From the Departments of 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
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ABSTRACT |
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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 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 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 PIP5KI Adenovirus containing PIP5KI 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) 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 TNF 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 PIP5KI Microscopy--
Apoptotic index was determined using DAPI
staining or PIP5KI Cleavage of Purified Recombinant PIP5KI In Vitro PIP5K Assay--
The kinase assay buffer had a final
concentration of 1 µCi of [ 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).
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.
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 PIP5KI
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 PIP5KI
PIP5KI
Transient overexpression of PIP5KI
To estimate how much PIP2 is required to protect against
apoptosis, we used adenovirus-mediated infection to introduce
PIP5KI
This level of PIP2 overproduction protected HeLa cells from
TNF The Anti-apoptotic Effect of PIP5KI
We inhibited PI 3-kinase with wortmannin (Fig. 5B).
Wortmannin did not block the anti-apoptotic effect of PIP5KI
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
Targeting of Akt to the plasma membrane was monitored by
immunofluorescence microscopy. PIP5KI PIP5KI
Endogenous PIP5KI Caspase 3 Cleavage of PIP5KI
PIP5KI
Caspase cleavage of regulatory proteins often results in a loss or gain
of function (36). PIP5KI Caspase 3 Cleavage of PIP5KI
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 PIP5KI (PIP5KI
), which synthesizes PIP2, suppressed apoptosis,
whereas a kinase-deficient mutant did not. Protection by the wild-type
PIP5KI
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 PIP5KI
was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KI
in vitro. Mutation of the P4 position
(D279A) of the PIP5KI
caspase 3 cleavage consensus prevented
cleavage in vitro, and during apoptosis in
vivo. Significantly, the caspase 3-resistant PIP5KI
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 PIP5KI
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(PIP5KI
) (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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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 PIP5KI
(Ref. 19)) were generated
using a QuickChange site-directed mutagenesis kit (Stratagene). The
PIP5KI
cDNA constructs were subcloned into pET-28c(+) (Novagen)
with a hexahistidine tag at the 5' end. Recombinant PIP5KI
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).
or
-gal were used to infect cells
according to protocol described by Shibaski et al. (21).
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.
. 10 ng/ml cycloheximide (CHX) or 0.2 µg/ml actinomycin D was added to
enhance the apoptotic effect of TNF
. 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.
were detected with an affinity purified rabbit
anti-PIP5KI
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).
-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.
-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.
Digestion by Caspases
--
PIP5KI
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).
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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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.
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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.
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.
(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 TNF
.
overexpression had no effect on basal apoptosis. However, PIP5KI
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
-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.
PIP5KI
overexpression protects against apoptosis. A and
B, effects of PIP5KI
on apoptosis in procaspase
9-overexpressing cells. A, HEK293 cells were transfected
with blank or PIP5KI
-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 PIP5KI
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 PIP5KI
; lane 3,
PIP5KI
alone. C-9, caspase 9; C-3, caspase 3. There was no detectable variation in protein loading, based on the
equivalent intensity of
-tubulin band in these lanes (data not
shown). The result shown is representative of two independent
experiments. C, effects of wild-type and D279A PIP5KI
overexpression on apoptosis induced by treatment with TNF
/CHX for
3 h. PIP5KI
and
-gal cDNAs were cotransfected at a 2:1
weight ratio. Left panel, transfected HEK293
cells that expressed
-gal were detected with X-gal and examined for
morphological signs of apoptosis. Data shown are the mean ± S.E.
of two independent experiments.
overexpression alters the actin cytoskeleton (18, 19) and may
also have an impact on the other components of the phosphoinositide
cycle. To determine whether PIP5KI
overexpression suppresses
apoptosis by inhibiting caspases, we monitored the activation of
cotransfected procaspase 9. Western blotting showed that PIP5KI
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 PIP5KI
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.
in HEK293 cells also protected
against apoptosis induced by TNF
(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.
. 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 PIP5KI
overexpression on PIP3 synthesis and Akt
activation. A, 32P incorporation into
phospholipids, analyzed by thin layer chromatography. HeLa cells were
infected with PIP5KI
or
-gal adenovirus and labeled with
32P. Phospholipid standards were used to identify the
32P-labeled lipids. B, HeLa cells infected with
PIP5KI
or
-gal adenovirus were treated with 200 nM
wortmannin or carrier (dimethyl sulfoxide) for 30 min at 37 °C. They
were then exposed to TNF
/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
-gal or
PIP5KI
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-PIP5KI
. HEK293 cells were transfected with 0.5 µg of
GFP-AktPH either alone or with 0.5 µg of Myc-PIP5KI
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 PIP5KI
. iii
and iv, localization of Myc-PIP5KI
and GFP-AktPH,
respectively, in a starved cell cotransfected with both
cDNAs.
