Giovanni Monaco

Elke Decrock

Haidar Akl

Raf Ponsaerts

Tim Vervliet

Tomas Luyten

Marc De Maeyer

Ludwig Missiaen

Clark W Distelhorst

Humbert De Smedt

Jan B Parys

Luc Leybaert

Ph.D Geert Bultynck
email: geert.bultynck@med.kuleuven.be

Selective regulation of IP 3 receptors by the BH4 domain of Bcl-2 versus Bcl-Xl

Keywords: Apoptosis, Anti-apoptotic Bcl-2-family members, Calcium signaling, Endoplasmic reticulum, Intracellular Ca 2+ -release channels

Anti-apoptotic Bcl-2 targets the inositol 1,4,5-trisphosphate receptor (IP 3 R) via its BH4 domain, thereby suppressing IP 3 R Ca 2+ -flux properties and protecting against Ca 2+dependent apoptosis. Here, we directly compared IP 3 R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound IP 3 R's modulatory domain nor inhibited IP 3 -induced Ca 2+ release (IICR) in permeabilized and in intact cells.

We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP 3 R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP 3 R binding and inhibition. This difference in IP 3 R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their anti-apoptotic action. While both BH4-Bcl-2 and BH4-Bcl-Xl had anti-apoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP 3 Rs. In agreement with the IP 3 R-binding properties, the anti-apoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 severely reduced its binding to the IP 3 R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP 3 Rs and Ca 2+ -driven apoptosis by these functional domains. Mutating this residue affects Bcl-2's function in Ca 2+ signaling and apoptosis.

INTRODUCTION

Bcl-2-family members control cell survival by regulating programmed cell death and autophagy. This protein family is subdivided into pro-and anti-apoptotic proteins [START_REF] Chipuk | The BCL-2 family reunion[END_REF].

The founding member is Bcl-2, a 26-kDa protein localized in mitochondrial and endoplasmic-reticulum (ER) membranes. Members of this protein family are characterized by the presence of Bcl-2-homology domains (BH domains) [START_REF] Brunelle | Control of mitochondrial apoptosis by the Bcl-2 family[END_REF][START_REF] Chipuk | How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?[END_REF]. Proapoptotic proteins contain either 3 BH domains (BH1, BH2 and BH3) and are referred to as the multi-domain proteins, like the executioner proteins Bax and Bak, or a single BH domain (BH3) and are referred to as the BH3-only proteins. Anti-apoptotic proteins, like Bcl-2 and Bcl-Xl, are unique in this family of proteins, since they are characterized by an additional BH domain (BH4). However, recent studies suggested the presence of a conserved BH4 motif in both anti-and pro-apoptotic Bcl-2-family members [START_REF] Chipuk | The BCL-2 family reunion[END_REF][START_REF] Kvansakul | Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands[END_REF]. The BH1, BH3 and BH2 domains of the anti-apoptotic Bcl-2-family proteins form a hydrophobic pocket responsible for binding the BH3 domain of pro-apoptotic proteins, thereby counteracting their pro-apoptotic activity and preventing subsequent mitochondrial outer-membrane permeabilization (MOMP) [START_REF] Bertsch | The second messenger binding site of inositol 1,4,5-trisphosphate 3-kinase is centered in the catalytic domain and related to the inositol trisphosphate receptor site[END_REF].

Besides this hydrophobic cleft, the BH4 domain is also essential for Bcl-2's antiapoptotic activity [START_REF] Hunter | Functional dissection of the human Bc12 protein: sequence requirements for inhibition of apoptosis[END_REF][START_REF] Huang | The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4[END_REF]. Bcl-2 lacking its BH4 domain may act as a dominant negative inhibitor of Bcl-2 (6) and seems to display a Bax-like function, promoting cell death [START_REF] Cheng | Conversion of Bcl-2 to a Bax-like death effector by caspases[END_REF].

While MOMP and the subsequent release of apoptogenic factors including cytochrome c (CytC) from the mitochondrial intermembrane space are considered crucial events in the initiation of apoptosis, Ca 2+ signaling from the ER to the mitochondria seems to play an important role [START_REF] Thomenius | Bcl-2 on the endoplasmic reticulum: protecting the mitochondria from a distance[END_REF][START_REF] Oakes | The control of endoplasmic reticulum-initiated apoptosis by the BCL-2 family of proteins[END_REF]. Several pro-and anti-apoptotic Bcl-2 proteins localize at the ER membrane, where they modulate Ca 2+ -release events ((11, 12), and recently reviewed in [START_REF] Rong | Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis[END_REF][START_REF] Rizzuto | Ca 2+ transfer from the ER to mitochondria: when, how and why[END_REF][START_REF] Sammels | Intracellular Ca 2+ storage in health and disease: a dynamic equilibrium[END_REF]). Bcl-2 proteins thereby influence mitochondrial function due to the close proximity of ER and mitochondrial membranes and the formation of inter-organelle protein-complexes involving the IP 3 R and voltage-dependent anion channel (VDAC) [START_REF] Pinton | Calcium and apoptosis: ERmitochondria Ca 2+ transfer in the control of apoptosis[END_REF][START_REF] Giorgi | Structural and functional link between the mitochondrial network and the endoplasmic reticulum[END_REF][START_REF] Hayashi | MAM: more than just a housekeeper[END_REF][START_REF] Joseph | IP(3) receptors in cell survival and apoptosis: Ca 2+ release and beyond[END_REF]. Ca 2+ transfer via the ER-mitochondria connection plays a bimodal role in cell survival [START_REF] Spat | High-and low-calcium-dependent mechanisms of mitochondrial calcium signalling[END_REF][START_REF] Decuypere | IP 3 Receptors, Mitochondria, and Ca 2+ Signaling: Implications for Aging[END_REF][START_REF] Decuypere | The IP(3) receptor-mitochondria connection in apoptosis and autophagy[END_REF]. Small oscillatory Ca 2+ signals promote cell survival by stimulating mitochondrial function and bio-energetics, whereas a larger or more persistent mitochondrial [Ca 2+ ] increase promotes cell death by provoking opening of the mitochondrial permeability transition pore and MOMP [START_REF] Rizzuto | Ca 2+ transfer from the ER to mitochondria: when, how and why[END_REF][START_REF] Pinton | Calcium and apoptosis: ERmitochondria Ca 2+ transfer in the control of apoptosis[END_REF][START_REF] Hayashi | MAM: more than just a housekeeper[END_REF][START_REF] Bathori | Ca 2+ -dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC)[END_REF][START_REF] Csordas | Structural and functional features and significance of the physical linkage between ER and mitochondria[END_REF]. Thus, both promoting cytosolic Ca 2+ oscillations as well as blunting cytosolic Ca 2+ transients underpins the anti-apoptotic action of Bcl-2-family members [START_REF] Jones | The proapoptotic factors Bax and Bak regulate T cell proliferation through control of endoplasmic reticulum Ca 2+ homeostasis[END_REF].

A plethora of studies from different research groups showed that the IP 3 R plays a central role in this paradigm as a target of Bcl-2, Bcl-Xl and Mcl-1 [START_REF] Oakes | Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum[END_REF][START_REF] Chen | Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate[END_REF][START_REF] Zhong | Bcl-2 differentially regulates Ca 2+ signals according to the strength of T cell receptor activation[END_REF][START_REF] White | The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP 3 R[END_REF][START_REF] Foskett | Bcl-xL Regulation of InsP 3 Receptor Gating Mediated by Dual Ca 2+ Release Channel BH3 Domains[END_REF][START_REF] Eckenrode | Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca 2+ signaling[END_REF]. However, the localization of their interaction sites on the IP 3 R as well as the physiological roles of these interactions remains unsolved. Different, not mutually exclusive, mechanisms for the control of IP 3 R activity by Bcl-2-family members have been proposed: i) Increasing the ratio of anti-over pro-apoptotic Bcl-2 proteins caused IP 3 R sensitization, provoking a Ca 2+ leak through the channel [START_REF] Oakes | Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum[END_REF]; ii) Bcl-Xl interacted with the C-terminus of all IP 3 R isoforms and sensitized IP 3 R activity, promoted spontaneous Ca 2+ oscillations, enhanced mitochondrial bio-energetics and augmented resistance against apoptotic triggers [START_REF] White | The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP 3 R[END_REF][START_REF] Li | Apoptosis regulation by Bcl-x(L) modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating[END_REF]. iii) In a series of studies, evidence was found for a direct inhibition of the IP 3 R by Bcl2 with no change in the steady-state ER Ca2+ levels [START_REF] Chen | Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate[END_REF][START_REF] Zhong | Bcl-2 differentially regulates Ca 2+ signals according to the strength of T cell receptor activation[END_REF][START_REF] Hanson | Bcl-2 suppresses Ca 2+ release through inositol 1,4,5-trisphosphate receptors and inhibits Ca 2+ uptake by mitochondria without affecting ER calcium store content[END_REF]. In a recent study, the Bcl-2 interaction domain was identified in the central, modulatory region of the IP 3 R (a.a. 1389 to 1408) (34). Disturbing endogenous IP 3 R/Bcl-2 complexes using cellpermeable peptides that represented the Bcl-2-binding site (a.a. 1389-1408) on the IP 3 R, potentiated IICR and sensitized cells towards pro-apoptotic Ca 2+ signaling (34). The BH4 domain was identified as a necessary and sufficient mediator for the interaction with the IP 3 R [START_REF] Rong | The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP 3 receptor[END_REF][START_REF] Rong | Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor[END_REF]. The BH4 domain of Bcl-2 (BH4-Bcl-2) interacted with IP 3 Rs, suppressed IICR, and prevented pro-apoptotic Ca 2+ signaling during strong T-cell receptor activation. iv) Finally, Bcl-Xl was shown to reduce the expression levels of IP 3 Rs via a decreased binding of NFATc2 to the IP 3 R promoter [START_REF] Li | Bcl-X(L) affects Ca 2 + homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors[END_REF], thereby contributing to the reduced IICR observed in Bcl-Xl-overexpressing cells.

