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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2007 Oct 16;104(43):16834–16839. doi: 10.1073/pnas.0708189104

Type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase regulates stress-induced apoptosis

Jun Zou 1, Jasna Marjanovic 1, Marina V Kisseleva 1, Monita Wilson 1, Philip W Majerus 1,*
PMCID: PMC2040409  PMID: 17940011

Abstract

A recently discovered phosphatidylinositol monophosphate, phosphatidylinositol 5-phosphate (PtdIns-5-P), plays an important role in nuclear signaling by influencing p53-dependent apoptosis. It interacts with a plant homeodomain finger of inhibitor of growth protein-2, causing an increase in the acetylation and stability of p53. Here we show that type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), an enzyme that dephosphorylates phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), forming PtdIns-5-P in vitro, can increase the cellular levels of PtdIns-5-P. When HeLa cells were treated with the DNA-damaging agents etoposide or doxorubicin, type I 4-phosphatase translocated to the nucleus and nuclear levels of PtdIns-5-P increased. This action resulted in increased p53 acetylation, which stabilized p53, leading to increased apoptosis. Overexpression of type I 4-phosphatase increased apoptosis, whereas RNAi of the enzyme diminished it. The half-life of p53 was shortened from 7 h to 1.8 h upon RNAi of type I 4-phosphatase. This enzyme therefore controls nuclear levels of PtdIns-5-P and thereby p53-dependent apoptosis.

Keywords: acetylated p53, inositol signaling, nuclear translocation

Inositol lipids participate in a variety of intracellular signaling pathways including cytoskeletal dynamics, intracellular membrane trafficking, cell proliferation, and apoptosis (1, 2). In response to agonists, the phosphoinositide profile is modulated by phospholipases, lipid kinases, and lipid phosphatases. The lipid messengers transduce signals through binding to proteins with binding domains specific for different phosphoinositides.

The most recently discovered of the seven known phosphoinositides is phosphatidylinositol 5-phosphate (PtdIns-5-P), and its function is the least understood (3). The origin of PtdIns-5-P in cells was until recently unknown. A study of changes in the cellular levels of PtdIns-5-P suggested that PtdIns-5-P arises from the action of a phosphatase rather than a kinase (4). Our discovery of two phosphatases that convert PtdIns-4,5-P2 to PtdIns-5-P provides a route for synthesis of this lipid (5). Recently, it was suggested that PtdIns-5-P specifically interacts with a plant homeodomain (PHD) finger of inhibitor of growth protein-2 (ING2) protein, and that this interaction is required for ING2-dependent activation of p53, which leads to increased apoptosis (6). This suggestion was based on the finding that RNAi of ING2 or overexpression of the phosphatidylinositol phosphate kinase (PIPK) type IIβ, an enzyme that converts PtdIns-5-P to PtdIns-4,5-P2, decreases apoptosis. Thus, it was presumed that both ING2 and PtdIns-5-P were required for acetylation of p53, although cellular PtdIns-5-P was not measured in that study (6).

The ING2 is a member of the inhibitor of growth family and acts as a cofactor on the histone acetyltransferase complex that functions in chromatin remodeling and p53 acetylation and activation (7). Mutation of the PHD finger that renders PtdIns-5-P-binding defective abrogates p53 acetylation as well as p53-dependent apoptosis in response to DNA-damaging stimuli (6). The level of PtdIns-5-P can be modulated in the nucleus by using PIPKIIβ, which utilizes PtdIns-5-P as a substrate to generate PtdIns-4,5-P2 (8). Elevated nuclear levels of PtdIns-5-P promote an ING2-p53 interaction and accelerate p53-dependent cell death. Although nuclear PIPKIIβ converts PtdIns-5-P into PtdIns-4,5-P2, it is very likely that the PtdIns-4,5-P2 4-phosphatases oppose its effects and generate PtdIns-5-P from PtdIns-4,5-P2. Therefore, stress-induced increase in the nuclear PtdIns-5-P levels could also result from increased phosphatase activity. The two mammalian PtdIns-4,5-P2 4-phosphatases dephosphorylate PtdIns-4,5-P2, forming PtdIns-5-P. Overexpression of one of them, type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase (type I 4-phosphatase), resulted in a significant decrease in cellular PtdIns-4,5-P2, suggesting an effect on cellular PtdIns-5-P levels (5). In this report, we further investigate this enzyme and its product, PtdIns-5-P, in the context of cellular stress. Stress-induced elevation in nuclear PtdIns-5-P can at least partially be attributed to type I 4-phosphatase. Nuclear translocation of type I 4-phosphatase was observed upon cellular stress. As a consequence, nuclear levels of PtdIns-5-P increased and promoted cell death through ING2-enhanced p53 acetylation, stability, and activity.

