BH3-only subset ofproapoptotic BCL-2 family members sense diverse deathsignals and respond by initiating caspase activation(Puthalakath and Strasser, 2002). BH3-only moleculesmay achieve this by binding and inhibiting antiapoptoticBCL-2 family members or by directly activatingproapoptotic BAX and BAX (Letai et al., 2002). CertainBH3-only molecules, after activation, translocate tomitochondria and induce the oligomerization of BAXand BAX into predicted pores in the OMM, facilitatingegress of the protein contents of the intermembranespace. Consistent with this model, most BH3-onlyproteins localize to mitochondria, at least in part, andtrigger cyt.c release, which in all cases is inhibited byBCL-2 overexpression.

Given that representatives from all three groups of theBCL-2 family have been found at the ER, however, it islikely that the ratiometric interactions between anti-apoptotic and proapoptotic members that regulate themitochondrial apoptotic pathway likely also regulateapoptotic pathways at the ER (Figure 2). The BH3-onlyprotein BIK, for example, is located primarily at the ER(Mathai et al., 2002). BIK mRNA and protein areinduced by p53 in response to DNA damage oroncogenic stress (Mathai et al., 2002; unpublished),and ER BIK induces cyt.c release independent of anassociation with mitochondria or zVAD-sensitive cas-pases (Germain et al., 2002). An S9 fraction containingboth microsomes and cytosol, but not either fractionalone, isolated from BIK-treated H1299 cells (in thepresence of caspase inhibitors) can induce mitochondrialrelease of cyt.c in vitro. This suggests that BIK activatesfactors in both the ER and cytosol to induce mitochon-drial transformations (Germain et al., 2002). Thisactivity of BIK is influenced by BCL-2 at the ER sinceBIK/BCL-2 heterodimers can be crosslinked at thesurface of the ER and high BIK :BCL-2 ratios changethe set of proteins BCL-2 interacts with at the ER,resulting in Ca2þ release, egress of cyt.c from mitochon-dria, and apoptosis (unpublished). When in excess,however, ER-localized BCL-2 is able to protect againstBIK-induced apoptosis. The ratio of BH3-only and

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antiapoptotic BCL-2 family members at the ER, there-fore, may regulate downstream signals emitted from theorganelle, including Ca2þ signals and/or proximalzVAD-insensitive caspase activation, which in turnregulate mitochondrial release of cyt.c. Other BH3-onlyproteins, such as BIM and NOXA (Puthalakath et al.,1999; Oda et al., 2000), also partially locate to the ERand, therefore, may elicit similar effects.

By analogy to their known functions at mitochondria,it is also possible that certain BH3-only proteins induceBAX/BAK oligomerization in the ER membrane andmediate release of lumenal proteins that regulate caspaseactivation. Additionally, BAX/BAK pores might con-trol ER Ca2þ stores, influencing local [Ca2þ ]c and/orfacilitating mitochondrial Ca2þ uptake and sensitizationfor cyt.c release. This model is consistent with poreforming properties of BCL-2 proteins, their ability tomodulate movement of Ca2þ across the ER membrane,and the dependence of most BH3-only proteins onBAX/BAK to induce apoptosis (Cheng et al., 2001).Moreover, the ER undergoes profound dilation andunfolding of cisternae during apoptosis (Weller et al.,1995; Wu et al., 1999; Chang et al., 2000; Herrera et al.,2000; Johnson et al., 2000; Muriel et al., 2000; Zeng andXu, 2000) suggesting that ER homeostasis and volumecontrol is lost, perhaps due to membrane permeabilization.

Regulation of initiator caspases at the ER by BCL-2proteins

Other apoptotic models posit that of BCL-2 proteinsregulate caspase activation upstream or in parallel to the

mitochondrial pathway (Huang and Stasser, 2000) andthat mitochondria amplify weak initiator caspasesignals. As discussed above, caspase-12 activation atthe ER membrane clearly represents one mechanism ofmitochondria-independent initiator caspase activation.The ability of ER-localized BCL-2 family members todirectly regulate this caspase certainly deserves testing.Additionally, the novel procaspase-8 isoform, procas-pase-8L, like procaspase-12, is peripherally associatedwith the cytosolic face of the ER (Breckenridge et al.,2002). However, procaspase-8L processing seems to beregulated through an association with the BAP31complex. BAP31 is a ubiquitously expressed polytopicintegral membrane protein of the ER that associateswith related BAP29, components of the actomyosinnetwork, and BCL-2/BCL-xL (Adachi et al., 1996;Ng et al., 1997; Nguyen et al., 2000). Procaspase-8L,but not procaspase-8, is selectively recruited to theBAP31 complex during oncogenic E1A-inducedapoptosis, which coincides with its proteolytic proces-sing. This recruitment is dependent on procaspase-8L’sNex domain, a 59 amino acid stretch that extendscanonical procaspase-8/a at the N-terminus. E1A-induced processing (and presumably activation) ofprocaspase-8L is regulated by its association with BAPproteins since cells doubly deficient in Bap31 and Bap29do not process procaspase-8L in response to E1A andshow reduced caspase-8 and -3 activity and subsequentapoptosis. It is possible that the cytosolic tails of BAP31and BAP29 recruit factors that facilitate procaspase-8Lactivation. Importantly, BCL-2 overexpression does notinhibit recruitment of procaspase-8L to the BAP31complex but abrogates subsequent enzyme processingand apoptosis. Moreover, E1A-induced processing ofprocaspase-8L is subject to ratiometric regulation byBIK and BCL-2 located at the ER (unpublished),suggesting that it might be a target of BH3-initiatedcaspase activation. At present, it is not known whetherother initiator caspases respond to signals at the ER.Given that initiator procaspase-2 has been implicatedupstream of mitochondrial dysfunction during drug-induced apoptosis (Lassus et al., 2002) and that thiscaspase is located at Golgi and ER membranes in somecell types (Mancini et al., 2000), however, it isconceivable that procaspase-2 is also regulated byBCL-2 proteins at the ER.

