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. Author manuscript; available in PMC: 2010 Feb 11.
Published in final edited form as: Adv Enzyme Regul. 2008 Apr 29;48:10. doi: 10.1016/j.advenzreg.2008.04.001

The role of inositol signaling in the control of apoptosis

Philip W Majerus 1,*, Jun Zou 1, Jasna Marjanovic 1, Marina V Kisseleva 1, Monita P Wilson 1
PMCID: PMC2820388  NIHMSID: NIHMS76851  PMID: 18486622

Introduction

Intracellular signaling reactions are frequently carried out by changes in phosphorylation. There are three main systems of phosphorylation as shown in figure 1.

Fig. 1.

Fig. 1

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Systems of phosphorylation in cells.

The oldest and most simple is the cAMP/cGMP system which is conserved from bacteria to humans and is a relatively simple on/off switch. Next is the inositol phosphorylation system which does not exist in bacteria but is found in all eukaryotes. This system is more complex but manageable with 63 possible soluble inositol phosphates, over half of which have been found in cells to date. In addition there are 8 known inositol lipids as described in figure 2. The parent lipid is phosphatidylinositol which is esterified to diacylglycerol in the one position leaving 5 potential hydroxyl groups for further phosphorylation. Only the 3, 4, and 5 positions have been found to be phosphorylated in cells and inositol lipids with every possible combination of these phosphates have been identified in cells as shown in figure 3, depicting the current status of inositol signaling in humans. Initially it was thought that the phosphoinositides were simply precursors of the water soluble inositol phosphate messengers but it is now clear that the lipids themselves have diverse signaling functions (York, 2006; Gonzales and Anderson, 2006). In this report we will describe studies showing that both inositol phosphates and phosphoinositides are modulators of apoptosis. We recently defined the pathway for synthesis of inositol hexakisphosphate (InsP6) in mammalian cells as shown in figure 4 (Verbsky et al., 2005). Role of InsP6 in apoptosis

Fig. 2.

Fig. 2

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Phosphorylation of inositol lipids.

Fig. 3.

Fig. 3

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Cellular phosphoinositol metabolism.

Fig. 4.

Fig. 4

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The pathway for InsP6 synthesis in mammalian cells.

The precursor inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is formed by hydrolysis of PtdIns(4,5)P2 by one of the many phospholipase C enzymes. According to J. York (York, 2006) this reaction evolved for production of the higher inositol phosphates rather than to evoke calcium mobilization since fungi do not have IP3 receptors but do require InsP6 and other highly phosphorylated inositols for survival (York, 2006). In mammals, Ins(1,4,5)P3 is converted to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by inositol trisphosphate 3-kinase (E.C. 2.7.1.127) and further metabolized to inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) by inositolpolyphosphate 5-phosphatase (E.C. 3.1.1.56). Ins(1,3,4)P3 is then converted to inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) by inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) (E.C.2.7.1.159). This rather unique enzyme is able to phosphorylate its substrate in either the 5 or 6 position of the inositol ring although in cells the net effect is the formation of the tetrakisphosphate with the 6-position phosphorylated since any 5 phosphate containing product is rapidly converted back to Ins(1,3,4)P3 by the above pathway (Miller et al., 2005). 5/6-kinase is at a critical branch point of inositol phosphate metabolism which leads to the formation of all of the highly phosphorylated inositol phosphates including InsP6 and as noted below is the rate limiting step in InsP6 synthesis (Verbsky et al., 2005). Ins(1,3,4,6)P4 is then converted to inositol 1,3,4,5,6-pentakisphosphate (InsP5) by inositol phosphate multikinase (E.C. 2.7.1.151). InsP5 is then converted to InsP6 by InsP5-2-kinase (E.C. 2.7.1.155).

That 5/6-kinase is the regulated step in InsP6 synthesis is indicated by several lines of evidence: 1) overexpression of 5/6-kinase in cells increases the level of InsP6 greatly while RNAi of the enzyme reduces InsP6 levels in cells (Verbsky et al., 2005). 2) The enzyme is inhibited by many of the end products of highly phosphorylated inositols as shown in table 1.