Results

The Level of Cellular PtdIns-5-P Is Altered by Type I 4-Phosphatase in HEK-293 Cells.

We have identified two PtdIns-4,5-P2 4-phosphatases that hydrolyze PtdIns-4,5-P2 to PtdIns-5-P in vitro. The level of PtdIns-4,5-P2 was reduced by ≈20% in stable cell lines that overexpress type I 4-phosphatase (5). We predicted that this would increase cellular PtdIns-5-P. In HEK-293 TRex cell lines that overexpress type I 4-phosphatase, we examined PtdIns-5-P levels 24 h after induction of enzyme expression by using tetracycline. Levels of PtdIns-5-P were 1.54 ± 0.26, n = 8, compared with the normalized value in control cells of 1.0 ± 0.08, n = 7 (P < 0.05). Because both type I and type II 4-phosphatases convert PtdIns-4,5-P2 to PtdIns-5-P in vitro, we determined the effect of type II 4-phosphatase on PtdIns-5-P levels. We found no changes in total cellular PtdIns-5-P in the cell lines that were stably transfected with type II 4-phosphatase (data not shown).

Type I 4-Phosphatase Enhances Endogenous p53 Stability.

Interaction between PtdIns-5-P and the nuclear adaptor protein ING2 is presumed to stimulate p53 acetylation and therefore promote p53 activity and stability in response to cellular stress (6, 8, 9). We first examined the endogenous p53 levels in HeLa cells treated with siRNAs targeting both 4-phosphatases. The siRNA of type II 4-phosphatase did not reduce p53 expression (data not shown). However, when type I 4-phosphatase levels were reduced, a dramatic decrease in p53 levels in response to the genotoxic agent etoposide was seen (Fig. 1A). The greatest reduction in p53 levels was observed in cells treated with 100 μM etoposide for 24 h (Fig. 1A). We also examined the levels of the p53 downstream effector p21. Treatment of type I 4-phosphatase with siRNA also decreased the level of p21 relative to that in control siRNA-treated cells in the presence of 20 μM and 50 μM etoposide (Fig. 1A). At the highest concentration of etoposide, p21 was reduced even without RNAi, perhaps as the result of cytotoxicity. We also assessed the level of p53 in the cells that overexpress either type I or type II 4-phosphatase alone over a 24-h time course. Overexpression of type I 4-phosphatase increased endogenous p53 (Fig. 1B), whereas cells overexpressing type II 4-phosphatase showed no changes in p53 (data not shown).

Fig. 1.

Fig. 1.

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The type I 4-phosphatase regulates endogenous levels of p53. (A) HeLa cells were transfected with either control siRNA or type I and II 4-phosphatase (ptase) siRNA for 36 h and then treated with etoposide (Eto) at the indicated concentration for 24 h. Cell lysates were analyzed by Western blotting for poly(ADP-ribose) polymerase (PARP), p53 (antibody 9282), type I 4-phosphatase, β-tubulin, and p21. Numbers denote the intensity of p53 bands relative to those in cells treated with control siRNA and 20 μM etoposide. The intensity of the bands is shown in arbitrary units. (B) HEK-293 cells stably expressing FLAG-tagged type I 4-phosphatase were induced with tetracycline (TET) for 24 h, then incubated with 100 μM etoposide for the indicated times. Western blot analysis with antibodies against total p53, FLAG, and β-tubulin was performed. Numbers denote the intensity of p53 bands relative to corresponding uninduced cells.

In cells overexpressing type I 4-phosphatase, RT-PCR was used to measure p53 mRNA levels to rule out the possibility that increases in p53 levels after stress are due to increased transcription of p53 (data not shown). Changes in p53 are due to altered stability of the protein because the turnover rate of p53 in cells treated with etoposide is increased with type I 4-phosphatase siRNA, as shown in Fig. 2A. The results of a similar experiment using doxorubicin as a genotoxic agent are shown in Fig. 2B. The half-life of p53 was reduced from 7 h to 1.8 h by using siRNA of type I 4-phosphatase (Fig. 2C).

Fig. 2.

Fig. 2.

