characterization of cytoplasmic caspase-2 activation by induced proximity

Characterization of cytoplasmic caspase-2 activation by inducedproximity

Lisa Bouchier-Hayes1, Andrew Oberst1, Gavin P. McStay1, Samuel Connell1, Stephen W.G.Tait1, Christopher P. Dillon1, Jonathan M. Flanagan2, Helen M. Beere1, and Douglas R.Green1,*1 Department of Immunology St. Jude Children's Research Hospital, 262 Danny Thomas Place,Memphis, TN, U.S.A., 381052 Department of Hematology St. Jude Children's Research Hospital, 262 Danny Thomas Place,Memphis, TN, U.S.A., 38105

AbstractCaspase-2 is an initiator caspase, activated in response to heat shock and other stressors that induceapoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2activation complexes, such as the PIDDosome. We have adapted bimolecular fluorescencecomplementation (BiFC) to measure caspase-2 induced proximity in real time, in single cells. Non-fluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescentcomplex were fused to caspase-2 allowing visualization and kinetic measurements of caspase-2induced proximity after heat shock and other stresses. This revealed that the caspase-2 activationplatform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage,cytoskeletal disruption and other treatments. Activation, as measured by this approach, in responseto heat shock, was RAIDD-dependent and upstream of mitochondrial outer membranepermeabilization. Furthermore we identify Hsp90α as a key negative regulator of heat shock-inducedcaspase-2 activation.

IntroductionThe initiator caspases-8, -9 and -10 are responsible for cleavage and activation of theexecutioner caspases-3, and -7 that orchestrate apoptosis. Unlike executioner caspases, initiatorcaspases are activated by dimerization (Boatright et al., 2003; Donepudi et al., 2003). Upondimerization, initiator caspases undergo auto-cleavage that stabilizes the active enzyme (Popet al., 2007). Initiator caspases are generally activated by recruitment to large molecular weightprotein complexes or “activation platforms” that induce the proximity of caspase molecules,facilitating dimerization (Boatright and Salvesen, 2003). For example, caspase-8 is activatedwhen bound to the death inducing signaling complex (DISC) following death receptor ligation.In a similar fashion, caspase-9 is activated by the Apaf-1 apoptosome formed upon cytochromec release from the mitochondria (Creagh et al., 2003).

© 2009 Elsevier Inc. All rights reserved.*Correspondence: [email protected]'s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptMol Cell. Author manuscript; available in PMC 2010 September 25.

Published in final edited form as:Mol Cell. 2009 September 25; 35(6): 830–840. doi:10.1016/j.molcel.2009.07.023.

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Caspase-2 is also an initiator caspase but specific details of how, where and when it is activatedremain unclear. Unlike other initiator caspases, caspase-2 does not directly activate executionercaspases but instead acts via mitochondrial outer membrane permeabilization (MOMP) toinduce apoptosis (Guo et al., 2002). The caspase-2 activation platform appears to be a complexof proteins termed the PIDDosome, consisting of PIDD, the adaptor protein RAIDD andcaspase-2 (Tinel and Tschopp, 2004). RAIDD binds and induces dimerization of the pro-formof caspase-2 via interaction between the caspase recruitment domain (CARD) present in bothproteins (Duan and Dixit, 1997). A second caspase-2 activation platform has been identifiedto contain PIDD, caspase-2 and the nuclear serine/threonine kinase DNA-PKcs and does notrequire RAIDD (Shi et al., 2009). Caspase-2 was initially considered to be a component of theTNF receptor complex and hence has been reported to be activated independently of thePIDDosome through certain death receptor pathways (Droin et al., 2001; Duan and Dixit,1997; Lavrik et al., 2006; Wagner et al., 2004). Indeed, studies with PIDD-deficient cellssuggest that PIDD is not essential for caspase-2 activation (Manzl et al., 2009), suggesting thatalternate caspase-2 activation platforms may exist.

Caspase-2 deficient mice have a mild apoptotic phenotype, the defining feature of which is anexcess number of oocytes (Bergeron et al., 1998). However, caspase-2 deficient cells havebeen shown to be more resistant to apoptosis induced by cytoskeletal disruptors, such asvincristine and cytochalasin D (Ho et al., 2008), and heat shock (Tu et al., 2006). DHEA, aninhibitor of the pentose phosphate pathway, has been shown to activate caspase-2 in Xenopusoocytes, suggesting a role in metabolic pathways (Nutt et al., 2005). In neurons, caspase-2 hasbeen shown to be activated during β-amyloid and trophic factor withdrawal-induced apoptosis(Stefanis et al., 1998; Troy et al., 2000) although in the latter case caspase-9 can compensatefor the lack of caspase-2 in knockout animals (Troy et al., 2001). Caspase-2 has also beenimplicated in both p53-dependent (Lassus et al., 2002; Robertson et al., 2002) and p53-independent DNA damage-induced apoptosis (Shi et al., 2009; Sidi et al., 2008). Howevermany of these observations are still considered quite controversial (Krumschnabel et al.,2009).