The massive surface area of the ER membrane mayprovide a platform for the assembly of caspaseregulatory complexes. This model parallels apoptoticregulation in C. elegans, wherein a BCL-2 protein(Ced-9) binds an adapter molecule (Ced-4) preventingit from activating a caspase (Ced-3). By binding Ced-9,BH3-only Egl-1 initiates apoptosis by displacing Ced-4(Metzstein et al., 1998). BCL-2 can compensate forCed-9 loss-of-function mutations in nematodes, indicat-ing that BCL-2 may be able to function in a similarmanner in mammalian cells (Hengartner and Horvitz,1994). For example, upregulation of BIK followingoncogenic stress increases the BIK :BCL-2 ratioat the ER, which might relieve the inhibitory effect ofBCL-2 on procaspase-8L processing. ER proteins

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Excess BH3

Excess BH3

Figure 2 BH3-driven apoptotic pathways at the ER. Diverseapoptotic stresses may increase the concentration of BH3-onlyproteins, such as BIK, at the ER. When in excess, BH3-onlyproteins may mobilize ER Ca2þ stores and sensitize mitochondriato undergo apoptotic transitions in response to a direct BH3 hit, oractivate other Ca2þ -dependent processes in the cytosol. BH3-onlyproteins might also stimulate other Ca2þ -indpendent pathwaysleading to cyt.c release from the mitochondria or facilitate localprocaspase-12, procaspase-8L, or procaspase-2 activation at theER surface. To accomplish these tasks, BH3-only proteins maysimply relieve BCL-2 repression of these processes or, additionally,they may play a second role in activating these pathways afterBCL-2 is repressed. It remains to be determined if the ratiometricinfluence of anti- and proapoptotic members also includesBAX,BAK

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such as the reticulon family members NSP-C andRTN-xs, which bind and inhibit BCL-2 and BCL-xL(Tagami et al., 2000), or p53-inducible genes that targetthe ER, including PIDD and Scotin (Lin et al., 2000;Bourdon et al., 2002), might also play a role in theregulation of caspase activation at the ER. Alterna-tively, high BH3/BCL-2 ratios may relieve BCL-2inhibition of some other apoptotic process at the ER,notably calcium homeostasis.

The various models for the function of BCL-2family proteins at the ER are not mutually exclusiveand one could imagine that, depending on the apoptoticstimulus, multiple signalling pathways could emergefrom the ER to either directly activate the mitochondrialpathway (as with BIK) via caspase-dependentand -independent means, or to sensitize mitochondriato a secondary direct BH3 hit at this organelle(as with ER Ca2þ release), or to synergize withmitochondrial cyt.c release to activate downstreamcaspases (as with the caspase-12/ER stress pathway)(Figure 2).

Caspase substrates located at the ER

Several ER proteins have now been identified as caspasesubstrates. The majority seem to be caspase-3 targetsand, therefore, their cleavage likely contributes to thecoordinated shutdown of normal cellular processesduring the execution phase of apoptosis.

BAP31

An exception, however, is BAP31, which in addition tobeing a regulator of procaspase-8L, is also a caspase-8substrate itself (Ng et al., 1997). The cytosolic tail ofhuman BAP31 contains two identical caspase recogni-tion sequences that are rapidly cleaved by caspase-8following stimulation of the Fas death receptor (Nguyenet al., 2000; Wang et al., 2003), and presumablyfollowing procaspase-8L activation at the ER (Breck-enridge et al., 2002). Fas initiates apoptosis by directlyrecruiting and activating procaspase-8 at the plasmamembrane. Caspase-8 in turn cleaves the BH3-onlymolecule, BID, generating tBID, which induces BAX/BAK-dependent cyt.c release from mitochondria andsubsequent activation of downstream caspases (Kors-meyer et al., 2000). In some cell types, Fas signallingdoes not require the participation of mitochondria forapoptotic execution because sufficient caspase-8 isactivated to permit direct cleavage and activation ofdownstream caspases (Scaffidi et al., 1998). Stableexpression of a caspase-resistant BAP31 (crBAP31)mutant, in which the caspase-8 recognition asp residueshave been substituted with ala, strongly inhibits Fas-induced apoptosis in KB epithelial cells (Nguyen et al.,2000). While crBAP31 had a very weak influence onFas-induced caspase activation or cleavage of effectorcaspase substrates, it strongly inhibited cyt.c release,