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The type I 4-phosphatase knockdown decreases the stability of endogenous p53. (A) HeLa cells were transfected with control or type I 4-phosphatase siRNA for 36 h. Before treatment with 150 μg/ml cycloheximide (CHX) for the times indicated, cells were pretreated with 100 μM etoposide for 4 h. The total p53 level from cell extracts was detected by using Western blotting with an anti-p53 antibody (9282). (B) Western blotting was performed as in A except the cells were pretreated with doxorubicin for 1 h. (C) The half-life of p53 was estimated based on the intensity of p53 bands at different time points shown in B.

Overexpression of Type I 4-Phosphatase Promotes p53 Acetylation and Cell Death, and Both Are Inhibited by PIPKIIβ.

We found that the level of p53 was increased by type I 4-phosphatase, which could be due to an increase in the levels of PtdIns-5-P leading to ING2-dependent p53 acetylation. We examined whether acetylation of p53 was enhanced in the presence of type I 4-phosphatase and whether a more active acetylated p53 leads to apoptosis. In TRex cells stably expressing type I 4-phosphatase after tetracycline induction, treatment with etoposide to induce apoptosis was examined, as shown in Fig. 3A. Compared with the uninduced cells, the type I 4-phosphatase-overexpressing cells became rounded and irregular and suffered a significant decrease in viability (Fig. 3A). Overexpression of type I 4-phosphatase upon tetracycline treatment by itself had no effect on cell viability because no morphological alteration was observed compared with the control cells in the absence of etoposide (Fig. 3A). Western blot analysis of HeLa cell extracts after etoposide treatment shows that expression of the type I 4-phosphatase increases acetylated p53 levels, whereas the cells cotransfected with PIPKIIβ have reduced acetylated p53 levels (Fig. 3C). The increase in acetylated p53 evoked increased apoptosis, as shown by the FACS analysis shown in Fig. 3B, in which cell death is measured as the fraction of cells in the sub-G1 compartment. The proportion in sub-G1 population was returned to control levels when PIPKIIβ was cotransfected with type I 4-phosphatase. The apoptotic cell death observed appeared to be correlated with the level of acetylated p53 because the increase in acetylated p53 when type I 4-phosphatase was used was attenuated by the overexpression of PIPKIIβ (Fig. 3 B and C). These data highlight the role of PtdIns-5-P in the control of the DNA damage response because the up-regulation of PtdIns-5-P by type I 4-phosphatase results in an increase in p53 acetylation and cell death.

Fig. 3.

Fig. 3.

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The type I 4-phosphatase enhances p53 acetylation and cell death, both of which are inhibited by PIPKIIβ. (A) The type I 4-phosphatase promotes cell death in HEK-293 cells. Stably transfected HEK-293 TRex cells expressing type I 4-phosphatase were grown in the presence or absence of tetracycline for 24 h before treatment with 100 μM etoposide for 24 h. (B) Expression of PIPKIIβ inhibits type I 4-phosphatase-mediated cell death in HeLa cells. The HeLa cells were transfected with vector (pcDNA4/TO), FLAG-tagged type I 4-phosphatase alone, or FLAG-tagged type I 4-phosphatase and PIPKIIβ. One day after transfection, apoptosis was induced with 100 μM etoposide treatment for 24 h. Cell death was measured by the percentage of cells in the sub-G1 population by using propidium iodide staining and FACS analysis. FL3-H, fluorescence channel 3 height. (C) The type I 4-phosphatase enhances p53 acetylation and is inhibited by PIPKIIβ. The HeLa cells were treated as in B and induced with the indicated concentration of etoposide for 24 h. The p53 immunoprecipitates (DO-1 antibody) obtained from cell extracts with equal amounts of total protein were analyzed by using Western blotting with antibodies against acetyl-p53 (Lys-382, antibody 2525s). Numbers denote the intensity of acetylated p53 bands relative to corresponding untreated cells. Western blots were also prepared with antibodies against hemagglutinin (HA)-tagged PIPKIIβ, FLAG-tagged type I 4-phosphatase, and β-tubulin (lower three blots).

Increase in Acetylation of p53 by Type I PtdIns-4,5-P2 4-Phosphatase Is ING2 Dependent.