Table 1.

Inhibition of calf brain 5/6-kinase by inositol polyphosphates

Inositol polyphosphate Ki
InsP6 37 μM
InsP5 2.5 μM
Ins(3,4,5,6)P4 30 nM
Ins(1,3,4,5)P4 3.5 μM
Ins(1,3,4,6)P4 3.5 μM
Ins(1,2,5,6)P4 52 μM
Ins(1,4,5)P3 31 μM
Ins(1,5,6)P3 2 μM
Ins(2,4,5)P3 15 μM
Ins(2,4)P2 290 μM
Ins(1,4)P2 95 μM
Ins(4,5)P2 225 μM
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The Ki was determined using the following equation: v = Vm[S]/Km(1 + [I]/Ki) + S

The most potent inhibitor of the enzyme is inositol 3,4,5,6-tetrakisphosphate which has a Ki several orders of magnitude greater than the others. This inositol phosphate is also a substrate for the enzyme (Yang et al., 2000). The inhibition constants shown in table 1 are similar to previously reported inhibition constants obtained using partially purified enzyme (Hughs et al., 1994). 3) Overexpression or RNAi of inositol phosphate multikinase that is downstream of 5/6-kinase has no effect on cellular levels of InsP5 or InsP6 (Verbsky et al., 2005). 4) Overexpression of InsP5 2-kinase only increases InsP6 levels to the extent of converting any InsP5 to InsP6 (Verbsky et al., 2005). 5) A mutation in a maize plant with low InsP6 levels was identified by positional cloning and it was found that the mutation was in the Zea mays 5/6-kinase gene (Shi et al., 2003).

When 5/6-kinase is transiently transfected into HEK 293 cells which have been labeled with [32P]-inorganic phosphate, immunoprecipitation of 5/6-kinase yields a phosphorylated enzyme (Sun et al., 2002). Phospho-amino acid analysis reveals that 5/6-kinase is phosphorylated on serine and tyrosine residues (fig. 5a). However, when recombinant 5/6-kinase is heterologously expressed in and purified from E. coli, and used in an in vitro phosphorylation assay, phosphoamino acid analysis of the resulting phosphorylated 5/6-kinase does not yield phosphorylated serine and tyrosine residues. As shown in figure 5a, digestion of autophosphorylated 5/6-kinase yields only inorganic phosphate. To further explore the nature of the phosphorylation, the chemical stability of autophosphorylated 5/6-kinase was assessed using the method described by Muimo et al. (Muimo et al., 2000). As shown in figure 5b, the phosphate bond is acid labile, base stable, and somewhat labile in the presence of neutral hydroxylamine. This pattern of stability is consistent with the bond being a phosphoramide, since acylphosphates are both acid and base labile, whereas phosphoserine and threonine residues are stable under both conditions.

Fig. 5.

Fig. 5

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Phosphorylation of 5/6-kinase. A. Phosphoamino acid analysis of phosphorylated 5/6-kinase immunoprecipitated from HEK 293 cells labeled with [32P]-inorganic phosphate (left panel) and that of purified 5/6-kinase incubated in an in vitro autophosphorylation reaction (right panel) under previously described conditions (Wilson et al., 2001). B. Chemical stability of autophosphorylated 5/6-kinase. Purified 5/6-kinase was allowed to autophosphorylate in the presence of [γ-32P]-ATP, incubated under various conditions for 10 min. at 30°C, run on SDS-PAGE, transferred to a PVDF membrane and autoradiographed. Incubation conditions include reaction with 20 mM Tris-HCl, pH 7.6 as a control (lane 1), reaction with 0.1 N HCl, pH 1.0 (lane 3), reaction with 0.1 N NaOH, pH 13 (lane 4), and 0.8 M hydroxylamine with 0.1 M acetate, pH 5.2 (lane 6). Lanes 2 and 5 are empty.

The nature of the amino acid residue phosphorylated by the enzyme is uncertain but seems most likely to be a histidine based on the above properties. This labile bond was also seen with immunoprecipitated endogenous enzyme as seen from the finding of inorganic phosphate after acid hydrolysis of the enzyme as shown in figure 5a.