Consistent with a role in DNA damage, caspase-2 has been shown to be a tumor suppressor(Ho et al., 2009). This may suggest that caspase-2 functions in the nucleus. Indeed, caspase-2has been observed in the nucleus and it has been suggested that this is the site of its activation(Baliga et al., 2003; Colussi et al., 1998; Paroni et al., 2002). For example, the DNA-PKcs-containing PIDDosome appears to activate caspase-2 in the nucleus following DNA damage(Shi et al., 2009). However, caspase-2 has also been shown to reside in the cytoplasm and atthe Golgi complex (Mancini et al., 2000) and may also be activated in these subcellularlocations.

A major contributing factor to the paucity of knowledge on the physiological activation ofcaspase-2 is that current methods are limited in their ability to measure the activation of theinitiator caspases. Caspase-2 undergoes autocatalytic processing when it is activated and isalso cleaved by caspase-3 (Slee et al., 1999) downstream of MOMP. However, such cleavagedoes not activate it (Baliga et al., 2004). Thus, monitoring cleavage alone is not sufficient toidentify caspase-2 as the apical caspase. Similarly, inhibitors and fluorogenic substrates basedon the preferred caspase-2 substrate sequence, VDVAD can respectively inhibit or be cleavedas efficiently by other caspases, potentially leading to spurious results (McStay et al., 2008).Furthermore, none of these methods can elucidate the localization or kinetics of caspase-2activation. Therefore we sought to use recruitment of caspase-2 to its activation platforms, theproximal step in the caspase-2 pathway (Baliga et al., 2004), as a readout for caspase-2activation.

Bouchier-Hayes et al.

Mol Cell. Author manuscript; available in PMC 2010 September 25.

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In order to measure caspase-2 recruitment to activation platforms accurately, we adapted theBimolecular Fluorescence Complementation (BiFC) technique. BiFC uses split fluorescentproteins that alone are not fluorescent but when fused to interacting proteins associate to formthe fluorescent molecule (Shyu et al., 2006). In this study we have fused caspase-2 to the N-terminal half and to the C-terminal half of Venus fluorescent protein. Following recruitmentof caspase-2 to its activation platform, induced proximity of caspase-2 occurs and,consequently, association of the two halves of Venus is enforced. Induced proximity ofcaspase-2 is required for its dimerization and is measured as increased Venus fluorescence.Using this approach, we characterize the kinetics and localization of caspase-2 activation andreveal an unexpected role for Hsp90 in the regulation of heat stress-induced caspase-2dimerization.

ResultsTo study the recruitment of caspase-2 to its activation platforms, its subsequent inducedproximity and activation during apoptosis, we used the BiFC assay described in theintroduction. We fused the CARD domain of caspase-2 (C2-CARD, aa 1-122) to each of thesplit Venus proteins, Venus C 155-239 and Venus N 1-173. We transiently expressed the C2-CARD BiFC pair in Hela cells and investigated the ability of the proteins to undergo BiFCupon co-expression of the PIDDosome components, PIDD and RAIDD (Figure 1A, B). UponPIDD expression, almost half of the cells became Venus-positive indicating induction of BiFCof the C2-CARD pair. Expression of RAIDD induced C2-CARD BiFC in the majority of cells,while neither the CARD containing protein, Apaf-1, nor the death domain containing protein,FADD, induced BiFC above background levels. When RAIDD expression was titrated to levelswhere only a small amount of BiFC was observed, PIDD was able to synergize with RAIDDto restore C2-CARD BiFC in the majority of cells (Figure 1C, 1D). Thus, we were able toinduce BiFC of C2-CARD by reconstituting the PIDDosome, indicating that the BiFC observedrepresents caspase-2 induced proximity upon recruitment to its activation platform.

We have previously shown that heat shock activates caspase-2 (Tu et al., 2006) and we thereforedetermined if heat shock can similarly induce BiFC of caspase-2. Following heat shock, cellsexpressing the C2-CARD BiFC pair became strongly Venus-positive (Figure 2A, 2B). Thiseffect was dose-dependent with respect to the amount of the BiFC pair expressed, while at themaximum amount of plasmid the level of spontaneous Venus re-association remained low.

To determine if the CARD of caspase-2 accurately recapitulated the behavior of the intactprotein, we generated Venus C (VC) and Venus N (VN) epitope-tagged full-length caspase-2(C2-FL, Figure 2C). The catalytic cysteine (C303) in caspase-2 was mutated to an alanine toprevent apoptosis upon expression of the caspase, to facilitate analysis. Caspase-2 contains aclassical nuclear localization sequence (NLS) in its prodomain (aa 131-138) that is not presentin C2-CARD (Baliga et al., 2003). Thus, we also generated Venus C and Venus N-taggedversions of a fragment of caspase-2 containing the prodomain including the NLS (C2-Pro, aa1-147). When we expressed each of these BiFC pairs in Hela cells they all induced BiFCfollowing heat shock (Figure 2D and Supplemental Figure S1A) suggesting that the NLS isnot required for caspase-2 induced proximity.

