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 Ca(2+). PKB elicits its effects through the phosphorylation and inactivation of proapoptotic factors. Ca(2+) stimulates many prodeath pathways, among which is mitochondrial permeability transition. We identified Ca(2+) release through inositol 1,4,5-trisphosphate receptor (InsP(3)R) intracellular channels as a prosurvival target of PKB. We demonstrated that in response to survival signals, PKB interacts with and phosphorylates InsP(3)Rs, significantly reducing their Ca(2+) release activity. Moreover, phosphorylation of InsP(3)Rs by PKB reduced cellular sensitivity to apoptotic stimuli through a mechanism that involved diminished Ca(2+) flux from the endoplasmic reticulum to the mitochondria. In glioblastoma cells that exhibit hyperactive PKB, the same prosurvival effect of PKB on InsP(3)R was found to be responsible for the insensitivity of these cells to apoptotic stimuli. We propose that PKB-mediated abolition of InsP(3)-induced Ca(2+) release may afford tumor cells a survival advantage.
- SourceAvailable from: Paolo PintonFrontiers in oncology. 01/2014; 4:276.
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ABSTRACT: VDAC1, an outer mitochondrial membrane (OMM) protein, is crucial for regulating mitochondrial metabolic and energetic functions and acts as a convergence point for various cell survival and death signals. VDAC1 is also a key player in apoptosis, involved in cytochrome c release and interactions with anti-apoptotic proteins. Recently, we demonstrated that various pro-apoptotic agents induce VDAC1 oligomerization and proposed that a channel formed by VDAC1 oligomers mediates cytochrome c release. As VDAC1 transports Ca(2+) across the OMM and because Ca(2+) has been implicated in apoptosis induction, we addressed the relationship between cytosolic Ca(2+) levels ([Ca2+]i), VDAC1 oligomerization and apoptosis induction. We demonstrate that different apoptosis inducers elevate cytosolic Ca(2+) and induce VDAC1 over-expression. Direct elevation of [Ca(2+)]i by the Ca(2+)-mobilizing agents A23187, ionomycin and thapsigargin also resulted in VDAC1 over-expression, VDAC1 oligomerization and apoptosis. In contrast, decreasing [Ca(2+)]i using the cell-permeable Ca(2+)-chelating reagent BAPTA-AM inhibited VDAC1 over-expression, VDAC1 oligomerization and apoptosis. Correlation between the increase in VDAC1 levels and oligomerization, [Ca(2+)]i levels and apoptosis induction, as induced by H2O2 or As2O3, was also obtained. On the other hand, cells transfected to overexpress VDAC1 presented Ca(2+)-independent VDAC1 oligomerization, cytochrome c release and apoptosis, suggesting that [Ca(2+)]i elevation is not pre-requisite for apoptosis induction when VDAC1 is over-expressed. The results suggest that Ca(2+) promotes VDAC1 over-expression by an as yet unknown signaling pathway, leading to VDAC1 oligomerization, ultimately resulting in apoptosis. These findings provide new insight into the mechanism of action of existing anti-cancer drugs involving induction of VDAC1 over-expression as a mechanism for inducing apoptosis. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease.Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor
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ABSTRACT: Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease.Biochimica et Biophysica Acta 04/2014; · 4.66 Impact Factor
Phosphorylation of inositol 1,4,5-trisphosphate
receptors by protein kinase B/Akt inhibits
Ca2?release and apoptosis
Tania Szado*, Veerle Vanderheyden†, Jan B. Parys†, Humbert De Smedt†, Katja Rietdorf*, Larissa Kotelevets‡,
Eric Chastre‡, Farid Khan§, Ulf Landegren¶, Ola So ¨derberg¶, Martin D. Bootman*?, and H. Llewelyn Roderick*?**
Laboratories of *Molecular Signaling and§Protein Technologies, The Babraham Institute, Cambridge CB2 3AT, United Kingdom; **Department
of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom;†Laboratory of Molecular and Cellular
Signaling, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N1, B-3000 Leuven, Belgium;
‡Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite 773, Centre de Recherche Bichat Beaujon, Faculte ´ de Me ´decine X. Bichat,
Universite ´ Paris 7 Denis Diderot, 75018 Paris, France; and¶Department of Genetics and Pathology/Molecular Medicine,
The Rudbeck Laboratory, University of Uppsala, Se-75185 Uppsala, Sweden
Communicated by Michael J. Berridge, The Babraham Institute, Cambridge, United Kingdom, December 3, 2007 (received for review November 1, 2007)
Imbalance of signals that control cell survival and death results in
pathologies, including cancer and neurodegeneration. Two path-
ways 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 phosphor-
ylation and inactivation of proapoptotic factors. Ca2?stimulates
many prodeath pathways, among which is mitochondrial perme-
ability 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, phos-
phorylation of InsP3Rs by PKB reduced cellular sensitivity to apo-
ptotic 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 prosur-
vival 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.