In the present study we have directly compared the cellular mechanism and the role of the BH4 domains of Bcl-2 and Bcl-Xl. Our results indicate that although the BH4 domain of Bcl-2 and Bcl-Xl are very similar in sequence and structure, a difference in one single residue between BH4-Bcl-2 and BH4-Bcl-Xl critically determined its IP 3 R binding, suppression of IP 3 R activity and protection against IP 3 R-driven apoptosis.

RESULTS

BH4-Bcl-2 and BH4-Bcl-Xl differentially regulate IICR

A schematic, linear representation of the structure of the anti-apoptotic Bcl-2 and of a monomeric subunit of the IP 3 R is provided in Fig. 1A, depicting Bcl-2's interaction via its BH4 domain with the central, modulatory domain of the IP 3 R. The position of the Bcl-2-binding site is indicated, while IDP (IP 3 R-Derived Peptide) represents a peptide with a sequence corresponding to Bcl-2-binding site on the IP 3 R1 and therefore able to disrupt Bcl-2/IP 3 R interaction in a competitive way. The inhibitory action of Bcl-2's BH4 domain on the IP 3 R can be completely abolished using IDP. Importantly, IDP does not interfere with other BH4-domain targets of Bcl-2, like VDAC and calcineurin [START_REF] Rong | The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP 3 receptor[END_REF]. Thus, IDP is an excellent tool to decipher the role of IP 3 R in the observed actions of BH4-Bcl-2 on intracellular Ca 2+ signaling and cell death. Here, we directly compared the effect of the BH4 domain of Bcl-2 and Bcl-Xl on IICR using a unidirectional 45 Ca 2+ -flux assay in permeabilized mouse embryonic fibroblasts (MEF cells). In these experiments, the nonmitochondrial Ca 2+ stores of saponin-permeabilized wild-type MEF cells were loaded to steady state with 45 Ca 2+ . After adding 4 μM thapsigargin, the efflux of 45 Ca 2+ was followed in the presence of 1 mM EGTA. This assay allows the quantitative assessment of Ca 2+ -efflux properties under unidirectional conditions in the absence of ER and mitochondrial Ca 2+ -uptake activity. The data were plotted as a function of time as either the Ca 2+ content (Fig. 1B) or the fractional loss, i.e. the amount of Ca 2+ leaving the store in a 2-min time period divided by the total store Ca 2+ content at that time point (Fig. 1C). Fig. 1B shows that, due to an inherent Ca 2+ leak, the Ca 2+ content of the stores slowly decreased over time, while adding IP 3 (3 μM) 10 min after the start of the efflux accelerated the decrease in Ca 2+ content due to IICR. This is observed as a steep increase in the fractional loss (Fig. 1C). As shown in Fig. 1B, the ER Ca 2+ stores contain at that moment about 50 % of their initial load. The BH4 domains of Bcl-2 (BH4-Bcl-2; a.a. and Bcl-Xl (BH4-Bcl-Xl; a.a. were produced as synthetic peptides. In addition, two control peptides were designed: a scrambled version of the BH4 domain of Bcl-2 (BH4-Bcl-2SCR) and a "binding-deficient" version of BH4-Bcl-2 in which the surfaceaccessible residues were altered (BH4-Bcl-2BIND). The effect of these BH4-domain peptides on IICR was tested by pre-incubating the peptides 4 min before exposing the stores to IP 3 . The 45 Ca 2+ -flux assays showed that BH4-Bcl-2 caused a potent concentration-dependent inhibition of IICR (Fig. 1B and Fig. 1C). Importantly, BH4-Bcl-2 did not alter the Ca 2+ -leak rate from the ER in the absence of IP 3 . This is evident from the data in Fig. 1B and Fig. 1C, in which BH4-Bcl-2 does not affect the leak or fractional loss in the period before the addition of IP 3 . In contrast to BH4-Bcl-2, BH4-Bcl-2BIND and BH4-Bcl-2SCR did not inhibit IICR (Fig. 1D and Supplemental Fig. 1A-B). BH4-Bcl-2 inhibited IICR with an IC 50 of ~ 30 μM and an IC 100 of ~ 100 μM (Fig. 1D).

Strikingly, the BH4 domain of Bcl-Xl did not inhibit IICR (estimated IC 50 > 500 μM) (Fig. 1D and Supplemental Fig. 1C). The unidirectional 45 Ca 2+ flux experiments described above were performed in the absence of cytosolic Ca 2+ . We repeated the experiments using more physiological conditions, like 300 nM free Ca 2+ (Supplemental Fig. 2B), 1 μM free Ca 2+ (Supplemental Fig. 2C) and 1 mM Mg-ATP (Supplemental Fig. 2D) and examined the effect of 40 μM BH4-Bcl-2 on IICR. Importantly, under all these conditions, 40 μM BH4-Bcl-2 provoked an inhibition of IICR of about 50 % compared to the vehicle-treated conditions.

Next, we investigated whether the differential regulation of IP 3 Rs by BH4-Bcl-2 and BH4-Bcl-Xl could be observed in C6-glioma cells, a cellular model optimized for in situ electroporation of membrane impermeable molecules [START_REF] Vuyst | In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling[END_REF][START_REF] Decrock | Connexin 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model[END_REF]. We loaded BH4-Bcl-2 or BH4-Bcl-Xl (both 20 μM) by electroporation in C6 cells and examined the response to IP 3 liberated from its caged precursor (50 μM; Fig. 1E and Fig. 1F) by UV-flash photolysis. We found that BH4-Bcl-2 was able to inhibit IICR events in intact cells, whereas BH4-Bcl-Xl did not. Again, BH4-Bcl-2SCR and BH4-Bcl-2BIND control peptides did not display any effect on IICR. Furthermore, the inhibition by BH4-Bcl-2 was completely prevented by loading the cells with IDP (20 μM), indicating a competitive binding of the BH4 domain to the IP 3 R. Similar results were obtained using ATP (1 μM), a physiological agonist known to provoke IP 3 R-mediated Ca 2+ release in these cells (Fig. 1G-H). Finally, we examined whether these BH4-domain peptides affected the ER Ca 2+ -store content. Hence, we assessed the thapsigargin (2.5 μM)releasable Ca 2+ in these cells 1 min after exposing the cells to EGTA (1 mM) and found no differences among the different conditions (Fig. 1I-J). Hence, the decreased ATPinduced Ca 2+ release in the presence of BH4-Bcl-2 was not due to decreased ER Ca 2+store content.

These results indicate that BH4-Bcl-2 and BH4-Bcl-Xl differentially regulate IP 3 R function independently of the ER Ca 2+ -store content: BH4-Bcl-2 inhibited IICR in permeabilized and intact cells, whereas BH4-Bcl-Xl did not.

BH4-Bcl-2 and BH4-Bcl-Xl differentially bind to the central, modulatory domain of the IP 3 R

Previously, we have shown that the inhibitory action of the BH4 domain of Bcl-2 on IICR was due to direct binding to the central, modulatory domain of the IP 3 R (35), Fig. 1A). Therefore, we compared the binding of BH4-Bcl-2 and BH4-Bcl-Xl to GST-Dom3 (i.e. a.a. 923-1581 of IP 3 R1, fused to GST) (Fig. 2A). This protein fragment corresponds to a tryptic subdomain, which can be obtained by limited proteolysis of the IP 3 Rs, and which is located in the central part of the modulatory and coupling domain of the IP 3 R [START_REF] Yoshikawa | Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor. Structural and functional coupling of cleaved ligand binding and channel domains[END_REF]. Using GST-pull-down assays, we found that BH4-Bcl-2 strongly and specifically interacted with GST-Dom3. In contrast to BH4-Bcl-2, no specific binding of BH4-Bcl-Xl to GST-Dom3 was observed. However, we consistently noticed a higher non-specific binding of BH4-Bcl-Xl to GST than for BH4-Bcl-2. The reason for this is unclear. In addition, by altering the surface-accessible residues in BH4-Bcl-2 [START_REF] Rong | Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor[END_REF], we created a mutant version of BH4-Bcl-2 (BH4-Bcl-2BIND) that was not pulled down by GST-Dom3 (Fig. 2A), indicating that these residues are important for interaction with the IP 3 R.

The normalized binding activity of the three peptides to GST-Dom3 is shown in Fig. 2B.