We next explored if caspase-2 induced proximity was induced by other apoptotic stimuli,specifically those reported to activate caspase-2. As before, we transiently expressed the C2-CARD BiFC pair in Hela cells and observed induction of BiFC under different conditions(Figure 2E, 2F, Supplemental Figure S2). Treatment with TNF or etoposide, resulted in anincrease in the number of Venus-positive cells over background although the cells showedmarginal brightness (Figure 2E). The tubulin disruptors, taxol, vincristine and colchicine, alsocaused an increase in the number of positive cells. These stimuli induced similar levels of BiFC



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when the C2-Pro or C2-FL pair was expressed under similar conditions (Supplemental FigureS1B, S1C, S1D). In contrast, the actin disruptor cytochalasin B had little effect, whilecytochalasin D showed an increase over background (Figure 2F, Supplemental Figure S2). Thepentose phosphate pathway inhibitor, DHEA, also induced considerable C2-CARD BiFC(Supplemental Figure S2). Treatment with anti-Fas, which induces substantial apoptosis inHela cells (data not shown), did not induce BiFC (Supplemental Figure S2), indicating that theinduced proximity observed only occurs in response to certain stresses reported to activatecaspase-2, including DNA damage, cytoskeletal disruption and metabolic stress (Ho et al.,2008; Nutt et al., 2005; Robertson et al., 2002). Of the conditions analyzed, heat shock resultedin the most robust BiFC response and thus we proceeded to rigorously characterize theregulation of caspase-2 activation induced by heat stress.

Using time-lapse confocal microscopy, we analyzed the kinetics of caspase-2 recruitment toactivation platforms in Hela cells expressing the C2-CARD Venus pair during heat shock-induced apoptosis. C2-CARD complexes were detected as fluorescent punctate spots in thecytoplasm as early as 5 hr after heat shock that increased in intensity over time (Figure 3A,Supplemental Movie S1). Similar kinetics were observed when the C2-Pro or C2-FL pair wereexpressed (Supplemental Figure S3). To clarify the origin and destination of these punctatespots within the cell we increased the resolution of the time-lapse imaging by taking a numberof confocal sections through the z-plane of the cell. Analysis of BiFC in the 3D time-lapseshowed that the punctate spots originated at the periphery of the cell and translocated toaccumulate in a region adjacent to the nucleus (Figure 3B, Supplemental Movie S2). Theaverage intensity of Venus in each cell was measured at each time point, and showed a steadyincrease in the fluorescence starting at approximately 5 hr after heat shock (Figure 3C). Inuntreated cells no increase in Venus intensity was observed (Figure 3C). Similarly, cellsexposed to a non-lethal heat shock of 42°C failed to induce an appreciable amount of bi-fluorescence. Thus this effect was specific to heat shock at 45°C, the temperature required toinduce apoptosis in Hela cells, demonstrating that the kinetic events we observed representheat shock-induced capase-2 induced proximity in real time.

The results in Figures 3 and S3 suggest that caspase-2 activation platforms occur in the cytosolafter heat shock. However, numerous reports suggest that caspase-2 is activated in the nucleus(Baliga et al., 2003; Paroni et al., 2002). Using immunofluorescence, we found that, consistentwith previous reports, endogenous caspase-2 is localized to the Golgi apparatus, the nucleusand the cytosol ((Mancini et al., 2000); Supplemental Figure S4). After heat shock, caspase-2remained associated with the Golgi apparatus, although the Golgi itself became dismantled.To more accurately determine the subcellular compartment where induced proximity ofcaspase-2 occurs, we transiently expressed each of the BiFC pairs in Hela cells and investigatedthe subcellular localization of caspase-2 BiFC. Following heat shock the C2-FL, C2-Pro andC2-CARD BiFC pairs each appeared as a series of punctate spots in the cytoplasm (Figure 4Aand Supplemental Movies S3-6). The 3D images of the cells clearly show that caspase-2induced proximity did not occur in the nucleus and was distinctly cytoplasmic for C2-CARD,C2-FL and C2Pro. We observed similar patterns of caspase-2 induced proximity in responseto treatment with vincristine, taxol and colchicine (Figure 4B and Supplemental Figure S5)indicating that cytoskeletal disruption also activates caspase-2 in the cytosol. Finally, treatmentwith etoposide also resulted in a cytosolic rather than nuclear localization of caspase-2 inducedproximity (Supplemental Figure S5). These results indicate that caspase-2 activation generallyoccurs in the cytoplasm rather than the nucleus as detected by this approach.

Given that we observed efficient caspase-2 BiFC in response to heat shock and that we havepreviously shown that heat shock-induced activation of caspase-2 is RAIDD dependent (Tu etal., 2006), we investigated the dependency of casapse-2 induced proximity on RAIDD. Theintroduction of two point mutations, D83A and E87A into the CARD domain of caspase-2 has



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been reported to disrupt the interaction between caspase-2 and RAIDD (Duan and Dixit,1997). Co-immunoprecipitation experiments showed that these mutations attenuated thebinding of caspase-2 to RAIDD rather than completely disrupting it (Figure 5A). Consistentwith the weaker binding of C2-CARD to RAIDD, the level of BiFC after heat shock was greatlyreduced when the C2-CARD mutant was expressed compared to that of the wild type C2-CARD (Figure 5B, 5C). This result suggested that caspase-2 induced proximity requires itsbinding to RAIDD. This result also indicates that the BiFC observed is due to a RAIDD-CARD/C2-CARD interaction and not a C2-CARD/C2-CARD interaction.

