Phosphorylation of inositol 1,4,5-trisphosphate receptors by protein kinase B/Akt inhibits Ca²⁺ release and apoptosis

Abstract

Imbalance of signals that control cell survival and death results in pathologies, including cancer and neurodegeneration. Two pathways that are integral to setting the balance between cell survival and cell death are controlled by lipid-activated protein kinase B (PKB)/Akt and Ca²⁺. PKB elicits its effects through the phosphorylation and inactivation of proapoptotic factors. Ca²⁺ stimulates many prodeath pathways, among which is mitochondrial permeability transition. We identified Ca²⁺ release through inositol 1,4,5-trisphosphate receptor (InsP₃R) intracellular channels as a prosurvival target of PKB. We demonstrated that in response to survival signals, PKB interacts with and phosphorylates InsP₃Rs, significantly reducing their Ca²⁺ release activity. Moreover, phosphorylation of InsP₃Rs by PKB reduced cellular sensitivity to apoptotic stimuli through a mechanism that involved diminished Ca²⁺ flux from the endoplasmic reticulum to the mitochondria. In glioblastoma cells that exhibit hyperactive PKB, the same prosurvival effect of PKB on InsP₃R was found to be responsible for the insensitivity of these cells to apoptotic stimuli. We propose that PKB-mediated abolition of InsP₃-induced Ca²⁺ release may afford tumor cells a survival advantage.

Protein kinase B (PKB) is a central player in regulating many signaling pathways controlling cell metabolism, growth, and survival (1, 2). PKB elicits these effects by phosphorylating and regulating the activity of downstream targets such as glycogen synthase kinase 3β and Bad, or via transcription factors such as Forkhead (1, 3). Because of this critical role of PKB, gain or loss of function is manifest in major disease phenotypes such as cancer and type 2 diabetes (1, 4–6).

Ca²⁺ released from the endoplasmic reticulum (ER) through inositol 1,4,5-trisphosphate (InsP₃) receptors (InsP₃Rs) plays a key role in regulating physiological processes (7). However, under pathological conditions, InsP₃-induced Ca²⁺ release (IICR) can be subverted to promote cell death pathways (8–10). The importance of IICR in cell death is underlined by the uncovering of functional interactions with a number of proteins with known proapoptotic and antiapoptotic activity. Notable among these are Bcl-2, Bcl-X_L, and cytochrome c (11–14). PKB has also recently been shown to phosphorylate the InsP₃R, with consequences for cell survival (15).

We investigated whether cross-talk between the phosphatidylinositol 3-kinase (PI3K)/PKB and InsP₃/Ca²⁺ signaling pathways regulated how cells responded to death-inducing stimuli. We determined that PKB-mediated phosphorylation of InsP₃R results in a decrease in the magnitude of IICR and resultant flux of Ca²⁺ from the ER to mitochondria. Moreover, we show that this decrease in Ca²⁺ flux caused by PKB-mediated phosphorylation of InsP₃Rs contributes to protection from the effects of apoptotic stimuli. This prosurvival action of PKB was also apparent in a glioblastoma cell line (U87) that exhibits increased PKB activity caused by a deletion in the gene encoding the phosphatidylinositol 3,4,5 trisphosphate (PIP3) phosphatase, PTEN. Together, these results contribute to a mechanism by which PKB and IICR interact to regulate cell death and survival.

Results

Ca²⁺ Release from Intracellular Stores Is Regulated by PKB.

Growth factor status and genetic factors that enhance PKB activity significantly impact on cell fate [supporting information (SI) Fig. 7]. On the other hand, the occurrence of Ca²⁺ signals during the cell death process, and the protection from apoptosis afforded by buffering intracellular Ca²⁺, places Ca²⁺ at a central position in promoting cell death (SI Fig. 7).

