From the Department of Chemistry and Biochemistry, Revelle College
and School of Medicine, University of California, San Diego,
La Jolla, California 92093-0601
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INTRODUCTION |
Cytosolic phospholipase A2
(cPLA2)1 has
unique structural and regulatory properties within the PLA2
superfamily (1, 2). Current widespread interest in the cytosolic
phospholipase A2 stems from its putative role as a key
enzyme involved in the inflammatory response, a provider of arachidonic
acid, the precursor for prostaglandins and leukotrienes (3, 4). This
enzyme was also implicated in the regulation of several other processes
such as platelet activation, cell proliferation, and the generation of
several second messengers (3-5). A mobilization of intracellular
Ca2+ was implicated in the translocation of
cPLA2 to cellular membranes resulting in the subsequent
specific liberation of arachidonic acid from the sn-2
position of phospholipids (6, 7). This scenario of activation of
cPLA2 was validated by the discovery that the N terminus of
this enzyme contains an autonomous calcium and lipid binding
domain, CaLB (7), homologous to the C2 domain (8) of protein kinase C
and several other proteins shown to associate with lipid membranes in a
Ca2+-dependent fashion (7, 9). Both the native
enzyme and its CaLB domain were shown to associate with fragments of
cellular membranes and synthetic lipid vesicles at physiologically
relevant Ca2+ concentrations (6, 7, 10, 11). Thus far, the
CaLB domain is the only recognized regulatory domain of
cPLA2 (5).
It was noted early on (12) that several anionic phospholipids
including phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2) activated cPLA2. This
stimulatory effect was hypothesized to result from an enhancement of
the partitioning of cPLA2 into lipid membranes caused in
general by all anionic lipids. We have now employed an approach that
was specifically designed for the characterization of proteins that
bind to membranes through multiple attachment points to demonstrate
that cPLA2 binds in a 1:1 stoichiometry with high affinity
and specificity to PtdIns(4,5)P2 in lipid vesicles, and the
effect is quite distinct from that of other anionic lipids. Furthermore, the resulting increase in membrane affinity is accompanied by a quantitative increase in enzymatic activity. In addition, an
apparent functional similarity between cPLA2 and
phospholipase C
1 (PLC
1) now allows us to
propose the location of a pleckstrin homology (PH) domain in
cPLA2.
It is now recognized that many of the key proteins involved in signal
transduction possess multimodular structures (13). Already hundreds of
multimodular proteins have been identified that contain domains such as
the PH (14, 15), C1 (16), C2 (8, 16), src homology-2 (13),
and src homology-3 (13) domains. These modules can
specifically bind certain membrane proteins (src homology-2,
src homology-3, C2, and PH domains) and specific membrane
lipids (C1, C2, and PH domains). This fact highlights the need for
studies of multimodular membrane-binding proteins that take into
account the specific properties of the interactions occurring within
the two-dimensional surface of cellular membranes (17) as demonstrated
here for cPLA2.
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EXPERIMENTAL PROCEDURES |
Materials--
PtdIns(4,5)P2 and Triton X-100 as
well as horseradish peroxidase-coupled anti-rabbit IgG antibody were
purchased from Calbiochem. D-myo-Inositol-1,4,5-triphosphate
(Ins(1,4,5)P3) and
D-myo-inositol-1,3,4,5-tetraphosphate (Ins(1,3,4,5)P4) were purchased from Cayman Chemical Co.
(Ann Arbor, MI).
L-
-1-Palmitoyl-2-(arachidonoyl)phosphatidylcholine (PAPC) (52 mCi/mmol) was provided by NEN Life Science Products. All
other phospholipids were supplied by Avanti (Birmingham, AL). Salts and
buffers, purchased from Fisher, listed the actual lot analysis
indicating the amount of contaminating Ca2+. Fatty
acid-free bovine serum albumin (BSA) was purchased from Sigma. EGTA was
supplied by Fluka. Immobilon-P membrane was purchased from Millipore.
The enhanced chemiluminescence kit and the HyperFilm were supplied by
Amersham Corp. Purified cPLA2, both the wild type (18) and
the Ser-228
Ala mutant (19), as well as a monoclonal antibody
against cPLA2 (10), were a generous gift of Drs. Ruth M. Kramer and John D. Sharp, Lilly. Phosphatidylinositol 3-phosphate
(PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol
3,4,5-trisphosphate (PtdIns(3,4,5)P3) (20) were generous
gifts of Dr. Ching-Shih Chen, University of Kentucky (Lexington,
KY).
Binding Assay--
The association of cPLA2 with
large unilamellar vesicles (LUVs) (pore diameter 0.2 µm) was
quantitated in a sucrose-loaded vesicle assay originally devised for
the study of PLC
1 (21). In brief, LUVs were produced by
extrusion (22) in 170 mM sucrose, 20 mM PIPES,
pH 6.8. The lipid composition varied depending on the experiment.