5/6-kinase also has protein kinase activity phosphorylating proteins involved in the cellular response to stress including c-Jun, IkBα, ATF2 and p53 (Wilson et al, 2001). This suggested to us that 5/6-kinase might play a role in the apoptotic response to cellular stress. Further support for this suggestion came from the fact that 5/6-kinase from E. histolytica was cloned as a protein induced by cellular stress (Field et al., 2000).

We examined the role of 5/6-kinase in apoptosis induced by tumor necrosis factor α or FAS by both overexpressing the enzyme in HEK 293 cells or by RNAi of the endogenous enzyme in these cells (Sun et al., 2003). We found that overexpression of 5/6-kinase protects cells from apoptosis as shown in figure 6 while RNAi enhances it (data not shown). We found however that these effects were not mediated by the protein kinase activity of the enzyme but rather its ability to phosphorylate its inositol phosphate substrate. We found that overexpression of InsP5 2-kinase, the final enzyme in the pathway to InsP6, also protects cells from apoptosis and RNAi of this enzyme enhances apoptosis indicating that the effects noted emanated from either the altered levels of InsP6 or one of the inositol pyrophosphates formed from InsP6 (Verbsky and Majerus, 2005). We have also found that InsP6 is essential for life in mammals since deletion of either InsP5 2-kinase or inositol phosphate multikinase in mice has an embryonic lethal phenotype (Verbsky et al., 2005b, Frederick et al., 2005).

Fig. 6.

Fig. 6

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Apoptosis induced by TNFα in HEK 293 cells expressing 5/6-kinase or vector alone. Cells were treated with TNFα (1 ng/ml) and CHX (0.5 μg/ml) for 6 hours, then stained with APOPercentage (Biocolor, Belfast, Northern Ireland) as per manufacturer’s instructions.

Role of Phosphatidylinositol 5-phosphate in apoptosis

Phosphatidylinositol 5-phosphate (PtdIns5P) is the most recently discovered inositol lipid and its function is least understood (Rameh et al., 1997). We now describe studies showing that this lipid plays a critical role in controlling p53-dependent apoptosis (Zou et al., 2007). PtdIns5P is formed in mammalian cells by the action of PtdIns(4,5)P2 4-phosphatase (no E.C. number as yet) (type I 4-phosphatase) (Ungewickell et al., 2005) as shown in figure 3. Thus PtdIns(4,5)P2 is metabolized to both PtdIns4P and PtdIns5P.

We have also shown that PtdIns5P is a substrate for phospholipase Cγ1 (E.C. 3.1.4.11) forming inositol 1,5-bisphosphate (Ins(1,5)P2) as its product (unpublished). PtdIns5P is a good substrate for the enzyme with Km (120 μM) and Vm (15 μmoles/min/mg protein) similar to those found using PtdIns(4,5)P2 as substrate (data not shown). Furthermore the Ins(1,5)P2 product is further metabolized by inositol polyphosphate 1-phosphatase (E.C. 3.1.3.57) to form inositol 5-phosphate. This gives rise to two previously unknown inositol phosphates whose functions in cell signaling remain to be discovered.