Because of the central roles of both PKB and Ca²⁺ in regulating cell death, we investigated whether PKB-mediated phosphorylation of InsP₃Rs, and suppression of Ca²⁺ signals, contributed to the prosurvival role of PKB. Inducible overexpression of constitutively active PKB (CA-PKB) promoted the phosphorylation of InsP₃Rs (Fig. 1A and SI Fig. 8 for characterization of CA-PKB-expressing cell lines). The example Ca²⁺ traces and the histogram of the percentage of responding cells in Fig. 1 Bi and Bii, respectively, illustrate the significant inhibition of histamine-induced Ca²⁺ release by CA-PKB overexpression. The time from agonist addition to peak response (latency) and the percentage of cells exhibiting Ca²⁺ oscillations after application of 100 μM histamine was also reduced in CA-PKB-expressing cells (SI Fig. 9 Ai and Aii). Kinase dead (KD)-PKB had no effect on agonist-induced Ca²⁺ signals (Fig. 1B and SI Fig. 9A). Ca²⁺ release stimulated by cell permeant InsP₃ (InsP₃-BM) was also reduced by CA-PKB overexpression, indicating that PKB was directly modulating IICR (Fig. 1C and SI Fig. 9B). The PKB-mediated inhibition of agonist-induced Ca²⁺ release was not caused by a decrease in ER luminal Ca²⁺ content because the integrated cytosolic Ca²⁺ transient [area under the curve (AUC)] induced by the irreversible sarco/ER ATPase (SERCA) pump inhibitor thapsigargin was unaffected by CA-PKB expression (29,860 ± 1,118 nM·s vs. 31,220 ± 767.7 nM·s; P > 0.05 in YFP- or CA-PKB-expressing cells, respectively).

Fig. 1.

IICR and mitochondrial Ca²⁺ uptake is regulated by PKB. (A) CA-PKB expression promotes phosphorylation of InsP₃R1. (Upper) An autoradiograph of ³²P-labeled InsP₃Rs. (Lower) Immunoblots (IB) of proteins present in the lysates used as input for the IPs. InsP₃Rs were immunoprecipitated from control cells (−) or cells expressing CA-PKB as shown (+). (Bi and Bii) Histamine-induced Ca²⁺ signals in HeLa cells expressing CA-PKB (n = 89 cells), YFP (n = 64 cells), or KD-PKB (n = 74 cells). (Bi) Typical responses from individual HeLa cells stimulated with the histamine concentrations (μM) shown. (Bii) Averaged data showing the effect of CA-PKB or KD-PKB expression on the proportion of responding cells. (C) Typical Ca²⁺ responses recorded in control and CA-PKB-expressing cells stimulated with InsP₃ ester (InsP₃-BM). (Di) Average background subtracted, mitochondrial Rhod-2 fluorescence changes in control and CA-PKB expressing HeLa cells. (Dii) Confocal images of Rhod-2 fluorescence in HeLa cells stimulated with 1 μM ATP. Images captured at the indicated time points are shown. (E and F) (Upper) Autoradiographs of ³²P-labeled InsP₃Rs. (Lower) Immunoblots (IB) of the indicated proteins in the lysates used as input for the IPs. (E) InsP₃R phosphorylation in HeLa cell transfected with PKB siRNA or control siRNA. (F) Effect of LY294002 onInsP₃R phosphorylation in HeLa cells grown under serum-replete normal growth conditions. (G) Impact of PKB siRNA or control siRNA on the amplitude of Ca²⁺ responses induced by the concentrations of histamine (μM) indicated. (H) Amplitude of Ca²⁺ responses induced by the concentrations of histamine (μM) shown in control and serum-starved HeLa cells. The data represent mean ± SEM. * indicates P < 0.05. NS indicates not significant.

As Ca²⁺ released from InsP₃Rs is taken up in a privileged manner by mitochondria, mitochondrial Ca²⁺ levels represent an exquisitely sensitive measure of Ca²⁺ released from ER-localized InsP₃Rs. Using confocal imaging of mitochondrially compartmentalized Rhod-2 (16), a significantly lower level of agonist-induced mitochondrial Ca²⁺ uptake was observed in CA-PKB-expressing cells compared with controls (Fig. 1 Di and Dii).

Because serum-starvation sensitized cells to apoptotic stimuli (SI Fig. 7), we investigated whether InsP₃Rs were phosphorylated under normal serum-replete growth where PKB is tonically active. Reduction of PKB expression by siRNA, or activity, using LY294002 (inhibits PI3K, which lies directly upstream of PKB) significantly decreased ³²P-labeling of InsP₃Rs to a level that was not detectable above background (Fig. 1 E and F).