The ING2 has been shown to stimulate acetylation of p53 on Lys-382 and induce apoptosis (10), which was dependent on PtdIns-5-P binding (6). Therefore, we determined whether the ING2 protein was required for type I 4-phosphatase to promote acetylation of p53. We measured the level of acetylated p53 in the presence of type I 4-phosphatase after the depletion of the ING2 with siRNAs (Fig. 4A). The ING2 depletion leads to a decrease in the level of acetylated p53, which was previously elevated as the result of overexpression of type I 4-phosphatase (Fig. 4B). Because depletion of ING2 markedly abrogates p53 acetylation and p53-dependent apoptosis in response to cell stress (6), type I 4-phosphatase is likely to mediate acetylation and apoptosis of p53 through the function of the PtdIns-5-P–ING2 complex.

Fig. 4.

Fig. 4.

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The type I 4-phosphatase enhances p53 acetylation and is inhibited by ING2 siRNA. (A) Cells were transfected with ING2 for 24 h and then treated with control or ING2 siRNA (#1, Ambion ID: 116979; #2, Ambion ID: 116981; #3, see Materials and Methods for sequence) for another 24 h. Cell lysates were analyzed by using Western blotting for ING2. (B) Stably transfected HEK-293 TRex cells expressing FLAG-type I 4-phosphatase were grown in the presence (type I 4-ptase) or absence of tetracycline for 24 h before transfection with control or ING2 siRNA. Twenty-four hours after transfection, cells were induced with 100 μM etoposide, and 0.5 μM trichostatin A was added into the medium 5 h before harvesting the cells. The p53 was immunoprecipitated (antibody 393) from cell extracts containing equal amounts of total protein. The amount of p53 acetylated at Lys-382 was detected by Western blotting (Lys-382, antibody 2371). Numbers denote the intensity of acetylated p53 bands relative to that in corresponding control siRNA-transfected cells.

Distribution of Type I PtdIns-4,5-P2 4-Phosphatase Was Altered upon Cellular Stress.

We have previously demonstrated that type I 4-phosphatase is ubiquitously expressed and colocalizes with the endosomal-specific marker LAMP1 in an overexpression system (5). However, given that the main components of the ING2-p53 pathway are found in the nucleus, it is possible that some of type I 4-phosphatase protein is localized in the nucleus as well, especially when cells are confronted with cellular stress. We isolated intact nuclei by using a detergent-free method (11), and the efficiency of subcellular fractionation was verified by using immunoblotting with tubulin and histone H4 antibodies (Fig. 5A). Consistent with our previous report (5), type I 4-phosphatase is predominately located in the cytosol (Fig. 5A). However, exposure to etoposide for 4 h resulted in a significant increase of type I 4-phosphatase in the nuclear fraction, with both the endogenous and overexpression system. Because the total cellular amount of type I 4-phosphatase appeared unchanged, an increase in nuclear type I 4-phosphatase likely resulted from the redistribution of this enzyme. The nuclear fraction of 4-phosphatase increased from 15% to 40% of the total in response to cellular stress.

Fig. 5.

Fig. 5.

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Translocation of type I 4-phosphatase into the nucleus upon cellular stress increases nuclear levels of PtdIns-5-P. (A) The HeLa cells transfected with vector (pcDNA4/TO) or type I 4-phosphatase were treated with 100 μM etoposide for 4 h, and the cells were then fractionated into cytosolic (C) and nuclear (N) fractions. The level of type I 4-phosphatase in each fraction was analyzed by Western blotting with anti-type I 4-phosphatase antibody. Tubulin and histone H4 were detected with corresponding antibodies and used as internal loading controls and markers for fractionation. (B) The nuclear level of PtdIns-5-P increases in response to cellular stress. The HeLa cells transfected with vector (pcDNA4/TO) or type I 4-phosphatase were cultured in 150-mm plates and treated with etoposide for 4 h, and the intact nuclei were isolated. A mass assay was performed to determine the nuclear PtdIns-5-P content as described in the legend to Fig. 1. Synthetic PtdIns-5-P (100 pmol) was used as the loading control, and 32P-labeled PtdIns-4,5-P2 was confirmed by using HPLC. (C) Quantification of PtdIns-5-P levels shown in B. The results are presented as relative mass compared with that in vector-transfected cells (1.00 ± 0.13, n = 8, versus 1.59 ± 0.23, n = 8, unpaired t test, P < 0.05).

Type I 4-Phosphatase Increases Nuclear PtdIns-5-P upon Cellular Stress.

