Role of the inositol 1,4,5-trisphosphate receptor/Ca2+-release channel in autophagy
Cell Communication and Signaling volume 10, Article number: 17 (2012)
Autophagy is an important cell-biological process responsible for the disposal of long-lived proteins, protein aggregates, defective organelles and intracellular pathogens. It is activated in response to cellular stress and plays a role in development, cell differentiation, and ageing. Moreover, it has been shown to be involved in different pathologies, including cancer and neurodegenerative diseases. It is a long standing issue whether and how the Ca2+ ion is involved in its regulation. The role of the inositol 1,4,5-trisphosphate receptor, the main intracellular Ca2+-release channel, in apoptosis is well recognized, but its role in autophagy only recently emerged and is therefore much less well understood. Positive as well as negative effects on autophagy have been reported for both the inositol 1,4,5-trisphosphate receptor and Ca2+. This review will critically present the evidence for a role of the inositol 1,4,5-trisphosphate receptor/Ca2+-release channel in autophagy and will demonstrate that depending on the cellular conditions it can either suppress or promote autophagy. Suppression occurs through Ca2+ signals directed to the mitochondria, fueling ATP production and decreasing AMP-activated kinase activity. In contrast, Ca2+-induced autophagy can be mediated by several pathways including calmodulin-dependent kinase kinase β, calmodulin-dependent kinase I, protein kinase C θ, and/or extracellular signal-regulated kinase.
What is autophagy?
Autophagy is the name for a group of lysosomal degradation processes conserved throughout evolution [1, 2]. It is responsible for the disposal of cellular material that cannot be degraded by the ubiquitin-proteasome system, like long-lived proteins, other macromolecules and even entire organelles. Depending on the delivery mechanism of this material to the lysosomes, autophagy is divided into three main types: microautophagy, chaperone-mediated autophagy and macroautophagy [2, 3].
The latter is the best-studied form of autophagy and involves the formation of a typical double-membrane cistern, named phagophore, which surrounds and ultimately engulfs the cytoplasmic material to be degraded. The resulting vesicle is the autophagosome, which can fuse with late endosomes or lysosomes, leading to the breakdown of its content [1, 4]. Because macroautophagy is the main focus of this review, it will further be referred to as autophagy.
Appropriate autophagy levels are not only needed for cellular homeostasis (removing of aggregated proteins and damaged organelles) [5, 6] but also for development, cell differentiation, ageing and tumor suppression [1, 6, 7].
Depending on the type of stress faced by cells, autophagy can either provide the cell with building blocks and energy (e.g. during starvation) or help the cell to cope with potentially damaging elements (e.g. aggregated proteins, viral infection and other intracellular pathogens) [1, 4, 6, 8, 9]. Thus, autophagy ensures cell survival, but if the stress persists for a longer time, it can lead to cell death, also termed programmed cell death type 2 . Cell death is however not the normal outcome of autophagy, and the concept has been proposed that cell death may occur along with autophagy rather than executed by autophagy [11, 12].
In order to avoid uncontrolled or excessive levels of autophagy, the process is tightly regulated. More than 30 autophagy genes (atg) have hereby been identified as essential regulators [1, 13]. The mammalian target of rapamycin (mTOR) is an important upstream negative regulator of the canonical autophagy pathway. In normal conditions, mTOR hyperphosphorylates Atg13, thereby inhibiting its activity. In conditions where mTOR is inhibited, e.g. subsequently to activation of the upstream AMP-activated kinase (AMPK), the resulting active Atg13 forms a complex with Atg1/Unc-51-like kinase 1 (ULK1) and FIP200, the focal adhesion kinase family interacting protein of 200 kDa . In addition, mTOR also directly phosphorylates ULK1 at S757, thereby preventing the interaction between AMPK and ULK1. During starvation, AMPK directly activates ULK1 by phosphorylation on S317 and S777 [15, 16]. The ULK1 complex together with the class III phosphatidylinositol 3-kinase complex (PtdIns3K Complex III), which mainly consists of PtdIns3K (Vps34), Vps15, Atg6/Beclin 1 and Atg14/Barkor , are necessary for phagophore formation (Figure 1).
Beclin 1 plays a central and critical role in these initial steps as a platform protein, recruiting other regulatory proteins to the PtdIns3K Complex III . It was originally identified as a 60 kDa Bcl-2-interacting protein . Structurally, it consists of an N-terminal domain containing a BH3 domain, a central coiled-coil domain, and a C-terminal evolutionarily conserved domain. Although other functions are possible , its best-characterized function is its role in autophagy; moreover, in contrast to the other BH3-only proteins, it does not promote apoptosis [13, 21, 22]. In normal conditions, Beclin 1 is neutralized by binding through its BH3 domain to the hydrophobic cleft of the anti-apoptotic Bcl-2-protein family members Bcl-2, Bcl-Xl, Mcl-1 and Bcl-w. Their interaction can be dynamically regulated by various mechanisms, allowing the release of Beclin 1 and subsequent activation of the PtdIns3K Complex III during autophagy-inducing conditions . A first mechanism involves phosphorylation of either Bcl-2 or Beclin 1. Bcl-2 can be phosphorylated by c-Jun NH2-terminal kinase-1 (JNK1) , and Beclin 1 by death-associated protein kinase (DAPK) . Either phosphorylation opposes the interaction between the two proteins. Also a number of regulatory proteins can modulate the interaction of Beclin 1 with Bcl-2 . For instance, under hypoxic conditions, BNIP3 will bind Bcl-2 and Bcl-Xl through its BH3 domain, thereby dissociating Beclin 1 from them and triggering autophagy . After starvation and reactive oxygen species production, HMGB1, the High Mobility Group Box 1 protein, translocates to the cytosol, where it can disrupt the Beclin 1/Bcl-2 complex and thus induce autophagy . Another protein that can play a role in this process is Nutrient-deprivation Autophagy Factor-1 (NAF-1). NAF-1 binds both Bcl-2 and the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), stabilizing the Beclin 1/Bcl-2 interaction and inhibiting the induction of autophagy . In addition, other Beclin 1-associated proteins also can enhance (Ambra-1, UVRAG or Bif-1) or inhibit (Rubicon) Beclin 1’s autophagy-stimulating functions . Furthermore, recent work identified the importance of the intracellular localization and membrane recruitment of Beclin 1 for its role in autophagy. It appears that although both Beclin 1 and Bcl-2 are also found at the mitochondria, inhibition of Beclin 1’s function in autophagy mainly depends on Bcl-2 that is located at the endoplasmic reticulum (ER) . In addition, membrane anchoring of Beclin 1 seems a critical factor in its ability to induce autophagy. Beclin 1 was recently shown to bind to lipid membranes containing either cardiolipin or other lipids favoring a negative curvature, which may be related to the formation of omegasomes, the early precursors of autophagosomes . Three aromatic amino acids (F359, F360 and W361) responsible for this interaction have been identified and seemed critical for Beclin 1’s role in autophagy. Indeed, Beclin 1 mutants lacking these three aromatic residues fail to bind lipid membranes and are impaired in their ability to rescue autophagy in Beclin 1-deficient cells.