The tonic activity of endogenous PKB also regulated agonist-induced Ca²⁺ release. As would be expected of an InsP₃R inhibitor, reduction of endogenous PKB expression by siRNA, or its activity by serum starvation, resulted in an enhancement of agonist-induced Ca²⁺ release (Fig. 1 G and H). Together, these data suggest that phosphorylation of InsP₃R by PKB significantly reduces its sensitivity to InsP₃. Moreover, InsP₃Rs are phosphorylated by endogenous, tonically active PKB (in serum), CA-PKB, and PKB that had been activated by physiological stimuli.

S2681 in InsP₃R1 Regulates IICR.

To further isolate the contribution of PKB phosphorylation of InsP₃Rs to Ca²⁺ release, IICR was quantitated in cells overexpressing InsP₃Rs in which the phosphorylatable serine in the PKB consensus site was mutated to an unreactive alanine (S2681A in InsP₃R1) (15). COS-7 cells expressing InsP₃R1^S2681A had greater ATP-induced Ca²⁺ responses than their InsP₃R1^wt-expressing counterparts (Fig. 2Ai, Aii, and Aiii; the peak, AUC, and latency of the Ca²⁺ signal are shown). These effects were not caused by differences in expression levels of the wild-type or mutated receptors or differences in their intracellular distribution (SI Figs. 10B and 11 and data not shown). COS-7 cells were used for these experiments because they express low levels of endogenous InsP₃R1 and have been successfully used to study InsP₃R function (17). Moreover, in these cells, heterologously expressed wild-type InsP₃Rs can be overexpressed at a level significantly greater than endogenous receptors, are correctly targeted, and exhibit significantly increased agonist-induced Ca²⁺ release and decreased latency of the Ca²⁺ transient (SI Fig. 11).

Fig. 2.

InsP₃R1-S2681 regulates agonist-induced Ca²⁺ release. (A i–iii) Averaged data of ATP-induced Ca²⁺ signals in COS-7 cells expressing InsP₃R1^wt (n = 293 cells) or InsP₃R1^S2681A (n = 192 cells). Peak Ca²⁺ response (Ai), AUC (Aii), and latency (Aiii) are shown for the ATP concentrations (μM) indicated. (B i and ii) Carbachol-induced Ca²⁺ responses in M3-expressing DT40 cells transiently transfected with InsP₃R1^wt (n = 35 cells) or InsP₃R1^S2681A (n = 44 cells). * indicates that the data are statistically significant (P < 0.05).

Agonist-induced Ca²⁺ release was also monitored in M3 muscarinic receptor-expressing DT40 InsP₃R triple knockout chicken B lymphocyte cell line (DT40 TKO) (18). The peak and AUC of the Ca²⁺ signal induced by carbachol was significantly greater in cells transiently transfected with InsP₃R1^S2681A than cells transfected with InsP₃R1^wt (Fig. 2 Bi and Bii). Together, the data derived from HeLa (Fig. 1), COS-7, and DT40 cells support the conclusion that phosphorylation of InsP₃R1-S2681 by PKB was inhibitory to Ca²⁺ release.

PKB Interacts with InsP₃R1.

Because protein kinases often reside in a complex with their substrates, whether PKB and InsP₃Rs interact was next investigated. Using coimmunoprecipitation (co-IP), an interaction between overexpressed CA-PKB and endogenous full-length InsP₃Rs was detected in HeLa cells (Fig. 3 Ai and Aii). KD-PKB did not interact with InsP₃R1 (data not shown). PKB also interacted with a YFP-tagged ER-localized NH₂-terminally truncated InsP₃R1 that encompassed only the amino acids COOH-terminal of transmembrane domains 5 (YFP-CT) (Fig. 3B).

Fig. 3.