The metabolism of nuclear phosphoinositides may be independent of their cytosolic counterparts (2). Jones et al. (8) have demonstrated that nuclear PtdIns-5-P rises upon stress stimulation as a consequence of inhibition of PIPKIIβ as the result of phosphorylation at Ser-326. Given that type I 4-phosphatase is redistributed into the nucleus in response to cellular stress, we hypothesized that type I 4-phosphatase might be responsible for controlling nuclear PtdIns-5-P. We measured the level of nuclear PtdIns-5-P in both resting and induced cells by using PIPKIIα to form 32P-labeled PtdIns-4,5-P2, as described in Materials and Methods. Interestingly, despite the fact that the total cellular PtdIns-5-P was increased by using type I 4-phosphatase, the amount of nuclear PtdIns-5-P was too low to detect under resting conditions (Fig. 5B Upper), suggesting that the nuclear fraction of PtdIns-5-P represents only a small fraction of the total PtdIns-5-P. However, a 4-h treatment with etoposide led to an increase in nuclear PtdIns-5-P, in both vector-transfected and type I 4-phosphatase-transfected cells (Fig. 5B Lower). The increase in nuclear PtdIns-5-P in cells overexpressing type I 4-phosphatase was 50% greater than that of the vector-transfected cells (Fig. 5C).

Discussion

We have found that type I 4-phosphatase regulates nuclear PtdIns-5-P levels, which in turn mediate p53-dependent apoptosis through interaction with ING2 in response to genotoxic stress. The redistribution of type I 4-phosphatase into the nucleus demonstrates the role of nuclear PtdIns-5-P in phosphoinositide-mediated stress response signaling. These findings are summarized in the model shown in Fig. 6.

Fig. 6.

Fig. 6.

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A model of the type I 4-phosphatase-mediated PtdIns-5-P stress response pathway. In response to cellular stress, type I 4-phosphatase is translocated into the nucleus, where it hydrolyzes PtdIns-4,5-P2 into PtdIns-5-P. The elevated nuclear PtdIns-5-P facilitates the ING2–p53 apoptotic pathway by promoting ING2-dependent p53 acetylation. The p53 activity and stability are enhanced, and apoptotic cell death is therefore increased.

PtdIns-5-P represents less than 10% of total phosphatidylinositol monophosphates, and previously it was unclear as to how it was formed. Myotubularins and PIKfyve are the mammalian enzymes previously shown to increase cellular levels of PtdIns-5-P (12, 13), but they do not provide a route for the net synthesis of PtdIns-5-P. We have shown here that type I 4-phosphatase is capable of increasing the cellular level of PtdIns-5-P in vivo in an overexpression system. It is likely that this modulation is the result of direct hydrolysis because we demonstrated earlier that total cellular PtdIns-4,5-P2 is depleted (5). Given the fact that high levels of PtdIns-4,5-P2, which is the substrate of type I 4-phosphatase, are widely distributed in both cytoplasm and the nucleus, it is possible that this dephosphorylating process may be the predominate synthetic pathway for PtdIns-5-P.

Several PHD fingers containing nuclear proteins are PtdIns-5-P-binding ligands (6, 14, 15). It has been shown that ING2 is a nuclear receptor for PtdIns-5-P in response to stress, and this interaction and its consequences have been studied extensively (6, 8, 16). The only known regulator of PtdIns-5-P in apoptotic events identified previously is PIPKIIβ (8). Here we demonstrate that type I 4-phosphatase regulates cellular and nuclear levels of PtdIns-5-P and plays an important role in the PtdIns-5-P-mediated apoptotic process.

The PHD domain in general transcription factor IIH, a RNA polymerase II component, is a binding partner of PtdIns-5-P. The activation domain of the transcriptional activator VP16 also binds to the same site. Therefore, PtdIns-5-P may alter transcription by binding-site competition (14). The ING2 also binds histone H3 trimethyllysine, which marks chromatin at sites of repression of gene transcription. However, this binding site appears to be distinct from that of PtdIns-5-P because mutations in ING2 that inhibit binding to histone H3 trimethyllysine do not block PtdIns-5-P binding or ING2 effects on apoptosis. The PtdIns-5-P not only may up-regulate the p53 apoptosis pathway, but also may regulate a set of proliferation genes that are under the control of several PHD-containing proteins. Moreover, all five members of the ING family have been implicated in p53 function (17), PtdIns-5-P binding (8), and the acetylation of chromatin through interaction with specific histone acetyltransferase–deacetylase complexes (18). We speculate that ING2 suppressor proteins are key components linking chromatin modulation with p53-dependent tumor suppression and that nuclear PtdIns-5-P is the phosphatidylinositol messenger in this process.

