Regulation of Apoptosis by Phosphatidylinositol 4,5-Bisphosphate Inhibition of Caspases, and Caspase Inactivation of Phosphatidylinositol Phosphate 5-Kinases*

Marisan MejillanoDagger §, Masaya YamamotoDagger §, Andrew L. RozelleDagger §, Hui-Qiao SunDagger , Xiaodong Wang, and Helen L. YinDagger ||

From the Departments of Dagger  Physiology and  Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Received for publication, August 10, 2000, and in revised form, October 5, 2000



    ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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REFERENCES

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP2 levels in mixed lipid vesicles. Caspase 3 binding to PIP2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha  (PIP5KIalpha ), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP2 is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P4 position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KIalpha inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.



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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Phosphoinositides have major roles in intracellular signaling and cell proliferation. The D3 phosphorylated phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate (PIP3)1 and PI(3,4)P2, have been clearly implicated in the promotion of cell survival. They stimulate the phosphorylation of Akt/PKB (1), a serine/threonine kinase that inactivates multiple components of the apoptotic machinery (2-4). The D4 phosphorylated phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), has not been directly shown to promote cell survival, although it may contribute in a number of ways indirectly. PIP2 is a substrate for phosphoinositide 3-kinase that synthesizes the pro-survival D3 lipids (5), and it is a bona fide signaling molecule that regulates the actin cytoskeleton, vesicular trafficking, channel and transporter activities, and nuclear functions (6). PIP2 synthesis is increased by growth factors (7), by thrombin (8), and by integrin signaling (9). In addition, PIP2 inhibits gelsolin, a caspase substrate (10) that is a major effector of cytoskeletal changes (11). Recently, it was reported that PIP2 complexed with gelsolin inhibits caspase 3 and caspase 9, but not caspase 8 (12), and that PIP2 alone does not inhibit caspases.

We now report that PIP2 alone inhibits initiator and executioner caspases in the two major apoptotic cascades. These cascades start with death receptor activation of procaspase 8 or mitochondrial activation of procaspase 9, and both converge on procaspase 3. We also present in vivo evidence for the roles of PIP2 in apoptotic signaling. Human type I phosphatidylinositol phosphate 5 kinase alpha  (PIP5KIalpha ) (13-15) protects against apoptosis in both pathways, and it is inactivated by caspase 3 cleavage during apoptosis. These results suggest a novel feedforward amplification mechanism for maintaining the balance between phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.


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Lipids

PIP2 was from either Calbiochem or Roche Molecular Biochemicals. PI(3,4)P2 and PIP3 were gifts of C. S. Chen (University of Kentucky, Lexington, KY). PI(4)P, PS, and PC were purchased from Avanti Lipids. Phosphoinositide micelles and mixed vesicles were prepared by probe sonication (16).

Plasmids and Recombinant Proteins

Apaf-1 and procaspase 9 were expressed in Sf20 cells and purified (17). Recombinant caspase 3 and caspase 8 were purified from bacteria. PCMV2 procaspase 9 vector was as described by Li et al. (17). The human PIP5KIalpha cDNA was generated by PCR from a HeLa cell cDNA pool. It was subcloned into pGEM-T (Promega) and an expression vector (pCMV5) containing a Myc tag at the 5' end (18). The caspase 3-resistant mutant (D279A) and kinase-deficient mutant (D270A) (equivalent to Asp-227 of mouse PIP5KIalpha (Ref. 19)) were generated using a QuickChange site-directed mutagenesis kit (Stratagene). The PIP5KIalpha cDNA constructs were subcloned into pET-28c(+) (Novagen) with a hexahistidine tag at the 5' end. Recombinant PIP5KIalpha was purified from a nickel-nitrilotriacetic acid-agarose column (Qiagen). Human plasma gelsolin was expressed in Escherichia coli and purified by ion exchange chromatography (16). The GFP-AktPH cDNA was a gift of T. Balla (National Institutes of Health, Bethesda, MD) (20).

Adenovirus containing PIP5KIalpha or beta -gal were used to infect cells according to protocol described by Shibaski et al. (21).

Caspase Activity Assays

Caspase Activation in Cell Extracts-- HeLa cells were broken by Dounce homogenization in a hypotonic buffer. The lysate was centrifuged at 100,000 × g. The high speed supernatant contained mitochondria-derived cytochrome c, and the caspase 9 cascade was activated by incubation with 1 mM dATP at 30 °C for 1 h (22).

Apoptosome Activation Assay-- Procaspase 9 was first incubated with PIP2 micelles on ice for 10 min in a buffer containing 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM PMSF). Apoptosomes were assembled by mixing procaspase 9 with Apaf-1, cytochrome c (0.01 µg/µl), dATP (100 µM), and MgCl2 (2.5 mM) and incubating for 1 h at 30 °C (17, 23).