PKB interacts with InsP₃R1. (A i and ii) Co-IPs of full-length InsP₃R1 with PKB. A line is shown in the images where different parts of the same gel have been grouped. (Ai) IPs using anti-InsP₃R1 antibody or preimmune IgG were performed on lysates prepared from HeLa cells expressing YFP or CA-PKB. (Aii) Immunoblot showing the proteins present in the lysates used as input for the IP in Ai. (B) Co-IP of PKB with the InsP₃R1 COOH terminus. IPs using anti-GFP antibody or preimmune IgG control from lysates prepared from cells transfected with YFP-CT and/or CA-PKB DNA. (Top) An immunoblot (IB) to detect co-IP of PKB. (Middle and Bottom) Immunoblots of the proteins present in the lysates used for IP. (C i and ii) In situ proximity ligation assay in HeLa cells. (Ci) The red pseudocoloration indicates an interaction between endogenous InsP₃R1 and PKB. The ER is indicated by the CLNX staining in green. Nuclei are shown by the blue DAPI staining. (Cii) Quantitation of proximity ligation assay (n = 50 cells per condition). The bars represent the mean intensity ± SEM of AlexaFluor 561 fluorescence. * denotes that the data are significantly different (P < 0.05).

Because PKB is activated at the plasma membrane, but the majority of InsP₃Rs reside on the ER, we next set out to establish whether activated endogenous PKB could gain access to InsP₃Rs on the ER. Using an in situ proximity ligation assay (19), a significant InsP₃R-PKB interaction was detected in cells grown under serum-replete normal growth conditions (see images and histogram of mean cellular fluorescence intensity, Fig. 3 Ci and Cii). An insulin-dependent increase in PKB-InsP₃R1 interaction was also detected (Fig. 3 Ci and Cii). Controls that either included the InsP₃R antibody immunizing peptide in the staining procedure or replacing the PKB probe with a probe for the Max protooncogene were negative (Fig. 3Cii). Colocalization analysis revealed that the sites where PKB and InsP₃R1 interacted [rolling circle amplification (RCA) product present] overlapped with the distribution of calnexin, and therefore were localized to the ER (Pearson's correlation coefficient = 0.44 ± 0.01). Using classical colocalization analysis of confocal images of immunostained cells, we found that the distribution of both total and phosphorylated active PKB also overlapped with endogenous InsP₃Rs (SI Fig. 12). Together, these data show that active PKB is present not only at the plasma membrane where it is activated, but is also localized in the cytosol, where it interacts with InsP₃Rs.

Phosphorylation of InsP₃Rs by PKB Inhibits Apoptosis.

To specifically test the role of PKB-dependent phosphorylation of InsP₃Rs onapoptosis, menadione-induced apoptosis was measured in COS-7 cells expressing either InsP₃R1^wt or InsP₃R^S2681A. This cell death stimulus was used because its action was sensitive to InsP₃-metabolizing 5′-phosphatase expression and therefore is IICR-dependent (Fig. 4Ai). Cells expressing InsP₃R1^S2681A exhibited significantly higher levels of menadione-induced apoptosis than cells expressing InsP₃R1^wt (Fig. 4Aii). A similar effect was also observed when staurosporine was used to induce apoptosis (SI Fig. 13).

Fig. 4.

InsP₃R1^S2681A-expressing cells exhibit increased mitochondrial Ca²⁺ uptake and apoptosis. (A i and ii) Sub-G₁ DNA content of COS-7 transfected with the indicated expression vectors and treated with 50 μM menadione (experiments performed in triplicate on 3 separate days). (Bi) Menadione-induced mitochondrial Ca²⁺ uptake in COS-7 cells transfected with InsP₃R1^WT (gray trace) or InsP₃R^S2681A (black trace). (Bii) Proportion of InsP₃R1^WT- expressing (n = 23) or InsP₃R^S2681A-expressing (n = 25) cells that showed a significant mitochondrial response to menadione. * denotes that the data are significantly different (P < 0.05).

We next tested the idea that PKB-mediated regulation of InsP₃R activity and Ca²⁺ flux from the ER to the mitochondria contributed to the mechanism by which PKB elicited its prosurvival effects. Significantly, menadione-induced mitochondrial Ca²⁺ increases were considerably reduced in cells overexpressing InsP₃R1^S2681A, but not cells expressing InsP₃R1^wt (Fig. 4B).

PKB Regulation of IICR and Apoptosis in Glioblastoma Cells.