mitochondrial depolarization, cytoskeletal reorganiza-tion, and membrane blebbing. The fact that crBAP31inhibits these events in the face of caspase activationsuggests that, at least in these intact epithelial cells, theER exerts a restraint on apoptotic mitochondrialtransition and cytoplasmic restructuring that isovercome by BAP31 cleavage. Interestingly, crBAP31did not inhibit Fas-induced cleavage of BID ordownstream insertion of BAX into mitochondria,but strongly inhibited subsequent activation andoligomerization of BAX and BAK (Wang et al., 2003).To exert this antiapoptotic effect on mitochondria,BAP31 requires an association with the putativeion channel protein of the ER, A4. Although speculativeat present, it is possible that BAP31–A4 complexmight regulate Ca2þ flux between the ER andmitochondria during Fas-mediated apoptosis, which inturn sensitizes mitochondria to tBID (see below).Indeed, dynamic ER Ca2þ spikes have been implicatedin mediating TNF-a-induced cyt.c release (Pu et al.,2002).

Caspase-8 cleavage of BAP31 generates a membraneembedded p20 fragment that induces cell death whenexpressed ectopically for prolonged periods (Ng et al.,1997). Adenoviral expression of p20 induces an im-mediate release of Ca2þ from the ER, which is followedby mitochondrial recruitment of Drp1, a criticalmediator of OMM fission, and dramatic fragmentationand fission of the mitochondrial network into smallpunctiform organelles (Breckenridge et al., 2003). p20seems to regulate the fission machinery through Ca2þ

signals because inhibiting ER–mitochondria Ca2þ

transmission prevents Drp1 redistribution and mito-chondrial fission. Interestingly, ER Ca2þ signals havealso been shown to regulate mitochondrial restructuringduring ceramide-induced apoptosis (Pinton et al., 2001).Given that Drp1-dependent mitochondrial fission is arequirement for cyt.c release during apoptosis (Franket al., 2001), p20 might assist other caspase-8 substrates,such as tBID, to induce mitochondrial cristae restruc-turing (Scorrano et al., 2002) and cyt.c release duringapoptosis. Accordingly, mitochondria that had under-gone p20-induced fission were strongly sensitized tocaspase-8-induced cyt.c release. Collectively these dataare consistent with the ‘two-hit’ model described byHajnoczky and co-workers (Szalai et al., 1999), in whichan ER Ca2þ signal cooperates with a direct BH3-onlyprotein hit on mitochondria to efficiently promotethe mitochondrial release of cyt.c. Of note, crBAP31inhibits Fas-induced ER Ca2þ release and mitochondrialfission (our unpublished data). These opposing effectsof crBAP31 and p20 on ER Ca2þ homeostasis andmitochondrial integrity are independent of eachother since reconstitution of crBAP31 into Bap31-nullcells (which cannot generate p20) still preventscaspase-8-induced cyt.c release, and p20 still inducesits proapoptotic effects in the absence of Bap31.Thus, caspase cleavage of BAP31, like other antiapop-totic proteins (Cheng et al., 1997; Clem et al., 1998),converts it from an inhibitor to activator of apoptosis(Figure 3).

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SREBPs

Cleavage of other ER proteins by downstreamexecutioner caspases might contribute to morpho-logical progression of apoptosis or in some casessimply disable cellular processes at the ER duringcell suicide. For example, caspase-3 cleavage of thesterol-regulatory element binding proteins (SREBPs)SREBP-1 and SREBP-2 upregulates sterol responsegenes, which may affect lipid rearrangementsand/or morphological changes of the plasma membraneduring apoptosis (Higgins and Ioannou, 2001). SREBPsare ER membrane-bound transcription factors thatare normally transported to the Golgi in sterol-depletedcells, where they undergo sequential cleavage bySite-1 and -2 proteases (DeBose-Boyd et al., 1999).This cleavage releases an N-terminal basic helix–loop–helix leucine zipper transcription factor domain tothe nucleus that activates transcription of thegenes involved in sterol metabolism, such as the LDLreceptor and HMG-CoA reductase. Caspase cleavage ofSREBPs occurs at the ER during apoptosis, indepen-dent of cellular sterol levels, and liberates anactive transcription factor to the nucleus (Wang et al.,1996).

SRP 72

Caspase cleavage of the 72-kDa component of the signalrecognition particle (SRP 72) might shutdown or alterthe translation of secretory proteins during apoptosis.SRP is composed of six polypeptides and a 7S structuralRNA, which binds the signal sequence of newlysynthesized polypeptides as they surface from theribosome, arrests translation elongation until the com-plex has bound the SRP receptor at the ER membrane,and reinitiates translation elongation and translocationinto the ER lumen or membrane (Keenan et al., 2001).SRP 72 is thought to help facilitate binding of SRP tothe SRP receptor and translocation of polypeptidesacross the ER membrane. Caspase-3 cleavage of SRP-72removes a short conserved, and highly serine phos-

phorylated C-terminal fragment that may be importantfor regulation in vivo, although the truncated proteincan still function in vitro (Utz et al., 1998).