signaling ? cell death ? cancer
survival (1, 2). PKB elicits these effects by phosphorylating and
regulating the activity of downstream targets such as glycogen
synthase kinase 3? and Bad, or via transcription factors such as
Forkhead (1, 3). Because of this critical role of PKB, gain or loss of
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-XL,
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 phosphatidyl-
inositol 3-kinase (PI3K)/PKB and InsP3/Ca2?signaling pathways
regulated how cells responded to death-inducing stimuli. We de-
termined that PKB-mediated phosphorylation of InsP3R results in
a decrease in the magnitude of IICR and resultant flux of Ca2?
in Ca2?flux caused by PKB-mediated phosphorylation of InsP3Rs
contributes to protection from the effects of apoptotic stimuli. This
rotein kinase B (PKB) is a central player in regulating many
signaling pathways controlling cell metabolism, growth, and
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
Ca2?Release from Intracellular Stores Is Regulated by PKB. Growth
factor status and genetic factors that enhance PKB activity signif-
icantly impact on cell fate [supporting information (SI) Fig. 7]. On
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
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?re-
lease 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 ?M 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).
Author contributions: T.S., V.V., J.B.P., H.D.S., M.D.B., and H.L.R. designed research; T.S.,
V.V., K.R., M.D.B., and H.L.R. performed research; K.R., L.K., E.C., F.K., U.L., O.S., and H.L.R.
contributed new reagents/analytic tools; T.S., V.V., J.B.P., H.D.S., M.D.B., and H.L.R. ana-
lyzed data; and M.D.B. and H.L.R. wrote the paper.
The authors declare no conflict of interest.
?To whom correspondence may be addressed: E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
February 19, 2008 ?
vol. 105 ?
no. 7 ?
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 compartmen-
talized 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).
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 decreased32P-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-
reduction of endogenous PKB expression by siRNA, or its activity
Ca2?release (Fig. 1 G and H). Together, these data suggest that
phosphorylation of InsP3R by PKB significantly reduces its sensi-
tivity to InsP3. Moreover, InsP3Rs are phosphorylated by endoge-
nous, 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 phosphor-
ylatable serine in the PKB consensus site was mutated to an
unreactive alanine (S2681A in InsP3R1) (15). COS-7 cells express-
ing InsP3R1S2681Ahad greater ATP-induced Ca2?responses than
their InsP3R1wt-expressing counterparts (Fig. 2Ai, Aii, and Aiii; the
were not caused by differences in expression levels of the wild-type
or mutated receptors or differences in their intracellular distribu-
tion (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 sig-
nificantly greater than endogenous receptors, are correctly tar-
32P-labeled InsP3Rs. (Lower) Immunoblots (IB) of proteins present in the lysates
or cells expressing CA-PKB as shown (?). (Bi and Bii) Histamine-induced Ca2?
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)
Images captured at the indicated time points are shown. (E and F) (Upper)
Autoradiographs of32P-labeled InsP3Rs. (Lower) Immunoblots (IB) of the indi-
cated 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
Ca2?responses induced by the concentrations of histamine (?M) indicated. (H)
Amplitude of Ca2?responses induced by the concentrations of histamine (?M)
*indicates P ? 0.05. NS indicates not significant.
IICR and mitochondrial Ca2?uptake is regulated by PKB. (A) CA-PKB
293 cells) or InsP3R1S2681A(n ? 192 cells). Peak Ca2?response (Ai), AUC (Aii),
and latency (Aiii) are shown for the ATP concentrations (?M) indicated. (B i
and ii) Carbachol-induced Ca2?responses in M3-expressing DT40 cells tran-
siently transfected with InsP3R1wt(n ? 35 cells) or InsP3R1S2681A(n ? 44 cells).
*indicates that the data are statistically significant (P ? 0.05).