Once the phagophore is formed, its further elongation depends on the formation of the Atg5-Atg12-Atg16L1 complex  and the lipidation with phosphatidylethanolamine of microtubule-associated protein 1 light chain 3 (Atg8/LC3) to LC3-II. The lipid tail enables LC3-II insertion into the membrane . The resulting autophagosomes are subsequently transported along microtubules via a dynein-dependent mechanism. Final fusion with endosomes and lysosomes is regulated by ESCRTIII, SNAREs, Rab7 and class C Vps proteins .
Interestingly, the processes of apoptotic cell death and of pro-survival autophagy are interrelated in a complex way. They can have antagonistic, additive or even synergistic effects, depending on cell type and conditions . This interplay is also evident from the molecular interactions occurring between apoptosis- and autophagy-related proteins, including the Beclin 1/Bcl-2 interaction. Also the pro-apoptotic tumor suppressor p53 has regulatory effects on autophagy, while p62 is not only involved in the delivery of cargo to the autophagosomes, but also in caspase 8 activation . Many Atg proteins also serve as caspase substrates , whereby the cleaved C-terminal fragment of Beclin 1 even gains pro-apoptotic functions . A similar switch from pro-autophagic to pro-apoptotic functions were found upon calpain-mediated cleavage of Atg5 , indicating many levels of interaction between those two pathways. In addition, Ca2+ is a well-known regulator of many intracellular processes, including apoptosis [36, 37]. Especially the IP3R, a Ca2+ channel mainly located in the ER, plays hereby a crucial role [38, 39]. More recently, it appeared that the IP3R may also play an important role in the control of autophagy, though the available data on the role of Ca2+ and the IP3R in autophagy are at least partially contradictory [40, 41].
In this review, we will therefore critically review the available evidence on the role of the ER Ca2+ store and in particular of the IP3R in the regulation of autophagy. While the role of the IP3R in controlling apoptosis is already well-established, its function in regulating autophagy only quite recently emerged. The available data however not only support a role for the IP3R and for IP3-induced Ca2+ release in the control of autophagy, but indicate that this role can depend on the exact conditions and so either has a preventing or an enhancing role with respect to autophagy.
Role of the IP3R in autophagy
Importance of intracellular Ca2+ in autophagy
The role of Ca2+ in the regulation of autophagy has been investigated since 1993 , and the first study already indicated a complex role for Ca2+ as both an increase and a decrease in the cytosolic [Ca2+ suppressed autophagy. This complexity was not directly resolved as further reports suggested that intracellular Ca2+ signaling and Ca2+-handling proteins inhibited autophagy, while other reports indicated a stimulatory role for Ca2+ (reviewed in ). Although Ca2+ release from the ER has hereby a major role, it is important to note that also other intracellular compartments may contribute to the control of autophagic flux by Ca2+, including the lysosomes. For instance, in rat astrocytes, nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to trigger Ca2+ release from these acidic compartments via the two-pore channels and so to induce autophagy . Interestingly, the leucine-rich repeat kinase-2 can induce autophagy by, directly or indirectly, activating this pathway, leading to an increase in cytosolic [Ca2+, possibly also activating Ca2+-induced Ca2+ release from the ER, and finally activating calmodulin-dependent kinase kinase β (CaMKKβ) and AMPK .
In any case, there is no doubt that intracellular Ca2+ signals affect autophagy with hereby a prominent role for the ER as the main intracellular Ca2+ store and the IP3R as the most ubiquitously expressed intracellular Ca2+-release channel. Three genes encode an IP3R, leading to the expression of three IP3R isoforms (IP3R1, IP3R2, IP3R3). All IP3R isoforms are activated upon cell stimulation, phospholipase C activation and subsequent IP3 production. IP3 diffuses into the cytoplasm and binds and activates the IP3R, leading to IP3-induced Ca2+ release. The IP3R isoforms vary in a number of properties, including their affinity for IP3 and their regulation mechanisms. Main regulatory factors are the cytosolic and the luminal [Ca2+, ATP, their phosphorylation status and their interaction with regulatory proteins [45–47]. The subsequent complex spatio-temporal Ca2+ signals occurring in the cell regulate many intracellular processes, including cell death [36–39, 48].