Subsequently, the LUVs were suspended in 5 volumes of solution
containing 100 mM KCl, 20 mM PIPES, pH 6.8 (referred to hereafter as the "standard binding solution"), and
centrifuged at 150,000 × g for 15 min at 25 °C. The
supernatant was discarded, and the pellet was resuspended in KCl/PIPES
buffer. Enzyme (0.5 µg/ml) and lipid vesicles (0.1 to 1 mM total lipid) were suspended in KCl/PIPES buffer with 0.1 mg/ml BSA, 0.5 mM EGTA and CaCl2 at a
concentration required to achieve the desired concentration of free
ion, calculated according to a published algorithm (23). This
suspension was allowed to equilibrate for 10 min and was then
centrifuged again for 15 min at 25 °C at 150,000 × g. The contents of each tube were separated into a top (75%
total volume) and a bottom fraction. To ensure identical conditions
during deposition of the sample on the Immobilon-P membrane, both
fractions were restored to the original volume (0.75 ml) and the
composition of the original sample, including the lipid but not the
enzyme content. Equal aliquots of these fractions, as well as of
several protein suspensions of known concentration, 0.30 ml each, were spotted on an Immobilon-P membrane and subsequently immunoblotted and
detected according to a standard enhanced chemiluminescence protocol
(Amersham Corp.). The signal was quantitated by scanning densitometry
and converted into protein mass using cPLA2 standards included in the same Immobilon-P membrane. The amount of protein associated with vesicles was calculated as described elsewhere (24). As
quantitated by liquid scintillation counting of 14C-labeled
PAPC, routinely included in all lipid mixtures, approximately 95% of
the lipid was found in the bottom fraction. The apparent membrane
affinity of cPLA2, Kapp, used to
quantitate the association of this enzyme with vesicles is defined as
the reciprocal of the total lipid concentration at which 50% of the
protein is associated with vesicles. Due to a large excess of lipid
over protein in the experiments reported here, this quantity can be
treated as a molar partition coefficient of the protein into lipid
(25).
Activity Assay--
The activity of cPLA2 toward
PAPC was quantitated in a modified Dole assay (26) as described
previously (27). LUVs were prepared by extrusion, using the same
procedure as described in the binding assay, using either standard
binding solution (with the addition of BSA at 1 mg/ml) or "standard
micelle assay solution" depending on the comparisons necessary;
however, they contained a larger fraction of 14C-labeled
PAPC, to ensure approximately 200,000 cpm per assay sample.
Triton X-100/phospholipid mixed micelles were prepared by adding an
appropriate aliquot of the detergent to a suspension of multilamellar
lipid vesicles to achieve the final total (lipid + detergent)
concentration of 4 mM in the assay. Unless indicated otherwise, the reaction was carried out at 40 °C in a solution containing 100 mM KCl, 0.2 mM
CaCl2, 1 mg/ml BSA, and 1 mM dithiothreitol (Cleland's reagent), 20 mM HEPES, pH 7.5, the standard
micelle assay solution. The dependence of the activity on the
cPLA2 concentration was found to be linear in both vesicles
and micelles.
The kinetics of cPLA2 activity were analyzed using the dual
phospholipid kinetic model, Equation 1 (17, 28),
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(Eq. 1)
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where Xs is the molar fraction of the
substrate; S is the bulk concentration of phospholipid,
Ks is the dissociation constant from the interface,
calculated per phospholipid only; Vmax is the
maximal rate of hydrolysis, and Km is the
interfacial Michaelis constant.
When the concentration of the substrate in the interface is far below
saturation (Xs
Km) this
model can be simplified to Equation 2,
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(Eq. 2)
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where the enzymatic efficiency Eff = Vmax/Km and the fraction of
the enzyme associated with micelles, Xa = S/(Ks + S).
Secondary Structure Prediction--
The secondary structure was
predicted using three different algorithms incorporated into the
program DNAsis (Hitachi Software Engineering America, Ltd., San
Francisco, CA) or using the service offered by the Protein Design
Group, EMBL (Heidelberg, Germany).
Data Analysis--
Unless indicated otherwise, experiments were
performed in two independent sets, each in duplicate. Results are
presented as the mean ± S.D. (not shown when the bar size is
smaller than the symbol). Kinetic and binding models were fitted to the
data with a weighted least-squares procedure. All other lines shown
were smoothed curves through the data to allow for ready visual
comparisons. Although all results are internally consistent for each
figure and table, the range of PtdIns(4,5)P2 activation of
cPLA2 varies between Tables II and III and Fig. 6 for
several reasons. (i) The intrinsic activity of cPLA2 on
PAPC varies between pure PAPC vesicles and TX-100 mixed micelles. (ii)
The enzyme undergoes a gradual decrease in specific activity over
storage time. (iii) PtdIns(4,5)P2 also degrades over
storage time and is light-sensitive. (iv) Some experiments resulted in
as high as 25% substrate depletion due to the dramatic activation of
PtdIns(4,5)P2 which tended to lower the apparent fold
activation.
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RESULTS |
Membrane Association--
We investigated the effect of
PtdIns(4,5)P2, phosphatidylethanolamine (PE),
phosphatidylinositol (PI), and phosphatidylserine (PS) on the
association of recombinant human cPLA2 with LUVs composed primarily of PAPC. From among this group of lipids,
PtdIns(4,5)P2 proved to be particularly effective at
increasing vesicle association. To avoid alterations in the chemical
composition of the vesicles over the time course of the binding
experiment, binding studies were performed using a Ser-228
Ala
mutant of cPLA2, which is devoid of enzymatic activity
(19). The affinity of this mutant for PAPC vesicles with or
without 0.3 mol % PtdIns(4,5)P2 was the same as the
native protein (data not shown). Fig. 1
demonstrates that as little as 0.1 mol %
PtdIns(4,5)P2 was sufficient to cause a measurable increase
in the association of the enzyme with LUVs. Furthermore, at higher
mol % PtdIns(4,5)P2, the ratio of membrane-bound to free
enzyme increased linearly with the mole fraction of
PtdIns(4,5)P2 in the membrane, i.e. 1 order of
magnitude increase in mol % PtdIns(4,5)P2 was accompanied
by a numerically identical increase in the membrane affinity. This
suggests that the enzyme and PtdIns(4,5)P2 form a 1:1
complex at the membrane interface. The simplest model of binding to a
specific ligand at the interface (25, 29, 30) is shown in Equation 3,
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(Eq. 3)
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where K1 is the experimentally determined
value of the membrane association constant in the absence of
PtdIns(4,5)P2, Xp is the mol %
PtdIns(4,5)P2 in the membrane, and Kd is the interfacial dissociation constant for the
PtdIns(4,5)P2/enzyme complex. A fit of Equation 3 for to
the dependence of membrane association of cPLA2 on mol %
PtdIns(4,5)P2 (Fig. 1) yielded a Kd of
0.05 ± 0.02 mol %. In other words, just 1 molecule of
PtdIns(4,5)P2 per 2,000 lipid molecules in the membrane
(~360,000 lipid molecules per LUV) is sufficient to double the
affinity of cPLA2 for the lipid bilayer.