Fluorogenic Caspase Activity Assay-- Enzyme activity was determined by measuring the release of AFC from synthetic substrates at 37 °C. Recombinant hamster caspase 3 or human caspase 8 (between 6 to 100 nM) was incubated with 267 µM Ac-DEVD-AFC or Ac-IETD-AFC (Enzyme Systems Products), respectively, in 25 mM Hepes, pH 7.0, 80 mM KCl, 1 mM EGTA. Results were analyzed as described by Zhou et al. (24). Inhibition rates were calculated from progress curves that are generated by adding caspase to a fluorogenic substrate in the presence of 0-20 µM phosphoinositide. The rate for the uninhibited reaction (Vo) was obtained from the linear portion of the time course of the reaction, and the rate for the inhibited reaction (Vi) was determined from the steady state formation of the product. The apparent Ki(app) (apparent inhibition constant), was derived from the slope of the [(Vo/Vi- 1] versus PIP2 curve. Ki, the inhibition constant, was calculated using the equation Ki = (Ki)app/(1 + [S]/Km), where [S] is the substrate concentration. Km, the Michaelis constant for substrate cleavage, was calculated in the range of 5-200 µM for Ac-IETD-AFC, and of 10-700 µM for Ac-DEVD-AFC using the Lineweaver-Burke plot.

Lipid Binding Assay

Mixed lipid vesicles containing 90% PC and 10% of phosphoinositides or PS were prepared by probe sonication in water, and added to caspase 3. The final reaction mixture contained 30 µM amount of the test phospholipid (phosphoinositide or PS) and 2.5 µM caspase 3 in 20 mM Hepes, pH 7.5, 110 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM PMSF, and 0.2 mg/ml bovine serum albumin. Bovine serum albumin was included to reduce nonspecific binding. After a 30-min incubation at room temperature, the samples were centrifuged at 100,000 × g for 30 min at room temperature. The supernatants were collected, and the pellets were resuspended to the original volume. Equivalent volumes were loaded onto SDS gels.

Apoptosis Assays

Transfection-- HeLa and HEK293 cells were transfected using LipofectAMINE Plus and LipofectAMINE (Life Technologies, Inc.), respectively. 2 µg of total DNA was used in all cases. Cells were analyzed within 24 h after transfection.

Apoptosis Induction-- Apoptosis was induced by transfection of pCMV2-FLAG-procaspase 9 (for 24 h), or with apoptotic inducers. These include 1 µM staurosporine or 50 ng/ml TNFalpha . 10 ng/ml cycloheximide (CHX) or 0.2 µg/ml actinomycin D was added to enhance the apoptotic effect of TNFalpha . The latter was used in some experiments, because cycloheximide is no longer available commercially. 20 µM z-DEVD-fmk (Enzyme Systems Products) or 200 nM wortmannin (Sigma) was added to cells 30 min prior to addition of the apoptosis inducers when indicated.

Western Blotting-- Floating and adherent cells were collected, washed with phosphate-buffered saline and lysed with radioimmune precipitation assay buffer (50 mM Hepes, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, protease inhibitor mixture (Roche Molecular Biochemicals), 2 mM phenylmethylsulfonyl fluoride). In some cases, phosphatase inhibitors (50 mM sodium fluoride, 1 mM orthovanadate, 45 mM pyrophosphate) were included. Samples were subjected to SDS-polyacrylamide gel electrophoresis and used for Western blotting. Endogenous and overexpressed PIP5KIalpha were detected with an affinity purified rabbit anti-PIP5KIalpha antibody (gift of R. A. Anderson, University of Wisconsin, Madison, WI) and with anti-c-Myc (Santa Cruz), respectively. Other antibodies used are: anti-caspase 3 (Transduction Laboratories), anti-caspase 9 (17) (PharMingen), anti-Akt and anti-phospho-Akt (New England Biolabs), anti-PARP p85 (Promega), and anti-FLAG (Sigma). Immunoreactive bands were detected using the enhanced chemiluminescence system (ECL, Bio-Rad).

Microscopy-- Apoptotic index was determined using DAPI staining or beta -gal staining. For DAPI staining, cells were fixed in formaldehyde and stained with 1 µg/ml DAPI, and with anti-FLAG to detect cells overexpressing procaspase 9. beta -Gal-transfected cells were identified after staining with X-gal. Blue round cells with irregularly shaped nuclei (apoptotic) and blue spread cells (nonapoptotic) in randomly chosen fields were counted in a blinded fashion. More than 500 cells were counted per condition. The two methods gave comparable results.

PIP5KIalpha Digestion by Caspases

Cleavage of Purified Recombinant PIP5KIalpha -- PIP5KIalpha was incubated with caspase or buffer for 60 min at 37 °C in 20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM NaEDTA, 1 mM NaEGTA, 1 mM dithiothreitol, and 0.1 mM PMSF (caspase 3 buffer).