A role for PtdIns5P in p53-dependent apoptosis has been suggested from previous studies. Gozani and co-workers showed that the nuclear factor ING2 was required for efficient acetylation of p53 (Gozani et al., 2003). Acetylation of p53 stabilizes the molecule preventing its proteosomal degradation thereby enhancing apoptosis. RNAi of ING2 reduced apoptosis. Transfection of cells with phosphatidylinositol phosphate kinase (PIPK) type IIβ (E.C. 2.7.1.149) which converts PtdIns5P to PtdIns(4,5)P2 also decreases apoptosis leading the authors to conclude that PtdIns5P formed a complex with ING2 which acted as a cofactor for p53 acetylation, although PtdIns5P levels were not measured in that study. This work was done prior to the discovery of type I 4-phosphatase and thus the source of PtdIns5P was not known. We have now shown that tetracycline-inducible stable HEK 293 cells overexpressing type I 4-phosphatase have a 20% reduction in PtdIns(4,5)P2 levels and have a 50% increase in PtdIns5P (Ungewickell et al., 2005; Zou et al., 2007). When these cells are subjected to genotoxic stress using either etoposide or doxorubicin, p53-dependent apoptosis is increased. Induction of type I 4-phosphatase expression with tetracycline in these cells increases PtdIns5P levels and promotes p53 stability thereby increasing apoptosis. Conversely RNAi of the enzyme decreases p53 stability and inhibits apoptosis. We measured the half life of p53 in control cells and in cells treated with an RNAi construct against type I 4-phosphatase by inhibiting protein synthesis with cycloheximide and Western blotting for p53 at various times thereafter. In control cells the half life of p53 was 7 hours while in RNAi treated cells it was reduced to 1.8 hours. The ability of type I 4-phosphatase to stabilize p53 was ING2 dependent since the effect was lost upon RNAi of ING2. The results are consistent with the idea that PtdIns5P forms a complex with ING2 and both are required for acetylation and stabilization of p53. We measured acetylation of p53 directly using antibodies specific for acetylated p53 to confirm the above hypothesis. Type I 4-phosphatase is primarily cytosolic where it is found bound to the surface of late endosomal/lysosomal membranes (Ungewickell et al., 2005). Since ING2 and p53 are nuclear proteins we investigated the possibility that cellular stress caused a redistribution of type I 4-phosphatase to the nucleus. HeLa cells were treated with 100 μM etoposide for 4 hours and cells were harvested and nuclei were isolated. We found that prior to etoposide treatment 10% of cellular type I 4-phosphatase was nuclear which increased to 40% of the total after etoposide. The total level of type I 4-phosphatase in cells was unchanged so this represents a migration of the enzyme into the nucleus. This nuclear import effected a 50% increase in nuclear PtdIns5P. Our interpretation of these results is summarized in figure 7.

Fig. 7.

Fig. 7

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Model depicting the PtdIns-5-P stress response mediated by type I 4-phosphatase.

Within the nucleus PtdIns5P levels are determined by the opposing actions of type I 4-phosphatase which forms PtdIns5P and that of PIPK type IIβ which converts it to PtdIns(4,5)P2. Upon genotoxic stress type I 4-phosphatase migrates to the nucleus where it effects an increase in PtdIns5P which complexes with ING2 to promote acetylation of p53, stabilizing it and thereby increasing apoptosis. It is not clear how type I 4-phosphatase is able to get into the nucleus. It does not contain any canonical nuclear localization sequences and it does contain putative membrane spanning regions (one or two). Perhaps it gains access to the nucleus by some type of vesicle trafficking wherein a vesicle fuses with the nuclear membrane. Future experiments will be directed at resolving these questions.

Summary

Inositol signaling reactions are very broad in scope affecting many cellular functions. In this report we describe experiments showing that two distinct parts of this system play pivotal roles in an important cellular event, namely apoptosis. Apoptosis is important for organ development and also for controlling cell survival after various stresses which include DNA damage and other proapoptotic stimuli such as tumor necrosis factor α. We show that the inositol phosphate InsP6 or one of its pyrophosphate metabolites determines the extent of apoptosis following tumor necrosis factor α treatment whereby increased cellular levels of InsP6 protect from apoptosis and decreased levels promote it. Cellular levels of InsP6 are determined by the activity of 5/6-kinase since this is the rate limiting enzyme in production of the highly phosphorylated inositol phosphates including InsP6. A lipid inositol metabolite PtdIns5P is also critical in regulating the activity of p53-dependent apoptosis. This phospholipid is formed in cells by the action of type I 4-phosphatase on PtdIns(4,5)P2. PtdIns5P stabilizes p53 by promoting its acetylation in complex with the nuclear factor ING2. Upon genotoxic stress type I 4-phosphatase migrates to the nucleus where it catalyzes the formation of PtdIns5P to stabilize p53 and increase apoptosis.

Acknowledgments

We thank Peter Nicholas and Cecil Buchanan who aided in much of the work described here.

Footnotes

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