IP3 receptor and GRP94

IP3R1 and 2 are cleaved by caspase-3, and IP3R3undergoes calpain proteolysis during apoptosis, whichablates IP3-induced Ca2þ flux (Hirota et al., 1999; Diazand Bourguignon, 2000). In the light of the importantrole of IP3R signalling during the early stages ofapoptosis (Jayaraman and Marks, 1997), caspase-mediated proteolysis might represent functional down-regulation of this channel during the execution phase ofapoptosis. Similarly, calpain proteolysis of the Ca2þ -binding ER chaperone GRP94 may also affect Ca2þ

signalling during apoptosis (Reddy et al., 1999). Over-expression of GRP94 confers resistance to apoptosis,and antisense knockdown of GRP94 sensitizes cells toetoposide-induced apoptosis.

Presenilins and APP

Caspases cleave presenilins and the amyloid precursorprotein (APP), which are involved in the pathogenesis ofAlzheimer’s disesase (AD). Presenilin-1 (PS1) and -2(PS2), are highly homologous integral membraneproteins of the ER and Golgi that are mutated in mostaggressive early-onset forms of familial AD (Wellingtonand Hayden, 2000). Both presenilins have eight pre-dicted transmembrane domains, and a cytosolic hydro-philic loop between transmembrane domains 6 and 7.Cleavage within the cytosolic loop by endoproteasesgenerates stable C-terminal and N-terminal fragments,which are the functionally active form of the protein.Although the exact function of the presenilins is notknown, PS1 and PS2 modulate APP cleavage by g-secretase and the sensitivity of cells to apoptosis (Selkoe,1999). Overexpression of full-length wt PS1 and PS2 ortheir AD mutants sensitizes cells to apoptosis (Wolozinet al., 1996; Wellington and Hayden, 2000) by increasing

CASPASE-8

tBID

BAX/BAK BAX/BAKOligomers

BAP31 p20 BAP31

cyt. c, etc

Ca2+

Drp1Mito fission

ER-mediatedMitochondrial Sensitizaton

MitochondrialExecution

Figure 3 Cleavage of BAP31 at the ER sensitizes mitochondria to caspase-8-initiated release of cyt.c to the cytosol in intact cells.Activation of caspase-8 leads to simultaneous processing of BID and BAP31. tBID activates cristae remodelling and BAK/BAXoligomerization in the OMM providing a conduit for cyt.c efflux across, enhancing cytic release the membrane. Cleavage of BAP31generates p20, which triggers ER Ca2þ release and Drp1-dependent mitochondrial fission. In contrast, full-length BAP31 preventsFas-induced mitochondrial fission and cyt.c release. ER-driven Ca2þ signals are likely to be modulators of apoptotic mitochondrialtransition rather than obligatory events

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the ER Ca2þ load and agonist-induced ER Ca2þ release(Mattson et al., 2001) or by binding BCL-2 (Albericiet al., 1999; Passer et al., 1999). Conversely, antisenseknockdown of PS2 or expression of the mature C-terminal fragments of PS2 confers protection toapoptosis (Wellington and Hayden, 2000). PS1 andPS2 are cleaved within their C-terminal fragments bycaspases, which abrogates their antiapoptotic activity.Interestingly, caspase cleavage of PS2 is regulated byphosphorylation of two Ser residues overlapping thecleavage sites at Asp326 and Asp329. Phosphorylation ofthese residues inhibits caspase-3 cleavage in vitro, andmutation of the Ser residues to Asp or Glu (which mimicthe phosphorylated state) blocks caspase cleavage in vivoand enhances the antiapoptotic function of PS2 (Walteret al., 1999). AD-causing mutations in presenilins shiftthe normal processing pattern of APP by secretasestoward the production of the amyloidogenic and toxicAb-42 peptide, which is linked to AD. In addition,caspase-3 cleavage of APP at VEVD720 and caspase-6cleavage at VNLD653 in patients harboring the SwedishAPP mutation increases the amount of APP that isprocessed to Ab-42 (Gervais et al., 1999; Wellington andHayden, 2000). Lu et al. (2000) reported that the shortintracellular C-terminal fragment arising from caspasecleavage at VEVD720 is also proapoptotic and mightcontribute to AD pathogenesis. Therefore, caspasecleavage of presenilins and APP creates an apoptoticamplification cycle, accelerating cell death in theneurons of AD patients: caspase cleavage of APPincreases the generation of Ab-42, which is proapoptoticand in turn leads to further caspase activation and APPprocessing, and caspase cleavage of presenilins increasesthe sensitivity of neurons to apoptosis induced by Ab-42(Wellington and Hayden, 2000). Interestingly, Ab-42-induced apoptosis seems to be dependent on caspase-12activation at the ER (Nakagawa et al., 2000), althoughit is not clear how Ab-42 exerts this effect.