To confirm the results obtained with GST-pull-down assays, we assessed the binding of GST-Dom3 to BH4-Bcl-2 and BH4-Bcl-Xl in a more quantitative manner using surface plasmon resonance (SPR). Therefore, we developed biotinylated BH4-Bcl-2 and BH4-Bcl-Xl. Their scrambled counterparts were used as a control. These peptides were immobilized on a streptavidin-coated sensor chip. We subsequently monitored the binding of GST-Dom3 (Fig. 3A-C) and GST (Supplemental Fig. 3A-C and Fig. 3E) to the sensor chip. In all experiments, background signals obtained from the reference flow cell containing the scrambled biotinylated version of either BH4-Bcl-2 or BH4-Bcl-Xl, were subtracted to generate response curves. In each sensorgram, the association phase is plotted. Control GST (up to 40 μM) did not display any specific binding towards either biotin-BH4-Bcl-2 or biotin-BH4-Bcl-Xl (Supplemental Fig. 3A-C). GST-Dom3 displayed a concentration-dependent increase in resonance units, in the biotin-BH4-Bcl-2-containing flow cell, upon addition of the purified GST-Dom3 protein as analyte (Fig. 3A). This indicates a specific binding towards biotin-BH4-Bcl-2 with estimated K d of about 1 μM (Fig. 3C). In the biotin-BH4-Bcl-Xl-containing flow cell (Fig. 3B), GST-Dom3 injection resulted in significantly less increase in resonance units, indicating a very low affinity (Fig. 3C).

In addition, we monitored the binding of GST-Dom3 to biotin-BH4-Bcl-2BIND corrected for binding to its scrambled counterpart (Fig. 3D). In this case, we noticed a slightly higher binding to the scrambled version of biotin-BH4-Bcl-2BIND, resulting in negative values in the SPR plots. Clearly, GST-Dom3 did not specifically bind to biotin-BH4-Bcl-2BIND in contrast to its binding to biotin-BH4-Bcl-2.

Since the Bcl-2-binding site (a.a. 1389 to 1408) identified by Distelhorst and co-workers (34) is largely conserved among the different IP 3 R isoforms, we also compared the binding properties of the GST-Dom3 (3.3 μM) derived from IP 3 R1, IP 3 R2 (a.a. 913-1562) and IP 3 R3 (a.a. 910-1427) (Fig. 3E and Fig. 3F). GST (3.3 μM) is shown as a control. We found that the Dom3 from all three IP 3 R isoforms bound to biotin-BH4-Bcl-2 with a roughly similar potency (Fig. 3E and Fig. 3F). The quality of the purified GSTfusion proteins used for this SPR analysis was controlled using SDS-PAGE (Fig. 3G).

Identification of residues in BH4-Bcl-2 responsible for binding and inhibiting IP 3 Rs

Next, we investigated which residues in BH4-Bcl-2 are responsible for interaction with IP 3 Rs. Therefore, based on a bio-informatics approach (36), we successively changed the various predicted surface-accessible residues into alanines (Fig. 4A) and investigated their properties towards inhibiting IICR in unidirectional 45 Ca 2+ flux experiments using permeabilized MEF cells. The experiments were carried out as described in Fig. 1. The highly quantitative nature of these experiments allowed an assessment of the contribution of each of the residues in binding and inhibiting IP 3 Rs. For this analysis, we used 60 μM of the different BH4-Bcl-2 mutant peptides. This concentration, when used for wild-type BH4-Bcl-2, reduced the IICR to about 20% of its control value (Fig. 4B). From this analysis it appeared that BH4-Bcl-2 acts as a discontinuous binding domain, in which every surface-accessible residue (Asp10, Arg12, Lys17, His20, Tyr21, Gln25, Arg26, Tyr28) contributed to the inhibition of IICR. Quantitative analysis indicated that particularly the mutations to alanine of Lys17, His20, Tyr21 or Arg26 largely abolished the inhibitory properties of BH4-Bcl-2. This indicates that these four residues are critical in binding and inhibiting IP 3 Rs. However, we can not exclude that changing these residues into an Ala alters the α-helical properties of BH4-Bcl-2, thereby indirectly affecting the binding of BH4-Bcl-2 to IP 3 Rs. Therefore, we analysed the predicted αhelical structure and content using PSIPRED vs3.0 (Supplemental Fig. 4). All Ala mutants displayed similar predicted α-helical properties as the wild-type BH4-Bcl-2.

Nevertheless, we cannot exclude that some of these Ala mutations may have minor effects on the folding and the secondary order structure of the BH4-domain.

Sequence alignment identifies a difference of one critical residue that is essential for the differential IP 3 R-inhibitory properties of BH4-Bcl-2 and BH4-Bcl-Xl

Sequence alignment shows that Lys17 (K17) in BH4-Bcl-2 corresponds to Asp11 (D11) in BH4-Bcl-Xl (Fig. 5A), thereby introducing a charge change in the middle of the alphahelical structure [START_REF] Rong | The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP 3 receptor[END_REF][START_REF] Muchmore | Xray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death[END_REF][START_REF] Lee | Evidence for alpha-helical conformation of an essential N-terminal region in the human Bcl2 protein[END_REF]. To investigate the effect of such change, we made a mutant BH4-Bcl-2 peptide in which we replaced Lys17 by an aspartate (BH4-Bcl-2 K/D) and a mutant BH4-Bcl-Xl peptide in which we replaced Asp11 by a lysine (BH4-Bcl-Xl D/K).

Strikingly, we found that BH4-Bcl-2 K/D lost its inhibitory action on IICR, whereas BH4-Bcl-Xl D/K became active and imposed a strong inhibition of IICR (Fig. 5B-C). Dose-response curves (Fig. 5D) indicated that BH4-Bcl-2 and BH4-Bcl-Xl D/K inhibited IICR with an IC 50 of 30 and 60 μM, respectively. In contrast, BH4-Bcl-Xl and BH4-Bcl-2 K/D were largely ineffective. The effects of the BH4-domain mutants on IICR were also observed in intact C6-glioma cells using caged IP 3 (Fig. 5E-F). In contrast to BH4-Bcl-2, BH4-Bcl-2 K/D did not inhibit IICR in intact cells, while in contrast to BH4-Bcl-Xl, BH4-Bcl-Xl D/K was able to significantly reduce IICR. We confirmed these findings in intact C6-glioma cells using the physiological agonist ATP (1 μM; Fig. 5G), while in the same conditions none of the BH4-domain peptides did alter the amount of thapsigargin (2.5 μM) -releasable Ca 2+ from the ER, indicating that the ER Ca 2+ -store content is not affected by the peptides (Fig. 5H).

The importance of this critical residue was further confirmed by SPR experiments using biotin-BH4-Bcl-2 K/D and biotin-BH4-Bcl-Xl D/K immobilized on a streptavidin-coated sensor chip. Scrambled peptides were again used as negative controls. Background binding to these reference flow cells was subtracted to generate sensorgrams as described above. Consistent with the functional data obtained in permeabilized and intact cells, BH4-Bcl-2 K/D largely lost its ability to interact with GST-Dom3 (Fig. 5I), whereas BH4-Bcl-Xl D/K became able to bind GST-Dom3 (Fig. 5J).

Taken together, these data indicate that one single residue difference between BH4-Bcl-2 and BH4-Bcl-Xl can explain their differential action on IP 3 Rs.

BH4-Bcl-2 and BH4-Bcl-Xl differentially affect CytC-and STS-induced apoptosis

In a next series of experiments, we assessed whether BH4-Bcl-2 and BH4-Bcl-Xl displayed a difference in protection against apoptotic cell death in C6-glioma cells. Cell death was triggered by loading the cells with CytC (10 μM) or by incubation with STS (2 μM). CytC was co-loaded with dextran Texas Red (DTR; 100 μM) to identify the zone of cells loaded with CytC. The apoptotic index was determined by measuring caspase activity using the in situ CaspACE-fluorescent marker and by examining DNA fragmentation using DAPI staining, 25 min post-CytC loading or 6 hrs after STS exposure. The apoptotic index was expressed relative to the condition in which the apoptotic trigger was applied in the absence of BH4 peptides (vehicle), which was set to 100%.

We first examined whether CytC interaction with the IP 3 R contributed to CytC-induced apoptosis in the C6 cell model. CytC-IP 3 R interaction is part of a positive feedback loop that is known to enhance cell death by promoting ER Ca 2+ release through IP 3 R channels [START_REF] Boehning | Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis[END_REF](44)(45). We therefore examined CytC-induced cell-death in the absence and presence of a peptide matching the C-terminal CytC-binding site on IP 3 Rs (IP 3 RCYT; 50 μM). When cells were loaded with CytC in the presence of IP 3 RCYT, CytC-induced cell death decreased to about 50 -60% of the control value (Fig. 6A). These results indicate that part of the CytC-induced cell death was due to CytC binding to the IP 3 Rs, thereby enhancing cell death via a deadly loop involving the IP 3 R. Subsequently, we tested the different BH4 domains of Bcl-2 and Bcl-Xl and a scrambled negative control, in this cell system (all peptides at 20 μM in the loading solution). From these experiments, it is clear that both BH4-Bcl-2 and BH4-Bcl-Xl were able to protect against CytC-induced cell death. However, BH4-Bcl-2 was significantly more potent in protecting than BH4-Bcl-Xl (Fig. 6B). The BH4-Bcl-2SCR control did not protect at all. Importantly, changing Lys17 into Asp in BH4-Bcl-2 (BH4-Bcl-2 K/D) reduced the level of protection, thereby resembling BH4-Bcl-Xl. By contrast, changing Asp11 into Lys in BH4-Bcl-Xl (BH4-Bcl-Xl D/K) increased cell survival, thereby resembling BH4-Bcl-2. Again, these results point toward a critical role of the Lys17 in BH4-Bcl-2 and of the Asp11 in BH4-Bcl-Xl being responsible for the differential properties of both domains.