This study also provides a link between p53 posttranslational modification and PtdIns-5-P. p53 is specifically acetylated at several lysine residues in the C-terminal regulatory domain by using CBP/p300, which requires ING2 as a cofactor (7). Acetylation of p53 induces a conformational change that enhances its sequence-specific DNA-binding activity (19). Our experiments show that elevated PtdIns-5-P promotes the acetylation of p53 and that this effect is abrogated by reducing ING2 protein levels with siRNA, indicating that ING2 is the signal receptor in this event. Acetylation appears to be very important for p53 stability by protecting it from degradation (20). In our overexpression and siRNA knockdown experiments, the stability of p53 correlated with the levels of type I 4-phosphatase under stress conditions.

Type I 4-phosphatase is redistributed into the nuclear compartment upon stress. Nuclear transport of macromolecules uses nuclear localization signals, which facilitate the interaction with cytoplasmic receptor proteins. However, no typical nuclear localization signal sequences with a cluster of positive amino acids (21) have been identified in type I 4-phosphatase, suggesting that the type I 4-phosphatase might bind to another nuclear localization signal-bearing protein to be transported. A key in future studies will be identification of type I 4-phosphatase binding partners and the investigation of their effects on enzymatic activity and/or intracellular localization.

Materials and Methods

Reagents and Chemicals.

All chemicals and reagents, unless specifically noted, were purchased from Sigma–Aldrich (St. Louis, MO). The [γ-32P]ATP was purchased from MP Biomedicals (Solon, OH).

Antibodies and Fluorescent Probes.

The following antibodies were used: anti-type I 4-phosphatase as described (5). For anti-p53, monoclonal antibodies DO-1 (Calbiochem, San Diego, CA), polyclonal antibody 9282 (Cell Signaling, Danvers, MA), and FL393 (Santa Cruz Biotechnology, Santa Cruz, CA) were used; rabbit anti-acetylated human p53 (K382) polyclonal antibody was from Trevigen (catalog no. 2371; Gaithersburg, MD) and from Cell Signaling (catalog no. 2525s). Rabbit anti-ING2 antibody was provided by Curtis Harris (National Cancer Institute, Bethesda, MD). Mouse monoclonal anti-FLAG epitope antibody was from Sigma, and polyclonal anti-βtubulin ab6046 was from Abcam (Cambridge, MA). Anti-histone H4, rabbit polyclonal ab10158 was from Abcam, and anti-HA epitope, mouse monoclonal HA-7, was from Sigma–Aldrich.

Plasmid, Cell Culture, Transfection, and Treatment.

The ING2 cDNA in the pFLAG-CMV-6v vector was provided by Curtis Harris at the National Cancer Institute. The PIPKII α and β isoforms in the CMV5-HA vector were described previously (22). The HeLa cells were maintained in culture using 10% FBS in Dulbecco's modified Eagle's medium. Stably transfected HEK-293 TRex cells in a tetracycline-inducible vector (Invitrogen, Carlsbad, CA) expressing type I and II 4-phosphatase (5) were maintained in Dulbecco's modified Eagle's medium with 10% FBS (Tet system approved; Clontech, Mountain View, CA), 2 mM glutamine, 5 μg/ml blasticidin (Invitrogen), 0.3 mg/ml zeocin (Invitrogen), 100 units/ml penicillin G, and 10 μg/ml streptomycin. Protein expression was induced with 0.5 μg/ml tetracycline. The HeLa cells were transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. siRNA transfections were done by using a Nucleofector kit (Amaxa, Gaithersburg, MD). All experiments were performed 1–3 days after transfection. The siRNA duplexes were obtained from Ambion, Austin, TX) and sequences are: type I 4-phosphatase as described (5); control siRNA (luciferase) duplex: sense 5′-CUUACGCUGAGUACUUCGAdTdT; antisense, 5′-UCGAAGUACUCAGCG UAAGdTdT; ING2 siRNA sequence: sense 5′-GGCAAGACAAAUGGAGUUUCA; antisense: 5′-UAACUCCAUUUGUCUUGCCCG. Unless noted, apoptosis was induced by either 100 μM etoposide or 5 μg/ml doxorubicin, for the indicated times. For detection of acetylated p53, cells were treated with 0.5 μM trichostatin A for 5 h.

Subcellular Fractionation and PtdIns-5-P Mass Measurement.

