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INTRODUCTION |
Although phosphatidylinositol 4,5-bisphosphate
(PIP2)1
constitutes only
1% of the lipids in the plasma membrane of
a typical mammalian cell, it plays many important roles in signal
transduction and cell biology (reviewed in Ref. 1). For example,
PIP2 is the source of three second messengers:
diacylglycerol, inositol 1,4,5-trisphosphate (IP3), and
phosphatidylinositol 3,4,5-trisphosphate (reviewed in Refs. 2
4).
PIP2 also recruits proteins containing pleckstrin homology
(PH) (reviewed in Ref. 5) and other domains (reviewed in Ref. 6) to the
plasma membrane and is a cofactor for the activation of phospholipase D
(reviewed in Refs. 7, 8). Finally, it is required for exocytosis
(reviewed in Ref. 9) and activates several different ion channels
(reviewed in Ref. 10). How can PIP2 play so many different
roles? The pool of PIP2 hydrolyzed by
phosphoinositide-specific phospholipase C (PLC) may be sequestered in
cholesterol-enriched lipid "rafts" (11), or the enzymes that
synthesize PIP2 may be concentrated in specific regions of
the plasma membrane (4). Alternatively, proteins may reversibly bind
much of the PIP2 in the plasma membrane, blunting changes
in the level of free PIP2 that would otherwise occur. We
explore here the hypothesis that myristoylated alanine-rich protein
kinase C substrate (MARCKS) not only binds a significant fraction of
the PIP2 in the plasma membrane, but also releases it upon
binding by Ca2+/calmodulin or phosphorylation by protein
kinase C (PKC).
MARCKS (reviewed in Refs. 12 and 13) is a ubiquitous PKC (14)
substrate, present at high concentration in many cell types; for
example, its concentration in brain is
10 µM (13, 15),
comparable to the concentration of PIP2. It has two
conserved regions required for membrane binding: a myristoylated N
terminus and a basic effector domain (residues 151
175). The mechanism by which MARCKS binds to the plasma membrane is well understood (reviewed in Refs. 16 and 17); the N-terminal myristate inserts into
the bilayer and the cluster of basic residues in the effector domain
interacts electrostatically with acidic lipids. When the effector
domain of MARCKS binds to Ca2+/calmodulin or is
phosphorylated by PKC, its interaction with acidic lipids is reversed
(reviewed in Refs. 12, 13, 16, and 17).
Previous work suggested that the effector domain of MARCKS interacts
with PIP2; both MARCKS and its effector domain peptide, MARCKS-(151
175), inhibit the PLC-catalyzed hydrolysis of
PIP2 in vesicles, and this inhibition is reversed by
Ca2+/calmodulin binding or PKC phosphorylation of
MARCKS-(151
175) (18). Although the simplest interpretation of these
results is that the effector domain binds strongly to PIP2,
it is difficult to rule out potential artifacts because
MARCKS-(151
175) also aggregates the vesicles. For example, the
putative large lateral domains formed in membranes by MARCKS-(151
175)
(18, 19) are probably an aggregation-related artifact (20).
The objective of this study was to test our hypothesis more rigorously.
First, we investigated whether MARCKS-(151
175) inhibits the
PLC-catalyzed hydrolysis of PIP2 in phospholipid
monolayers, a system that eliminates the possibility of artifacts due
to vesicle aggregation. Second, we measured directly the binding of
MARCKS-(151
175) to bilayers containing PIP2 using two
independent techniques. Next, we conducted competition and
electrophoretic mobility experiments to investigate the stoichiometry
of the complex formed between MARCKS-(151
175) and PIP2.
Finally, we examined the importance of nonspecific electrostatic
interactions by comparing the specificity of MARCKS-(151
175) for
PI(4,5)P2 versus PI(3,4)P2 and
measuring the effect of salt on the binding. The results all support
the hypothesis that the effector domain of MARCKS binds with high affinity to PIP2 in membranes; under "Discussion," we
consider the biological implications of this strong interaction.
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EXPERIMENTAL PROCEDURES |
Materials--
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine
(PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine
(PS),
1-palmitoyl-2- oleoyl-sn-glycero-3-phosphatidylethanolamine
(PE),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine, phosphatidylinositol (PI), and cholesterol were purchased from Avanti
Polar Lipids (Alabaster, AL). The ammonium salt of
L-
-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2 or native PI(4,5)P2) was
either purchased from Roche Molecular Biochemicals (Mannheim, Germany)
or purified from bovine brain extract (Sigma) as described
elsewhere (21). Dipalmitoyl phosphatidylinositol 4-phosphate (PI(4)P)
was from Calbiochem (La Jolla, CA). The ammonium salt of dipalmitoyl
phosphatidylinositol 4,5-bisphosphate (dipalmitoyl
PI(4,5)P2) and dipalmitoyl phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) were from Matreya (Pleasant
Gap, PA).