In Vitro PIP5K Assay-- The kinase assay buffer had a final concentration of 1 µCi of [gamma -32P]ATP, 180 µM ATP, 70 µM PI(4)P, and a 1:1 w/w ratio of PI(4)P/PS vesicles (13). The reaction was allowed to proceed at 37 °C and stopped at timed intervals. Samples were extracted with CHCl3:MeOH:HCl, spotted on thin layer chromatography (TLC) plates together with unlabeled PIP2 standards. Phospholipids were resolved with 1-propanol:H2O:NH2OH (65:15:20) and detected by autoradiography. PIP2 standard was visualized with iodine vapor.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Effects of PIP2 on Caspase Activation in Cell Extracts and in in Vitro Reconstituted Apoptosomes-- The mitochondrial pathway was activated by adding dATP to cell extracts to initiate the Apaf-1/procaspase 9/cytochrome c apoptosome cascade (17, 22, 23). In untreated cytosolic extracts, procaspase 3 was present as an inactive 32-kDa zymogen (Fig. 1A, left panel, lane 1). dATP generated a 17-kDa band corresponding to the larger caspase 3 subunit, and a decrease in the intensity of the procaspase 3 band (lane 3). Pretreatment of extracts with PIP2 completely inhibited procaspase 3 processing (lane 2).



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Fig. 1.   Effects of PIP2 and PIP3 on procaspase processing in cell-free systems. High speed supernatants of HeLa cell extracts were incubated with dATP to initiate caspase activation, and samples were blotted with anti-caspase antibodies. A, 3 µM PIP2 micelles inhibit procaspase processing. Pro, procaspase; C3, caspase 3; C9, caspase 9. The smaller caspase subunit for each caspase was not detected in cell extracts under these conditions. B, extracts were treated with 3 µM each of PIP2 micelles (M), 10% PIP2/PC vesicles (V), and PIP3 micelles and Western blotted with anti-caspase 3. C, procaspase 9 activation in an in vitro reconstituted apoptosome system. Apoptosomes were assembled by mixing purified procaspase 9 that was preincubated with 0, 5, or 20 µM PIP2 (lanes 1-3), with Apaf-1, cytochrome c, and dATP. Samples were Western blotted with anti-procaspase 9.

Since procaspase 3 is a substrate for caspase 9, the lysates were also blotted with anti-caspase 9 (Fig. 1A, right panel). Procaspase 9 (47 kDa) was detected in the naive lysate (lane 1), and dATP converted all of the procaspase 9 to a 37-kDa mature form. PIP2 blocked procaspase 9 cleavage, suggesting that PIP2 acted at the level of the initiator caspase. 3 µM PIP2/PC vesicles were as inhibitory as 3 µM PIP2 micelles (Fig. 1B, lanes 3 and 4). 3 µM PIP3 also decreased pro-caspase 3 cleavage, although to a lesser extent (lane 5). Thus, PIP2 and PIP3 inhibit caspase processing, and inhibition occurs in a physiologically relevant milieu.

PIP2 also inhibited procaspase 9 activation in an in vitro reconstituted apoptosome system (23). In the presence of Apaf-1, cytochrome c, and dATP, procaspase 9 was cleaved into two smaller polypeptides (Fig. 1C). Cleavage was dependent on Apaf-1. 5 µM PIP2 partially inhibited procaspase 9 processing, and 20 µM PIP2 inhibited it completely. PIP2 may suppress procaspase 9 activation by inhibiting caspase 9 as soon as it is processed. In this way, further autoprocessing would be blocked. The alternative possibility that PIP2 inhibits procaspase 9 binding to Apaf-1 is less likely, but has not been ruled out. The instability of purified recombinant caspase 9 in vitro (25) precluded detailed analysis of the effect of PIP2 on caspase 9 activity.

Characterization of PIP2 Interaction with Purified Caspases-- PIP2 inhibited purified caspases 8 and 3 in a dose-dependent manner (Fig. 2, A and B). We used progress curves to calculate an inhibition constant (Ki), according to the method described by Zhou et al. (24). This method is used extensively to estimate the Ki of many inhibitors of apoptosis (26, 27). Among the phosphoinositides tested, PIP2 was most potent (Table I). PIP3 inhibited with a 10-fold higher Ki than PIP2. PI(4)P had no effect (Fig. 3A), so its Ki cannot be calculated. Inositol trisphosphate, the inositol polyphosphate that is equivalent to PIP2 except that it has no diacylglycerol chain, was not inhibitory at high concentrations (>33 µM, data not shown). Thus, caspases 8 and 3 are able to discriminate between phosphoinositide stereoisomers, and caspase inhibition requires the phosphoinositol headgroup and the diacylglycerol chain. These characteristics are similar to that of some, but not all, phosphoinositide-binding proteins (28, 29). Since PIP2 is much more abundant than PIP3 (40-fold by one estimate) (30), PIP2 is likely to be the predominant inhibitor of caspases in quiescent cells, although PIP3 may also directly inhibit caspases in proliferating cells.