Intact nuclei were separated by using a detergent-free method (11). The PtdIns-5-P mass assay was conducted as described by Roberts et al. (4) with some minor modifications. (i) The supporting matrix was glyceryl controlled pore glass, 120–200 mesh (Sigma–Aldrich). (ii) Phosphatidylinositols were eluted by using 2:6:3 (vol/vol) CHCl3/CH3OH/2 M triethylamine bicarbonate. The freshly prepared triethylamine bicarbonate was generated by bubbling CO2 through 2 M triethylamine bicarbonate until the pH was 7.

Analysis of p53 Turnover.

p53 stability experiments were performed in HeLa cells treated with control siRNA or type I 4-phosphatase siRNA for 36 h. Cells were then treated with doxorubicin or etoposide for 1 h or 4 h, respectively. Cycloheximide (150 μg/ml) was then added to halt protein synthesis. Cells were harvested at the indicated time points. Cell lysates were analyzed by using immunoblotting. Quantification was performed by using an ID image analysis system (Eastman Kodak, Rochester, NY).

Immunoprecipitation and Western Blotting.

Whole-cell lysates were prepared in radioimmunoprecipitation assay buffer (150 mM NaCl/10 mM Tris, pH 7.2/0.1% SDS/1% deoxycholate/1% Nonidet P-40/5 mM EDTA) containing Complete Protease Inhibitor Mix (Roche, Nutley, NJ), Halt Phosphatase Inhibitor Mixture (Pierce, Rockford, IL), and 10 μM trichostatin A. Cell lysates (500 μg of total protein) were precleared with staphylococcal protein A beads (IPA300; Repligen, Waltham, MA) and incubated with 1 μg of affinity-purified antibodies overnight at 4°C, followed by incubation at 4°C with protein A-agarose beads for 2 h. Immunoprecipitated complexes were washed four times with PBS and eluted in SDS sample buffer by boiling for 4 min, electrophoresed through SDS/polyacrylamide gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis. The appropriate horseradish peroxidase-conjugated secondary antibody and the SuperSignal West Pico chemiluminescent substrate (Pierce) were used to visualize the bands. Where indicated, bands were compared by using densitometry of Western blots with an Image Station 440 CF (Eastman Kodak, Rochester, NY), and the data were analyzed by using Kodak 1D V.3.5.4 software (Kodak Scientific Imaging System, Rockville, MD).

FACS.

Propidium iodide staining was carried out as follows. Harvested cells were washed in PBS and fixed in cold 70% ethanol. After 30-min fixation at 4°C, cells were washed twice with ice-cold PBS and spun at 1,100 × g, and the supernatant was discarded. The solution (500 μl, 100 μg/ml RNase, 0.1% Triton X-100, 20 μg/ml propidium iodide) was added, the samples were subjected to FACS (FACScan; Becton Dickinson, Franklin Lakes, NJ), and the data were analyzed by using CellQuest software (Becton Dickinson).

Acknowledgments

We thank Dr. Curtis Harris for the gifts of ING2 expression vectors and ING2 antibody. We acknowledge the contributions to this work of Peter Nicholas and Cecil Buchanan for their helpful assistance and Dr. Shao-Chun Chang for invaluable advice. This work was supported by National Institutes of Health Grant HL 16634 (to P.W.M.) and American Heart Association Grant 0730350N (to M.V.K.).

Abbreviations

ING2

inhibitor of growth protein-2

PHD

plant homeodomain

PIPK

phosphatidylinositol-phosphate kinase

PtdIns-5-P

phosphatidylinositol 5-phosphate

PtdIns-4,5-P2

phosphatidylinositol 4,5-bisphosphate

type I 4-phosphatase

type I phosphatidylinositol-4,5-bisphosphate 4-phosphatase.

Footnotes

The authors declare no conflict of interest.