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Fig. 1.
PtdIns(4,5)P2 increases the
affinity of cPLA2 for lipid vesicles. The membrane
affinity was quantitated using the apparent membrane association
constant, Kapp, defined as the reciprocal of the
total lipid concentration required for half-maximal association of the
protein with the membrane. The experiment was performed in the standard
binding solution at 2 µM free Ca2+ under
conditions described under "Experimental Procedures." Large unilamellar vesicles were composed of PAPC and indicated mol % PtdIns(4,5)P2. Each point represents the average of at
least two independent determinations at two different phospholipid
concentrations (1 and 0.2 mM, except for the point at 3 mol % PtdIns(4,5)P2 which used 0.5 and 0.1 mM
phospholipid). Due to the large excess of lipid over protein, the ratio
of membrane-bound to free enzyme was proportional to the total lipid
concentration for all lipid compositions used. The line represents a
fit of Equation 3 to the data. The interfacial dissociation constant
for the PtdIns(4,5)P2-enzyme complex, Kd = 0.05 ± 0.02 mol %, was determined from the fit of
Kapp to the mol % PtdIns(4,5)P2 in
the membrane, Xp. The experimentally determined
value of the membrane association constant in the absence of
PtdIns(4,5)P2, K1 = 550 ± 80 M 1, was used in this fit.
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Inositol (1,4,5)-trisphosphate, a polar moiety of
PtdIns(4,5)P2 liberated in the cell by PLC-catalyzed
hydrolysis (31), had no apparent effect on the
PtdIns(4,5)P2-induced increase in membrane affinity for up
to a 10-fold molar excess over this phospholipid (data not shown). As
illustrated in Table I, the high affinity binding of cPLA2 to PtdIns(4,5)P2 was
sufficient to cause a measurable association with
PAPC/PtdIns(4,5)P2 vesicles in the presence of EGTA
([Ca2+] <2 nM). As in the presence of
Ca2+, the affinity of cPLA2 for
PAPC/PtdIns(4,5)P2 vesicles increased linearly with mol %
PtdIns(4,5)P2. In contrast to PtdIns(4,5)P2, PI
displayed little effect on the association of the enzyme with lipid
vesicles even at a 10-fold higher mol % than
PtdIns(4,5)P2 (Table I). Lack of any appreciable effect of
a mono-anionic lipid, PI, on the Ca2+-induced association
of cPLA2 with lipid membrane was consistent with the lack
of the effect of another anionic lipid, PS, on the Ca2+
dependence of cPLA2 binding to lipid vesicles reported
earlier (11). We have decided to compare the effect of PS on the
association of cPLA2 under experimental conditions
identical to those used in characterization of the interaction of this
phospholipid with the Ca2+-dependent protein
kinase C (24), another enzyme that associates with lipid membranes in a
Ca2+-dependent manner (9, 32). Both of these
proteins share a homologous CaLB/C2 domain (7, 33) shown to associate
with lipid membranes in the presence of Ca2+ (11).
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Table I
Effect of various phosphatidylinositols, phosphatidylethanolamine, and
free [Ca2+] on the affinity of cPLA2 for PAPC
vesicles
Total lipid concentration was 1 mM. Vesicles were composed
of mixtures of PAPC with the indicated mol % of the added
phospholipid.
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To allow for a direct comparison, we studied cPLA2 under
conditions that were identical to those employed with the
Ca2+-dependent protein kinase C,
i.e. using vesicles composed of POPC:PS mixtures. As
illustrated in Fig. 2, the fraction of
vesicle-bound enzyme declined with an increasing mol % PS. Thus, in
contrast to the case with PtdIns(4,5)P2, the mono-anionic
lipids, PI and PS, displayed little or even an opposite effect on the
association of cPLA2 with lipid vesicles. Surprisingly,
under the same experimental conditions, the membrane affinity of
cPLA2 for vesicles composed of a 1:1 mixture of PAPC:PE was
about 6-fold that of vesicles composed entirely of PAPC (Table I).

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Fig. 2.
PS decreases the binding of cPLA2
to lipid membranes. Large unilamellar vesicles were composed of
the indicated mol % of PS in POPC. The fraction of vesicle-associated
enzyme was determined as described under "Experimental Procedures."
The standard binding solution was used at 0.2 mM
CaCl2 and a total lipid concentration of 1 mM.
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Activity--
We have also tested the effect of
PtdIns(4,5)P2, PI, PS, and PE on the activity of the enzyme
toward PAPC in the same vesicles as used in the binding assay (Fig.