To further investigate the dependence of caspase-2 activation on its interaction with RAIDDduring heat shock, we expressed the C2-CARD, C2-Pro and C2-FL pairs in RAIDD-deficientmouse embryonic fibroblasts (MEF). Similar to our observations in Hela cells, when wild typeMEF were subjected to heat shock, the number of Venus-positive cells increased in a dose-dependent manner with respect to the amount of the caspase-2 pair expressed. However, in theabsence of RAIDD, the percentage of Venus-positive cells was greatly reduced in each case(Figure 5D, Supplemental Figure S6). When RAIDD was reintroduced into RAIDD-deficientcells by transient transfection, its expression restored the C2-CARD BiFC induced by heatshock to wild type levels (Figure 5E, 5F). Together, these results demonstrate that caspase-2requires RAIDD for induced proximity. Thus we conclude that the activation platform thatrecruits caspase-2 after heat shock includes the adaptor protein RAIDD.

The anti-apoptotic proteins Bcl-2 and Bcl-xL are potent inhibitors of heat shock inducedapoptosis (Cuende et al., 1993), suggesting that MOMP is required in this pathway. Asexpected, Hela cells expressing Bcl-xL were protected from heat shock-induced apoptosis(Figure 6A). However, when the C2-CARD pair was expressed in Hela cells expressing Bcl-xL the increase in BiFC intensity over time after heat shock was identical to that observed forHela cells (Figure 6B). The inability of Bcl-xL to block C2-CARD induced proximity isconsistent with previous reports that caspase-2 activation occurs upstream of MOMP (Bonzonet al., 2006; Tu et al., 2006). To formally test this, we fused the catalytic domains (aa 110-436)of caspase-2 to FV (F36V), a modified FK506 binding protein (FKBP). Addition of FKBP toa protein enables conditional dimerization by addition of a ligand containing two FK506moieties (AP20187) to the medium. Enforced dimerization of caspase-2 induced apoptosis inthese cells and this death was completely inhibited by Bcl-xL (Figure 6C). Therefore activationof caspase-2 occurs upon dimerization of the caspase, inducing apoptosis in a Bcl-xL inhibitablemanner. Thus, caspase-2 activation is upstream of MOMP and apoptosis.

During apoptosis, the mitochondrial intermembrane space proteins cytochrome c, Omi andSmac are released simultaneously (Munoz-Pinedo et al., 2006). Therefore, to furthercharacterize the temporal relationship between caspase-2 activation and MOMP, we expressedthe C2-CARD pair in Hela cells expressing Omi fused to the fluorescent protein mCherry.Using time-lapse microscopy, we compared the onset of C2-CARD BiFC to the release ofOmimCherry from the mitochondria. As shown in Figure 6D and 6E, induction of BiFC startsapproximately 4 hours after heat shock while the onset of MOMP occurs 6-10 hr later(Supplemental Movie S7). It is of note that not all cells undergo MOMP during the timeframeof the time-lapse experiments although the BiFC is induced in these cells at similar times (inthe example shown in Figure 6E, right, MOMP did not occur, despite caspase-2 dimerization).Thus recruitment of caspase-2 to its activation platform alone may not be sufficient to committhe cell to apoptosis, indicating regulatory steps upstream of MOMP.

To identify some of these regulatory elements upstream of MOMP, we further explored therelationship between heat shock-induced stress and caspase-2 activation. To determine ifcaspase-2 induced proximity was regulated by heat-induced transcription of pro-apoptoticfactors, we heat shocked cells expressing the C2-CARD BiFC pair in the presence of the protein



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synthesis inhibitor cycloheximide. Rather than inhibiting heat shock-induced C2-CARD BiFC,we observed that the same proportion of cells became Venus-positive when cells were heatedin the presence and absence of cycloheximide. (Figure 7A). This indicated that caspase-2induced proximity does not require heat shock-induced transcriptional upregulation of pro-apoptotic proteins to proceed. This suggests that assembly of caspase-2 activation platformssuch as the PIDDosome is not due to heat shock-induced expression of its components, suchas PIDD or RAIDD.

Nevertheless, it is well established that heat regulates the transcription of the heat shockproteins (Hsps) that can promote cell survival (Li and Werb, 1982; Strasser and Anderson,1995; Subjeck et al., 1982). Stress-induced expression of Hsps is mediated primarily by thetranscription factor heat shock factor-1 (HSF-1), which is protective against lethal heat stress(McMillan et al., 1998). As predicted, HSF-1−/− MEF were much more sensitive to heat shock-induced apoptosis compared with wild type MEF (Figure 7B). We expressed the C2-CARDBiFC pair in HSF-1+/+ and HSF-1−/− cells and subjected them to heat shock. At temperaturesthat are non-lethal in wild type cells (43°C), a greater proportion of HSF-1-deficient cellsbecame Venus-positive compared to wild type cells (Figure 7C). We observed similar resultsin cells expressing C2-Pro BiFC or C2-FL BiFC (Supplemental Figure S7A and S7B). Thisresult indicated that HSF-1 inhibits heat shock-induced caspase-2 activation.