To assess whether the difference in potency between BH4-Bcl-2 and BH4-Bcl-Xl is due to their differential action on the IP 3 R, the BH4-domain peptides were loaded together with IDP. IDP consisting of the Bcl-2-binding site on IP 3 R1 will compete with the IP 3 R for the binding of Bcl-2. Strikingly, IDP completely abolished the protective action of BH4-Bcl-2. In contrast, the BH4-Bcl-Xl-mediated protection against CytC-induced cell death was not influenced by the presence of IDP (Fig. 6C). These results indicate that in contrast to BH4-Bcl-Xl, BH4-Bcl-2 acted via binding to the IP 3 R to protect against apoptosis.

The effect of CytC on cell death depends on its positive feedback on the activity of the IP 3 R, which however occurs at a late post-mitochondrial stage of apoptosis. We therefore also used STS, which acts upstream of the mitochondria and CytC release and initiate apoptosis via ER Ca 2+ -release-dependent mechanisms [START_REF] Eckenrode | Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca 2+ signaling[END_REF](45)[START_REF] Nutt | Bax-mediated Ca 2+ mobilization promotes cytochrome c release during apoptosis[END_REF][START_REF] Assefa | Caspase-3-induced truncation of type 1 inositol trisphosphate receptor accelerates apoptotic cell death and induces inositol trisphosphate-independent calcium release during apoptosis[END_REF][START_REF] Kruman | Calcium and reactive oxygen species mediate staurosporine-induced mitochondrial dysfunction and apoptosis in PC12 cells[END_REF][START_REF] Verbert | Caspase-3truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling[END_REF]. STS (2 μM; 6 hrs) led to apoptosis with a minor fraction of these cells displaying secondary necrosis (Fig. 6D). Primary necrosis was not observed, since all propidium iodide (PI) positive cells were also characterized by activated caspases. As shown in Fig. 6E, the results with STS were similar to those obtained with CytC loading. Both BH4-Bcl-2 and BH4-Bcl-Xl protected against STS-induced cell death, but BH4-Bcl-2 was significantly more potent than BH4-Bcl-Xl. BH4-Bcl-2SCR did not protect at all. Importantly, BH4-Bcl-2-mediated protection against STS was completely suppressed when IDP was loaded together with the BH4-Bcl-2 peptide, whereas the effect of BH4-Bcl-Xl was independent of the presence of IDP. These data provide again strong evidence that BH4-Bcl-2 but not BH4-Bcl-Xl exerts its protective effect against cell death by inhibiting IP 3 R activity.

As an additional control, we used PAC-1 (150 μM, 16 hrs), a direct procaspase-3activating compound, which acts downstream of CytC [START_REF] Putt | Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy[END_REF]. These conditions caused apoptosis in ~15 % of the cell population. We found that BH4-Bcl-2 and BH4-Bcl-Xl did not protect against PAC-1-induced cell death, confirming that the target of these peptides is indeed upstream of caspase-3 (Fig. 6F).

In addition, we loaded BH4-Bcl-2 and BH4-Bcl-Xl together in C6-glioma cells, but no additive action regarding their protection against CytC-and STS-induced cell death was found (Fig. 6G and Supplemental Fig. 5). Furthermore, the protective effects of both BH4-Bcl-2 as well as BH4-Bcl-Xl against CytC-induced apoptosis were ablated when CytC's positive feedback loop on the IP 3 R was prevented by co-loading IP3RCYT, peptide (Fig. 6H).

Collectively, these data indicate that the BH4 domains of Bcl-2 and Bcl-Xl both protect against apoptosis by acting on different targets upstream of caspase activation. While BH4-Bcl-2's protective effect was stronger and largely dependent on its interaction with a specific binding site on the IP 3 R, BH4-Bcl-Xl's protective effect is independent of binding to the IP 3 R. Nevertheless, in these paradigms BH4-Bcl-Xl likely acts on a target that functions downstream of the IP 3 R-signaling cascade.

Full-length Bcl-2 K/D is less efficient in binding IP 3 Rs, inhibiting IP 3 R activity and protecting against Ca 2+ -dependent apoptosis

Finally, we tested whether the critical Lys17 identified in BH4-Bcl-2 plays a crucial role in the IP 3 R-inhibitory action of full-length Bcl-2. Therefore, we mutated Lys17 into Asp in full-length 3xFLAG-Bcl-2, creating 3xFLAG-Bcl-2 K/D. First, we compared the GST-Dom3-binding properties of 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D by expressing these proteins in COS-7 cells. Cell lysates were prepared and used in GST-pull down assays using purified GST-Dom3 (Fig. 7A). Strikingly, we found that the binding 3xFLAG-Bcl-2 K/D to GST-Dom3 was severely compromised compared to wild-type 3xFLAG-Bcl-2. Quantitative analysis revealed that 3xFLAG-Bcl-2 K/D binding to GST-Dom3 was about ~80% less than the binding observed for 3xFLAG-Bcl-2 (Fig. 7B).

Next, we examined the effect of 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D overexpression in COS-7 cells on ATP-induced Ca 2+ release. We applied 1 μM ATP, which is a submaximal dose for triggering Ca 2+ signals, in Fura-2-loaded COS-7 cells. We cotransfected an mCherry plasmid in a ratio of mCherry plasmid/3xFLAG plasmid of 1/3 and monitored the Ca 2+ signals in mCherry-transfected cells (Supplemental Fig. 6). In each experiment, 10 cells were selected and Ca 2+ Finally, we examined the effect of 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 overexpression on the protection against apoptotic stimuli, like STS. We used the cleavage of poly-(ADPribose)-polymerase (PARP), a downstream target of activated caspase-3, to monitor STSinduced apoptosis in transfected COS cells (Fig. 7H). We found that 3xFLAG-Bcl-2 overexpression significantly reduced PARP cleavage upon STS treatment, compared to empty vector transfected cells. Importantly, 3xFLAG-Bcl-2 K/D overexpression was much less potent than 3xFLAG-Bcl-2 in preventing STS-induced PARP cleavage.

Quantification of the ratio of cleaved over full-length PARP from three independent experiments revealed a significant difference between 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D (Fig. 7I).

We have also confirmed these findings in stable cell lines. Therefore, we used WEHI7.2 cells, which have very low levels of endogenous Bcl-2 and we created stable WEHI7.2 cell lines expressing either Bcl-2 or Bcl-2 K/D. First, using Western-blotting analysis with a Bcl-2 antibody, we show that both stable cell lines display similar levels of Bcl-2 and Bcl-2 K/D (Fig. 8A). Next, we monitored apoptosis using three independent experimental approaches: Western-blotting analysis with PARP antibodies (Fig. 8B), Annexin V-FITC/propidium iodide (PI) FACS analysis (Fig. 8C-D) and caspase-3activity assay (Fig. 8E). In all assays, Bcl-2 overexpression protected WEHI7.2 cells against STS-induced apoptosis, while Bcl-2 K/D was less effective than wild-type Bcl-2 in protecting these cells against STS. Quantitative analysis of the STS-induced apoptosis of the different WEHI7.2 cells (five independent experiments) indicated that although both Bcl-2 and Bcl-2 K/D overexpression decreased the number of apoptotic cells upon STS treatment, Bcl-2 K/D is clearly less effective (Fig. 8D-E). These data correlate well with the IP 3 R binding and Ca 2+ -signaling results obtained in COS cells. This indicates that Lys17 is an important residue in the BH4 domain of full-length Bcl-2 for mediating Bcl-2-dependent protection against STS-induced cell death.

DISCUSSION

The major findings of this study are that i) BH4-Bcl-2 and BH4-Bcl-Xl, although very similar in primary sequence and secondary structure [START_REF] Rong | The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP 3 receptor[END_REF][START_REF] Muchmore | Xray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death[END_REF][START_REF] Lee | Evidence for alpha-helical conformation of an essential N-terminal region in the human Bcl2 protein[END_REF], act differentially on IP 3 Rs, on IICR and on Ca 2+ -dependent apoptosis, ii) one critical residue that has an opposite charge in BH4-Bcl-2 versus BH4-Bcl-Xl is responsible for their distinct biological properties, iii) mutating this residue in the BH4 domain of full-length Bcl-2 decreases its ability to bind and inhibit IP 3 Rs and to protect against apoptotic stimuli. BH4-Bcl-2 bound to the central, modulatory domain of the IP 3 R, thereby inhibiting IICR and protecting against IP 3 R-dependent apoptosis. In contrast, BH4-Bcl-Xl did not bind to the central, modulatory domain of the IP 3 R and did not inhibit IICR. Nevertheless, BH4-Bcl-Xl exerted an anti-apoptotic activity, which however was significantly smaller than for BH4-Bcl-2, and was independent of an action on IP 3 Rs.