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Fig. 2.   Effects of PIP2 micelles on caspase 8 and 3 activities. A and B, representative progress curves for caspase 8 and caspase 3, respectively. Caspases were added to a mixture of tetrapeptide-AFC substrates and PIP2 micelles. AFC generation after substrate cleavage was plotted against time. The different symbols denote PIP2 concentrations used. A, caspase 8 (60 nM). Circles, 0 µM; squares, 1 µM; triangles, 3 µM; diamonds, 5 µM; hexagons, 20 µM. B, caspase 3 (30 nM). Symbols (from top to bottom) denote 0, 0.5, 1, 5, and 10 µM. A' and B', [(Vo/Vi- 1] was plotted as a function of PIP2 concentration. C, gelsolin competed with caspase 8 for PIP2. Caspase 8 (60 nM) was added to solutions containing Ac-IETD-AFC and 10 µM gelsolin preincubated with PIP2 micelles (10 and 20 µM, closed squares and gray diamonds; the two curves overlap) or to PIP2 micelles with no gelsolin (10 and 20 µM, open squares and diamonds). Closed circles indicate control, with caspase 8 and substrate, and no other addition. Closed triangles indicate caspase 8, substrate, and gelsolin in the absence of PIP2. D, gelsolin competed with caspase 3 for PIP2. 30 nM caspase 3 was incubated with 4 µM gelsolin and 10 µM PIP2. Symbols are as in C.


                              
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Table I
Inhibition constants (Ki) for caspases



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Fig. 3.   Effects of PIP2 vesicles on caspase 3 activity. A, 4 µM 10% PIP2/PC (triangles) inhibited caspase 3, while 20 µM PI(4)P (squares) and PS (10% PS/PC vesicles; diamonds) did not. Circles, caspase 3 in the absence of lipids. B, PIP2/PC vesicles were inhibitory at low fractional concentrations (1% (open symbols) and 4% (closed triangle) with PC). Circles, caspase 3 alone; open diamonds, circles, and squares, caspase 3 with 0.5, 5, and 10 µM PIP2 in vesicles containing 1% PIP2 and 99% PC; closed triangles, caspase 3 with 2 µM PIP2 in vesicles containing 4% PIP2 and 96% PC. C, caspase 3 binding to lipid vesicles. Mixed lipid vesicles containing 90% PC, and 10% of one of the following: PIP3, PIP2, PI(3,4)P2, PS (lanes 1-4, respectively, at a final concentration of 30 µM) were incubated with 2.5 µM caspase 3 and collected by high speed centrifugation. Equivalent fractions of supernatants and pellets were analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.

The inhibitory effect of PIP2 was reduced by gelsolin, a well characterized PIP2-binding protein (11, 28). This was demonstrated using either caspase 8 or caspase 3. Gelsolin had minimal effect on caspase 8 activity by itself (Fig. 2C). However, gelsolin prevented PIP2 from inhibiting caspase 8. We tested a range of PIP2 concentrations (5-20 µM). We consistently observed reduced caspase 8 inhibition by PIP2 in the presence of gelsolin (data not shown). Likewise, gelsolin blocked the inhibitory effect of PIP2 on caspase 3 (Fig. 2D).

Our results are different from that of Azuma et al. (12) in two respects. First, they found that PIP2 does not inhibit caspase on its own. However, PIP2 becomes inhibitory when complexed with gelsolin. Second, they reported that, although the PIP2:gelsolin complex inhibits caspase 3 and caspase 9, it did not inhibit caspase 8. Their results suggest that gelsolin may enhance PIP2 regulation of caspases, while ours indicate that gelsolin competes with caspases for PIP2. We cannot explain why our results were different. The Azuma group prepared unilamellar lipid vesicles using an extrusion technique (12) and observed no inhibition of caspase activity between 0.25 and 2 µM PIP2. They did not show results at higher doses. We used PIP2 from two other sources (Calbiochem and Roche Molecular Biochemicals), prepared micelles, and mixed vesicles by probe sonication. We found a dose-dependent inhibition of caspase activity beginning at 2 µM PIP2 presented as mixed micelles. Our PIP2 had no detectable impurities, based on a high pressure liquid chromatography analysis that can distinguish between the PIP2 from PI(3,4)P2, PI(4)P, and PIP3.2 Another potential explanation is that the different assay conditions may affect the outcome. We use a much higher gelsolin:PIP2 ratio to observe inhibition. Decreasing the gelsolin:PIP2 ratio, however, did not promote caspase activity. We have also ruled out that differences in KCl or divalent ion concentration or pH account for our different findings. Although the reason for the discrepancies between the two groups has not yet been resolved, both studies highlight the potential role of PIP2 in caspase regulation. Competition and cooperation among PIP2-binding proteins have been documented previously (31), and cross-talk between them may add another level of complexity to their regulation in vivo.

PIP2 was also inhibitory when presented to caspases in mixed vesicles with PC (Fig. 3, A and B). In contrast, 10% phosphatidylserine (PS), 90% PC vesicles and PI(4)P vesicles were not inhibitory even at high concentrations (Fig. 3A). 2 µM PIP2 was inhibitory when presented as 4% PIP2, 96% PC vesicles (Fig. 3B). PIP2 was still inhibitory at even higher dilution (1% PIP2, 99% PC), although more PIP2 was required. A similar dependence on PIP2 fractional concentration has been reported for several other PIP2-binding proteins (32).