The IP3R suppresses autophagy
A first study implicating the role of the IP3R in autophagy was based on the use of Li+. Li+ induced autophagy by inhibiting inositol monophosphatase, and subsequently decreasing IP3 levels. Autophagy was induced in an mTOR-independent manner, as no decrease in phosphorylation of mTOR substrates was observed, and it was proposed that the IP3R acted as an inhibitor of autophagy. This finding was confirmed in another study, demonstrating that in HeLa cells chemical inhibition of IP3Rs with xestospongin B (XeB), a potent and selective IP3R antagonist  or suppression of IP3R expression using siRNA also induced autophagy .
To further investigate the role of the IP3R, several groups investigated the properties of the IP3R triple knock out chicken DT40 B lymphocytes (TKO cells) originally developed by T. Kurosaki . These cells displayed higher levels of autophagic markers than their wild-type counterparts in two studies [53, 54], but not in a third one . The variation between those results may however be due to the exact experimental conditions, as these cells appear extremely sensitive to nutrient supply . Anyway, in both studies demonstrating higher basal autophagy levels in the TKO cells, heterologous expression of either IP3R1 or IP3R3, but not of the type 2 ryanodine receptor, another ER Ca2+-release channel, suppressed autophagic levels [53, 54].
It was proposed that the control of autophagy by the IP3R depended on the binding of Beclin 1 to the IP3R . In this scaffolding model, the IP3R facilitates the binding of Beclin 1 to Bcl-2 by recruiting both proteins. This model is attractive, because Beclin 1 and Bcl-2 have been proposed to target distinct IP3R regions with Beclin 1 binding to the IP3-binding domain  and Bcl-2 predominantly binding in the middle of the modulatory and transducing domain [56, 57]. Moreover, it was proposed that XeB would dissociate Beclin 1 from the IP3R/Bcl-2 complex and so induce autophagy . According to this model, the IP3R Ca2+-channel function would not be involved. Correlating with this, siRNA-mediated knock down of Beclin 1 did neither affect histamine-induced Ca2+ release nor the steady-state ER Ca2+ content .
Control of autophagy by the IP3R but independently of IP3-induced Ca2+ release was however not confirmed in other studies. TKO cells expressing channel-dead IP3R mutants (IP3R1 D2550A, IP3R3 D2550A or T2591A) had, in contrast to those expressing wild-type IP3R1 or IP3R3, similar levels of autophagic markers as control TKO cells [53, 54]. This strongly suggests that Ca2+ release through the IP3R is critical for the inhibition of autophagy by the IP3R, and it was proposed that this was related to a regulation of mTOR activity .
The group of K. Foskett performed a very detailed study to clarify the inhibitory effect of the IP3R on autophagy induction . It is well established that a fraction of the IP3Rs are present in ER domains forming close associations with the mitochondria . These domains are structurally stabilized by various proteins, and allow efficient transfer of Ca2+ ions from the ER to the mitochondria [48, 59–61]. The study by Cárdenas et al.  showed in TKO cells increased glucose and decreased O2 consumption, pyruvate dehydrogenase inhibition and AMPK activation. These observations suggest a mechanism in which constitutive Ca2+ release through IP3Rs fuels into the mitochondria, thereby augmenting mitochondrial bio-energetics and ATP production . Also in neuroblastoma cells these essential Ca2+ signals could be abolished via siRNA-mediated knock down or XeB-mediated inhibition of IP3Rs, leading to a decreased ATP production, an increased AMP/ATP ratio and subsequent AMPK activation and autophagy stimulation. As mTOR activation appeared unaffected, a non-canonical AMPK-dependent stimulation of autophagy was proposed . A possible pathway is e.g. via direct phosphorylation of ULK1 by AMPK .
In follow-up on these results, recent data indicate that Bax inhibitor-1 (BI-1) overexpression promotes autophagy by decreasing ER Ca2+ store content, decreasing thereby IP3R-mediated Ca2+ transfer to the mitochondria, O2 consumption, and ATP production, and thus leading to AMPK stimulation and autophagy induction . Moreover, BI-1 overexpression in TKO cells was without effect, further demonstrating that BI-1 induced autophagy via a pathway requiring the IP3R.
Although it is not yet clearly established by which pathway AMPK activation leads to autophagy induction in response to IP3R inhibition, it is clear that conditions suppressing IP3R activity cause autophagy induction via a mitochondrial pathway . Under a normal situation however, basal autophagy levels are kept minimal through a Ca2+- and IP3R-dependent mechanism, although an additional scaffold function for the IP3R cannot be excluded. Moreover, the relation between the IP3R and mTOR activity may form a feedback loop, as it was shown that the various IP3R isoforms can be phosphorylated and stimulated by mTOR [63–65] (Figure 1).
The IP3R can promote autophagy
In contrast to the previous, other results indicated that intracellular Ca2+ signaling, and hence potentially the IP3R, can activate autophagy. Several of those studies [66–69] were based on the use of thapsigargin, a potent sarco/endoplasmic reticulum Ca2+- ATPase inhibitor. Treatment of cells with thapsigargin results in an increase in their cytosolic [Ca2+ and increased autophagy. Similar results were found for other treatments that increased [Ca2+ (reviewed in ). As the induction of autophagy by Ca2+-mobilizing agentia was counteracted by BAPTA-AM but was not affected in cells deficient for proteins involved in the unfolded protein response (IRE1α, eIF2α, ATF6) , it can be assumed that the induction of the autophagic flux was not consecutively to ER stress, an event triggered by the depletion of the ER Ca2+ stores . Although this does not exclude a priori a role for Ca2+ originating from other compartments, the existing data support an important role for the ER Ca2+ stores in autophagy induction and in a number of studies a direct role for the IP3R herein was even presented.