3). The presence of modest mol %
PtdIns(4,5)P2 in lipid vesicles resulted in a substantial increase of enzymatic activity of cPLA2 (Fig. 3). Notably,
this increase was much higher than could result solely from a
PtdIns(4,5)P2-induced increase in the fraction of the
enzyme associated with lipid vesicles. The percentage of
membrane-associated enzyme rose from about 30% to about 95% when the
mol % PtdIns(4,5)P2 rose from 0 to 3%. In contrast, the
activity rose by a factor of 55 (Fig. 3). At Ca2+ levels
approximating those of a quiescent cell, 70 nM, 1 mol % PtdIns(4,5)P2 caused an increase in cPLA2
activity from 7 ± 2 to 260 ± 80 nmol/min/mg. This increase
was equally dramatic as that seen at 2 µM
Ca2+ (Fig. 3). The presence of 1 mol %
PtdIns(4,5)P2 was sufficient to elicit measurable enzymatic
activity in the absence of exogenous Ca2+ (Fig.
4A). The activity under these
conditions was linear with time. In contrast, at 2 µM
free Ca2+ the linear character of the activity was
sustained only for the first few minutes of the reaction (Fig.
4B). Note, however, that as was the case for
PtdIns(4,5)P2, the Ca2+-induced increase in
enzymatic activity of cPLA2 (Fig. 4, A versus B)
was about 10-fold larger than the increase in the percentage of
membrane-bound enzyme, from about 10 to just over 90% (Table I and
Fig. 1). The same time course was observed at a 20 times lower enzyme
concentration (data not shown).

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Fig. 3.
The rate of hydrolysis catalyzed by
cPLA2 increases with the mol % PtdIns(4,5)P2
in the membrane. Large unilamellar vesicles were composed of the
indicated mol % PtdIns(4,5)P2 in PAPC. The standard
activity assay was performed using the standard binding solution at 2 µM free Ca2+ and 1 mM total
lipid. To ensure that the progress of hydrolysis remained in the linear
region, all assays were performed for 1 and 2 min, each in duplicate.
Each point represents mean ± S.D. The solid line
represents the fit of a single-site activator model to the data with
Amax = 17 ± 3 µmol min 1
mg 1 and C50 = 6.4 ± 1.4 mol %.
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Fig. 4.
Hydrolysis at low and high
[Ca2+]. Hydrolysis was measured using the standard
binding solution with 0.5 mM EGTA ([Ca2+] <2
nM) (A) or 2 µM free
Ca2+ (B) at 25 °C and 1 mM total
lipid, 1:99 PtdIns(4,5)P2:PAPC in the forms of LUVs.
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Interestingly, the rate of hydrolysis did not diminish continuously
over time. For times longer than 10 min, the rate appeared to achieve
another steady value at approximately 1/3 that of the rate in the first
2 to 3 min. Nevertheless, the experimental
condition-dependent activity made it difficult to study the
kinetics and lipid specificity in the activation of cPLA2.
Fortunately, as demonstrated in Fig. 5,
the time course was linear even at high activity conditions when the
phospholipids were dissolved in Triton X-100 to form detergent/lipid
mixed micelles. This linearity suggests also that cPLA2
does not preferentially hydrolyze PtdIns(4,5)P2 over
PAPC.

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Fig. 5.
Triton X-100 preserves the linearity of the
progress of hydrolysis. Large unilamellar vesicles composed of
1:99 PtdIns(4,5)P2:PAPC assayed directly ( ) or prior to
the assay were solubilized in 3-fold molar excess of Triton X-100
( ). The standard micelle assay solution, containing 200 µM free Ca2+, was used at 1 mM
total lipid.
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Fig. 6A demonstrates that in
the presence of approximately 1 molecule of PtdIns(4,5)P2
per micelle (0.75 mol % of total lipid + detergent), the initial rate
of hydrolysis of PAPC by cPLA2 was essentially linear with
the molar percentage of PAPC and about 40 times higher than in the
absence of PtdIns(4,5)P2 (Fig. 6B). In contrast,
a similar percentage of PI displayed little effect on the hydrolysis of
PAPC (Fig. 6B). The non-linear character of the kinetics
observed at the lower percentage of PAPC and in the absence of
PtdIns(4,5)P2 (Fig. 6B) is consistent with the dual phospholipid kinetic model (17, 28). An increase in the mol %
substrate increases both the fraction of the enzyme associated with the
micelles and the rate of catalysis, since the enzyme is far from being
saturated by the substrate. In contrast, PtdIns(4,5)P2 causes most of the enzyme to associate with micelles even at the lowest
mol % PAPC. Thus, the linear kinetics shown in A reflects only the change in the interfacial concentration of the substrate.

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Fig. 6.
PtdIns(4,5)P2, but not PI,
increases the enzymatic efficiency of cPLA2. A,
mixed micelles of Triton X-100 contain 0.75 mol %
PtdIns(4,5)P2 and the mol % of PAPC indicated.
B, mixed micelles contained none ( ) or 1 mol % ( )
PI. The activity was determined after 15 min incubation at 40 °C.
Solid lines represent a fit of Equation 2 to the data.
A, enzymatic efficiency, Eff = 0.33 ± 0.07 µmol min 1 mg 1
mol % 1. Due to the essentially linear dependence of the
initial rate on the mol % PAPC, only the Eff
could be reliably determined from the fit of the model to the data. The
dissociation constant from the interface, Ks, has a
negligible effect on the determination of Eff.
B, Eff = 8 ± 1 10 3 µmol min 1 mg 1
mol % 1 and Ks = 0.7 ± 0.2 mM.