Caspase 3 binding to PIP2 and PIP3 was demonstrated by cosedimentation with mixed lipid vesicles (Fig. 3C). Consistent with the lack of inhibition by PI(3,4)P2/PC and PS/PC, caspase 3 did not cosediment with these vesicles. Since caspases do not have recognizable pleckstrin homology domains (PH), which mediate phosphoinositide binding in many proteins (29), their PIP2 binding domains remain to be identified. PIP2-binding proteins that do not have a recognizable PH domain often bind PIP2 through lysine- and arginine-rich regions (34).3

Protection against Apoptosis by PIP5KI Overexpression-- To determine whether PIP2 inhibits caspases in vivo, we overexpressed human type I PIP5KIalpha (13) by transient overexpression and by adenovirus-mediated infection, and examined the effect on apoptosis. Apoptosis was induced by overexpression of procaspase 9 or by treatment with TNFalpha .

Overexpression of procaspase 9 (17) allowed us to bypass the upstream aspects of apoptotic signaling, and to focus on the effects of PIP2 on procaspase 9 processing and its downstream sequelae. HEK293 cells were used for transient expression studies, because they can be readily cotransfected with multiple expression vectors at high frequency. Control-transfected HEK293 cells had a low level of apoptosis (between 10% and 14%), and PIP5KIalpha overexpression had no effect on basal apoptosis. However, PIP5KIalpha overexpression reduced the percentage of apoptotic procaspase 9-transfected cells significantly (Fig. 4A). Apoptosis was assayed by nuclear condensation as visualized by DAPI staining. Similar results were obtained using morphological criteria upon cotransfection with beta -galactosidase (data not shown). In contrast, a kinase-deficient mutant (D270A) (19) did not protect against apoptosis (Fig. 4A), nor did it induce apoptosis in the absence of apoptotic stimuli (data not shown). These results confirm that the kinase activity is required for protection against apoptosis.



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Fig. 4.   PIP5KIalpha overexpression protects against apoptosis. A and B, effects of PIP5KIalpha on apoptosis in procaspase 9-overexpressing cells. A, HEK293 cells were transfected with blank or PIP5KIalpha -containing vectors and procaspase 9 (3:1 weight ratio). Twenty-four hours later, they were stained with anti-FLAG (to detect procaspase 9 expression) and with DAPI. Procaspase 9-overexpressing cells that were apoptotic were scored by the presence of condensed DAPI-stained nuclei. Background apoptosis observed in cells not transfected with procaspase 9 (between 10% and 15%) was subtracted, and the values shown are mean ± S.E. of three independent experiments. D279A is the caspase 3-resistant mutant, and D270A is the kinase-deficient mutant. Overexpression of the wild-type (wt) or mutant PIP5KIalpha had no detectable effect on apoptosis in the absence of apoptotic stimuli (data not shown). B, Western blotting. Lane 1, lysate from HEK293 cells transfected with procaspase 9; lane 2, procaspase 9 and PIP5KIalpha ; lane 3, PIP5KIalpha alone. C-9, caspase 9; C-3, caspase 3. There was no detectable variation in protein loading, based on the equivalent intensity of beta -tubulin band in these lanes (data not shown). The result shown is representative of two independent experiments. C, effects of wild-type and D279A PIP5KIalpha overexpression on apoptosis induced by treatment with TNFalpha /CHX for 3 h. PIP5KIalpha and beta -gal cDNAs were cotransfected at a 2:1 weight ratio. Left panel, transfected HEK293 cells that expressed beta -gal were detected with X-gal and examined for morphological signs of apoptosis. Data shown are the mean ± S.E. of two independent experiments.

PIP5KIalpha overexpression alters the actin cytoskeleton (18, 19) and may also have an impact on the other components of the phosphoinositide cycle. To determine whether PIP5KIalpha overexpression suppresses apoptosis by inhibiting caspases, we monitored the activation of cotransfected procaspase 9. Western blotting showed that PIP5KIalpha overexpression increased the ratio of procaspase 9 to caspase 9 from 1.9 to 8.8 (Fig. 4B, right panel). This is consistent with the attenuation of procaspase 9 processing. Moreover, downstream apoptotic events were also suppressed; more procaspase 3 and less caspase 3-digested PARP (the p85 fragment) were recovered. Thus, the apoptotic index and biochemical evidence suggest that PIP5KIalpha overexpression suppresses the activation of the apical caspase 9, and the downstream effector caspase 3 as well. These results are consistent with protection from apoptosis by PIP2 inhibition of caspases.

Transient overexpression of PIP5KIalpha in HEK293 cells also protected against apoptosis induced by TNFalpha (Fig. 4C). Since this death receptor apoptotic cascade is initiated by procaspase 8 activation, and PIP2 inhibits caspase 8 activity in vitro (Fig. 2A), the observed reduction in apoptotic index is likely to be mediated by blocking caspase 8 activation.