In a first study, it was assumed that the Cd2+-induced autophagy was linked to Ca2+ mobilization via IP3Rs . The involvement of IP3Rs was deduced from indirect evidence based on the use of the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) and the siRNA-mediated knock down of Ca2+/calmodulin-dependent phosphatase calcineurin putatively acting on the IP3R. However, 2-ABP is not specific for the IP3R [71, 72], and it has not yet been clarified in which circumstances calcineurin exactly regulates IP3R activity [46, 73].
A second study indicating a role for the IP3R in autophagy was performed in the slime mold Dictyostelium discoideum . Because this organism lacks the apoptotic machinery, autophagy can be studied in the absence of any interference by apoptosis . Importantly, in this organism, the differentiation factor DIF-1 led from starvation-induced autophagy to autophagic cell death, which did not occur when the unique IP3R gene was inactivated by random insertional mutagenesis or when IP3-induced Ca2+ signaling was blocked by BAPTA-AM .
Finally, in mammalian cell lines, we recently determined that the induction of autophagy triggered by nutrient starvation occurred concomitantly with a remodeling of the proteins involved in ER Ca2+ homeostasis and dynamics, thereby sensitizing multiple Ca2+-handling mechanisms. During the initial phase of the autophagy response, the levels of the Ca2+-binding chaperones calreticulin and GRP78/BiP increased and ER Ca2+ leak decreased, resulting in an elevation of the steady state ER Ca2+ levels, while the sensitivity of the IP3R towards IP3 increased . This sensitization of the Ca2+-flux properties of the IP3R was not only indirectly due to the increased ER Ca2+-store content, but also involved direct effects through increased Beclin 1 binding to the IP3R. Functional experiments suggested that predominantly the N-terminal part of Beclin 1, containing the BH3 domain, was involved in this stimulation. Moreover, Beclin 1 appears to interact with the N-terminal part of the IP3R, especially in its suppressor domain (a.a. 1–225) . This domain regulates at the one hand the affinity of the IP3R for IP3 and at the other hand interacts with and regulates its C-terminal Ca2+-channel domain . Importantly this sensitization of the IP3R is essential for the proper induction of autophagy upon nutrient starvation, as cells loaded with BAPTA-AM displayed an impaired autophagic flux . This is in agreement with results from other groups demonstrating that autophagy induction in response to various autophagic triggers, including nutrient deprivation, was also inhibited by BAPTA-AM [78–81]. Results obtained with the IP3R inhibitor XeB were more complex, because XeB induced autophagic flux in normal cells, but suppressed autophagic flux in starved cells . This points towards a dual role for IP3R function in autophagy depending on the cellular condition. In normal cells, IP3Rs suppress autophagic flux by fueling Ca2+ into the mitochondria to sustain ATP production, thereby preventing AMPK activity [41, 54]. In nutrient-deprived cells, however, IP3Rs are required to promote Ca2+-signaling events that are critical for up-regulating autophagic flux [40, 75].
As Bcl-2 can suppress IP3-induced Ca2+ release [56, 82, 83], it may be argued that IP3R sensitization by Beclin 1 is an indirect effect, due to its effects on Bcl-2, e.g. by dissociating Bcl-2 from IP3Rs. However, a Beclin 1 mutant unable to bind Bcl-2  remained able to sensitize IP3-induced Ca2+ release in vitro, indicating that these events were not due to a suppression of the inhibitory effect of Bcl-2. Nevertheless, in a cellular context, Bcl-2 seems to play an important role in tethering Beclin 1 at the ER membranes in the proximity of the IP3R channel .
Autophagy can be positively or negatively regulated by the IP3R
Taken together these various results indicate a complex action of the IP3R in autophagy regulation, whereby depending on the state of the cells IP3-induced Ca2+ release can suppress or promote autophagy (Figure 1). This complex behavior probably also explains in part the contradictory results obtained in cells treated with thapsigargin or BAPTA-AM. Indeed, differing cellular conditions, concentrations of the applied chemicals and incubation times could underlie the different results obtained in different studies . Finally, also the localization of the IP3Rs and the subcellular localization of the resulting Ca2+ signals (cytosol or mitochondria) may determine the specific outcome on autophagy. In addition, it can be expected that regulators of the IP3R may impinge on the cellular autophagy levels by modulating IP3-induced Ca2+ release. Main regulators in this context are obviously Beclin 1 and Bcl-2, though their exact regulation by associated proteins or phosphorylation events remains to be explored. Moreover, as these same proteins control apoptosis [6, 48], it is extremely important to dissect their relative role and activation mechanisms in both processes.
Downstream targets of Ca2+ in Ca2+-induced autophagy
From the above, it is clear that IP3-induced Ca2+ release can induce autophagy. However, the Ca2+-dependent mechanisms involved and the target(s) of the intracellular Ca2+ signal remain elusive, although different mechanisms have already been proposed (Figure 1).
Based on experiments using inhibitors of CaMKKβ (STO-609) or of AMPK (compound C) as well as siRNA-mediated knock down of these enzymes, it was proposed that autophagy induced by thapsigargin or other Ca2+-mobilizing agents was mediated via CaMKKβ, thereby activating its downstream target AMPK [66, 79, 85]. The latter is a negative regulator of mTOR while a positive regulator of ULK1, both resulting in the induction of autophagy. However, since thapsigargin also induced mTOR inhibition and autophagy in AMPK knock-out cells , although to a lesser extent, an AMPK-independent pathway for autophagy induction is also likely present. In follow up of this study, a recent study elegantly demonstrated that CaMKI was also activated and played a role in autophagy . In particular, it was shown that CaMKI stimulated the formation autophagosomes in a pathway involving PtdIns3K Complex III but independently of AMPK (Figure 1). In this respect, it is important to mention that Vps34, a component of the PtdIns3K Complex III, was already reported to be activated by Ca2+ and calmodulin , although this finding was later disputed .