Many cancers exhibit increased PKB activity as a result of mutation or deletion of the gene encoding the lipid phosphatase PTEN (6). This mechanism underlies the resistance to apoptotic stimuli of the U87 glioblastoma cell line (20). Re-expression of PTEN in these cells (U87^PTEN) decreases PKB activity (Fig. 5A) and rescues their sensitivity to an apoptotic stimulus (SI Fig. 14). We investigated whether InsP₃R was a target for the prosurvival effect of enhanced PKB activity in these cells. InsP₃R1 immunoprecipitated from U87 cells were more highly phosphorylated than similarly treated U87^PTEN cells (Fig. 5B). IICR, stimulated by endothelin (ET), was lower in U87 cells than in U87^PTEN cells (Fig. 5C). Furthermore, mitochondrial Ca²⁺ increases after exposure to menadione were significantly lower in the U87 cells than the U87^PTEN cells (Fig. 5D). Both menadione–induced mitochondrial Ca²⁺ uptake and apoptosis in the U87^PTEN cells were inhibited by expression of InsP₃ 5′-phosphatase (Fig. 5 E and F), indicating that these processes depended on IICR. Thus, the U87 cells are less sensitive to apoptosis because phosphorylation of their InsP₃Rs reduces Ca²⁺ release and subsequent transfer of Ca²⁺ to the mitochondria.

Fig. 5.

InsP₃R phosphorylation results in decreased agonist-induced Ca²⁺ release and menadione-induced mitochondrial Ca²⁺ uptake in PTEN-deficient, U87 glioblastoma cells. (A) Immunoblots of the indicated proteins in lysates prepared from U87 and U87^PTEN cells (indicated by PTEN − and +, respectively). (B) (Top) Autoradiograph of ³²P-labeled InsP₃Rs immunoprecipitated from U87 and U87^PTEN cells. (Middle and Bottom) The proteins present in the lysate used as input for the IP. (C i–iii) ET-induced Ca²⁺ release in U87 cells (black; n = 190 cells) and U87^PTEN cells (gray; n = 183 cells). (Ci) Typical traces of ET-induced Ca²⁺ release in U87 and U87^PTEN cells. (Cii) Proportion of responding cells. (Ciii) Peak Ca²⁺ response. (Di) Menadione-induced mitochondrial Ca²⁺ uptake in U87 cells (black trace; n = 25) and U87^PTEN cells (gray trace; n = 14). (E) Proportion of YFP-expressing (n = 50) and InsP₃ 5′-phosphatase-expressing (n = 50) U87^PTEN cells that exhibited menadione-induced increases in mitochondrial Ca²⁺. (F) Menadione-stimulated cell death in U87^PTEN cells depended on InsP₃Rs. U87 and U87^PTEN cells were adenovirally infected with InsP₃ 5′-phosphatase or YFP, and subsequently stimulated with 50 μM menadione (experiments performed in triplicate on 3 separate days). * indicates that the data are significantly different (P < 0.05).

Discussion

In this study, we have identified and characterized a mechanism by which the prosurvival kinase PKB protects cells from apoptosis-inducing stimuli that engage the InsP₃R/Ca²⁺-dependent cell death pathway. We also provide evidence for a fundamental role of this mechanism in regulating the sensitivity of a cancer cell line to apoptotic stimuli.

The data presented here shows that agonist-induced Ca²⁺ release is inhibited by PKB-mediated phosphorylation of InsP₃Rs. Significantly, agonist-induced Ca²⁺ release was regulated by endogenous tonically active PKB and CA-PKB that had been overexpressed. Moreover, increased cellular PKB activity significantly impacted the agonist-induced increase in mitochondrial Ca²⁺, which is a proximal sensor of Ca²⁺ release through InsP₃Rs (21, 22). Because PKB activation had no effect on ER store loading or InsP₃R expression levels, but prevented Ca²⁺ release in response to cell-permeant InsP₃, we concluded that PKB was directly regulating InsP₃R activity. This conclusion was further supported by the enhanced agonist-induced Ca²⁺ release observed in cells expressing mutated InsP₃Rs that could not be phosphorylated by PKB compared with cells overexpressing wild-type receptors.