References

  • 1.Payrastre B, Missy K, Giuriato S, Bodin S, Plantavid M, Gratacap M. Cell Signaling. 2001;13:377–387. doi: 10.1016/s0898-6568(01)00158-9. [DOI] [PubMed] [Google Scholar]
  • 2.Gonzales ML, Anderson RA. J Cell Biochem. 2006;97:252–260. doi: 10.1002/jcb.20655. [DOI] [PubMed] [Google Scholar]
  • 3.Rameh LE, Tolias KF, Duckworth BC, Cantley LC. Nature. 1997;390:192–196. doi: 10.1038/36621. [DOI] [PubMed] [Google Scholar]
  • 4.Roberts HF, Clarke JH, Letcher AJ, Irvine RF, Hinchliffe KA. FEBS Lett. 2005;579:2868–2872. doi: 10.1016/j.febslet.2005.04.027. [DOI] [PubMed] [Google Scholar]
  • 5.Ungewickell A, Hugge C, Kisseleva M, Chang SC, Zou J, Feng Y, Galyov EE, Wilson M, Majerus PW. Proc Natl Acad Sci USA. 2005;102:18854–18859. doi: 10.1073/pnas.0509740102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Gozani O, Karuman P, Jones DR, Ivanov D, Cha J, Lugovskoy AA, Baird CL, Zhu H, Field SJ, Lessnick SL, et al. Cell. 2003;114:99–111. doi: 10.1016/s0092-8674(03)00480-x. [DOI] [PubMed] [Google Scholar]
  • 7.Pedeux R, Sengupta S, Shen JC, Demidov ON, Saito S, Onogi H, Kumamoto K, Wincovitch S, Garfield SH, McMenamin M, et al. Mol Cell Biol. 2005;25:6639–6648. doi: 10.1128/MCB.25.15.6639-6648.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Jones DR, Bultsma Y, Keune WJ, Halstead JR, Elouarrat D, Mohammed S, Heck AJ, D'Santos CS, Divecha N. Mol Cell. 2006;23:685–695. doi: 10.1016/j.molcel.2006.07.014. [DOI] [PubMed] [Google Scholar]
  • 9.Li M, Luo J, Brooks CL, Gu W. J Biol Chem. 2002;277:50607–50611. doi: 10.1074/jbc.C200578200. [DOI] [PubMed] [Google Scholar]
  • 10.Nagashima M, Shiseki M, Miura K, Hagiwara K, Linke SP, Pedeux R, Wang XW, Yokota J, Riabowol K, Harris CC. Proc Natl Acad Sci USA. 2001;98:9671–9676. doi: 10.1073/pnas.161151798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Clarke JH, Letcher AJ, D'Santos CS, Halstead JR, Irvine RF, Divecha N. Biochem J. 2001;357:905–910. doi: 10.1042/0264-6021:3570905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Tronchere H, Laporte J, Pendaries C, Chaussade C, Liaubet L, Pirola L, Mandel JL, Payrastre B. J Biol Chem. 2004;279:7304–7312. doi: 10.1074/jbc.M311071200. [DOI] [PubMed] [Google Scholar]
  • 13.Sbrissa D, Ikonomov OC, Deeb R, Shisheva A. J Biol Chem. 2002;277:47276–47284. doi: 10.1074/jbc.M207576200. [DOI] [PubMed] [Google Scholar]
  • 14.Di Lello P, Nguyen BD, Jones TN, Potempa K, Kobor MS, Legault P, Omichinski JG. Biochemistry. 2005;44:7678–7686. doi: 10.1021/bi050099s. [DOI] [PubMed] [Google Scholar]
  • 15.Alvarez-Venegas R, Sadder M, Hlavacka A, Baluska F, Xia Y, Lu G, Firsov A, Sarath G, Moriyama H, Dubrovsky JG, Avramova Z. Proc Natl Acad Sci USA. 2006;103:6049–6054. doi: 10.1073/pnas.0600944103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Huang W, Zhang H, Davrazou F, Kutateladze TG, Shi X, Gozani O, Prestwich GD. J Am Chem Soc. 2007;129:6498–6506. doi: 10.1021/ja070195b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Campos EI, Chin MY, Kuo WH, Li G. Cell Mol Life Sci. 2004;61:2597–2613. doi: 10.1007/s00018-004-4199-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Doyon Y, Cayrou C, Ullah M, Landry AJ, Cote V, Selleck W, Lane WS, Tan S, Yang XJ, Cote J. Mol Cell. 2006;21:51–64. doi: 10.1016/j.molcel.2005.12.007. [DOI] [PubMed] [Google Scholar]
  • 19.Sterner DE, Berger SL. Microbiol Mol Biol Rev. 2000;64:435–459. doi: 10.1128/mmbr.64.2.435-459.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Rodriguez MS, Desterro JM, Lain S, Lane DP, Hay RT. Mol Cell Biol. 2000;20:8458–8467. doi: 10.1128/mcb.20.22.8458-8467.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Chook YM, Blobel G. Curr Opin Struct Biol. 2001;11:703–715. doi: 10.1016/s0959-440x(01)00264-0. [DOI] [PubMed] [Google Scholar]
  • 22.Kisseleva M, Feng Y, Ward M, Song C, Anderson RA, Longmore GD. Mol Cell Biol. 2005;25:3956–3966. doi: 10.1128/MCB.25.10.3956-3966.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

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