-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 PIP5KI
did not act by increasing
PIP3 synthesis.
Was Not Mediated through PI
3-Kinase or Akt Activation--
Although we did not detect an increase
in PIP3 synthesis in PIP5KI
overexpressing cells, it is
important to use other assays to rule out the possibility that
PIP5KI
protects against apoptosis through PIP3 and Akt.
. This
treatment increased apoptosis in TNF
-treated control-infected
cells, but not in PIP5KI
-infected cells. Therefore, the
anti-apoptotic effect of PIP5KI
was independent of PI 3-kinase.
-gal
adenovirus- and PIP5KI
adenovirus-infected cells (Fig.
5C).
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 PIP5KI
is
partly cytosolic, partly punctate, and partly plasma
membrane-associated (Fig. 5D, iii) (18). In
contrast, GFP-AktPH remains cytosolic in the starved, PIP5KI
-overexpressing cell (Fig. 5D, iv). Only
13.9% of the PIP5KI
-overexpressing cells have membrane-associated
GFP-AktPH, a value indistinguishable from that of cells not
overexpressing PIP5KI
(14.3%). Taken together, our data indicate
that PIP5KI
does not protect against apoptosis by activating Akt.
Therefore, PIP2 inhibition of caspases is a more likely
primary mechanism for the protection by PIP5KI
.
Cleavage during Apoptosis--
We noticed that the
68-kDa Myc-PIP5KI
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 PIP5KI
is cleaved during apoptosis to generate these fragments. Staurosporine, which activates the mitochondrial apoptotic pathway, decreased the intensity of the full-length Myc-PIP5KI
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
tubulin
bands. Cleavage of PIP5KI
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.
PIP5KI is a caspase
3 substrate in vivo and in
vitro. A, cleavage of overexpressed and
endogenous Myc-PIP5KI
. Left panel, HeLa cells
transfected with wild-type or caspase 3-resistant (D279A) Myc-PIP5KI
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-
tubulin. Right panel, HeLa
cells (not transfected) were treated with TNF
/CHX for 6 h.
Endogenous PIP5KI
was detected with affinity-purified
anti-PIP5KI
. B, cleavage of overexpressed and endogenous
PIP5KI
by caspase 3 in cell extracts. Extracts prepared from cells
transfected with Myc-PIP5KI
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.
was also degraded during apoptosis. TNF
/CHX
decreased the intensity of the 65-kDa PIP5KI
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.
--
The involvement of caspase 3 in PIP5KI
cleavage was confirmed by adding recombinant caspase 3 to
HeLa extracts prepared from cells transfected with Myc-PIP5KI
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 PIP5KI
is cleaved by a
caspase 3-dependent pathway during apoptosis. PIP5KI
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 PIP5KI
is accessible to caspases.
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 PIP5KI
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
PIP5KI . A, identification of
the caspase 3 cleavage site. Purified, recombinant His-tagged wild-type
(wt) and D279A PIP5KI
were incubated with caspase 3 for
60 min at 37 °C, and Western blotted with anti-PIP5KI
.
Lane 1, untreated hPIP5KI
; 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 PIP5KI
. B,
in vitro kinase assay. PIP5KI
was incubated with caspase
3 or buffer for 60 min at 37 °C. Digestion of PIP5KI
was
monitored by Western blotting (data not shown). The digested and
undigested kinase were incubated with PI(4)P and
[
-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.
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
PIP5KI
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 PIP5KI
causes a
loss-of-function. Sequence analyses show that this cleavage site is
conserved in the equivalent mouse isoform (mouse PIP5KI
; 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 PIP5KI
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.
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 PIP5KI
cleavage is an integral part of apoptotic
signaling, we compared the ability of wild-type myc-PIP5KI
and the
D279A mutant to suppress procaspase 9 overexpression-induced or
TNF
/CHX-induced apoptosis. In each case, apoptosis was suppressed
considerably more by the D279 mutant than by the wild-type PIP5KI
(Fig. 4, A and C). Western blotting showed that
these two isoforms were expressed at comparable levels (Fig.
4D). Therefore, PIP5KI
inactivation by caspase 3 decreases its ability to protect against apoptosis, and this may
promote the initiation/progression of the apoptotic cascade.
; 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-PIP5KI
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;
-gal,
-galactosidase;
PC, phosphatidylcholine;
PS, phosphatidylserine;
GFP, green fluorescent
protein;
CHX, cycloheximide;
PMSF, phenylmethylsulfonyl fluoride;
TNF
, tumor necrosis factor
;
PARP, poly (ADP-ribose) polymerase;
AFC, 7-amino-4-trifluoromethyl coumarin;
DAPI, 4,6-diamidino-2-phenylindole.
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