The data presented here contrasts with that previously reported by Khan et al. (15), which indicated that mutation of S2681A did not affect IICR. Possible explanations for these discrepancies are that in the study of Khan et al. increases in intracellular Ca²⁺ were presented as a normalized change in fluorescence but not absolute Ca²⁺ values. Thus, they could only conclude that there was no effect of InsP₃R1 S2681 phosphorylation on the EC₅₀ for Ca²⁺ release. A further possibility is that because of the low tonic level of PKB activity in DT40 cells (SI Fig. 15 and ref. 23), the wild-type InsP₃R would not have been highly phosphorylated, and thus the effect of InsP₃R1^S2681A would have been small. Unlike Khan et al. (15), we were able to detect a small, but significant, effect of the S2681A mutation on Ca²⁺ release in DT40 cells, although it was less than that observed in COS-7 cells. A possible explanation for our ability to detect an effect of S2681A mutation upon Ca²⁺ release is that unlike Khan et al., we used DT40 cells stably expressing muscarinic receptors rather than cells in which muscarinic receptors were transiently transfected.

Our data show that during the life cycle of normally dividing cells InsP₃Rs are tonically phosphorylated by PKB. Stimulation of serum-starved cells with insulin or FBS also promoted PKB-dependent phosphorylation of InsP₃Rs (SI Fig. 10A). Experiments performed here also corroborated the identification of S2681 as the serine residue phosphorylated by PKB in InsP₃R1 by Khan et al. (15) (SI Fig. 10 B and C) and demonstrated that S2681 was the site for phosphorylation by endogenous PKB that had been physiologically activated. We also showed in vitro phosphorylation of this site in recombinant InsP₃R1 COOH terminus by recombinant PKB, indicating that PKB was sufficient to catalyze the phosphorylation of InsP₃Rs, and that no accessory factors are required. We report that InsP₃R3 is also phosphorylated by PKB, both in vitro by recombinant active PKB (SI Fig. 10D) and after insulin stimulation in intact cells (data not shown). Together, our data satisfies the criteria laid out by Manning and Cantley (1) for a protein to constitute a PKB substrate. Interestingly, in the absence of activated PKB, no InsP₃R phosphorylation was observed. Because InsP₃Rs are a substrate for a number of other protein kinases, including PKA and CaMKII (24, 25), it might be expected that basal activity of these enzymes may also lead to a low level of phosphorylation of InsP₃R1. However, in the studies cited above, it appears that unless stimulated appropriately little InsP₃R phosphorylation is detected. The remarkable degree of sequence conservation through evolution of the PKB consensus site in InsP₃Rs is suggestive of a fundamental role of phosphorylation of this region in InsP₃R function. The COOH terminus of InsP₃Rs is critically important in the regulation of channel gating and is a hotspot for interactions with other proteins, including huntingtin-associated protein, Bcl-X_L, protein phosphatase 1a, and cytochrome c (12, 14, 26–28). Akin to other protein kinases/substrate relationships (29), we also detected an interaction between PKB and the InsP₃R. Using an in situ proximity ligation technique, we detected this interaction between endogenous PKB and InsP₃Rs in cells either maintained in serum-containing medium or stimulated with insulin. Significantly, this PKB–InsP₃R association occurred throughout the ER, and not only at the plasma membrane where PKB is primarily activated. Activated PKB was, however, detected throughout the cell. Although PIP₃ has previously been localized to the ER (30), we have shown a clear visualization of endogenous activated PKB interacting with one of its substrates in a location distal from the plasma membrane.

By investigating the effect of mutagenesis of the PKB consensus site in InsP₃R1 on cell death and mitochondrial Ca²⁺ increases stimulated by agents that cause apoptosis in an IICR-dependent manner (14, 31), we also determined that IICR was a bona fide prosurvival target of PKB. The relevance of these findings to disease was shown in the U87 glioblastoma cancer cell line that exhibits increased PKB activity (20). In these cells, we detected increased InsP₃R phosphorylation and decreased agonist-induced Ca²⁺ release when compared with derivatives of these cells in which PTEN was re-expressed. Furthermore, unlike their PTEN-expressing derivatives, the U87 cells were also recalcitrant to menadione-induced apoptosis and did not exhibit any mitochondrial Ca²⁺ uptake after menadione treatment. From these data we conclude that phosphorylation of InsP₃Rs by hyperactive PKB was significantly responsible for the lack of sensitivity of the U87 cells to apoptotic stimuli.