One potential means for this inhibition is through HSF-1-mediated expression of a heat shockprotein, such as Hsp90α (the inducible form of Hsp90 (Xiao et al., 1999)). To investigate this,we blocked Hsp90 function by adding a pharmalogic inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; (Sharp and Workman,2006)). Treatment of HSF-1+/+ cells expressing the C2-CARD, C2-Pro or C2-FL BiFC pairwith 17-DMAG did not lead to a significant increase in Venus-positive cells (Figure 7D, 7E,Supplemental Figure S7A, S7B). However, when we mildly heat stressed HSF-1+/+ cells inthe presence of 17-DMAG, the proportion of cells displaying BiFC was comparable to thatobserved in HSF-1−/− cells subjected to heat shock (Figure 7D, 7E, Supplemental Figure S7A,S7B). In Hela cells we observed that treatment with 17-DMAG alone led to a modest amountof C2-CARD induced proximity (Supplemental Figure S7C). When we combined 17-DMAGwith mild heat stress at 43°C, that does not normally induce C2-CARD dimerization, werestored the level of BiFC to that observed when the cells were heat shocked at 45°C. Thisstrongly suggests that activation of HSF-1 by heat induces the expression of Hsp90 that, inturn, can inhibit induced proximity of caspase-2.

To determine directly if Hsp90 can inhibit caspase-2 activation, we investigated the ability ofHsp90 to inhibit processing of caspase-2 in vitro. We heated Jurkat lysates to 37°C to inducespontaneous PIDDosome assembly (Tinel and Tschopp, 2004) in the presence or absence ofHsp90α recombinant protein. Hsp90α completely blocked caspase-2 processing after 30 minand, after 1 hr; only the p31 intermediate fragment was detected while the p19 and p12fragments that represent complete cleavage were not produced (Supplemental Figure S6D).Therefore, we concluded that Hsp90α is an efficient inhibitor of caspase-2 activation in vitro.As we have noted, cleavage of caspase-2 is not a demonstration that it has been activated.However, these results are consistent with our findings in cells that HSP90α interferes, directlyor indirectly, with the activation of caspase-2.

To further investigate if caspase-2 activity is inhibited by Hsp90α, we used RNAi to specificallyknockdown Hsp90α. We achieved approximately 50% knockdown of Hsp90α without anyeffect on Hsp90β (Figure 7F). Under these conditions we found that siRNA mediatedknockdown of Hsp90α had a similar effect to 17-DMAG treatment, such that it sensitized cellsto formation of the caspase-2 activation platform at 43°C as detected by BiFC (Figure 7G).Together these results strongly suggest that Hsp90α negatively regulates caspase-2 activation.



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DiscussionDetecting initiator caspase activation is both difficult and frequently inaccurate using existingmethods. In this study, we took advantage of the fact that initiator caspase activation is drivenby induced proximity (Boatright et al., 2003; Read et al., 2002; Salvesen and Dixit, 1999) todevelop a real time technique to monitor initiator caspase activation in single cells. Using thisapproach, we visualized caspase-2 induced proximity in cells in response to heat shock and anumber of other stimuli reported to activate caspase-2 (Figure 2).

Of the initiator caspases, the regulation of caspase-2 activation is the least well characterized.However, similar to caspase-8 and caspase-9, it has been shown that cleavage of the caspaseis not required for enzymatic activity (Baliga et al., 2004). Since caspase-2 can be cleaved byactive executioner caspases (Slee et al., 1999), monitoring the kinetics of cleavage of caspase-2does not truly reflect its activation state. Therefore we have developed a method that directlymeasures engagement of caspase-2 by its activation platform and consequently is a specificand accurate method of measuring initiator caspase activation.

The refolding of split Venus fragments and its associated fluorescence is bright, highlyphotostable (Shyu et al., 2006) and rapid (Hu et al., 2002; Schmidt et al., 2003). This allowedus to accurately pinpoint the onset of caspase-2 induced proximity as well as the subcellulardistribution of activated caspase-2. We detected caspase-2 induced proximity as early as fivehours after heat shock (Figure 3C). The delay between caspase-2 activation and onset of MOMPvaried from 4-10 hours (Figure 6D, 6E). Despite caspase-2 BiFC being detected in the majorityof cells, not all of these cells underwent MOMP. This could be due to inefficient cleavage ofBid by caspase-2 compared to caspase-8 (Bonzon et al., 2006). Alternatively, this could resultfrom negative regulation of caspase-2 by the components of the assay system.

Caspase-2 BiFC appeared as fluorescent dots in the cytoplasm that are not associated withmitochondria (Figure 3). These fluorescent structures likely represent caspase-2 activationplatforms and are similar to the aggregates observed in the nucleus when caspase-2-GFP isoverexpressed, (Baliga et al., 2003). It has been proposed that the latter aggregates mayrepresent caspase-2 recruitment to PML bodies in the nucleus (Sanchez-Pulido et al.,2007;Tang et al., 2005). Alternatively, these aggregates may represent sites of the nuclearPIDDosome that contains DNA-PKcs, which seems to function primarily in the maintenanceof a cell cycle checkpoint rather than apoptosis (Shi et al., 2009). If caspase-2 is recruited tosuch structures, our results show that it is not brought into sufficient proximity to allow BiFC.Furthermore, this complex reportedly does not require RAIDD, while our results clearly showthat the recruitment of caspase-2 to activation platforms in the cytoplasm induced by heat shockrequires RAIDD (Figure 5).