In a study using hepatocytes and fibroblasts, thapsigargin induced autophagy through ER stress without inhibiting mTOR activity. Phosphorylation of protein kinase C θ (PKCθ) was hereby critical . Treatment with BAPTA-AM decreased both PKCθ phosphorylation and autophagy, demonstrating that Ca2+ is needed for PKCθ phosphorylation, though the mechanism remains elusive. Preliminary data indicate that the phospholipase C inhibitor U73122 partially inhibited ER stress-induced autophagy , underpinning a role for the IP3R. Interestingly, in amino acid starvation-induced autophagy no role for PKCθ was found, suggesting that this pathway is preferentially used during ER stress.
In mesangial cells, Cd2+ treatment triggers Ca2+ release from the ER, putatively by activation of the IP3R, and induces both autophagy and apoptosis. Extracellular signal-regulated kinase (ERK) activation was observed, and inhibition of ERK selectively suppressed the autophagic response, but not the apoptotic cell death response . As ERK activity was inhibited by BAPTA-AM, this study suggests a role for ERK in Ca2+-induced autophagy, although the target of ERK was not elucidated. It is hereby interesting to note that ERK may be involved in the phosphorylation of Bcl-2, thereby leading to Beclin 1 release from Bcl-2 [28, 88]. Moreover, previous studies had also demonstrated that ERK can be involved in autophagy through regulation of Gα-interacting protein  or of the microtubuli .
The induction of autophagy by cytosolic Ca2+ may therefore depend of one or more of these mechanisms, but the situation might also be more complex, as several other proteins regulating autophagy are known to be influenced by Ca2+. These proteins include DAPK, which is regulated by calmodulin and which can phosphorylate Beclin 1, thereby dissociating it from Bcl-2 and inducing autophagy . Finally, members of the S100 Ca2+-binding protein family  as well as eEF-2 kinase, responsible for the phosphorylation of eukaryotic elongation factor (eEF)-2  have also be implicated in the regulation of autophagy.
Taken together, these data indicate that an induction or a regulation of autophagy by Ca2+ is very plausible and that depending on the cell type or the cell state various mechanisms can be involved.
In conclusion, the available data indicate that ER Ca2+- store content, the IP3R and IP3-induced Ca2+ release act on the autophagic process. Under normal conditions, a basal level of IP3-induced Ca2+ release from the ER to the mitochondria is responsible for a certain level of ATP production, sufficient for keeping AMPK inactive and therefore precluding induction of autophagy. If Ca2+ transfer to the mitochondria decreases below a certain threshold, ATP synthesis is not anymore guaranteed, AMP/ATP ratio increases and AMPK is activated, leading to autophagy. Under stress conditions however, Beclin 1 may interact with the IP3R, leading to an increased IP3-induced Ca2+ release. At least part of the released Ca2+ diffuses to the cytosol where it can stimulate the induction of autophagy via a not yet completely understood pathway. This pathway may either involve CaMKKβ and AMPK, or develop in an AMPK-independent way. Whatever the mechanism, this dual regulation of autophagy by the IP3R and Ca2+ is of paramount importance for the determination of cell fate. As autophagy is important in pathological situations as e.g. cancer and neurodegenerative diseases [1, 6, 8], the correct understanding of autophagy regulation by Ca2+ may lead to important therapeutical consequences.
1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)
B-cell lymphoma 2
Calmodulin-dependent kinase I
Calmodulin-dependent kinase kinase β
Death-associated protein kinase
Extracellular signal-regulated kinase
Inositol 1,4,5-trisphosphate receptor
c-Jun NH2-terminal kinase-1
Microtubule-associated protein 1 light chain 3
Mammalian target of rapamycin
Nutrient-deprivation autophagy factor-1
Protein kinase C
- TKO Cells:
IP3R triple knock out chicken DT40 B lymphocytes
Unc-51-like kinase 1
Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW, Jimenez-Sanchez M, Korolchuk VI, Lichtenberg M, Luo S, Massey DCO, Menzies FM, Moreau K, Narayanan U, Renna M, Siddiqui FH, Underwood BR, Winslow AR, Rubinsztein DC: Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev. 2010, 90: 1383-1435.
Chen Y, Klionsky DJ: The regulation of autophagy - unanswered questions. J Cell Sci. 2011, 124: 161-170.
Cuervo AM: Autophagy: many paths to the same end. Mol Cell Biochem. 2004, 263: 55-72.
Singh R, Cuervo AM: Autophagy in the cellular energetic balance. Cell Metab. 2011, 13: 495-504.
Lee JY, Yao TP: Quality control autophagy: A joint effort of ubiquitin, protein deacetylase and actin cytoskeleton. Autophagy. 2010, 6: 555-557.
Wirawan E, Vanden Berghe T, Lippens S, Agostinis P, Vandenabeele P: Autophagy: for better or for worse. Cell Res. 2012, 22: 43-61.
Mizushima N, Levine B: Autophagy in mammalian development and differentiation. Nat Cell Biol. 2010, 12: 823-830.
Levine B, Kroemer G: Autophagy in the pathogenesis of disease. Cell. 2008, 132: 27-42.
Sridhar S, Botbol Y, Macian F, Cuervo AM: Autophagy and disease: always two sides to a problem. J Pathol. 2012, 226: 255-273.
Galluzzi L, Maiuri MC, Vitale I, Zischka H, Castedo M, Zitvogel L, Kroemer G: Cell death modalities: classification and pathophysiological implications. Cell Death Differ. 2007, 14: 1237-1243.