In summary, we provide evidence for a mechanism by which InsP₃R-mediated apoptosis is inhibited and by which PKB elicits its prosurvival effects (Fig. 6). This model also predicts that PKB constitutes a link between the cellular environment and growth factor status and can dynamically control IICR to determine cell fate. The significant prosurvival effect of PKB-mediated phosphorylation of InsP₃Rs places regulation of IICR high up in the hierarchy of the prosurvival targets of PKB. Indeed, IICR is an upstream signal in the activation of other proapoptotic pathways such as those mediated by Bad and calpains (9). Although our data focus on the role of PKB modulation of IICR in apoptosis, it is likely that this functional interaction also impacts other aspects of Ca²⁺ signaling. The recent descriptions of role for IICR in controlling autophagy (32) and cellular metabolism (12), which are also targets for the PKB pathway, support this idea. In addition, our data contribute to the developing model that regulation of IICR is a nexus at which multiple signaling pathways converge to determine the physiological output of a given cellular stimulus.

Fig. 6.

Model depicting the functional interaction between PKB and InsP₃Rs. (A) Under normal growth conditions with physiological PKB activity InsP₃Rs are basally phosphorylated, transfer of Ca²⁺ to the mitochondria is minimal, and ATP synthesis is promoted. (B) In the absence of growth factors, PKB activity is reduced, and the level of InsP₃R phosphorylation is diminished. Thus, upon toxin application Ca²⁺ flux from the ER to the mitochondria is enhanced, causing permeability transition and cell death. (C) In situations with enhanced PKB activity, such as during cancer or the presence of growth factor stimulation, the level of InsP₃R phosphorylation is increased, thereby decreasing the flux of Ca²⁺ from the ER to the mitochondria in response to an apoptotic stimulus. Agonist-induced Ca²⁺ release is also suppressed under these conditions.

Materials and Methods

Materials.

Cell culture reagents, NuPage gels, AlexaFluor-conjugated secondary antibodies, Ca²⁺ indicator dyes, and pluronic were from Invitrogen. Blasticidin, Zeocin, and tetracycline-free FBS were from InvivoGen. Primary antibodies, unless otherwise stated, were from Cell Signaling Technology. All other chemicals, unless stated otherwise, were purchased from Sigma.

Generation of Expression Vectors.

Expression plasmids for myristoylated CA and KD (K179A) forms of PKB have been described (33). The cDNAs encoding CA-PKB and KD-PKB were subcloned into the tetracycline-inducible expression vector pcDNA4-TO (Invitrogen), and pcDNA4-TO-YFP was created by subcloning the YFP cDNA from pEYFP-C1 (Clontech) into pcDNA4-TO. InsP₃R1^S2681A was generated by QuikChange Mutagenesis (Stratagene) using pcDNA3-mouse InsP₃R1 (provided by K. Mikoshiba, University of Tokyo, Tokyo) as template. The COOH-terminal mutant S2681A (CT^S/A) was made as above. Constructs were verified by sequencing. YFP-tagged InsP₃R1 COOH terminus (YFP-CT; amino acids 2431–2749) has been described (34).

Generation of Stable Cell Lines.

Tet suppressor HeLa cells were from Invitrogen and maintained as prescribed. Stable PKB- and YFP-expressing cell lines were generated after transfection by using GeneJuice (Merck) or JetPEI (Qbiogene) according to the manufacturer's guidelines. Expression was induced with 1 μg/ml doxycycline for 24 h.

Transient Transfection.

HeLa and COS-7 cell lines were from ECACC and maintained as described (35). For knockdown of endogenous PKB, cells were transfected with validated siRNA (Cell Signaling Technology) by using Lipofectamine 2000 (Invitrogen). Scrambled siRNA was used as control. For DNA transfection, Lipofectamine 2000 or JetPei (Qbiogene) was used. For imaging studies, InsP₃R transfected cells were identified by cotransfection with pEYFP-C1 (Clontech) at a 1:5 molar ratio. GFP-tagged InsP₃ 5′-phosphatase was transduced by using adenovirus. Cells were infected at a multiplicity of infection of 100 plaque-forming units 24 h before analysis or treatment. A GFP-expressing adenovirus was used as control. Adenoviral vectors were prepared according to the supplier's instructions (Microbix). M3-muscarinic receptor-expressing DT40 InsP₃R triple knockout chicken B-lymphocytes were from David Yule (University of Rochester, Rochester, NY) and cultured as described (18). DT40 cells were transfected with an AMAXA Nucleofector with solution T and program B-023. In brief, 2 × 10⁶ cells were first resuspended in 100 μl of solution T. To the cells in solution T, 10 μg of empty vector or InsP₃R1 expression vector was added together with 3 μg of a DsRed expression vector, which was used a transfection marker.