Many of the aforementioned studies use immunofluorescence methods or overexpression ofGFP-tagged caspase-2 to detect the localization of caspase-2. In contrast to the BiFC approach,these techniques cannot distinguish between active and inactive caspase-2. Our resultsindicated that caspase-2 is not dimerized in the nucleus and that the pattern of caspase-2 BiFCis not dependent on the NLS (Figure 4). We do not rule out, however, that there is an alternativefunction for caspase-2 in the nucleus that is independent of its recruitment to activationplatforms or activation. The aggregates of caspase-2 we observed in the cytosol are likely tobe composed of the PIDDosome, providing an activation platform for caspase-2. Supportingthis hypothesis, caspase-1 has been shown to co-localize with its adaptor protein ASC to similarcytosolic structures (Stehlik et al., 2003).

Our results indicate that Hsp90 can negatively regulate caspase-2 dimerization in response toheat shock (Figure 7). Heat shock proteins have been implicated as regulators of other initiatorcaspases. Hsp70 and Hsp90 inhibit apoptosome assembly by binding to Apaf-1 (Beere et al.,



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2000;Pandey et al., 2000;Saleh et al., 2000). Similarly, Hsp90 binds and inhibits NALP3, acomponent of the inflammasome that regulates caspase-1 (Mayor et al., 2007). It is not clearhow Hsp90 inhibits caspase-2 activation, but based on its proposed mechanism of inhibitionof the other complexes, Hsp90 may operate in an analogous way by preventing PIDDosomeassembly through binding one of the components of the complex. We detected binding ofHsp90α to PIDD upon exogenous expression (data not shown) but this binding is not directevidence of a mechanism for Hsp90α-mediated inhibition of caspase-2. It is equally possiblethat Hsp90α inhibits caspase-2 induced proximity by some other means. A number of Hsp90client proteins, such as p53 and ChkI (Arlander et al., 2003;Blagosklonny et al., 1996), havebeen implicated in the regulation of caspase-2 activation (Baptiste-Okoh et al., 2008;Sidi etal., 2008) and thus Hsp90α may inhibit caspase-2 through the regulation of these or other clientproteins. Ongoing studies will help to resolve the mechanisms whereby this chaperoneinfluences the activation of caspase-2.

The Hsp90 inhibitor, 17-DMAG, sensitized cells to heat shock-induced caspase-2 activation(Figure 7D, 7E). 17-DMAG binds to and blocks the nucleotide-binding pocket of Hsp90,causing dissociation from and destabilization of its client proteins. 17-DMAG is currently inclinical trials as a potential anti-tumor therapy (Sharp and Workman, 2006). Sensitization tocaspase-2-induced apoptosis may contribute to the anti-tumor effects of 17-DMAG. Furthercharacterization of the PIDDosome is required to fully elucidate the role of Hsp90 in theregulation of caspase-2 activation.

These results demonstrate the ability of BiFC to accurately and specifically measurerecruitment of initiator caspases to their respective activation platforms. This powerfulapproach can be used to further dissect the more elusive aspects of caspase-2 regulation duringapoptosis. Clearly this approach can also be extended to other CARD containing proteins thatmay be activated by induced proximity as well as other protein-protein interactions.

Experimental ProceduresCell culture and induction of apoptosis—Human Embryonic Kidney (HEK) 293T andHela cells were grown in Dulbecco's Modified Essential Medium (DMEM, GIBCO BRL) andmouse embryonic fibroblasts (MEF) were grown DMEM supplemented with non-essentialamino acids, sodium pyruvate and 2-mercaptoethanol (55μM). All media were supplementedwith 2mM glutamine, antibiotics and 10% fetal bovine serum (FBS).

To induce apoptosis by heat shock, media on the cells was exchanged for media warmed tothe heat shock temperature and cells were placed in an incubator set at the same temperaturefor one hour. Cells were returned to 37°C for the times indicated. qVD-OPH (20μM, MPBiomedicals) was included to inhibit caspases.

Microscopy—Cells were imaged using a spinning disk confocal microscope (Zeiss). Helacells were plated on dishes containing coverslips (Mattek) 24 hr prior to treatment. For time-lapse experiments media on the cells was supplemented with Hepes (20mM) and 2-mercaptoethanol (55μM). Cells were allowed to equilibrate to 37°C in 5% CO2 prior tofocusing on the cells. Each experiment included control images of untreated cells to ensurethat the imaging conditions did not induce phototoxicity and cell division proceeded.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.



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AcknowledgementsWe thank M. Parsons and M. Pinkoski for careful reading of the manuscript and F. Llambi for invaluable discussion,Dr. C-D Hu (Purdue University, Indiana) for BiFC plasmids, Dr. T. Mak (U. Toronto) for RAIDD-deficient mice andDr I. Benjamin (University of Utah) for HSF-1-deficient MEF. This work was supported by NIH grant AI47891(DRG).

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Figure 1.C2-CARD BiFC is induced by components of the PIDDosome. (A) Hela cells were transientlytransfected with C2-CARD VN (20ng) and C2-CARD VC (20ng) along with 500ng ofexpression plasmids encoding PIDD, RAIDD, FADD or Apaf-1. All wells also receivedpshooter.dsRed-mito (10ng) as a reporter for transfection. 24 hr after transfection thepercentage of dsRed-mito positive (red) cells that were Venus-positive (green) was determinedfrom a minimum of 300 cells per well. Results represent triplicate counts with error barsrepresenting standard deviation (B) Representative confocal images of cells from (A) areshown. (C) Hela cells were transiently transfected as in (A) with the indicated amounts ofexpression plasmid. 48 hr after transfection the percentage Venus-positive cells wasdetermined as in (A). (D) Representative images of cells from (C) are shown. Scale barsrepresent 10μm.