InsP3R1-S2681 regulates agonist-induced Ca2?release. (A i–iii) Aver-
www.pnas.org?cgi?doi?10.1073?pnas.0711324105Szado et al.
geted, and exhibit significantly increased agonist-induced Ca2?
release and decreased latency of the Ca2?transient (SI Fig. 11).
Agonist-induced Ca2?release was also monitored in M3 mus-
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 InsP3R1S2681Athan cells transfected
with InsP3R1wt(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?
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).
interacted with a YFP-tagged ER-localized NH2-terminally trun-
cated InsP3R1 that encompassed only the amino acids COOH-
terminal of transmembrane domains 5 (YFP-CT) (Fig. 3B).
Because PKB is activated at the plasma membrane, but the
majority of InsP3Rs reside on the ER, we next set out to establish
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
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 caln-
exin, 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 distri-
bution of both total and phosphorylated active PKB also over-
lapped 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
ptosis, menadione-induced apoptosis was measured in COS-7 cells
expressing either InsP3R1wtor InsP3RS2681A. This cell death stim-
5?-phosphatase expression and therefore is IICR-dependent (Fig.
4Ai). Cells expressing InsP3R1S2681Aexhibited significantly higher
levels of menadione-induced apoptosis than cells expressing
InsP3R1wt(Fig. 4Aii). A similar effect was also observed when
staurosporine was used to induce apoptosis (SI Fig. 13).
to the mechanism by which PKB elicited its prosurvival effects.
Significantly, menadione-induced mitochondrial Ca2?increases
were considerably reduced in cells overexpressing InsP3R1S2681A,
but not cells expressing InsP3R1wt(Fig. 4B).
PKB Regulation of IICR and Apoptosis in Glioblastoma Cells. Many
cancers exhibit increased PKB activity as a result of mutation or
mechanism underlies the resistance to apoptotic stimuli of the U87
glioblastoma cell line (20). Re-expression of PTEN in these cells
(U87PTEN) 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
U87PTENcells (Fig. 5B). IICR, stimulated by endothelin (ET), was
lower in U87 cells than in U87PTENcells (Fig. 5C). Furthermore,
mitochondrial Ca2?increases after exposure to menadione were
Both menadione–induced mitochondrial Ca2?uptake and apopto-
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
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
are significantly different (P ? 0.05).
PKB interacts with InsP3R1. (A i and ii) Co-IPs of full-length InsP3R1
Szado et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?
sis in the U87PTENcells 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.
In this study, we have identified and characterized a mechanism by
which the prosurvival kinase PKB protects cells from apoptosis-
pathway. We also provide evidence for a fundamental role of this
mechanism in regulating the sensitivity of a cancer cell line to
is inhibited by PKB-mediated phosphorylation of InsP3Rs. Signif-
icantly, 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
mutated InsP3Rs that could not be phosphorylated by PKB com-
pared with cells overexpressing wild-type receptors.
The data presented here contrasts with that previously reported
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
of InsP3R1 S2681 phosphorylation on the EC50for 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
InsP3R1S2681Awould 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
uptake and apoptosis. (A i and ii) Sub-G1DNA content of COS-7 transfected
with the indicated expression vectors and treated with 50 ?M menadione
(experiments performed in triplicate on 3 separate days). (Bi) Menadione-
induced mitochondrial Ca2?uptake in COS-7 cells transfected with InsP3R1WT
ing (n ? 23) or InsP3RS2681A-expressing (n ? 25) cells that showed a significant
mitochondrial response to menadione.*denotes that the data are signifi-
cantly different (P ? 0.05).
InsP3R1S2681A-expressing cells exhibit increased mitochondrial Ca2?
release and menadione-induced mitochondrial Ca2?uptake in PTEN-
lysates prepared from U87 and U87PTENcells (indicated by PTEN ? and ?,
tated from U87 and U87PTENcells. (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 U87PTENcells (gray; n ? 183 cells). (Ci) Typical
traces of ET-induced Ca2?release in U87 and U87PTENcells. (Cii) Proportion of
responding cells. (Ciii) Peak Ca2?response. (Di) Menadione-induced mito-
chondrial Ca2?uptake in U87 cells (black trace; n ? 25) and U87PTENcells (gray
trace; n ? 14). (E) Proportion of YFP-expressing (n ? 50) and InsP3 5?-
phosphatase-expressing (n ? 50) U87PTENcells that exhibited menadione-
in U87PTENcells depended on InsP3Rs. U87 and U87PTENcells were adenovirally
infected with InsP35?-phosphatase or YFP, and subsequently stimulated with
50 ?M menadione (experiments performed in triplicate on 3 separate days).