Future perspective

Although many studies implicate a regulatory role forthe ER in apoptosis, the signalling pathways thatemerge from this organelle remain largely obscure. Inthe case of ER stress, where the apoptotic signaloriginates within the ER itself, it is clear that the ERhas its own complement of apoptotic accessories thatindependently activate caspases and mitochondrial

dysfunction. It remains to be determined whether someof these ER signalling pathways function in otherapoptotic programs. In contrast, ER Ca2þ signals thataffect the ability of mitochondria to release IMSproteins in intact cells may be a general component ofmany apoptosis pathways. It will be important tounderstand the nature of proapoptotic Ca2þ signals,how apoptotic signals converge on Ca2þ releasechannels, and how BCL-2 proteins regulate this process.Most models of cyt.c release stem from studies testingthe effect of recombinant BCL-2 family members onisolated mitochondria (or liposomes), where the dy-namic nature of the mitochondrial network and itscontacts with the ER and cytoskeleton are lost. Thesemay well impact important optimal steps in apoptotictransformations of mitochondria in intact cells, includ-ing remodelling of cristae (Scorrano et al., 2002) andorganelle fission (Breckenridge et al., 2003; Frank et al.,2001). In physiological settings, the function of proa-poptotic BH3-only proteins may be suboptimal andtheir ability to transform mitochondria into a statecompetent for efficient cyt.c release may largely dependon the alliance with costimulatory signals from the ER.

A better understanding of the ER in apoptosis mayhave to await the clarification of BCL-2 function at thislocation. Do BCL-2 proteins operate at the ER as theydo at mitochondria and influence membrane perme-ability? If so, and if proapoptotic BAX and BAK arealso involved, is the mechanism different from that atthe OMM (Kuwana et al., Cell, 2002)? BCL-2 proteinsmay also play novel roles at the ER, for example byregulating initiator caspase activation complex(es) ana-logous to Ced-9 in C. elegans. Since deregulation ofBCL-2 family members at the ER affects cell survivaloutcomes, an understanding of their functions at thissite could be important for developing new therapeuticsfor treating diseases such as cancer. To date, however,little has emerged in the way of elucidating the criticalER transitions during apoptosis initiation that mightimpact the immediate downstream pathways. In retro-spect this leads one to contemplate on what our currentvision of mitochondria in apoptosis would be had cyt.cnot been discovered as a cytosolic factor required forcaspase-3 activation in vitro.

Acknowledgements

DGB, MG, and JM are recipients of the Canadian Institutes ofHealth Research Doctoral award.

References

Adachi T, Schamel WW, Kim KM, Watanabe T, Becker B,Nielsen PJ and Reth M. (1996). EMBO J., 15, 1534–1541.

Alberici A, Moratto D, Benussi L, Gasparini L, Ghidoni R,Gatta LB, Finazzi D, Frisoni GB, Trabucchi M, GrowdonJH, Nitsch RM and Binetti G. (1999). J. Biol. Chem., 274,30764–30769.

Annis MG, Zamzami N, Zhu W, Penn LZ, Kroemer G,Leber B and Andrews DW. (2001). Oncogene, 20, 1939–1952.

Antonsson B and Martinou JC. (2000). Exp. Cell Res., 256,50–57.

Baud V and Karin M. (2001). Trends Cell Biol., 11, 372–377.Berridge MJ, Lipp P and Bootman MD. (2000). Nat. Rev.

Mol. Cell Biol., 1, 11–21.Bourdon JC, Renzing J, Robertson PL, Fernandes KN andLane DP. (2002). J. Cell Biol., 158, 235–246.

Boya P, Cohen I, Zamzami N, Vieira HL and Kroemer G.(2002). Cell Death Differ., 9, 465–467.

Regulation of apoptosisDG Breckenridge et al

8616

Oncogene

Breckenridge DG, Nguyen M, Kuppig S, Reth M and ShoreGC. (2002). Proc. Natl. Acad. Sci. USA, 99, 4331–4336.

Breckenridge DG, Stoovic M, Marcellus R and Shore GC.(2003). J. Cell. Biol., 160, 1115–1127.

Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, HardingHP, Clark SG and Ron D. (2002). Nature, 415, 92–96.

Carlberg M, Dricu A, Blegen H, Kass GE, Orrenius S andLarsson O. (1996). Carcinogenesis, 17, 2589–2596.

Cavalli AL, O’Brien NW, Barlow SB, Betto R, GlembotskiCC, Palade PT and Sabbadini RA. (2002). Am. J. Physiol.Cell Physiol., 6, 6.

Chang SH, Phelps PC, Berezesky IK, Ebersberger Jr ML andTrump BF. (2000). Am. J. Pathol., 156, 637–649.

Cheng EH, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A,Ueno K and Hardwick JM. (1997). Science, 278, 1966–1968.

Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, LindstenT and Korsmeyer SJ. (2001). Mol. Cell, 8, 705–711.

Clem RJ, Cheng EH, Karp CL, Kirsch DG, Ueno K,Takahashi A, Kastan MB, Griffin DE, Earnshaw WC,Veliuona MA and Hardwick JM. (1998). Proc. Natl. Acad.Sci. USA, 95, 554–559.

Csordas G, Madesh M, Antonsson B and Hajnoczky G.(2002). EMBO J., 21, 2198–2206.

DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, GoldsteinJL and Espenshade PJ. (1999). Cell, 99, 703–712.