D(+)-sn-1-O-[1-[6'-[[6-[(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl]amino]hexanoyl]-2-O-hexanoylglyceryl D-myo-phosphatidylinositol 4,5-bisphosphate
(NBD-PIP2) was from Echelon Research Laboratories Inc.
(Salt Lake City, UT). Labeled [dioleoyl-1-14C]-L-
-dioleoyl-phosphatidylcholine,
[inositol-2-3H]-L-
-phosphatidyl-D-myo-inositol
4,5-bisphosphate ([3H]PIP2), and
[ethyl-1,2-3H]N-ethylmaleimide
([3H]NEM) were from PerkinElmer Life
Sciences. Hog brain calmodulin was from Roche Molecular Biochemicals.
Recombinant human PLC-
1 expressed in Escherichia
coli, a generous gift from Dr. Mario J. Rebecchi, was purified as
described elsewhere (22). Recombinant PLC-
1 expressed in
Sf9 cells, a generous gift from Dr. Suzanne Scarlata, was
purified as described elsewhere (23).
Peptide Preparations--
The peptide corresponding to the
basic effector domain of bovine MARCKS (24), referred to as
MARCKS-(151
175), has the sequence acetyl-KKKKKRFSFKKSFKLSGFSFKKNKK-amide and was obtained from CASM, State University of New York (Stony Brook, NY).
[3H]NEM-CKKKKKRFSFKKSFKLSGFSFKKNKK-amide, referred to as
[3H]NEM-MARCKS-(151
175), was synthesized as described
previously (25). A peptide corresponding to MARCKS-(151
175) with
Phe-157 changed to Cys was labeled with acrylodan as described
previously (26); it is referred to as acrylodan-MARCKS-(151
175).
Monolayer Measurements--
We measured PLC-catalyzed hydrolysis
of PIP2 (reviewed in Ref. 27) in a monolayer as described
previously (28, 29). In a typical experiment, 60 µl of 55 µM PC/PS/[3H]PIP2 (66.5:33:0.5)
in chloroform was carefully deposited onto the surface of a solution
containing 100 mM KCl, 25 mM HEPES, 0.1 mM EGTA, pH 7.5, 15 ml in a 5-cm-diameter Teflon trough.
Once the chloroform had evaporated (10 min), we measured the surface pressure of the monolayer (typically 25 mN/m) using a square piece of
filter paper and a balance as described previously (30). MARCKS-(151
175) was then added to the subphase; the magnetic stir bar
at the bottom of the trough mixed MARCKS-(151
175) uniformly in the
subphase within
1 min. After 3 min, we added PLC-
1 or PLC-
1 (final concentration
0.1 nM),
mixed the subphase solution for another 3 min, then added 100 µM CaCl2 to the subphase to produce a free
concentration of Ca2+
2 µM, as measured
using a Ca2+ electrode in separate experiments. A free
Ca2+ concentration of 2 µM produces
significant activation of both PLC-
and -
on monolayers and other
systems (29, 31, 32). We collected 200-µl aliquots of the subphase at
different times after the addition of Ca2+ and measured the
radioactivity due to [3H]IP3. Unless
specified, all measurements were done at 25 °C with a monolayer
consisting of PC/PS/[3H]PIP2 (66.5:33:0.5) at
a surface pressure of
25 mN/m.
We did several control experiments. First, we determined that the
PLC-catalyzed hydrolysis of PIP2 in the absence of
Ca2+ was negligible (data not shown), as expected for the
Ca2+-dependent PLC-
and -
enzymes.
Second, we showed that the initial hydrolysis rate (the initial slope
of the hydrolysis curves, such as those shown in Fig. 1) was
proportional to PLC concentration (data not shown). Third, we confirmed
that increasing the surface pressure,
, decreased the hydrolysis
rate significantly (28, 29), presumably because a hydrophobic region in
the catalytic domain of PLC inserts into the monolayer (33). Fourth, we
determined that the percentage of inhibition of PIP2
hydrolysis was independent of PLC concentration; increasing the
PLC-
1 concentration 5-fold did not change the percentage
of inhibition produced by 150 nM MARCKS-(151
175) (data
not shown).
Other studies showed the initial rate of hydrolysis catalyzed by both
PLC-
1 and PLC-
1 depends on the mole
fraction of PIP2 (28, 31, 32, 34, 35). Thus, if
MARCKS-(151
175) binds to PIP2, decreasing the free
concentration of PIP2 in the monolayer, we expect the
initial rate of hydrolysis to decrease.