Using molecular and biophysical approaches, we pinpointed one critical residue for inhibition of IP 3 Rs in the sequence of BH4-Bcl-2 (Lys17) that was not conserved in BH4-Bcl-Xl (Asp11). This residue is of key importance for the specific action of BH4-Bcl-2 on the IP 3 R. Moreover, changing Asp11 in BH4-Bcl-Xl into a Lys induced IP 3 R binding and inhibition, leading to a BH4-Bcl-2-like function. Importantly, mutating this Lys17 into Asp in full-length Bcl-2 affected its biological function at the level of the IP 3 R.

Bcl-2 and Bcl-Xl both act at the mitochondrial and the ER membranes. Their bestdocumented anti-apoptotic function depends on the hydrophobic cleft formed by BH3, BH1 and BH2, which is responsible for the neutralization of the activity of pro-apoptotic Bax, Bak and BH3-only proteins at the mitochondria (2). In addition, these proteins also protect against cell death by regulating ER Ca 2+ dynamics via interaction with the IP 3 R 20 [START_REF] Oakes | Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum[END_REF][START_REF] Chen | Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate[END_REF][START_REF] Zhong | Bcl-2 differentially regulates Ca 2+ signals according to the strength of T cell receptor activation[END_REF][START_REF] White | The endoplasmic reticulum gateway to apoptosis by Bcl-X(L) modulation of the InsP 3 R[END_REF][START_REF] Li | Apoptosis regulation by Bcl-x(L) modulation of mammalian inositol 1,4,5-trisphosphate receptor channel isoform gating[END_REF]. Several reports have suggested that Bcl-2 predominantly inhibits proapoptotic Ca 2+ transients, whereas Bcl-Xl predominantly stimulates IP 3 R-mediated prosurvival Ca 2+ oscillations (27-29, 32, 34). Nevertheless, other reports showed that Bcl-2 too may enhance IP 3 R activity [START_REF] Oakes | Proapoptotic BAX and BAK regulate the type 1 inositol trisphosphate receptor and calcium leak from the endoplasmic reticulum[END_REF][START_REF] Eckenrode | Apoptosis protection by Mcl-1 and Bcl-2 modulation of inositol 1,4,5-trisphosphate receptor-dependent Ca 2+ signaling[END_REF] and/or stimulate Ca 2+ oscillations [START_REF] Chen | Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate[END_REF][START_REF] Palmer | Bcl-2-mediated alterations in endoplasmic reticulum Ca 2+ analyzed with an improved genetically encoded fluorescent sensor[END_REF]. Hence, until now, it was not clear whether Bcl-2 and Bcl-Xl displayed distinct functional properties towards regulating IP 3 Rs and thus Ca 2+ -regulated apoptosis or whether they were similar in their action. Since we recently showed that BH4-Bcl-2 was sufficient to protect against IP 3 R-mediated apoptosis, we now made a direct comparison of the BH4domain properties of Bcl-2 and Bcl-Xl by using synthetic peptides. Our study reveals a specific cellular function for the BH4 domain of Bcl-2 as a potent inhibitor of IICR and Ca 2+ -dependent apoptosis, which is not shared by the BH4 domain of Bcl-Xl, although both motifs are very similar in sequence and structure. Our data indicate that this is due to a critical charge difference in one of the surface-accessible amino-acid residues. As a result, BH4-Bcl-Xl did not inhibit Ca 2+ flux through the IP 3 R. Nevertheless, BH4-Bcl-Xl was also able to protect against cell death. However, this effect was significantly smaller than for BH4-Bcl-2 and was not due to inhibition of IICR. This was concluded from the observation that the IDP peptide that corresponds to the Bcl-2-binding site of the IP 3 R and counteracts the effect of BH4-Bcl-2, did not interfere with the protective function of BH4-Bcl-Xl. Finally, using exogenous expression in COS cells and in WEHI7.2 cells, we demonstrated that the role of Lys17 is important for the action of full-length Bcl-2 on the IP 3 R, since full length Bcl-2 K/D was much less efficient in binding and inhibiting IP 3 Rs as well as in protecting against apoptotic stimuli. We observed a weak binding of fulllength Bcl-2 K/D (i.e. about 20 % of the binding of wild-type Bcl-2) to the IP 3 R fragment, which may indicate that residues other than Lys17 may contribute to the binding of full-length Bcl-2 to the IP 3 R. This remaining binding of Bcl-2 K/D to IP 3 R may be responsible for the weak inhibitory property of this protein on IP 3 R-mediated Ca 2+ signaling and its protective effects against STS-induced apoptosis. However, the latter may also be related to Bcl-2 K/D's anti-apoptotic actions through its hydrophobic cleft and may therefore indicate that its ability to scaffold pro-apoptotic BH3-domain proteins is unaffected by this mutation in the BH4 domain. Moreover, recent molecular data indicate that the BH4 domain of Bcl-2 is directly involved in its homo-dimerization and also in the interaction with the pro-apoptotic Bax [START_REF] Zhang | Bcl-2 homodimerization involves two distinct binding surfaces, a topographic arrangement that provides an effective mechanism for Bcl-2 to capture activated Bax[END_REF][START_REF] Ding | Bcl-2 and Bax interact via the BH1-3 groove-BH3 motif interface and a novel interface involving the BH4 motif[END_REF]. In this respect, it is not surprising that Bcl-2 K/D retains some anti-apoptotic action.

Clearly, while Bcl-2 seems to exclusively interact with the central, modulatory domain of the IP 3 R (34), Bcl-Xl seems to interact with the C-terminal tail of the IP 3 R (29). Interestingly, the latter has been shown to be involved in the interaction with the suppressor domain and in the gating of IP 3 R channels [START_REF] Boehning | Direct association of ligand-binding and pore domains in homo-and heterotetrameric inositol 1,4,5-trisphosphate receptors[END_REF]. Moreover, it was recently proposed that the hydrophobic cleft is responsible for the interaction of Bcl-Xl with the C-terminal tail of the IP 3 R, which contains two putative BH3-like domains, enabling a stable interaction with Bcl-Xl and sensitizing the IP 3 R channel to IP 3 [START_REF] Foskett | Bcl-xL Regulation of InsP 3 Receptor Gating Mediated by Dual Ca 2+ Release Channel BH3 Domains[END_REF].

Besides the differential interaction with the IP 3 R, Bcl-2 and Bcl-Xl could also differ with respect to other previously identified targets of the BH4 domain of Bcl-2-family members, as calcineurin, VDAC, Raf-1, Ras, CED-4, paxillin and NF-κB, which all may play a role in apoptosis [START_REF] Rong | Targeting Bcl-2 based on the interaction of its BH4 domain with the inositol 1,4,5-trisphosphate receptor[END_REF]. A side-by-side comparison between Bcl-2 and Bcl-Xl will be required to examine whether there is a distinct or preferential action of either domain on these targets and whether the critical sequence difference between BH4-Bcl-2 and BH4-Bcl-Xl revealed in this study affects their activity or selectivity. A protective action of TAT-BH4-Bcl-Xl was recently reported on ischemic brain damage [START_REF] Donnini | Prevention of ischemic brain injury by treatment with the membrane penetrating apoptosis inhibitor, TAT-BH4[END_REF]. The protection and anti-apoptotic activity was linked to restoration of mitochondrial function and strong Akt activation. Considering the preferential targeting of Bcl-Xl to the mitochondrial outer membrane [START_REF] Kaufmann | Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane[END_REF], we speculate that the protective action of BH4-Bcl-Xl may be mediated by a modulation of mitochondrial targets, like VDAC (57), a mitochondrial outer membrane channel known to participate in mitochondrial Ca 2+ fluxes [START_REF] Bathori | Ca 2+ -dependent control of the permeability properties of the mitochondrial outer membrane and voltage-dependent anion-selective channel (VDAC)[END_REF][START_REF] Tan | VDAC closure increases calcium ion flux[END_REF] and apoptosis [START_REF] Madesh | VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release[END_REF][START_REF] Hajnoczky | Old players in a new role: mitochondriaassociated membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria[END_REF][START_REF] Shoshan-Barmatz | VDAC, a multi-functional mitochondrial protein regulating cell life and death[END_REF]. Hence, this difference in molecular targets fits well the distinct cellular and biological context, in which Bcl-2 and Bcl-Xl seem to operate [START_REF] Kaufmann | Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane[END_REF]. However, while BH4-Bcl-2 and BH4-Bcl-Xl seem to have distinct targets, our results suggest that these targets do not function in completely independent pathways, since we did not observe an additive protective effect against apoptotic stimuli when BH4-Bcl-2 and BH4-Bcl-Xl were combined and since the protective effect of BH4-Bcl-2 as well as BH4-Bcl-Xl against CytC-induced apoptosis was abolished in the presence of IP3RCYT.

It is therefore conceivable that Bcl-Xl may act through its BH4 domain on a target downstream of the IP 3 R, but we can not exclude other targets independently of the IP 3 R/Ca 2+ -signaling cascade.