Diaz F and Bourguignon LY. (2000). Cell Calcium, 27,315–328.

Distelhorst CW, Lam M and McCormick TS. (1996).Oncogene, 12, 2051–2055.

Fischer H, Koenig U, Eckhart L and Tschachler E. (2002).Biochem. Biophys. Res. Commun., 293, 722–726.

Frank S, Gaume B, Bergmann-Leitrar ES, Laitmer WW,Robert EG, Catez F, Smith CL and Youle RS. (2001). Dev.Cell, 1, 515–525.

Fujita E, Kouroku Y, Jimbo A, Isoai A, Maruyama K andMomoi T. (2002). Cell Death Differ., 9, 1108–1114.

Germain M, Mathai JP and Shore GC. (2002). J. Biol. Chem.,277, 18053–18060.

Gervais FG, Xu D, Robertson GS, Vaillancourt JP, Zhu Y,Huang J, LeBlanc A, Smith D, Rigby M, Shearman MS,Clarke EE, Zheng H, Van Der Ploeg LH, Ruffolo SC,Thornberry NA, Xanthoudakis S, Zamboni RJ, Roy S andNicholson DW. (1999). Cell, 97, 395–406.

Green DR and Reed JC. (1998). Science, 281, 1309–1312.Hacki J, Egger L, Monney L, Conus S, Rosse T, Fellay I andBorner C. (2000). Oncogene, 19, 2286–2295.

Hajnoczky G, Csordas G, Madesh M and Pacher P. (2000).Cell Calcium, 28, 349–363.

Harding HP, Zhang Y, Bertolotti A, Zeng H and Ron D.(2000). Mol. Cell, 5, 897–904.

Harding HP, Zhang Y and Ron D. (1999). Nature, 397,271–274.

He H, Lam M, McCormick TS and Distelhorst CW. (1997). J.Cell Biol., 138, 1219–1228.

Hengartner MO and Horvitz HR. (1994). Cell, 76, 665–676.Herrera PL, Harlan DM and Vassalli P. (2000). Proc. Natl.

Acad. Sci. USA, 97, 279–284.Higgins ME and Ioannou YA. (2001). J. Lipid Res., 42,1939–1946.

Hirota J, Furuichi T and Mikoshiba K. (1999). J. Biol. Chem.,274, 34433–34437.

Huang DC and Shamer A. (2000). Cell, 103, 839–842.Ichimiya M, Chang SH, Liu H, Berezesky IK, Trump BF andAmstad PA. (1998). Am. J. Physiol., 275, C832–C839.

Ito Y, Pandey P, Mishra N, Kumar S, Narula N, KharbandaS, Saxena S and Kufe D. (2001). Mol. Cell. Biol., 21,6233–6242.

Jayaraman T and Marks AR. (1997). Mol. Cell. Biol., 17,3005–3012.

Johnson VL, Ko SC, Holmstrom TH, Eriksson JF and ChewSC. (2000). J. Cell. Biol., 113, 2941–2953.

Kaufman RJ. (1999). Genes Dev., 13, 1211–1233.Keenan RJ, Freymann DM, Stroud RM and Walter P. (2001).

Annu. Rev. Biochem., 70, 755–775.Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ andSchlesinger PH. (2000). Cell Death Differ., 7, 1166–1173.

Kuwana T, Mackey MR, Perkins G, Ellison MH, Latterich M,Schmeiter R, Green DR and Newmeyer DD. (2002). Cell,111, 331–342.

Lam M, Dubyak G, Chen L, Nunez G, Miesfeld RL andDistelhorst CW. (1994). Proc. Natl. Acad. Sci. USA, 91,6569–6573.

Lassus P, Opitz-Araya X and Lazebnik Y. (2002). Science,297, 1352–1354.

Letai A, Bassik M, Walensky L, Sorcinelli M, Weiler S andKorsmeyer S. (2002). Cancer Cell, 2, 183.

Li C, Fox CJ, Master SR, Bindokas VP, Chodosh LA andThompson CB. (2002). Proc. Natl. Acad. Sci. USA, 99,9830–9835.

Li H, Kolluri SK, Gu J, Dawson MI, Cao X, Hobbs PD, LinB, Chen G, Lu J, Lin F, Xie Z, Fontana JA, Reed JC andZhang X. (2000). Science, 289, 1159–1164.

Lilliehook C, Chan S, Choi EK, Zaidi NF, Wasco W, MattsonMP and Buxbaum JD. (2002). Mol. Cell. Neurosci., 19,552–559.

Lin Y, Ma W and Benchimol S. (2000). Nat. Genet., 26,122–127.

Lu DC, Rabizadeh S, Chandra S, Shayya RF, Ellerby LM,Ye X, Salvesen GS, Koo EH and Bredesen DE. (2000). Nat.Med., 6, 397–404.

Ma Y and Hendershot LM. (2001). Cell, 107, 827–830.Mancini M, Machamer CE, Roy S, Nicholson DW,Thornberry NA, Casciola-Rosen LA and Rosen A. (2000).J. Cell Biol., 149, 603–612.

Mathai JP, Germain M, Marcellus RC and Shore GC. (2002).Oncogene, 21, 2534–2544.