Kroemer G, Levine B: Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008, 9: 1004-1010.
Shen S, Kepp O, Kroemer G: The end of autophagic cell death?. Autophagy. 2012, 8: 1-3.
Cao Y, Klionsky DJ: Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res. 2007, 17: 839-849.
Mizushima N: The role of the Atg1/ULK1 complex in autophagy regulation. Curr Opin Cell Biol. 2010, 22: 132-139.
Kim J, Kundu M, Viollet B, Guan KL: AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011, 13: 132-141.
Alers S, Loffler AS, Wesselborg S, Stork B: Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol. 2012, 32: 2-11.
He C, Levine B: The Beclin 1 interactome. Curr Opin Cell Biol. 2010, 22: 140-149.
Kang R, Zeh HJ, Lotze MT, Tang D: The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011, 18: 571-580.
Liang XH, Kleeman LK, Jiang HH, Gordon G, Goldman JE, Berry G, Herman B, Levine B: Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol. 1998, 72: 8586-8596.
Wirawan E, Lippens S, Vanden Berghe T, Romagnoli A, Fimia GM, Piacentini M, Vandenabeele P: Beclin1: a role in membrane dynamics and beyond. Autophagy. 2012, 8: 6-17.
Sinha S, Levine B: The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene. 2008, 27 (Suppl 1): S137-S148.
Ciechomska IA, Goemans GC, Skepper JN, Tolkovsky AM: Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function. Oncogene. 2009, 28: 2128-2141.
Decuypere JP, Parys JB, Bultynck G: Regulation of the autophagic Bcl-2/Beclin 1 interaction. Cells 2012, 1:284-312. in press
Wei Y, Pattingre S, Sinha S, Bassik M, Levine B: JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell. 2008, 30: 678-688.
Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, Sabanay H, Pinkas-Kramarski R, Kimchi A: DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy. EMBO Rep. 2009, 10: 285-292.
Marquez RT, Xu L: Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. Am J Cancer Res. 2012, 2: 214-221.
Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouyssegur J, Mazure NM: Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol. 2009, 29: 2570-2581.
Tang D, Kang R, Livesey KM, Cheh CW, Farkas A, Loughran P, Hoppe G, Bianchi ME, Tracey KJ, Zeh HJ, Lotze MT: Endogenous HMGB1 regulates autophagy. J Cell Biol. 2010, 190: 881-892.
Chang NC, Nguyen M, Germain M, Shore GC: Antagonism of Beclin 1-dependent autophagy by BCL-2 at the endoplasmic reticulum requires NAF-1. EMBO J. 2010, 29: 606-618.
Huang W, Choi W, Hu W, Mi N, Guo Q, Ma M, Liu M, Tian Y, Lu P, Wang FL, Deng H, Liu L, Gao N, Yu L, Shi Y: Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein. Cell Res. 2012, 22: 473-489.
Geng J, Klionsky DJ: The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series. EMBO Rep. 2008, 9: 859-864.
Mehrpour M, Esclatine A, Beau I, Codogno P: Overview of macroautophagy regulation in mammalian cells. Cell Res. 2010, 20: 748-762.
Norman JM, Cohen GM, Bampton ET: The in vitro cleavage of the hAtg proteins by cell death proteases. Autophagy. 2010, 6: 1042-1056.
Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, Roelandt R, De Rycke R, Verspurten J, Declercq W, Agostinis P, Vanden Berghe T, Lippens S, Vandenabeele P: Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis. 2010, 1: e18-
Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, Brunner T, Simon HU: Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol. 2006, 8: 1124-1132.
Berridge MJ, Lipp P, Bootman MD: The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol. 2000, 1: 11-21.
Berridge MJ, Bootman MD, Roderick HL: Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003, 4: 517-529.
Joseph SK, Hajnóczky G: IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond. Apoptosis. 2007, 12: 951-968.
Kiviluoto S, Akl H, Vervliet T, Bultynck G, Parys JB, Missiaen L, De Smedt H: IP3 receptor-binding partners in cell-death mechanisms. WIREs Membr Transp Signal. 2012, 1: 201-210.
Decuypere JP, Bultynck G, Parys JB: A dual role for Ca2+ in autophagy regulation. Cell Calcium. 2011, 50: 242-250.
Cárdenas C, Foskett JK: Mitochondrial Ca2+ signals in autophagy. Cell Calcium. in press
Gordon PB, Holen I, Fosse M, Rotnes JS, Seglen PO: Dependence of hepatocytic autophagy on intracellularly sequestered calcium. J Biol Chem. 1993, 268: 26107-26112.
Pereira GJ, Hirata H, Fimia GM, do Carmo LG, Bincoletto C, Han SW, Stilhano RS, Ureshino RP, Bloor-Young D, Churchill G, Piacentini M, Patel S, Smaili SS: Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes. J Biol Chem. 2011, 286: 27875-27881.
Gomez-Suaga P, Luzon-Toro B, Churamani D, Zhang L, Bloor-Young D, Patel S, Woodman PG, Churchill GC, Hilfiker S: Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP. Hum Mol Genet. 2012, 21: 511-525.
Foskett JK, White C, Cheung KH, Mak DO: Inositol trisphosphate receptor Ca2+ release channels. Physiol Rev. 2007, 87: 593-658.
Vanderheyden V, Devogelaere B, Missiaen L, De Smedt H, Bultynck G, Parys JB: Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation. Biochim Biophys Acta. 2009, 1793: 959-970.
Parys JB, De Smedt H: Inositol 1,4,5-trisphosphate and its receptors. Adv Exp Med Biol. 2012, 740: 255-280.