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Figure 2.Induced proximity of caspase-2 is induced by various stressors. (A) Hela cells were transientlytransfected with the indicated amounts of each of C2-CARD VC, C2-CARD VN andpshooter.dsRed-mito (10ng). 24 hr later cells were either left untreated or heated for 1 hr at45°C. All cells were treated with qVDOPH (20μM). Cells were assessed 24 hr after treatmentfor the percentage cells that were Venus-positive cells (green), determined from a minimumof 300 cells per well. Results represent triplicate counts with error bars representing standarddeviation. (B) Representative images of cells from (A) are shown. (C) Schematic representationof the caspase-2 BiFC constructs. (*) indicates the active cysteine that was mutated to alaninein C2-FL. (D) Hela cells were transiently transfected with expression plasmids with C2-CARD,C2-Pro (20ng of each) or C2-FL BiFC plasmid pair (100ng of each) along with pshooter.dsRed-mito (10ng). Cells were treated and assessed as in (A). (E) Hela cells were transientlytransfected with plasmids encoding C2-CARD VC (20ng), C2-CARD VN (20ng) and dsRed-mito (10ng) for 24 hr followed by treatment with anti-Fas (250ng/ml)/CHX (10μg/ml), TNF(10ng/ml)/CHX (10μg/ml), etoposide (5μM), taxol (10μg/ml) or vincristine (50μg/ml), in thepresence of qVD-OPH (20μM). Representative confocal images were taken 24 hr aftertreatment. (F) Cells were transfected as in (E) for 24 hr followed by treatment with etoposide(Etop, 5μM), TNF (10ng/ml)/CHX (10μg/ml), taxol (Tax, 1μg/ml) or heat shock (45°C, 1 hr),cytochalasin B (CytoB, 20μg/ml) or vincristine (50μg/ml), with qVD-OPH (20μM). Cells wereassessed as in (A). Scale bars represent 10μm



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Figure 3.Kinetics of heat shock-induced C2-CARD BiFC. (A) Hela cells were transiently transfectedwith plasmids encoding C2-CARD VN (20ng), C2-CARD VC (20ng) and dsRed-mito (10ng).24 hr later cells were incubated at 45°C for 1 hr plus qVD-OPH (20μM). The cells were returnedto 37°C on a confocal microscope and images were taken every 6 min for 16 hr. Frames fromthe movie show BiFC (green) of a representative cell. (B) Hela cells were transfected andtreated as in (A). Confocal images from a time-lapse with a 3 × 1.5μm z-stack are shown.(C) Hela cells were transfected as in (A). 24 hr later cells were either left untreated or heatedat 42°C or 45°C for 1 hr with qVD-OPH (20μM).The cells were returned to 37°C on a confocal microscope and images were taken every 15min for 16 hr. The average intensity of Venus was measured at each time point. Each line ofthe graph is an average of 21 (untreated), 35 (42°C), or 41 (45°C) individual cells and errorbars represent SEM. Scale bars represent 10μm.



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Figure 4.Heat shock-induced caspase-2 BiFC is cytoplasmic. (A) Hela cells were transiently transfectedwith expression plasmids encoding C2-FL VC (100ng), C2-FL VN (100ng) or with theindicated BiFC plasmid pairs (20ng of each) along with dsRed-mito (10ng). 24 hr aftertransfection cells were left untreated or heated at 45°C for 1 hr with qVD-OPH (20μM). Cellswere assessed for BiFC (green) 24 hr after treatment. (B) Hela cells were transfected as in (A)and 24 hr after transfection cells were either left untreated or treated with vincristine (25μg/ml) with qVD-OPH (20μM). Cells were assessed 24 hr after treatment. Nuclei were stainedwith Draq5 (blue). Images are 3D isosurface rendering reconstructions composed from 0.1μm



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(A) or 0.2μm (B) serial confocal images through the z-plane of the cell. Scale bars represent10μm.



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Figure 5.Heat shock-induced C2-CARD BiFC requires RAIDD. (A) D83A/E87A C2-CARD mutantbinds RAIDD with less efficiency than wild type. 293T cells were transiently transfected withplasmids encoding FLAG-RAIDD and HA-C2-CARD VC (WT), HA-C2-CARD VC D83A/E87A (MT) or a control protein (HAFRB-VC). FLAG-RAIDD was immunoprecipitated fromcell lysates with anti-FLAG-agarose and blotted for the presence of C2-CARD with an anti-HA antibody. (*) indicates immunoglobulin light chain. (B) Hela cells were transientlytransfected with plasmids encoding C2-CARD VN (20ng) and C2-CARD VC (20ng) or theD83A/E87A mutant C2-CARD BiFC pair (20ng of each) along with pshooter.dsRed-mito(10ng) and 24 hr later cells were incubated at 45°C for 1 hr with qVD-OPH (20μM). The cellswere returned to 37°C on a confocal microscope and images were taken every 10 min for 20hr. Each line of the graph represents the average intensity of Venus of 15 (untreated), 29 (WTC2-CARD, 1 hr 45°C), or 22 (MT C2-CARD, 1 hr 45°C) individual cells. Error bars representSEM. (C) Representative images from the time-lapse are shown. (D) RAIDD−/− MEF andRAIDD+/+ MEF from littermate embryos were transiently transfected with the indicatedamounts of expression plasmids encoding C2-CARD pair and dsRed-mito (10ng). 24 hr post-transfection cells were left untreated or heat shocked for 1 hr at 44°C with qVD-OPH (20μM).The percentage Venus-positive cells was determined at 24 hr from a minimum of 300 cells perwell. Results represent triplicate counts with error bars representing standard deviation. (E)RAIDD−/− and RAIDD+/+ MEF were transiently transfected with the plasmids encoding theC2-CARD Venus pair (250ng of each) and dsRedmito (10ng) with the indicated amounts ofan expression plasmid encoding RAIDD. 24 hr post-transfection cells were left untreated orheat shocked for 1 hr at 44°C with qVD-OPH (20μM). The percentage Venus-positive cells