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The dissociation constant of the enzyme from the PAPC/Triton X-100
micelles, 0.7 ± 0.2 mM, obtained from the fit of the
dual phospholipid kinetic model (17, 28), is very similar to that obtained from direct measurements on PAPC vesicles, 0.5 ± 0.1 mM, under otherwise identical conditions. Hence, even in
the absence of PtdIns(4,5)P2, approximately two-thirds of
the enzyme was already associated with PAPC vesicles or Triton X-100
mixed micelles.
The mixed micelle system proved to be valuable for investigating
the specificity of the PtdIns(4,5)P2-cPLA2
interaction compared with other phosphatidylinositol polyphosphates.
The effects of the structurally homologous PI, phosphatidylinositol
4-phosphate (PtdIns(4)P), PtdIns(3)P, PtdIns(3,4)P2,
PtdIns(4,5)P2, PtdIns(3,4,5)P3 were all
tested, and the results are summarized in Table
II. Significantly, none of these other
phosphatidylinositol polyphosphates activated cPLA2 higher
than PtdIns(4,5)P2 emphasizing the importance of the 4- and
5-phosphate together to achieve the highest activation.
Although it is clear that PtdIns(4,5)P2 enhances the
binding of cPLA2 to substrate-containing surfaces, it was
not known if there was a way to down-regulate this interaction as is
the case for PLC
1. Ins(1,4,5)P3, the
soluble head group of PtdIns(4,5)P2, binds tightly to the
PH domain of PLC
1 and thus down-regulates the enzymatic
activation caused by binding to PtdIns(4,5)P2 as mediated through the PH domain. To determine if this were also the case
for cPLA2, the effects of the soluble head groups of both
PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were
investigated on cPLA2 activity and its
PtdIns(4,5)P2 activation. Soluble
Ins(1,4,5)P3 and Ins(1,3,4,5)P4 were added at a
10- and 5-fold molar excess, respectively, relative to
PtdIns(4,5)P2. The results (summarized in Table
III) showed that neither species
affected the PtdIns(4,5)P2 activation of
cPLA2. Additionally the results showed that neither appreciably activated cPLA2 themselves.
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Table III
Effect of inositol polyphosphates on cPLA2 activity toward
PAPC/Triton X-100 mixed micelles in the absence and presence of PtdIns(4,5)P2
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In addition to PtdIns(4,5)P2 other phospholipids,
especially the anionic phospholipids, were reported to activate
cPLA2 (12). Table II clearly shows that while the anionic
phospholipids do activate cPLA2, it is by several orders of
magnitude less than the effect of PtdIns(4,5)P2.
Interestingly, PE which appeared to increase the affinity of
cPLA2 for PE:PAPC vesicles actually decreased the rate of
hydrolysis of PAPC in the phospholipid/Triton X-100 micelles (Fig.
7). However, the progress of hydrolysis
of PAPC was about 5-fold faster in 1:1 PAPC:PE vesicles than in
vesicles composed entirely of PAPC. Consistent with the effect of
Ca2+ and PtdIns(4,5)P2, the PE-induced increase
in the activity of cPLA2 was severalfold higher than the
accompanying increase in the association of the enzyme with lipid
vesicles, from approximately 70 to just over 90% of the enzyme bound
to vesicles under identical experimental conditions.

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Fig. 7.
PE enhances the activity of cPLA2
in large unilamellar vesicles but has no effect in Triton
X-100/phospholipid mixed micelles. cPLA2 activity over
time was measured in the presence of vesicles composed of 1:0 ( ) or
1:1 ( ) PAPC:PE or mixed micelles composed of 1:0:3 ( ) or 3:1:12
( ) PAPC:PE:Triton X-100 using the standard micelle assay solution at
1 mM total lipid.
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The stimulatory effects of PtdIns(4,5)P2 and PE on the
activity of cPLA2 were largely independent of one another,
consistent with the previously published data (12). Substitution of a
quarter of PAPC by PE in vesicles already containing 1 mol %
PtdIns(4,5)P2 caused a further 5-fold increase in the
activity of cPLA2 toward PAPC (Fig.
8), similar to that seen in the absence
of PtdIns(4,5)P2 (Fig. 7). This increase is consistent with
a 6-fold higher affinity of cPLA2 for membranes containing
50 mol % (Table I). In contrast, replacing half of the substrate by
unlabeled PS reduced the rate of hydrolysis by a factor of 2.

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Fig. 8.
PE and PtdIns(4,5)P2 enhance the
enzymatic activity of cPLA2 independently of each
other. The cPLA2 activity over time was measured in
large unilamellar vesicles composed of 1:99
PtdIns(4,5)P2:PAPC ( ), 1:50:49
PtdIns(4,5)P2:PS:PAPC ( ), or 1:25:74
PtdIns(4,5)P2:PE:PAPC ( ) using the standard binding
solution at 200 µM Ca2+ and 1 mM
total lipid.
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DISCUSSION |
Our results demonstrate that cPLA2 binds with high
affinity and specificity to PtdIns(4,5)P2 in a 1:1
stoichiometry. The PtdIns(4,5)P2-induced increase in
membrane affinity is accompanied by an equally large increase in the
enzymatic activity of cPLA2. Importantly, this increase in
the activity is at least an order of magnitude larger than the
concomitant increase in the fraction of membrane-bound enzyme. The
interaction of cPLA2 with PtdIns(4,5)P2 is
sufficiently strong to cause measurable effects both on the membrane
association and the activity of this enzyme in the absence of exogenous
Ca2+. cPLA2 appears to contain a region
resembling a PH domain. We demonstrate a striking difference in the
requirements for mono-anionic lipids in
Ca2+-dependent association with lipid membranes
between cPLA2 and other proteins that contain CaLB/C2
domains. We also discuss implications of this study for the
investigation of other multimodular signal transduction proteins.