Decuypere JP, Monaco G, Bultynck G, Missiaen L, De Smedt H, Parys JB: The IP3 receptor-mitochondria connection in apoptosis and autophagy. Biochim Biophys Acta. 2011, 1813: 1003-1013.
Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, Cook LJ, Rubinsztein DC: Lithium induces autophagy by inhibiting inositol monophosphatase. J Cell Biol. 2005, 170: 1101-1111.
Jaimovich E, Mattei C, Liberona JL, Cárdenas C, Estrada M, Barbier J, Debitus C, Laurent D, Molgó J: Xestospongin B, a competitive inhibitor of IP3-mediated Ca2+ signalling in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells. FEBS Lett. 2005, 579: 2051-2057.
Criollo A, Maiuri MC, Tasdemir E, Vitale I, Fiebig AA, Andrews D, Molgó J, Díaz J, Lavandero S, Harper F, Pierron G, di Stefano D, Rizzuto R, Szabadkai G, Kroemer G: Regulation of autophagy by the inositol trisphosphate receptor. Cell Death Differ. 2007, 14: 1029-1039.
Sugawara H, Kurosaki M, Takata M, Kurosaki T: Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J. 1997, 16: 3078-3088.
Khan MT, Joseph SK: Role of inositol trisphosphate receptors in autophagy in DT40 cells. J Biol Chem. 2010, 285: 16912-16920.
Cárdenas C, Miller RA, Smith I, Bui T, Molgó J, Müller M, Vais H, Cheung KH, Yang J, Parker I, Thompson CB, Birnbaum MJ, Hallows KR, Foskett JK: Essential regulation of cell bioenergetics by constitutive InsP3 receptor Ca2+ transfer to mitochondria. Cell. 2010, 142: 270-283.
Vicencio JM, Ortiz C, Criollo A, Jones AW, Kepp O, Galluzzi L, Joza N, Vitale I, Morselli E, Tailler M, Castedo M, Maiuri MC, Molgó J, Szabadkai G, Lavandero S, Kroemer G: The inositol 1,4,5-trisphosphate receptor regulates autophagy through its interaction with Beclin 1. Cell Death Differ. 2009, 16: 1006-1017.
Rong YP, Aromolaran AS, Bultynck G, Zhong F, Li X, McColl K, Matsuyama S, Herlitze S, Roderick HL, Bootman MD, Mignery GA, Parys JB, De Smedt H, Distelhorst CW: Targeting Bcl-2-IP3 receptor interaction to reverse Bcl-2's inhibition of apoptotic calcium signals. Mol Cell. 2008, 31: 255-265.
Monaco G, Decrock E, Akl H, Ponsaerts R, Vervliet T, Luyten T, De Maeyer M, Missiaen L, Distelhorst CW, De Smedt H, Parys JB, Leybaert L, Bultynck G: Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl. Cell Death Differ. 2012, 19: 295-309.
Rizzuto R, Brini M, Murgia M, Pozzan T: Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science. 1993, 262: 744-747.
Pinton P, Giorgi C, Siviero R, Zecchini E, Rizzuto R: Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene. 2008, 27: 6407-6418.
Rizzuto R, Marchi S, Bonora M, Aguiari P, Bononi A, De Stefani D, Giorgi C, Leo S, Rimessi A, Siviero R, Zecchini E, Pinton P: Ca2+ transfer from the ER to mitochondria: when, how and why. Biochim Biophys Acta. 2009, 1787: 1342-1351.
Fujimoto M, Hayashi T: New insights into the role of mitochondria-associated endoplasmic reticulum membrane. Int Rev Cell Mol Biol. 2011, 292: 73-117.
Sano R, Hou YC, Hedvat M, Correa RG, Shu CW, Krajewska M, Diaz PW, Tamble CM, Quarato G, Gottlieb RA, Yamaguchi M, Nizet V, Dahl R, Thomas DD, Tait SW, Green DR, Fisher PB, Matsuzawa S, Reed JC: Endoplasmic reticulum protein BI-1 regulates Ca2+-mediated bioenergetics to promote autophagy. Genes Dev. 2012, 26: 1041-1054.
MacMillan D, Currie S, Bradley KN, Muir TC, McCarron JG: In smooth muscle, FK506-binding protein modulates IP3 receptor-evoked Ca2+ release by mTOR and calcineurin. J Cell Sci. 2005, 118: 5443-5451.
Régimbald-Dumas Y, Frégeau MO, Guillemette G: Mammalian target of rapamycin (mTOR) phosphorylates inositol 1,4,5-trisphosphate receptor type 2 and increases its Ca2+ release activity. Cell Signal. 2011, 23: 71-79.
Frégeau MO, Régimbald-Dumas Y, Guillemette G: Positive regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by mammalian target of rapamycin (mTOR) in RINm5F cells. J Cell Biochem. 2011, 112: 723-733.
Høyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Maathiasen IS, Jäättelä M: Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and Bcl-2. Mol Cell. 2007, 25: 193-205.
Sakaki K, Wu J, Kaufman RJ: Protein kinase C θ is required for autophagy in response to stress in the endoplasmic reticulum. J Biol Chem. 2008, 283: 15370-15380.
Lam D, Kosta A, Luciani MF, Golstein P: The inositol 1,4,5-trisphosphate receptor is required to signal autophagic cell death. Mol Biol Cell. 2008, 19: 691-700.
Grotemeier A, Alers S, Pfisterer SG, Paasch F, Daubrawa M, Dieterle A, Viollet B, Wesselborg S, Proikas-Cezanne T, Stork B: AMPK-independent induction of autophagy by cytosolic Ca2+ increase. Cell Signal. 2010, 22: 914-925.
Wang SH, Shih YL, Ko WC, Wei YH, Shih CM: Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway. Cell Mol Life Sci. 2008, 65: 3640-3652.