Mattson MP, Chan SL and Camandola S. (2001). BioEssays,23, 733–744.

McCormick TS, McColl KS and Distelhorst CW. (1997).J. Biol. Chem., 272, 6087–6092.

McCullough KD, Martindale JL, Klotz LO, Aw TY andHolbrook NJ. (2001). Mol. Cell. Biol., 21, 1249–1259.

Metzstein MM, Stanfield GM and Horvitz HR. (1998). TrendsGenet., 14, 410–416.

Morishima N, Nakanishi K, Takenouchi H, Shibata Tand Yasuhiko Y. (2002). J. Biol. Chem., 277, 34287–34294.

Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC,Agid Y and Ruberg M. (2000). J. Comp. Neurol., 426,297–315.

Murphy AN, Bredesen DE, Cortopassi G, Wang E andFiskum G. (1996). Proc. Natl. Acad. Sci. USA, 93,

9893–9898.Nakagawa T and Yuan J. (2000). J. Cell Biol., 150, 887–894.Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BAand Yuan J. (2000). Nature, 403, 98–103.

Nakamura K, Bossy-Wetzel E, Burns K, Fadel MP, Lozyk M,Goping IS, Opas M, Bleackley RC, Green DR and MichalakM. (2000). J. Cell Biol., 150, 731–740.

Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW,Cromlish JA and Shore GC. (1997). J. Cell Biol., 139,327–338.

Nguyen M, Breckenridge DG, Ducret A and Shore GC.(2000). Mol. Cell. Biol., 20, 6731–6740.

Regulation of apoptosisDG Breckenridge et al

8617

Oncogene

Nutt LK, Chandra J, Pataer A, Fang B, Roth JA, Swisher SG,O’Neil RG and McConkey DJ. (2002). J. Biol. Chem., 277,20301–20308.

Nutt LK, Pataer A, Pahler J, Fang B, Roth J, McConkey DJand Swisher SG. (2001). J. Biol. Chem., 6, 6.

Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, YamashitaT, Tokino T, Taniguchi T and Tanaka N. (2000). Science,288, 1053–1058.

Pan Z, Bhat MB, Nieminen AL and Ma J. (2001). J. Biol.Chem., 276, 32257–32263.

Passer BJ, Pellegrini L, Vito P, Ganjei JK and D’Adamio L.(1999). J. Biol. Chem., 274, 24007–24013.

Patil C and Walter P. (2001). Curr. Opin. Cell Biol., 13, 349–355.

Pinton P, Ferrari D, Magalhaes P, Schulze-Osthoff K,Di Virgilio F, Pozzan T and Rizzuto R. (2000). J. CellBiol., 148, 857–862.

Pinton P, Ferrari D, Rapizzi E, Di Virgilio FD, Pozzan T andRizzuto R. (2001). EMBO J., 20, 2690–2701.

Pozzan T, Rizzuto R, Volpe P andMeldolesi J. (1994). Physiol.Rev., 74, 595–636.

Pu Y, Luo KQ and Chang DC. (2002). Biochem. Biophys. Rev.Commun., 279, 762–769.

Puthalakath H, Huang DC, O’Reilly LA, King SM andStrasser A. (1999). Mol. Cell, 3, 287–296.

Puthalakath H and Strasser A. (2002). Cell Death Differ., 9,505–512.

Rao RV, Castro-Obregon S, Frankowski H, Schuler M, StokaV, del Rio G, Bredesen DE and Ellerby HM. (2002). J. Biol.Chem., 277, 21836–21842.

Rao RV, Hermel E, Castro-Obregon S, del Rio G, EllerbyLM, Ellerby HM and Bredesen DE. (2001). J. Biol. Chem.,276, 33869–33874.

Reddy RK, Lu J and Lee AS. (1999). J. Biol. Chem., 274,28476–28483.

Reimertz C, Kogal D, Rami A, Crittenden T and Prehn JH.(2003). J. Cell Biol., 162, 587–597.

Rudner J, Lepple-Wienhues A, Budach W, Berschauer J,Friedrich B, Wesselborg S, Schulze-Osthoff K and Belka C.(2001). J. Cell Sci., 114, 4161–4172.

Ruiz-Vela A, Gonzalez de Buitrago G and Martinez AC.(1999). EMBO J., 18, 4988–4998.

Ruiz-Vela A, Serrano F, Gonzalez MA, Abad JL, Bernad A,Maki M and Martinez AC. (2001). J. Exp. Med., 194, 247–254.

Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, TomaselliKJ, Debatin KM, Krammer PH and Peter ME. (1998).EMBO J., 17, 1675–1687.

Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA,Mannella CA and Korsmeyer SJ. (2002). Dev. Cell, 2, 55–67.

Scorrano L, Oakes SA, Pjerman ST, Chang EII, CorncinelliMD, Pozzon T and Kursmeyer SJ. (2003). Science, 300,135–139.

Selkoe DJ. (1999). Nature, 399, A23–A31.Shen X, Ellis RE, Lee K, Liu CY, Yang K, Solomon A,Yoshida H, Morimoto R, Kurnit DM, Mori K andKaufman RJ. (2001). Cell, 107, 893–903.