PtdIns(4,5)P2 Binding--
The analysis of
PtdIns(4,5)P2-induced association of cPLA2 with
lipid membranes revealed that those two molecules form a 1:1 complex at
the interface with a dissociation constant of 0.05 mol %. This
binding appears to arise from a specific interaction rather than a
nonspecific electrostatic attraction. The presence of mono-anionic
lipids at concentrations assuring much higher charge density than that
produced by the mol % PtdIns(4,5)P2 employed here failed
to increase the affinity of the enzyme for lipid vesicles. Consistent
with the specific interaction was the fact that the PtdIns(4,5)P2-stimulated increase in affinity for the
interface was also seen in Triton X-100/phospholipid mixed micelles.
This observation makes it unlikely that the
PtdIns(4,5)P2-induced increase in the affinity of
cPLA2 for lipid vesicles has its origin in the altered
physical properties of the lipid bilayer.
Notably, the affinity of cPLA2 for
PtdIns(4,5)P2 appears to be quantitatively identical to
that of PLC
1 for this same lipid. Inclusion of 0.5 mol % PtdIns(4,5)P2 in lipid vesicles increased their
affinity for PLC
1 by a factor of 11 (21). With double the concentration (1 mol %) of PtdIns(4,5)P2 in PAPC
vesicles, the affinity of cPLA2 for the membrane increased
by a factor of 20 (Fig. 1), also double the factor seen for
PLC
1. Thus, it is safe to assume that the interfacial
dissociation constant of cPLA2 and PLC
1 from
PtdIns(4,5)P2 incorporated into lipid membranes is about
0.05 mol % as shown in Fig. 1 for cPLA2. However, in strong contrast with the case for PLC
1 (34),
Ins(1,4,5)P3 has no effect on the
PtdIns(4,5)P2-induced association of cPLA2 with phospholipid vesicles.
In contrast to protein kinase C with whom cPLA2 shares a
homologous CaLB/C2 domain (7, 33), its requirement for monoanionic lipids in Ca2+-dependent membrane association
is strikingly different. A PS-dependent decrease in the
membrane affinity of cPLA2 caused by PS contrasts very
strongly with the 1,000-fold increase in the affinity of protein kinase
C for vesicles whose PS content was raised from 10 to 50 mol % (35),
under otherwise identical experimental conditions to those employed
here. The binding of cPLA2 to PS vesicles is hardly
detectable, and the membrane-bound/free cPLA2 ratio
increases essentially in linear fashion with the mol % of POPC. Thus,
the enzyme appears to associate with a single molecule of PC. Whether
this interaction occurs through the CaLB or the catalytic domain of
cPLA2 remains to be resolved.
The increased affinity of cPLA2 for membranes containing PE
likely originates in the change of physical properties of lipid membranes induced by this phospholipid (36). This conclusion is
corroborated by the fact that in Triton X-100 micelles PE acts as a
neutral diluent (Fig. 7). Interestingly, diacylglycerols that share
with PE its ability to induce the same change in the physical
properties in the lipid bilayers (37) were also shown to increase the
activity of cPLA2 when constituting a large fraction of the
lipid mixture (12).
Activity--
The PtdIns(4,5)P2-induced increase in
the membrane affinity was accompanied by an equally large increase in
the enzymatic activity of cPLA2. Interestingly, the effect
of PtdIns(4,5)P2 on the enzymatic activity of
cPLA2, shown here, appears to be much larger than the
effect on the activity of PLC
1 (38), an enzyme with
essentially identical affinity for PtdIns(4,5)P2 (21). The
stimulatory effect of PtdIns(4,5)P2 was much larger than
that previously reported (12), presumably due to the earlier use of
partially pure cPLA2 on uncharacterized sonicated
liposomes.
Notably, the linear kinetics of cPLA2, as shown in Fig. 6,
clearly demonstrate that the enzyme interacts with only a single molecule of substrate. As demonstrated elsewhere (39), the nonlinear kinetics seen in phosphatidylmethanol:PAPC mixed vesicles and interpreted as the evidence for cooperativity in cPLA2/PAPC
interaction (40) has its origin in the limited miscibility of
phosphatidylmethanol and PAPC.
The PtdIns(4,5)P2-stimulated increase in enzymatic activity
was quite specific for this phosphatidylinositol. The structurally similar PtdIns(3,4,5)P3 activated cPLA2 to
roughly 60% the level of PtdIns(4,5)P2 (Table II), so it
seems there is some tolerance for the addition of the 3-phosphate into
the binding site. The fact that PtdIns(3,4)P2 activated
cPLA2 to only 63% that by PtdIns(4,5)P2 (Table
II) and that PtdIns(3)P activated cPLA2 to only 32% that by PtdIns(4)P (Table II) demonstrates the stereoselectivity of cPLA2 for PtdIns(4,5)P2 and tends to rule out
the involvement of PI-3-kinase in the regulation of
cPLA2.
It is clear that the stimulatory effect of other anionic lipids is much
smaller than the PtdIns(4,5)P2 stimulation (Table II). This
contrasts with an earlier report (12) where the effects of
PtdIns(4,5)P2, PI, PS, and phosphatidic acid were well
within the same order of magnitude.