Ca²⁺ Imaging.

Fura-2 imaging was performed as described (35). Experiments were performed on three coverslips of 20 or more cells per day on 3 separate days. Mitochondrial Ca²⁺ imaging with Rhod-2 was performed as described (16). Experiments were performed by using a VoxCell Scan confocal imaging system (Visitech Ltd) equipped with a Hamamatsu ORCA-ER camera. The confocal scan head was attached to either an Olympus IX70 inverted microscope configured with a ×40, 1.35 n.a. UAPO oil immersion objective (Figs. 4 and 5) or a Nikon TE2000 equipped with ×40, 1.3 n.a. oil immersion objective (Fig. 1). Image analysis was performed with ImageJ.

Immunoblotting.

Immunoblots were performed essentially as described (35). For detection of PKB, 15–40 μg of cell lysate was separated on 7% or 10% SDS/PAGE or 4–12% precast Bis-Tris NuPAGE gradient gels. Proteins were detected with polyclonal or monoclonal PKB antibodies (both diluted 1:1,000). β-Actin was used as a loading control (mAb 1:10,000; Abcam). Phospho-PKB (S473) (1:1,000) was detected after stripping of total PKB antibody. InsP₃R1 was detected by using a polyclonal antibody generated in house against the COOH terminus of the protein as described (36).

³²P Labeling of InsP₃Rs.

Labeling experiments were performed similar to that described (35). In this study, 300 μg of protein lysate prepared from 2 × 35-mm dishes per condition was used for IP.

In Situ Proximity Ligation Assay.

This assay was performed as described (19). Proximity probes were constructed by conjugating oligonucleotides to a monoclonal PKB antibody and a polyclonal InsP₃R1 antibody. A probe for the Max oncogenic transcription factor has been described (19) and was used as a control. Cells were counterstained with an anticalnexin (CLNX) polyclonal antibody (Sigma) and visualized by using an AlexaFluor 488-conjugated secondary antibody. Coverslips were mounted in Vectashield containing DAPI (blue) (Vector Laboratories). Images were captured with a Zeiss LSM 510 META confocal microscope using a plan apochromat ×60, 1.40 n.a. oil immersion objective configured with LSM software version 3.2. Cellular RCA products were quantitated with ImageJ. To this end, mean pixel intensity of region of interest of the image not covered by a cell was subtracted from the mean pixel intensity of a region of interest that was drawn around each cell. The data are presented as mean pixel intensity. The brightness of sample images was increased for presentation. Pearson's colocalization analysis was performed with Volocity software using the region of interest threshold option (37) (version 4.01; Improvision).

Co-IP of InsP₃Rs and PKB.

Lysates were prepared as for immunoblotting. Thirty micrograms of each lysate was retained for immunoblot analysis, and IPs were performed on 500–1,000 μg of the remaining protein as described (35). InsP₃Rs, YFP-tagged proteins, and PKB were immunoprecipitated by using 2 μl of anti-InsP₃R1 or anti-GFP polyclonal antibodies or 2 μl of monoclonal PKB antibody, respectively.

Induction of Apoptosis and FACS Analysis.

When experiments involved transfection or adenoviral infection, growth media were replaced with fresh media or media containing apoptosis-inducing agent after 24 h. Cells were treated for 6 h with 50 μM Menadione. Cells in the media were retained and pooled with remaining adherent cells that were harvested by trypsinization. Cells were collected by centrifugation at 1,200 × g for 5 min and fixed in 70% EtOH/PBS overnight. Cells were then pelleted, incubated for 1 h at 37°C in 600 μl of propidium iodide (PI) buffer (PBS, pH 7.4, 0.4 μg/ml PI, 0.4 μg/ml RNaseA, 0.3% IGEPAL), and analyzed by FACS. Data are presented as fold changes in sub-G₁ population compared with the experimental condition indicated.

Data Analysis.

Statistical analysis was calculated by using Student's t test, ANOVA, and posthoc Tukey test or χ² test. Data are presented as mean ± SEM. Significance was accepted at P < 0.05.