To estimate how much PIP2 is required to protect against apoptosis, we used adenovirus-mediated infection to introduce PIP5KIalpha . HeLa cells were used for these studies, because they are efficiently infected by adenovirus (greater than 98%), while HEK293 cells are not. High efficiency infection is required for accurate quantitation of the extent of PIP2 overproduction in the entire cell population. PIP2 synthesis, determined by thin layer chromatography of 32P-labeled phospholipids, was increased by 2.6-fold (Fig. 5A). This is accompanied by a decrease in PI(4)P, but not by an increase in PIP3.



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Fig. 5.   Effect of PIP5KIalpha overexpression on PIP3 synthesis and Akt activation. A, 32P incorporation into phospholipids, analyzed by thin layer chromatography. HeLa cells were infected with PIP5KIalpha or beta -gal adenovirus and labeled with 32P. Phospholipid standards were used to identify the 32P-labeled lipids. B, HeLa cells infected with PIP5KIalpha or beta -gal adenovirus were treated with 200 nM wortmannin or carrier (dimethyl sulfoxide) for 30 min at 37 °C. They were then exposed to TNFalpha /actinomycin D for 1.5 or 3 h. Cells were stained with DAPI, and apoptotic nuclei were scored. About 500 cells were counted for each condition per experiment. Data shown are mean ± S.E. of two independent experiments. C, Western blotting for phospho-Akt after infection of Hela cells with beta -gal or PIP5KIalpha adenovirus. Cells were serum-deprived in 0.5% fetal calf serum overnight, lysed in the presence of phosphatase inhibitors, and Western blotted with anti-Akt and anti-phospho-Akt. PDGF, platelet-derived growth factor. D, immunofluorescence localization of GFP-AktPH after insulin stimulation or coexpression with Myc-PIP5KIalpha . HEK293 cells were transfected with 0.5 µg of GFP-AktPH either alone or with 0.5 µg of Myc-PIP5KIalpha cDNA, and serum-deprived overnight. i and ii, localization of GFP-AktPH without and with 1 µM insulin for 10 min, respectively. Cells were not overexpressing PIP5KIalpha . iii and iv, localization of Myc-PIP5KIalpha and GFP-AktPH, respectively, in a starved cell cotransfected with both cDNAs.

This level of PIP2 overproduction protected HeLa cells from TNFalpha -induced apoptosis, as evidenced by the delay in the onset of apoptosis and the extent of apoptosis (Fig. 5B). The 40% decrease in apoptosis in adenovirus-infected cells is comparable to that for transiently transfected HEK293 cells (Fig. 4C). Protection by a moderate level of increased PIP2 production suggests that a physiologically attainable change in the turnover of PIP2 could affect the progression of apoptosis. In addition, the absence of a detectable increase in PIP3 production suggests that PIP5KIalpha did not act by increasing PIP3 synthesis.

The Anti-apoptotic Effect of PIP5KIalpha Was Not Mediated through PI 3-Kinase or Akt Activation-- Although we did not detect an increase in PIP3 synthesis in PIP5KIalpha overexpressing cells, it is important to use other assays to rule out the possibility that PIP5KIalpha protects against apoptosis through PIP3 and Akt.

We inhibited PI 3-kinase with wortmannin (Fig. 5B). Wortmannin did not block the anti-apoptotic effect of PIP5KIalpha . This treatment increased apoptosis in TNFalpha -treated control-infected cells, but not in PIP5KIalpha -infected cells. Therefore, the anti-apoptotic effect of PIP5KIalpha was independent of PI 3-kinase.

We monitored Akt activation by assessing Akt phosphorylation and translocation to the plasma membrane. Western blotting with a phospho-Akt-specific antibody and a pan-Akt antibody showed that there was no difference in the extent of Akt phosphorylation between beta -gal adenovirus- and PIP5KIalpha adenovirus-infected cells (Fig. 5C).

Targeting of Akt to the plasma membrane was monitored by immunofluorescence microscopy. PIP5KIalpha was cotransfected with the PH domain of Akt tagged to the green fluorescent protein (GFP-AktPH). As shown by others (35), GFP-AktPH is diffusely cytosolic in starved cells and is recruited to the plasma membrane after insulin stimulation (Fig. 5D, i and ii). Pronounced plasma membrane localization of GFP-AktPH is detected in 47.5% of the insulin-treated cells (186 cells counted). Overexpressed PIP5KIalpha is partly cytosolic, partly punctate, and partly plasma membrane-associated (Fig. 5D, iii) (18). In contrast, GFP-AktPH remains cytosolic in the starved, PIP5KIalpha -overexpressing cell (Fig. 5D, iv). Only 13.9% of the PIP5KIalpha -overexpressing cells have membrane-associated GFP-AktPH, a value indistinguishable from that of cells not overexpressing PIP5KIalpha (14.3%). Taken together, our data indicate that PIP5KIalpha does not protect against apoptosis by activating Akt. Therefore, PIP2 inhibition of caspases is a more likely primary mechanism for the protection by PIP5KIalpha .