Our study may have important therapeutic consequences for cancers that are dependent on high levels of Bcl-2 as chronic lymphocytic leukemia (CLL) and other hematologic malignancies [START_REF] Cory | The Bcl-2 family: roles in cell survival and oncogenesis[END_REF][START_REF] Tolcher | Targeting Bcl-2 protein expression in solid tumors and hematologic malignancies with antisense oligonucleotides[END_REF]. BH4-Bcl-2 and BH4-Bcl-Xl may have distinct targets characterized by distinct binding characteristics. Hence, while BH3-mimetic molecules do not discriminate between the hydrophobic cleft of Bcl-2 and Bcl-Xl, specifically targeting the BH4 domain of Bcl-2 with IDP may be a very promising approach to promote cell death via the induction of pro-apoptotic Ca 2+ signaling in Bcl-2-dependent malignancies [START_REF] Zhong | Induction of Ca 2+ -driven apoptosis in chronic lymphocytic leukemia cells by peptide-mediated disruption of Bcl-2-IP 3 receptor interaction[END_REF], while preserving essential biological functions of Bcl-Xl in other cells.

To conclude, our study is the first to reveal a difference in the cellular activity of the BH4 domain of Bcl-2 and Bcl-Xl, which is explained by a single residue difference between both domains. This change was responsible for the binding and inhibition of the IP 3 R by BH4-Bcl-2, but not by BH4-Bcl-Xl.

MATERIALS AND METHODS

Peptides

All synthetic peptides were obtained from Thermo Electron, Germany. Biotinylated peptides were obtained from Lifetein, USA. Table 1 provides an overview of the different peptides used in this study and their primary sequence.

Plasmid vector constructs

The pGEX-6p2 construct (Amersham Biosciences, GE healthcare) encoding amino acids 923-1581 of mouse IP 3 R1 Domain 3 was obtained as previously described [START_REF] Sienaert | Molecular and functional evidence for multiple Ca 2+ -binding domains in the type 1 inositol 1,4,5-trisphosphate receptor[END_REF].

Cell culture and transfections

Mouse embryonic fibroblasts (MEF cells) were cultured at 37°C in a 9% CO 2 incubator in DMEM/Ham's F12 medium (1:1) (Invitrogen, Belgium) supplemented with 10% fetal calf serum (Sigma-Aldrich), 3.8 mM L-glutamine (Glutamax, Invitrogen), 85 IU/ml penicillin and 85 µg/ml streptomycin (Invitrogen). C6-glioma cells were cultured in DMEM/Ham's F12 medium (1:1), containing 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone and 2 mM L-glutamine at 37°C, 5% CO 2 .

COS-1 cells were seeded at a density of 15000 cells/cm 2 and cultured in DMEM supplemented with 10% fetal bovine serum (Sigma-Aldrich) at 37 O C 10% CO 2 . Two days after plating, COS-1 cells, were transiently transfected with the empty p3xFLAG-Myc-CMV™-24 vector (Sigma-Aldrich) or containing the cDNA of Bcl-2 or of the Bcl-2 K/D mutant. The transfection procedure was performed by using JetPRIME TM transfection reagent (Polyplus Transfections, USA) and following manufacturer's recommendations. For Ca 2+ imaging experiments, Cos-1 cells were also co-transfected with a pcDNA 3.1-mCherry vector (Invitrogen) using a DNA ratio of 1:2 between pcDNA 3.1-mCherry and p3xFLAG-Myc-CMV™-24 vectors.

WEHI7.2 murine cells were grown as described previously (34) and nucleofected with either pSFFV-Neo, pSFFV-Neo-Bcl-2 or pSFFV-Neo-Bcl-2 K/D vectors, using the Amaxa nucleofector TM and the dedicated Mouse T cell Nucleofector Kit according to the manufacturer's instructions. Subsequently, resistant cells were stably selected with 1 mg/ml G418 (Invitrogen) as described in [START_REF] Chen | Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate[END_REF][START_REF] Dieken | Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis[END_REF], but no clonal expansion was performed.

Western-blot analysis and antibodies

Cos-1 and WEHI7.2 cells were lyzed in a buffer containing 25 mM Hepes, pH 7.5, 1%

Triton X-100, 10% glycerol, 0.3 M NaCl, 1.5 mM MgCl 2 , 1 mM DTT, 2 mM EDTA, 2 mM EGTA and protease inhibitor cocktail tablets (Roche, Switzerland). Samples protein concentration was determined by Bradford assay (Sigma-Aldrich) using Bovine Serum Albumin (BSA) as standard. Proteins (10-20 μg) were separated by NuPAGE 4-12% Bis/Tris SDS-polyacrylamide gels using MES/SDS-running buffer (Invitrogen) and transferred onto a polyvinylidene Fluoride (PVDF) membrane. After blocking with TBS containing 0.1% Tween and 5% non-fat dry milk powder, the membrane was incubated with the primary antibody overnight. Next, membranes were incubated for 1 hr with a secondary horseradish peroxidase (HRP)-conjugated antibody (dilution 1:2000 in 0.1% Tween/TBS). Protein detection was performed with Pierce® ECL Western Blotting Substrate (Thermo Fisher Scientific; p/a Perbio Science BVBA, Erembodegem, Belgium). Bands quantification was done with ImageJ software (rsbweb.nih.gov/ij/).

Primary antibodies used in this study are: mouse monoclonal ANTI-FLAG® M2-Peroxidase (HRP) (sigma-aldrich), rabbit polyclonal anti-PARP-1 (Alexis, USA), mouse monoclonal Anti-GAPDH clone GAPDH-71.1 (Sigma-aldrich), mouse monoclonal anti-Bcl-2 (C2) (Santa Cruz Biotechnology, USA).

Unidirectional 45 Ca 2+ -flux assay

Twelve-well clusters containing MEF cells were fixed on a thermostated plate at 30 o C on a mechanical shaker. The culture medium was aspirated, and the cells were permeabilized by incubating them for 10 min in a solution containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 2 mM MgCl 2 , 1 mM ATP, 1 mM EGTA and 20 µg/ml saponin. The nonmitochondrial Ca 2+ stores were then loaded for 45 min in 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM MgCl 2 , 5 mM ATP, 0.44 mM EGTA, 10 mM NaN 3 to prevent mitochondrial Ca 2+ uptake, and 150 nM free 45 Ca 2+ (28 µCi/ml). After reaching steady-state loading, cells were washed twice with 500 μl of efflux medium (120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 1 mM EGTA) supplemented with 4 μM thaspigargin (Enzo Life Sciences, USA) to block SERCA Ca 2+ -uptake activity. Then, efflux medium was added and replaced every 2 min. IP 3 (3 µM) was added for 2 min after 10 min of efflux. At the end of the experiment, all 45 Ca 2+ remaining in the stores was released by incubation with 1 ml of a 2% (w/v) sodium dodecyl sulfate solution for 30 min. Ca 2+ release was plotted as fractional loss (% / 2 min) as a function of time, as previously described (67-69).

Electroporation loading

In situ electroporation of monolayer cell cultures was performed as described before [START_REF] Vuyst | In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling[END_REF], according to a procedure that was optimized for cell death studies [START_REF] Decrock | Connexin 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model[END_REF]. They were placed 400 µm underneath a two-wire Pt-Ir electrode on the microscopic stage and electroporated in the presence of a tiny amount of electroporation solution (10 µl).

Electroporation was done with 50 kHz bipolar pulses applied as trains of 10 pulses of 2 ms duration each and repeated 15 times. The field strength was 100 V peak-to-peak applied over a 400 µm electrode separation distance. After electroporation, cells were thoroughly washed with HBSS-Hepes. using Slide-A-Lyzer with a cut-off of 3 kDa (Thermo Fisher Scientific, USA). After dialysis, the concentration of the purified GST-fusion proteins was determined using BCA Protein Assay Reagent (Thermo Fisher Scientific), and the quality and integrity were examined by SDS-PAGE and GelCode TM blue stain reagent (Thermo Fisher Scientific) prior to GST-pull downs or SPR analysis.

GST-pull downs

Equal amounts (30 µg) of the intact full-length GST-fusion proteins or parental GST For the pull downs with full-lenght-Bcl-2 proteins, 200 µg of cleared lysate from COS-1 cells transiently transfected with the 3xFLAG-Bcl-2-vector, 3xFLAG-Bcl-2-K/D or the empty vector, were incubated with GST-tagged proteins using the same protocol as above. Eluates (10 µl) were subjected to western blot and incubated with anti-FLAG HRP-coniugated antibody, diluted 1:4000 in 0.1% Tween/TBS.