The stimulatory effects of PtdIns(4,5)P2 and PE, reported
earlier (12), were suggested to arise from an increase in
the fraction in the enzyme associated with lipid vesicles. As
demonstrated here, the observed stimulatory effect of
PtdIns(4,5)P2, Ca2+, and PE on the
enzymatic activity of cPLA2 are at least an order magnitude
larger than could possibly arise from just the changes in the fraction
of enzyme associated with either vesicles or micelles. Thus, the
PtdIns(4,5)P2-induced increase in the enzymatic efficiency of cPLA2 might occur through allosteric effects. However,
the fact that Ca2+ and PE act in the same manner as
PtdIns(4,5)P2 does, makes this explanation less plausible.
The fact that Ins(1,4,5)P3 and Ins(1,3,4,5)P4 had little effect on either the membrane affinity or the enzymatic activity of cPLA2 (Table III) casts further doubt on the
presence of a PtdIns(4,5)P2-induced allosteric
activation.
Comparability of Dissociation Constants--
The comparison
between cPLA2 and PLC
1 was possible because
the binding of both cPLA2 and PLC
1 to
PtdIns(4,5)P2 was measured taking into account that
PtdIns(4,5)P2 is an integral part of the membrane. The
values for the dissociation constants for the protein and
PtdIns(4,5)P2 that follow the same convention as for soluble ligands are 0.5 µM for cPLA2
(herein), 1.7 µM for PLC
1 (21), 53 µM for mSos1 (41), and 30 µM for pleckstrin
(42). These values although readily used and compared in the literature are, however, valid only for the specific experimental conditions employed, such as the content of PS in the membrane for
PLC
1 (21, 43) and the concentration of Ca2+
for cPLA2, reflecting the effect of the multiple attachment
points on the binding of all of these proteins to membranes.
To illustrate the point that the values are not comparable, at 2 µM free Ca+2 the apparent dissociation
constant for the PtdIns(4,5)P2-cPLA2-vesicle complex is 0.5 µM. However, in the absence of exogenous
calcium, and in the presence of 0.5 mM EGTA as well as 30 µM PtdIns(4,5)P2 (3 mol % of 1 mM total phospholipid), only 23 ± 6% protein was associated with vesicles. Thus, under such conditions, the apparent dissociation constant of the
PtdIns(4,5)P2-cPLA2 complex would appear to be
more than 30 µM. (Under the same conditions, but in the
absence of PtdIns(4,5)P2, the percentage of the enzyme associated with PAPC vesicles did not differ significantly from 0.)
This means that not only are these values reported in units of molarity
not comparable among any other PtdIns(4,5)P2-binding proteins yet reported (except for cPLA2 and
PLC
1 which also present surface concentration units),
but also the expression of the dissociation constant in molarity units
casts doubt on those numbers and their physiological significance.
As can be seen from Equation 3, the concentration of the specific
ligand at which 50% of the protein will be associated with the surface
depends on the affinity of other modules for the membrane through
general or specific interactions; this is contained within K1. One additional caveat for
K1, rarely taken into account in the studies of
domains extracted from multimodular proteins, is that its value is
valid only for either the whole protein or a domain and that these
numbers are likely to be different. In contrast, the interfacial
Kd measured in surface concentration units
(mol %), calculated from the PtdIns(4,5)P2-induced
increase in the membrane affinity of cPLA2, does not depend
on any other mode of membrane binding, e.g.
Ca2+-induced membrane association. This should also hold
true for other proteins with multiple modes of interaction with the
membrane.
Putative PH Domain--
The apparent 1:1 stoichiometry,
specificity, and high affinity observed in the binding of
cPLA2 to vesicles containing PtdIns(4,5)P2 suggest that this enzyme contains a specific binding site for PtdIns(4,5)P2. The strong enhancement of enzymatic activity
by PtdIns(4,5)P2 in either lipid vesicles or Triton
X-100/phospholipid mixed micelles is consistent with this notion.
Notably, the effect of PtdIns(4,5)P2 on the association of
cPLA2 with lipid vesicles is quantitatively identical to
that reported for PLC
1 (21). The high affinity
PtdIns(4,5)P2-binding site was localized in PLC
1 within a PH domain (43, 44). Recently the PH
domain, for which structural information exists in only 6 of the more than 100 identified proteins, has been proposed to function as a
membrane-localizing domain through specific interactions with PtdIns(4,5)P2 based on the results of several proteins that
have been shown to associate with PtdIns(4,5)P2 (21,
41-43); yet only PLC
1 (21) binds with sufficient
affinity to be biologically relevant given the bioavailability of
PtdIns(4,5)P2.
However, a computer-aided search for a PH domain in cPLA2
was inconclusive, presumably due to the very low homology in the amino
acid sequence of this domain as noted previously (14). Nevertheless, we
noticed a large degree of similarity between a short stretch of amino
acids, 271-283, in cPLA2 with the sequence in
PLC
1 that comprises a part of the
PtdIns(4,5)P2-binding site. Fig.
9A illustrates that all four
key residues in this motif, shown to coordinate
PtdIns(4,5)P2 in the crystal structure of PLC
1 (44), have conservative or identical matches in
cPLA2. The one other key residue shown by mutational
analysis, Arg-37 (38), also readily aligned with cPLA2. A
characteristic part of this motif, the KXK sequence, was
shown to have the two lysines coordinating the 4- and 5-phosphate in
the inositol ring in both the regulatory (44) and catalytic (45)
PtdIns(4,5)P2-binding sites in PLC
1. A Trp
involved in the coordination of the 1-phosphate (44) has several basic
residues on a C-flank in both proteins.

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Fig. 9.