PIP5KIalpha Cleavage during Apoptosis-- We noticed that the 68-kDa Myc-PIP5KIalpha band was consistently less intense in cells cotransfected with procaspase 9 (Fig. 4B, left panel), and that two lower molecular weight bands appeared. We therefore investigated the possibility that PIP5KIalpha is cleaved during apoptosis to generate these fragments. Staurosporine, which activates the mitochondrial apoptotic pathway, decreased the intensity of the full-length Myc-PIP5KIalpha band by 46% (Fig. 6A), and generated a 37-kDa cleavage product. This decrease was not an artifact due to unequal loading, as indicated by the comparable intensity of the alpha -tubulin bands. Cleavage of PIP5KIalpha was prevented by the cell permeant caspase 3 inhibitor z-DEVD-fmk, indicating that it was mediated by caspase 3. 



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Fig. 6.   PIP5KIalpha is a caspase 3 substrate in vivo and in vitro. A, cleavage of overexpressed and endogenous Myc-PIP5KIalpha . Left panel, HeLa cells transfected with wild-type or caspase 3-resistant (D279A) Myc-PIP5KIalpha were treated with staurosporine for 6 h in the absence or presence of z-DEVD-fmk. Cell lysates were analyzed by Western blotting with anti-Myc and anti-alpha tubulin. Right panel, HeLa cells (not transfected) were treated with TNFalpha /CHX for 6 h. Endogenous PIP5KIalpha was detected with affinity-purified anti-PIP5KIalpha . B, cleavage of overexpressed and endogenous PIP5KIalpha by caspase 3 in cell extracts. Extracts prepared from cells transfected with Myc-PIP5KIalpha and from untransfected HeLa cells were incubated with recombinant caspase 3 for 60 min at 37 °C, in the presence or absence of 50 µM z-DEVD-fmk.

Endogenous PIP5KIalpha was also degraded during apoptosis. TNFalpha /CHX decreased the intensity of the 65-kDa PIP5KIalpha band by 36% (Fig. 6A). A 37-kDa band was generated, and this band was not observed when apoptosis was blocked with z-DEVD-fmk.

Caspase 3 Cleavage of PIP5KIalpha -- The involvement of caspase 3 in PIP5KIalpha cleavage was confirmed by adding recombinant caspase 3 to HeLa extracts prepared from cells transfected with Myc-PIP5KIalpha and from untransfected cells (Fig. 6B). In both cases, the major product has the same electrophoretic mobility as the band generated in apoptotic cells (Fig. 6A) and cleavage was blocked by z-DEVD-fmk. These results establish that PIP5KIalpha is cleaved by a caspase 3-dependent pathway during apoptosis. PIP5KIalpha was cleaved more extensively in the in vitro conditions than in apoptotic cells. This may be because, in intact cells, only a fraction of the total PIP5KIalpha is accessible to caspases.

PIP5KIalpha has a DIPDG sequence (residues 276-280) in its kinase core that conforms to the caspase 3 p1DXXDp4 cleavage consensus. We mutated Asp-279 to Ala to determine whether it is part of a bona fide caspase 3 cleavage site. At the lowest caspase 3 dose used, wild-type PIP5KIalpha was already partially cleaved into two major fragments (37 and 28 kDa) (Fig. 7A, lane 2). In contrast, the D279A mutant was resistant to caspase 3 even at a 10-fold higher concentration (lane 3). Furthermore, this mutant, when overexpressed in HeLa cells, was not detectably cleaved in staurosporine- or TNF/CHX-treated cells (Fig. 6A).



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Fig. 7.   Caspase 3 cleaves and inactivates PIP5KIalpha . A, identification of the caspase 3 cleavage site. Purified, recombinant His-tagged wild-type (wt) and D279A PIP5KIalpha were incubated with caspase 3 for 60 min at 37 °C, and Western blotted with anti-PIP5KIalpha . Lane 1, untreated hPIP5KIalpha ; lanes 2 and 3, with caspase 3, 10-fold higher amount in lane 3 than in lane 2; lane 4, same amount of caspase 3 as in lane 3, but with no PIP5KIalpha . B, in vitro kinase assay. PIP5KIalpha was incubated with caspase 3 or buffer for 60 min at 37 °C. Digestion of PIP5KIalpha was monitored by Western blotting (data not shown). The digested and undigested kinase were incubated with PI(4)P and [gamma -32]ATP at 37 °C, and reaction was stopped at timed intervals. 32P-Labeled PIP2 was detected by autoradiography of a thin layer chromatogram (top panel). Radioactivity associated with PIP2 was determined by scintillation counting and plotted as a function of time (bottom panel). Open circles, without (w/o) caspase 3; closed circles, with (w/) caspase 3. Results shown are representative of three independent determinations.