SPR measurements

SPR experiments were performed as described before [START_REF] Ponsaerts | Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity[END_REF]. The binding of GST-Dom3

  signals were monitored as F340/F380 and calibrated to obtain [Ca 2+ ]. Typical Ca 2+ responses in empty vector-, 3xFLAG-Bcl-2and 3xFLAG-Bcl-2 K/D-transfected cells are shown in Fig. 7C, Fig. 7D and Fig. 7E, respectively. As evident from these experiments, cells expressing 3xFLAG-Bcl-2 displayed blunted Ca 2+ signals in response to ATP in comparison to empty vector-or 3xFLAG-Bcl-2 K/D-transfected cells. This indicates that the Lys17 mutation into Asp critically affects Bcl-2's ability to inhibit IP 3 R-mediated Ca 2+ signals. Quantitative analysis of 6 independent experiments (70 to 90 cells) indicated that 3xFLAG-Bcl-2 was much more potent in inhibiting IP 3 R-mediated Ca 2+ signals than 3xFLAG-Bcl-2 K/D. The partial inhibition of 3xFLAG-Bcl-2 K/D on IP 3 R-mediated Ca 2+ signaling may be explained by the fact that 3xFLAG-Bcl-2 K/D retained some binding to the IP 3 R (~ 20 % compared to 3xFLAG-Bcl-2).

pcDNA3. 1 (

 1 -) mouse IP 3 R2 and rat IP 3 R3 were used as templates for the construction of pGEX-6p2 vectors encoding the corresponding regions of IP 3 R2 (a.a. 913-1562 ) and IP 3 R3 (a.a. 910-1427). Their respective coding regions were amplified by PCR using the ); IP 3 R2 Reverse (5′-GCGGCGGAATTCCTAGAGCGTGTTGACCTGGCTG-3′); IP 3 R3 Forward (5′-GCGGCGGGATCCAACGTGCGGAGGTCCATCCAGGG-3′); IP 3 R3 Reverse (5′-GCGGCGGAATTCTCACAGAGCGCTCATGTGGGCATC-3′). PCR products were purified, digested with BamHI and EcoRI (restrictions sites are underlined above) and ligated into BamHI-EcoRI-treated pGEX-6p2 vector. 3xFLAG-Bcl-2 and Bcl-Xl were generated by subcloning their respective full-lenght cDNAs into p3xFLAG-myc-CMV-24 vector (sigma-aldrich, Germany) at HindIII/BglII sites. The 3xFLAG-Bcl-2 K/D mutant was generated by PCR Site Directed Mutagenesis introducing GAT (D) to replace AAG (K) in wild-type Bcl-2 -donor DNA. Neo and pSFFV-Neo-Bcl-2 vectors were kindly provided from Prof. Clark W. Distelhorst. The pSFFV-Neo-Bcl-2 K/D vector was also obtained by PCR Site-Directed Mutagenesis. To introduce the desired point mutations, we designed and used by sequencing (Agowa AG, Germany).

  C6 cells were grown to near confluency on 13 mm-(apoptosis experiments) or 18 mm-(Fluo-3 Ca 2+ imaging) diameter glass coverslips. Cell monolayer cultures were washed three times with Hanks' balanced salt solution buffered with Hepes (HBSS-Hepes) supplemented with D-glucose (0.81 mM MgSO 4 , 0.95 mM CaCl 2 , 137 mM NaCl, 0.18 mM Na 2 HPO 4 , 5.36 mM KCl, 0.44 mM KH 2 PO 4 , 5.55 mM D-glucose, 25 mM Hepes, pH 7.4) and subsequently three times with a low-conductivity electroporation buffer (4.02 mM KH 2 PO 4 , 10.8 mM K 2 HPO 4 , 1.0 mM MgCl 2 , 300 mM sorbitol, 2.0 mM Hepes, pH 7.4).

Fluo- 3 4 -

 34 Ca 2+ imaging C6 cells were seeded on 18 mm-diameter glass coverslips and ester-loaded for 25 min with 5 µM Fluo-3-AM (Invitrogen) in HBSS-Hepes supplemented with 1 mM of BL21(DE3) Escherichia coli cells were transformed with pGEX-6p2 constructs containing cDNAs of IP 3 R1 Domain 3 (GST-Dom3 IP 3 R1 : a.a. 923-1581), IP 3 R2 Domain 3 analogue (GST-Dom3 IP 3 R2: a.a. 913-1562), IP 3 R3 Domain 3 analogue (GST-Dom3 IP 3 R3: a.a. 910-1427) or with the empty vector. The expressed proteins were purified as previously described (34, 70). All fusion proteins were affinity purified and dialyzed against standard phosphate-buffered saline (PBS) without added Ca 2+ or Mg 2+ (2.67 mM KCl, 1.47 mM KH 2 PO 4 , 137.93 mM NaCl, 8.06 mM Na 2 HPO

(

  control) were incubated in Interaction Buffer (50 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.5% BSA, and protease inhibitor cocktail, pH 7.0) with 30 µg of different BH4 domains (BH4-Bcl-2, BH4-Bcl-Xl or a non-binding mutant (BH4-Bcl-2BIND) and immobilized on glutathione-Sepharose 4B beads (GE Healthcare Europe GmbH, Belgium) via rotation in a head-over-head rotator for 2 h at 4°C. The beads were washed 4 times with modified Interaction Buffer (150 mM NaCl instead of 300 mM NaCl, without BSA) and complexed GST-fusion proteins were eluted by incubating the beads with 40 µl LDS® (Invitrogen) for 3 min at 95°C and collected after centrifuging at 500 g for 5 min. Eluates (10 µl) were subjected to western blot analysis and the total protein content was visualized by GelCode TM blue staining of the Gel.

Fura- 2

 2 and parental GST (control) to the wild-type or mutated BH4 domain of Bcl-2 or Bcl-Xl was analyzed by SPR at 25°C using a Biacore 2000 instrument. Equal amounts (200 ng or 58.2 pmol) of >80% pure biotinylated BH4 peptides were immobilized on 4 different flow cells of a streptavidin-coated sensor chip (BR-1000-32; Biacore; Uppsala, Sweden) using PBS supplemented with 0.005% P20 (Polysorbate-20) at pH 7.0. At least 3 independent sensor chips were used for the quantitative analysis.Measurements with GST-fusion proteins as analyte were performed in PBS at a flow rate of 30 µl/min. Different concentrations of the analyte (injection volume 120 µl) were used in a random order to assess binding, expressed in terms of Resonance Units (R.U.).Bound peptide was removed by injection of 5 µl regeneration buffer (25 mM NaOH, 0.002% SDS) at 10 µl/min. Background signals were obtained from the reference flow cell, containing the BH4-scrambled peptide, and were subtracted to generate response curves using Biaevaluation 3.0 software. In the analysis, only the association phase of the binding curve was taken into account. Data from 2-3 different sensorgrams for each condition were fitted by non-linear regression analysis to a Hill-Langmuir binding isotherm using Origin 7.0 (OriginLab Corporation, UK) software. Ca 2+ imaging Two days after co-transfection (mCherry + 3xFlag construct), Cos-1 cells were loaded with Fura2-AM (5 µM) (Biotium, Inc., USA) in modified Krebs buffer (135mM NaCl, 5.9mM KCl, 1.2mM MgCl 2 , 11.6mM Hepes, pH 7.3, 11.5mM glucose, 1.5mM Ca 2+ ) for 30 min, followed by de-esterification over 30 min 5 mM BAPTA (Invitrogen) was added before each measurement to buffer all free extracellular Ca 2+ . ATP (1 µM) or Thapsigargin (5 µM)-induced Ca 2+ rises were measured ratiometrically (excitation at 340 nm/380 nm, emission detection at 510 nm) using Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20x air objective and an high-speed digital camera (Axiocam Hsm, Zeiss). Intracellular cytoplasmic Ca 2+ concentrations ([Ca 2+ ] cyt ) were calculated from the equation:[Ca 2+ ] cyt (nM) = K d ×Q×(R-R min )/(R max -R). K d isthe dissociation constant of Fura-2 for Ca 2+ at room temperature (220 nM), Q is the fluorescence ratio of the emission intensity excited by 380nm in the absence of Ca2+ to that in the presence of saturating Ca 2+ , R is the fluorescence ratio, R min and R max are the minimal and maximal fluorescence ratios, respectively. R min was measured by perfusion with 50 mM EGTA in Ca 2+ -free Krebs solution and R max was obtained by perfusion with 5 µM ionomycin and 250 mM CaCl 2 . Only cells mCherry fluorescent (excitation 546 nm, emission 610 nm) and Fura-2 loaded were considered for data analysis. 10-15 cells per experiment were analyzed for a total of 80 cells per transfection condition on 3 different experimental days. Traces were normalized to baseline levels with Excel software (Microsoft Office) and for each experiment the average peak value (Δ[Ca 2+ ] cyt ) was determined.Apoptosis inductionFor CytC-induced-apoptosis experiments, C6 cells were loaded by in situ electroporation in the presence of 100 μM DTR (to identify the loaded zone), 20 μM of Bcl-2 peptides or 50 µM IP3RCYT peptide and 10 μM of CytC (Sigma). After electroporation, cells were kept in 200 μl culture medium for 25 min. For STS-and PAC-1-induced apoptosis experiments, cells were electroporated in the presence of 100 µM DTR and 20 µM Bcl-2 peptides. After electroporation, cells were kept in 200 µl culture medium containing 2 µM STS (Sigma-Aldrich) or 150 µM PAC-1 (Tocris Bioscience, UK) for 6 hours.For STS-induced apoptosis, Cos-1 cells transiently transfected with 3xFLAG-vectors and WEHI7.2 cells, stably transfected with the various pSFFV-Neo vectors, were treated for 6 hours with 1 µM STS (Sigma-aldrich).Detection of apoptosis by fluorescence microscopyGlioma cells were stained first with 10 μM of the CaspACETM FITC-VAD-FMK "In Situ Marker" (Promega Benelux, The Netherlands) in HBSS-Hepes for 40 min at 37°C. After fixing the cells with 4% paraformaldehyde (PFA) for 25 min at room temperature, nuclei were additionally stained for 5 min with 1 μg/ml DAPI (Sigma) in PBS supplemented with Ca 2+ and Mg 2+ (PBSD+). Cells were mounted with Vectashield fluorescent mounting medium (VWR International, Belgium) on glass slides.Five images (in each culture) were taken in the electroporated area using a Nikon TE300 epifluorescence microscope equipped with a 10X objective (Plan APO, NA 0.45 -Nikon) and a Nikon DS-5M camera (Nikon Benelux, Belgium). The number of caspase-positive cells and DNA-fragmented nuclei were counted in each image and expressed relative to the number of nuclei present and stated as the Apoptotic Index (AI) . Small groups of apoptotic bodies were counted as remnants of a single apoptotic cell. Analyses were carried out blinded, making use of custom-developed counting software. For the STSand PAC-1-treated cultures, five additional images were taken outside the electroporated area. The AI in the electroporated area was expressed relative to the AI outside the area.FACS analysis and microplate-based apoptosis assayWEHI7.2 cells were pelleted by centrifugation and incubated with Annexin-V fluorescein isothiocyanate (FITC) (Becton Dickinson, USA) and propidium iodide (PI) (invitrogen, Eugene, Oregon, USA). Single-cell suspensions were analyzed by FACSCanto (Becton Dickinson). Cell death by apoptosis and secondary necrosis were scored by quantifying the population of (Annexin-V)-FITC positive and (Annexin-V-FITC and PI) positive cells. Flow cytometric data were plotted and analyzed using BD FACSDiva Software (Becton Dickinson). Caspase-3 activity of STS-treated-WEHI7.2 cells was measured according to the protocol of the commercially available NucView™ 488 Caspase-3 Kit for live Cells (Biotium). Cells were transferred to a 96-well plate at a density of 400.000 cells/well and Fluorescence was monitored using a FlexStation 3 microplate reader (Molecular Devices, USA). Results were normalized as percentage decrease over control condition.