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was determined as in (D). (F) Representative images of cells from (E) are shown. Scale barsrepresent 10μm.



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Figure 6.Caspase-2 is activated by induced proximity upstream of mitochondria to induce apoptosis.(A) Hela cells or Hela cells stably expressing Bcl-xL were heated for 1 hr at 45°C. Apoptosiswas assessed by flow cytometry for Annexin V binding at the indicated times. Error barsrepresent standard deviation. (B) Hela or Hela.Bcl-xL cells were transiently transfected withplasmids encoding C2-CARD pair (20ng of each) along with dsRed-mito (10ng) and 24 hrlater were heated at 45°C for 1 hr with qVD-OPH (20μM). Cells were returned to 37°C on aconfocal microscope and images were taken every 15 min for 16 hr. The average intensity ofVenus was measured at each time point. Results are an average of 21 (Hela) and 21 (Hela-Bcl-xL) individual cells. Error bars represent SEM. (C) Hela or Hela.Bcl-xL cells that were stablyexpressing vector or caspase-2 fused to FKBP dimerization domain were left untreated, treatedwith ActD (1μM) or the dimerization drug (AP20187, 200nM) for 24 hr. Apoptosis wasassessed as in (A). (D) Hela cells stably expressing Omi-mCherry were transiently transfectedwith expression plasmids encoding C2-CARD VN (20ng) and C2-CARD VC (20ng). 24 hrlater cells were incubated at 45°C for 1 hr. The cells were returned to 37°C on a fluorescentmicroscope and images were taken every 6 min for 16 hr. Frames from the movie showrepresentative cells undergoing BiFC (green) prior to release of Omi-mCherry from themitochondria (red). Scale bars represent 10μm. (E) Graphs of three representative cells froma movie are shown. Each point on Omi-mCherry graph (red squares) represents the punctate/diffuse index and is scaled and aligned to each point on the caspase-2 BiFC graph (greencircles) that represents the average intensity of Venus in the cell at 10 min intervals. Arrowsshow the point of onset of MOMP.



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Figure 7.Hsp90α blocks caspase-2 activation. (A) Hela cells were transiently transfected with C2-CARDVC (20ng), C2-CARD VN (20ng) and dsRed-mito (10ng). 24 hr later cells were left untreatedor heated for 1 hr at 45°C for 24 hr in the presence or absence of cycloheximide (CHX, 10μg/ml) with qVD-OPH (20μM). Cells were assessed 24 hr later for the percentage of Venus-positive cells, determined from a minimum of 300 cells per well. Results represent triplicatecounts with error bars representing standard deviation. (B) HSF-1+/+ or HSF-1−/− MEF fromlittermate embryos were heat shocked at the indicated temperatures for 1 hr. 24 hr laterapoptosis was assessed by flow cytometry for Annexin V binding. (C) HSF-1+/+ andHSF-1−/− MEF were transiently transfected with the indicated amounts of expression plasmidsencoding the C2-CARD Venus pair and dsRed-mito (10ng). 24 hr post-transfection cells wereleft untreated or heat shocked for 1 hr at 43°C with qVD-OPH (20μM). The percentage ofVenus-positive cells was determined at 24 hr as in (A). (D) HSF-1+/+ and HSF-1−/− MEF weretransiently transfected with the plasmids encoding the C2-CARD Venus pair (250ng of each)and dsRed-mito (10ng). 24 hr post-transfection cells were left untreated or heat shocked for 1hr at 43°C with qVDOPH (20μM), with or without 17-DMAG as indicated. The percentageVenus-positive cells was determined at 24 hr as in (A). (E) Representative images of cells from(D) are shown. Scale bars represent 10μm. (F) Hela cells were transfected with siRNA targetinghuman Hsp90α (100nM) or control siRNA (100nM). 24 hr post-transfection cells were leftuntreated or heat shocked for 1 hr at 43°C. Lysates were made at 72 hrs and assessed forHsp90α and β expression. (G) Hela cells were transiently transfected with the indicatedamounts of expression plasmids encoding the C2-CARD Venus pair and dsRedmito (10ng)along with the indicated siRNA (100nM). 24 hr post-transfection cells were left untreated orheat shocked for 1 hr at 42°C or 1hr at 43°C with qVD-OPH (20μM). The percentage Venus-positive cells was determined at 24 hr as in (A).



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characterization of cytoplasmic caspase-2 activation by induced proximity

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