Caspase cleavage of regulatory proteins often results in a loss or gain of function (36). PIP5KIalpha that was partially digested by caspase 3 had decreased kinase activity (Fig. 7B) in an in vitro kinase assay. The rate of [32P]PIP2 generation, determined from the slope of the linear portion of the activity curve, was reduced by 50%. The decrease in activity correlates with the 44% cleavage of the PIP5KIalpha sample used (estimated by densitometry of a Western blot similar to that shown in Fig. 7A; actual data not shown). These results establish that caspase 3 cleavage of PIP5KIalpha causes a loss-of-function. Sequence analyses show that this cleavage site is conserved in the equivalent mouse isoform (mouse PIP5KIbeta ; note that the nomenclature for the human and mouse isoforms are reversed (Ref. 15)). It is not present in the other two known type I PIP5K isoforms, nor is it present in the other major class of phosphoinositide kinases, the type II kinases (37). Modeling from the crystal structure of a type II kinase (37) reveals that the DIPD site in PIP5KIalpha is likely to be on a solvent-exposed surface that is part of the conserved ATP-binding core of the type I and type II kinases.

Caspase 3 Cleavage of PIP5KIalpha Promoted Apoptosis-- Apoptosis induces the cleavage/inactivation of many proteins involved in signaling cell survival, but the physiological significance of these changes has not been established in most cases. At one extreme, some of these proteins may be merely innocent bystanders that happen to be cleaved during the execution phase of apoptosis, when the process is already irreversible. To determine whether PIP5KIalpha cleavage is an integral part of apoptotic signaling, we compared the ability of wild-type myc-PIP5KIalpha and the D279A mutant to suppress procaspase 9 overexpression-induced or TNFalpha /CHX-induced apoptosis. In each case, apoptosis was suppressed considerably more by the D279 mutant than by the wild-type PIP5KIalpha (Fig. 4, A and C). Western blotting showed that these two isoforms were expressed at comparable levels (Fig. 4D). Therefore, PIP5KIalpha inactivation by caspase 3 decreases its ability to protect against apoptosis, and this may promote the initiation/progression of the apoptotic cascade.

In conclusion, our results show that PIP2 is a direct inhibitor of initiator and effector caspases. PIP2 inhibits caspases at low absolute concentrations and at high dilutions in mixed lipid vesicles. PIP2 accounts for between 0.4% and 1% of total membrane lipids in cells (30), and PIP2 concentration in the plasma membrane is estimated to be between 4 and 10 µM. Local concentrations of PIP2 in membrane may be even higher, since lipids are differentially partitioned in membrane microdomains (38). PIP2 is therefore likely to be present at a high enough concentration to inhibit some caspases in cells. Additional studies will be required to determine how much caspase is associated with PIP2 in nonapoptotic and apoptotic cells. Although early studies suggest that caspases are predominantly cytosolic, recent studies show that some caspases are associated with mitochondrial, microsome, or nuclear fractions, and that they redistribute during apoptosis (39-41). Caspase binding to PIP2 may account for some of this association with intracellular organelles, which contain PIP2 (34).

The broad spectrum of caspase inhibition by PIP2 is different from that of previously identified endogenous protein inhibitors of apoptosis (such as XIAP and cIAP) (26, 42), and approaches that of the baculovirus protein p35 (24, 43), which has no mammalian counterpart. The importance of clamping caspase activity to a minimal level in normal living cells is underscored by the fact that caspase activation triggers a self-amplifying autocatalytic cascade and by the existence of multiple checkpoints for caspase activation (33, 36). Caspase inhibition may help to establish a threshold for apoptosis and to fine tune the balance between survival and death. The threshold may be lowered by caspase inactivation of PIP5KIalpha ; this would dissipate the pro-survival PIP2, release the PIP2 clamp on caspases, and tip the balance toward cell death.


    ACKNOWLEDGEMENTS

We thank the following scientists for their help in this work: R. A. Anderson for the rabbit anti-PIP5KIalpha antibody, C. S. Chen for phosphoinositides, E. S. Alnemri (Thomas Jefferson Medical School, Philadelphia, PA) for the pCMV2-FLAG-procaspase 9 cDNA, T. Balla (NICHHD, National Institutes of Health, Bethesda, MD) for the PH-AktPH cDNA, Y. Shibasaki for the adenovirus containing PIP5KI, and L. Cantley (Harvard Medical School, Cambridge, MA) for sharing information about the kinase-deficient PIP5KI mutant prior to publication.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM61203 and AR41940 and by Robert Welch Foundation Grant I-1200.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed. Fax: 214-648-7891; E-mail: helen.yin@utsouthwestern.edu.

Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007271200

2 D. T. Hilgemann, personal communication.

3 L. Feng, M. Mejillano, H. L. Yin, J. Chen, and G. D. Prestwich, unpublished work.


    ABBREVIATIONS

The abbreviations used are: PIP3, phosphatidylinositol 3,4,5-triphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4)P, phosphatidylinositol 4-phosphate; PIP5KI, phosphatidylinositol phosphate 5-kinase type I; fmk, fluoromethylketone; beta -gal, beta -galactosidase; PC, phosphatidylcholine; PS, phosphatidylserine; GFP, green fluorescent protein; CHX, cycloheximide; PMSF, phenylmethylsulfonyl fluoride; TNFalpha , tumor necrosis factor alpha ; PARP, poly (ADP-ribose) polymerase; AFC, 7-amino-4-trifluoromethyl coumarin; DAPI, 4,6-diamidino-2-phenylindole.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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