*indicates that the data are significantly different (P ? 0.05).
InsP3R phosphorylation results in decreased agonist-induced Ca2?
www.pnas.org?cgi?doi?10.1073?pnas.0711324105Szado et al.
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
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 physiolog-
ically 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
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 evolu-
tion 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-
to other protein kinases/substrate relationships (29), we also de-
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
at the plasma membrane where PKB is primarily activated. Acti-
vated PKB was, however, detected throughout the cell. Although
PIP3has previously been localized to the ER (30), we have shown
a clear visualization of endogenous activated PKB interacting with
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
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 mitochon-
drial 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
of the prosurvival targets of PKB. Indeed, IICR is an upstream
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 physiolog-
ical output of a given cellular stimulus.
Materials and Methods
Materials. Cell culture reagents, NuPage gels, AlexaFluor-conjugated secondary
antibodies, Ca2?indicator dyes, and pluronic were from Invitrogen. Blasticidin,
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
and KD-PKB were subcloned into the tetracycline-inducible expression vector
QuikChange Mutagenesis (Stratagene) using pcDNA3-mouse InsP3R1 (provided
by K. Mikoshiba, University of Tokyo, Tokyo) as template. The COOH-terminal
ing. 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)
doxycycline for 24 h.
Transient Transfection. HeLa and COS-7 cell lines were from ECACC and main-
tained as described (35). For knockdown of endogenous PKB, cells were trans-
fected 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
1:5 molar ratio. GFP-tagged InsP35?-phosphatase was transduced by using ade-
novirus. Cells were infected at a multiplicity of infection of 100 plaque-forming
(Microbix). M3-muscarinic receptor-expressing DT40 InsP3R triple knockout
NY) and cultured as described (18). DT40 cells were transfected with an AMAXA
(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
PKB activity, such as during cancer or the presence of growth factor stimula-
tion, 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
Szado et al.
February 19, 2008 ?
vol. 105 ?
no. 7 ?
Nucleofector with solution T and program B-023. In brief, 2 ? 106cells were first
resuspended in 100 ?l of solution T. To the cells in solution T, 10 ?g of empty
vector or InsP3R1 expression vector was added together with 3 ?g of a DsRed
expression vector, which was used a transfection marker.
Ca2?Imaging. Fura-2 imaging was performed as described (35). Experiments
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.
or 4–12% precast Bis-Tris NuPAGE gradient gels. Proteins were detected with
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 ?g 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 mono-
clonal PKB antibody and a polyclonal InsP3R1 antibody. A probe for the Max
Cells were counterstained with an anticalnexin (CLNX) polyclonal antibody
(Sigma) and visualized by using an AlexaFluor 488-conjugated secondary anti-
body. Coverslips were mounted in Vectashield containing DAPI (blue) (Vector
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
son’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 ?g of the remaining protein as described (35). InsP3Rs,
YFP-tagged proteins, and PKB were immunoprecipitated by using 2 ?l of anti-
InsP3R1 or anti-GFP polyclonal antibodies or 2 ?l of monoclonal PKB antibody,
Induction of Apoptosis and FACS Analysis. When experiments involved trans-
with 50 ?M Menadione. Cells in the media were retained and pooled with
remaining adherent cells that were harvested by trypsinization. Cells were col-
lected 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 ?l of
propidium iodide (PI) buffer (PBS, pH 7.4, 0.4 ?g/ml PI, 0.4 ?g/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.
was accepted at P ? 0.05.
ACKNOWLEDGMENTS. We thank Profs. K. Mikoshiba, C. Erneux (University of
(University Medical Center of Utrecht, Utrecht, The Netherlands) for cDNAs;
Stuart Conway (University of St. Andrews, St. Andrews, Scotland) for InP3-BM,
J. Hanson, E. Vermassen, G. Morgan, S. Walker, S. Cook, L. Bauwens, M. Taussig,
C. Taylor, and Y. Sun for help and discussion; and the Engineering and Physical
Sciences Research Council Mass Spectrometry service (Swansea, U.K.). This work
Grant P5/05, Concerted Actions of the Katholieke Universiteit Leuven Grant
99/08, The Babraham Institute, The Royal Society, Human Frontier Science Pro-
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www.pnas.org?cgi?doi?10.1073?pnas.0711324105Szado et al.