Bootman MD, Collins TJ, Mackenzie L, Roderick HL, Berridge MJ, Peppiatt CM: 2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release. FASEB J. 2002, 16: 1145-1150.
Bultynck G, Sienaert I, Parys JB, Callewaert G, De Smedt H, Boens N, Dehaen W, Missiaen L: Pharmacology of inositol trisphosphate receptors. Pflugers Arch. 2003, 445: 629-642.
Bultynck G, Vermassen E, Szlufcik K, De Smet P, Fissore RA, Callewaert G, Missiaen L, De Smedt H, Parys JB: Calcineurin and intracellular Ca2+-release channels: regulation or association?. Biochem Biophys Res Commun. 2003, 311: 1181-1193.
Giusti C, Tresse E, Luciani MF, Golstein P: Autophagic cell death: analysis in Dictyostelium. Biochim Biophys Acta. 2009, 1793: 1422-1431.
Decuypere JP, Welkenhuyzen K, Luyten T, Ponsaerts R, Dewaele M, Molgó J, Agostinis P, Missiaen L, De Smedt H, Parys JB, Bultynck G: IP3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated. Autophagy. 2011, 7: 1472-1489.
Iwai M, Michikawa T, Bosanac I, Ikura M, Mikoshiba K: Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors. J Biol Chem. 2007, 282: 12755-12764.
Uchida K, Miyauchi H, Furuichi T, Michikawa T, Mikoshiba K: Critical regions for activation gating of the inositol 1,4,5-trisphosphate receptor. J Biol Chem. 2003, 278: 16551-16560.
Brady NR, Hamacher-Brady A, Yuan H, Gottlieb RA: The autophagic response to nutrient deprivation in the hl-1 cardiac myocyte is modulated by Bcl-2 and sarco/endoplasmic reticulum calcium stores. FEBS J. 2007, 274: 3184-3197.
Vingtdeux V, Giliberto L, Zhao H, Chandakkar P, Wu Q, Simon JE, Janle EM, Lobo J, Ferruzzi MG, Davies P, Marambaud P: AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-β peptide metabolism. J Biol Chem. 2010, 285: 9100-9113.
Knöferle J, Koch JC, Ostendorf T, Michel U, Planchamp V, Vutova P, Tönges L, Stadelmann C, Brück W, Bähr M, Lingor P: Mechanisms of acute axonal degeneration in the optic nerve in vivo. Proc Natl Acad Sci U S A. 2010, 107: 6064-6069.
Pfisterer SG, Mauthe M, Codogno P, Proikas-Cezanne T: Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy. Mol Pharmacol. 2011, 80: 1066-1075.
Chen R, Valencia I, Zhong F, McColl KS, Roderick HL, Bootman MD, Berridge MJ, Conway SJ, Holmes AB, Mignery GA, Velez P, Distelhorst CW: 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. J Cell Biol. 2004, 166: 193-203.
Rong YP, Bultynck G, Aromolaran AS, Zhong F, Parys JB, De Smedt H, Mignery GA, Roderick HL, Bootman MD, Distelhorst CW: The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor. Proc Natl Acad Sci U S A. 2009, 106: 14397-14402.
Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B: Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 2005, 122: 927-939.
Law BY, Wang M, Ma DL, Al-Mousa F, Michelangeli F, Cheng SH, Ng MH, To KF, Mok AY, Ko RY, Lam SK, Chen F, Che CM, Chiu P, Ko BC: Alisol B, a novel inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase pump, induces autophagy, endoplasmic reticulum stress, and apoptosis. Mol Cancer Ther. 2010, 9: 718-730.
Vergne I, Chua J, Deretic V: Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade. J Exp Med. 2003, 198: 653-659.
Yan Y, Flinn RJ, Wu H, Schnur RS, Backer JM: hVps15, but not Ca2+/CaM, is required for the activity and regulation of hVps34 in mammalian cells. Biochem J. 2009, 417: 747-755.
Liu Y, Yang Y, Ye YC, Shi QF, Chai K, Tashiro S, Onodera S, Ikejima T: Activation of ERK-p53 and ERK-mediated phosphorylation of Bcl-2 are involved in autophagic cell death induced by the c-Met inhibitor SU11274 in human lung cancer A549 cells. J Pharmacol Sci. 2012, 118: 423-432.
Ogier-Denis E, Pattingre S, El Benna J, Codogno P: Erk1/2-dependent phosphorylation of Gα-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells. J Biol Chem. 2000, 275: 39090-39095.
vom Dahl S, Dombrowski F, Schmitt M, Schliess F, Pfeifer U, Haussinger D: Cell hydration controls autophagosome formation in rat liver in a microtubule-dependent way downstream from p38MAPK activation. Biochem J. 2001, 354: 31-36.
Cheng Y, Yang JM: Survival and death of endoplasmic-reticulum-stressed cells: Role of autophagy. World J Biol Chem. 2011, 2: 226-231.
Work performed in the author’s laboratory on the topic was supported by Grant GOA/09/12 and OT START1/10/044 from the Research Council of the K.U. Leuven, and by grant G.0731.09 N from the Research Foundation Flanders (FWO).
The authors declare that they have no competing interests.
JBP, JPD and GB wrote the manuscript. All authors approved the final version.
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Parys, J.B., Decuypere, JP. & Bultynck, G. Role of the inositol 1,4,5-trisphosphate receptor/Ca2+-release channel in autophagy. Cell Commun Signal 10, 17 (2012). https://doi.org/10.1186/1478-811X-10-17
- Inositol 1,4,5-trisphosphate receptor
- Beclin 1
- AMP-activated kinase
- Mammalian target of rapamycin
- Calmodulin-dependent kinase kinase β