Sidrauski C, Chapman R and Walter P. (1998). Trends CellBiol., 8, 245–249.

Sugawara H, Kurosaki M, Takata M and Kurosaki T. (1997).EMBO J., 16, 3078–3088.

Szalai G, Krishnamurthy R and Hajnoczky G. (1999). EMBOJ., 18, 6349–6361.

Tagami S, Eguchi Y, Kinoshita M, Takeda M and TsujimotoY. (2000). Oncogene, 19, 5736–5746.

Torgler CN, Mariastella T, Raven T, Aubry J-P, Brown R andMeldrum E. (1997). Oncogene, 4, 263–271.

Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A,Bar-Sagi D, Jones SN, Flavell RA and Davis RJ. (2000).Science, 288, 870–874.

Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JSand Walter P. (2000). Cell, 101, 249–258.

Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, HardingHP and Ron D. (2000). Science, 287, 664–666.

Utz PJ, Hottelet M, Le TM, Kim SJ, Geiger ME, van VenrooijWJ and Anderson P. (1998). J. Biol. Chem., 273, 35362–35370.

Van de Craen M, Vandenabeele P, Declercq W, Van denBrande I, Van Loo G, Molemans F, Schotte P, VanCriekinge W, Beyaert R and Fiers W. (1997). FEBS Lett.,403, 61–69.

Walter J, Schindzielorz A, Grunberg J and Haass C. (1999).Proc. Natl. Acad. Sci. USA, 96, 1391–1396.

Wang B, Nguyen M, Breckenridge DG, Stojanovic M,Clemons PA, Kuppig S and Shore GC. (2003). J. Biol.Chem., 278, in press.

Wang HG, Pathan N, Ethell IM, Krajewski S, Yamaguchi Y,Shibasaki F, McKeon F, Bobo T, Franke TF and Reed JC.(1999). Science, 284, 339–343.

Wang KK. (2000). Trends Neurosci., 23, 20–26.Wang NS, Unkila MT, Reineks EZ and Distelhorst CW.(2001). J. Biol. Chem., 276, 44117–44128.

Wang X, Zelenski NG, Yang J, Sakai J, Brown MS andGoldstein JL. (1996). EMBO J., 15, 1012–1020.

Wang XZ, Harding HP, Zhang Y, Jolicoeur EM, Kuroda Mand Ron D. (1998). EMBO J., 17, 5708–5717.

Waterhouse NJ, Finucane DM, Green DR, Elce JS, Kumar S,Alnemri ES, Litwack G, Khanna K, Lavin MF and WattersDJ. (1998). Cell Death Differ., 5, 1051–1061.

Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopou-lou V, Ross AJ, Roth KA, MacGregor GR, Thompson CBand Korsmeyer SJ. (2001). Science, 292, 727–730.

Weller M, Malipiero U, Groscurth P and Fontana A. (1995).Exp. Cell Res., 221, 395–403.

Wellington CL and Hayden MR. (2000). Clin. Genet., 57, 1–10.Wolozin B, Iwasaki K, Vito P, Ganjei JK, Lacana E,Sunderland T, Zhao B, Kusiak JW, Wasco W andD’Adamio L. (1996). Science, 274, 1710–1713.

Wood DE and Newcomb EW. (1999). J. Biol. Chem., 274,8309–8315.

Wood DE, Thomas A, Devi LA, Berman Y, Beavis RC, ReedJC and Newcomb EW. (1998). Oncogene, 17, 1069–1078.

Wu F, Lukinius A, Bergstrom M, Eriksson B, Watanabe Yand Langstrom B. (1999). Eur. J. Cancer, 35, 1155–1161.

Xu Q and Reed JC. (1998). Mol. Cell, 1, 337–346.Ye J, Rawson RB, Kumnro R, Chan K, Done UP, Prymes R,BrownMG and Goldstein JL. (2000). Mol. Cell, 6, 1355–1364.

Yoneda T, Imaizumi K, Oono K, Yui D, Gomi F, Katayama Tand Tohyama M. (2001). J. Biol. Chem., 276, 13935–13940.

Yoshida H, Haze K, Yanagi H, Yura T and Mori K. (1998).J. Biol. Chem., 273, 33741–33749.

Yoshida H, Matsui T, Yamamoto A, Okada T and Mori K.(2001). Cell, 107, 881–891.

Youn HD, Sun L, Prywes R and Liu JO. (1999). Science, 286,790–793.

Zeng YS and Xu ZC. (2000). Neurosci. Res., 37, 113–125.Zhu L, Ling S, Yu XD, Venkatesh LK, Subramanian T,Chinnadurai G and Kuo TH. (1999). J. Biol. Chem., 274,33267–33273.

Zinszner H, Kuroda M, Wang X, Batchvarova N, LightfootRT, Remotti H, Stevens JL and Ron D. (1998). Genes Dev.,12, 982–995.

Zong WA, Li C, Hatzivassilion G, Lmdrtem T, Yu QC, YuanI and Thompson CB. (2003). J. Cell Biol., 162, 59–69.

Regulation of apoptosisDG Breckenridge et al

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