A, the key residues for
PtdIns(4,5)P2 binding were observed within the
PLC 1 PH domain, and many of these key residues can also
be found in cPLA2. Amino acids observed in the crystal
structure to contact inositol 1,4,5-trisphosphate for
PLC 1 are shown in red (44). Amino acids found
by mutagenesis to be critical for PtdIns(4,5)P2 binding in
PLC 1 are underlined (38). The residues from
cPLA2 that match the known key residues in
PLC 1 are shown in pink. B,
sequence alignment of PtdIns(4,5)P2 binding PH domains. The
sequence-based multiple alignment (14) of PH domains is shown for
PLC 1 (residues 22-129), human N-terminal pleckstrin (residues 5-100), and mouse brain -spectrin (residues 2197-2305) with gaps under PLC 1 (Arg-38 and Cys-48) as derived from
the structure-based alignment (44). The -strands and -helices of
PLC 1, derived from the crystal structure, are shown as
arrows and rectangles, respectively. The
highlighted residues indicate conservative matches in the
alignment of at least 3 of 4 of the aligned PH domains. These residues
are colored red for polar/charged, purple for
nonpolar, violet for aromatic residues, and
yellow for identical residues in at least the three known PH
domains. The Chou-Fasman prediction of secondary structure for
cPLA2 is presented under the sequences with B/b for
-strand and A/a for -helix with capital letters as
more probable. The boxed residues are emphasized in
A.
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In contrast, as pointed out earlier (44), pleckstrin (42), spectrin
(46), or mSos1 (41) bind PtdIns(4,5)P2 with lower affinity
and specificity than PLC
1 (44) or now cPLA2.
The former proteins have only one basic residue flanking the equivalent
tryptophan and lack the KXK motif.
Starting from this motif, we have attempted to align this part of
cPLA2 with the PH domains of the three proteins that were shown to associate with vesicles containing PtdIns(4,5)P2
(Fig. 9B). The alignment included the standard constraints
for PH domain alignments as follows: (i) the PH domain is presented in
six conserved modules; (ii) the six modules have a minimum size
separated by variable spacers; (iii) the basic residues in the region
from cPLA2 271-290, the universal Trp of module 6, as well
as the polar/non-polar character of the residues were used as the basis
for the alignment for all modules (14).
In addition, we checked the secondary structure prediction for this
putative PH domain, testing first the available algorithms (DNAsis from
Hitachi Software Engineering America, Ltd., San Francisco and service
offered by the Protein Design Group, EMBL, Heidelberg, Germany) by
comparing the predicted and experimentally determined structure of the
PH domain of PLC
1. The Chou-Fasman method (47) proved to
be the most accurate for PLC
1 and was subsequently used
to predict the secondary structure of the putative PH domain in
cPLA2. As illustrated in Fig. 9B, this predicted
structure is quite similar to the known structure of the PH domain from PLC
1 (44) and PH domains in general (14).
Considering all these factors, the region of cPLA2
presented in Fig. 9B has much more similarity to the PH
domain of PLC
1 and the N-terminal PH domain of
pleckstrin than those from many other proteins included in the PH
domain consensus (14, 15, 48). There are 11 identical residues among
the PH domains of both cPLA2 and pleckstrin and
cPLA2 and PLC
1. For comparison, 15 residues
are identical among the PH domains of PLC
1 and
pleckstrin. Thus, it is very likely that the region shown in Fig.
9B constitutes a PH domain. Although the existence of this
PH domain in cPLA2 is also a likely explanation for the
specificity, high affinity binding, and activating properties of
PtdIns(4,5)P2 to cPLA2, further structural
studies and mutagenesis studies are required to identify the site(s) of
interaction between PtdIns(4,5)P2 and cPLA2 as
well as the important chemical moieties involved in the interaction.
Functional Implications and Conclusions--
The presence of
PtdIns(4,5)P2 in membranes targeted by cPLA2 is
likely to increase both the fraction of membrane-associated enzyme as
well as its specific activity. Several recent studies demonstrated that
PtdIns(4,5)P2 can be synthesized in cellular membranes
"on demand" and in a compartmentalized fashion (see Ref. 49 for
review). If PtdIns(4,5)P2 synthesis also occurs on demand
in the nuclear and endoplasmic reticulum membranes, which are targeted
by cPLA2 (5), then PtdIns(4,5)P2 might be involved in the up-regulation of cPLA2 activity. At
Ca2+ levels approximating those of a quiescent cell,
PtdIns(4,5)P2 displays a particularly dramatic effect on
the activity of cPLA2. That may lead to alternative modes
of cPLA2 activation that do not require Ca2+
mobilization, as suggested recently (50).
In conclusion, we have demonstrated that cPLA2 binds with
high affinity, specificity, and 1:1 stoichiometry to
PtdIns(4,5)P2 in lipid vesicles and that there is a
resulting quantitative increase in the enzymatic activity. We
demonstrate also that quantitative approaches that take into account
the two-dimensional nature of ligand presentation are essential to
elucidate the structural and functional relationship in
multimodular membrane-binding proteins that play key roles in signal
transduction.
We thank Drs. Ruth Kramer and John D. Sharp
of Lilly Research Laboratories, Indianapolis, IN, for a generous gift
of both the wild type and the Ser-228
Ala mutant of
cPLA2. We thank Dr. Ching-Shih Chen of the University of
Kentucky, Lexington, KY, for a generous gift of PtdIns(3)P,
PtdIns(3,4)P2, and PtdIns(3,4,5)P3. We also
thank our research group for constructive comments on this manuscript
and encouragement throughout the process.