Figure 1 .

 1 Figure 1. Although the BH4 domain of Bcl-2 and Bcl-Xl are similar in sequence and structure, they differentially regulate IP 3 R-mediated Ca 2+ flux in permeabilized and intact cells. A, Schematic presentation of the anti-apoptotic Bcl-2-family members and the IP 3 R. The N-terminal BH4 domain is unique for anti-apoptotic members. The secondary structure and primary sequence of BH4-Bcl-2 and BH4-Bcl-Xl are very

Figure 3 .

 3 Figure 3. The central, modulatory domain of IP 3 R1 binds to immobilized biotin-BH4-Bcl-2, but not to biotin-BH4-Bcl-Xl, in SPR experiments. A-C, Sensorgrams are obtained after background correction for binding to the respective scrambled versions of the biotinylated BH4-domain peptides. The association phase is shown upon addition of different concentrations of purified GST-Dom3 to biotin-BH4-Bcl-2 (A) or biotin-BH4-Bcl-Xl (B). A typical experiment is shown. C, The quantitative analysis of the binding properties of GST-Dom3 to either biotin-BH4-Bcl-2 or biotin-BH4-Bcl-Xl is shown. Values were obtained from 3 independent experiments and are plotted as mean ± SEM. D, Sensorgrams representing the association phase of different concentrations of GST-Dom3 for the binding to either biotin-BH4-Bcl-2 or biotin-BH4-Bcl-2BIND. All curves are background corrected for binding to the respective scrambled peptides. E,

Figure 4 .

 4 Figure 4. Determination of the residues in BH4-Bcl-2 critical for inhibiting IICR. A, Zoomed picture of the BH4 domain of Bcl-2 showing its surface-accessible residues, is

Figure 5 .

 5 Figure 5. BH4-Bcl-2 and BH4-Bcl-Xl differ in one critical amino acid, which determines their effect on the IP 3 R. A, Aligned sequence of the BH4 domain of Bcl-2 and Bcl-Xl with indication of the surface-accessible residues is shown. The amino acids for which the mutation to alanine had the largest effect on the inhibitory effect of the BH4 domain, are indicated in red. The critical residue Lys17 in BH4-Bcl-2 is not conserved in BH4-Bcl-Xl. The sequences of these BH4-domain peptides are indicated. B, A typical experiment of a unidirectional 45 Ca 2+ -flux assay in permeabilized MEF cells, comparing the effect of BH4-Bcl-2, BH4-Bcl-Xl, BH4-Bcl-2 K/D and BH4-Bcl-Xl D/K

Figure 6 .

 6 Figure 6. BH4-Bcl-2, but not BH4-Bcl-Xl, protects against CytC-and STS-induced

Acknowledgements

This work was supported by the Research Foundation-Flanders (F.W.O.) grant nos. G.0604.07N to HDS, G.0788.11N to GB and MDM, and G.0134.09N to L.L.), by the Research Council of the K.U. Leuven via the Concerted Actions program (GOA /09/012) and via an OT START (SRT/10/044), and by the Interuniversity Attraction Poles Program (Belgian Science Policy; P6/28 to HDS, JBP and LM and P6/31 to L.L.), and by National Institutes of Health RO1 CA085804 (CWD). G.M. and E.D. were supported by a Ph.D. fellowship from Research Foundation-Flanders (F.W.O.). H.A. was supported by a post-doctoral fellowship from the Research Foundation-Flanders (F.W.O.). The authors wish to thank Kirsten Welkenhuyzen for their excellent technical assistance. We greatly appreciated the help of Dr. K. Mikoshiba (Lab. for Developmental Neurobiology, Brain Science Institute, RIKEN, Japan), Dr. D. Boehning (Dept. of Neuroscience and Cell Biology, University of Texas, Medical Branch, Texas, USA) and Dr. P. Zimmermann (Dept. of Human Genetics, K.U. Leuven), respectively, for providing the IP 3 R2expression plasmid, IP3RCYT peptide and the use of the BIAcore 2000.

Data and statistical analysis

Data are expressed as mean ± SEM, unless a typical experiment is shown (mean ± SD). Statistically significant differences were considered at P<0.05 (single symbols), P< 0.01 (double symbols) and P<0.001 (triple symbols) after using a two-tailed paired Student's t test (Excel Microsoft Office) or one-way ANOVA and a Bonferroni post-test using Origin7.0.

probenecid (Sigma-Aldrich) and 0.01% pluronic F-127 (Invitrogen) at 37°C, followed by de-esterification over 15 min. Subsequently cells were loaded with 100 µM DTR (Invitrogen) and 20 µM Bcl-2 peptides using the in situ electroporation technique as described above. For UV flash photolysis experiments, 50 µM caged IP 3 (Invitrogen) was also included in the loading solution. Imaging was carried out using an inverted fluorescence microscope equipped with a 40x oil-immersion objective and an intensified CCD camera (Extended Isis camera, Photonic Science, UK).

In the case of stimulation by ATP, cells were superfused for 1 min with HBSS-Hepes followed by 8 min with 1 µM of ATP (Sigma) in HBSS-Hepes while for thapsigargin experiments, cells were superfused for 1 min with Ca 2+ -free HBSS-Hepes containing 1 mM EGTA followed by 7 min with 2.5 µM thapsigargin (invitrogen) in the same buffer.

For IP 3 photoliberation experiments, cells were, after 10 sec, spot illuminated with 1-kHz pulsed UV light (349 nm UV laser Explorer, Spectra-Physics, Newport) during 20 ms (20 pulses of 90 μJ energy measured at the entrance of the microscope epifluorescence tube).

The UV flash was applied at five different places along the electroporated area per dish.

Images (1/sec) were generated with software written in Microsoft Visual C ++ 6.0. Fluorescence intensity changes in different cells (at least 20) were analyzed with customdeveloped FluoFrames software. For UV flash photolysis experiments, fluorescence intensity changes in all cells in a pre-defined 15.9 mm 2 region were analyzed. Ca 2+ concentration, [Ca 2+ ], changes were quantified as the area under the curve (AUC) of the separate Ca 2+ traces. N is equal to the number of dishes. 3xFLAG-Bcl-2 K/D is significantly different from 3xFLAG-Bcl-2. G, Quantitative analysis of the area under the curve of the TG-induced Ca 2+ signal in these cells. H, Western-blot analysis using anti-PARP antibodies for monitoring PARP cleavage upon STS treatment (1 μM for 6 hrs) in 3xFLAG-vector, 3xFLAG-Bcl-2 and 3xFLAG-Bcl-2 K/D-expressing COS cells. GAPDH was used as a loading control. I, Quantification of the immunoreactive bands of the ratio of the cleaved over the full-length PARP in the different transfected COS cell populations from three independent experiments. The ratio of cleaved over full-length PARP obtained for control cells were set at 100 % and the other ratio's were normalized to this value. Both 3xFLAG-Bcl-2 as well as 3xFLAG-Bcl-2 K/D significantly (*) prevent STS-induced apoptosis compared to the empty vector control, but 3xFLAG-Bcl-2 K/D is significantly ($) less potent than 3xFLAG-Bcl-2. We wish to remark that the results obtained with 3xFLAG-Bcl-2 K/D were borderline significantly (*!) different from the empty vector control (p=0.051).

Preparation of GST-fusion Proteins