Vesicle Preparations--
We used multilamellar vesicles (MLVs),
large unilamellar vesicles (LUVs), and sucrose-loaded LUVs, prepared as
described in detail elsewhere (20, 36). MLVs for the electrophoretic
mobility measurements were prepared by drying the lipid mixture on a
rotary evaporator, adding a solution containing 100 mM KCl,
1 mM MOPS, pH 7.0, and gently swirling the solution for
several min. LUVs for the fluorescence binding measurements were
prepared by drying the lipid mixture on a rotary evaporator, hydrating
the lipids in a solution containing 100 mM KCl, 1 mM MOPS, pH 7.0, then taking the MLVs through five cycles
of freezing and thawing followed by 10 extrusion cycles through a stack
of two polycarbonate filters (0.1-µm-diameter pore size) using a
Lipex Biomembranes Extruder (Vancouver, British Columbia, Canada) (37).
Sucrose-loaded LUVs for the centrifugation binding measurements were
prepared in a similar manner, except that the lipid film was hydrated
in a solution containing 176 mM sucrose, 1 mM
MOPS, pH 7.0. After extrusion, the solution outside the sucrose-loaded
LUVs was exchanged for 100 mM KCl, 1 mM MOPS,
pH 7.0.
Centrifugation Binding Measurements--
We measured the binding
of [3H]NEM-MARCKS-(151
175) to sucrose-loaded
PC/PIP2 LUVs using the centrifugation technique described previously (36). Sucrose-loaded PC/PIP2 LUVs were mixed
with 2 nM [3H]NEM-MARCKS-(151
175) and
centrifuged at 100,000 × g for 1 h. We measured
the radioactivity of the supernatant and the pellet to calculate the
percentage of [3H]NEM-MARCKS-(151
175) bound. We
minimized loss of the peptide to the tube in these experiments (and the
fluorescence experiments described below) by pre-treating the tube with
sonicated PC vesicles, which coats the tube surface with PC;
MARCKS-(151
175) binds only weakly to PC vesicles (Fig. 2), but
strongly to plastic.
Binding measurements were also done at several different salt
concentrations (100
500 mM KCl). Because the iso-osmotic
sucrose solution corresponding to [KCl] > 200 mM is
viscous and difficult to extrude, we used a mixture of sucrose and KCl
as an internal solution for the vesicles.
To describe the binding of the peptide to lipid vesicles without making
assumptions about the absorption mechanism, we use a molar partition
coefficient, K, as described previously (38, 39).
[P]m/[L] = K [P], where
[P]m is the molar concentration of
peptide bound to the membrane, [L] is the molar concentration of lipid accessible to the peptide (
one-half of the
total lipid concentration for these LUVs because the peptide interacts
only with the outer leaflet of the bilayer; the vesicles are not
permeable to the peptide, which is added to a solution of preformed
vesicles), and [P] is the molar concentration of free
peptide in the bulk aqueous phase. For most of our binding measurements
(e.g., Fig. 2), [L]
[P]m. Defining
[P]tot as the sum of bound and free peptide
molar concentrations, we get Equation 1.
|
(Eq. 1)
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Fluorescence Binding Measurements--
We determined the molar
partition coefficient, K, for the binding of
acrylodan-MARCKS-(151
175) to PC/PIP2 LUVs using a
fluorescence technique described in detail elsewhere (20). Briefly,
acrylodan is a polarity-sensitive fluorescent dye with an excitation
peak at
370 nm and an emission peak at
520 nm in water (40). When the acrylodan-labeled peptide binds to a membrane, the fluorophore inserts into the bilayer, shifting the emission peak to
455 nm and
increasing the fluorescence intensity. We added
acrylodan-MARCKS-(151
175) to PC/PIP2 LUVs and measured
the fluorescence of the mixture at 455 nm, corrected as described
elsewhere (20), to calculate the percentage of bound peptide at a given
lipid concentration.
Stoichiometry Measurements--
We obtained information about
the stoichiometry of the peptide-PIP2 complex by examining
how the fraction of peptide bound decreases when increasing
concentrations of peptide are added to a solution of sucrose-loaded
PC/PIP2 LUVs. We used the centrifugation technique with a
constant lipid concentration (10,000 nM accessible lipid in
the form of sucrose-loaded PC/PIP2 (99.7:0.3) LUVs or 30 nM accessible PIP2) to obtain the results shown
in Fig. 3. About 80% of MARCKS-(151
175) is bound when the peptide is
present at low concentration (e.g. 2 nM). As the
peptide concentration increases, it binds a significant fraction of the
accessible PIP2 and the fraction of bound peptide
decreases. We performed the experiments with both
[3H]NEM-MARCKS-(151
175) and a mixture of
[3H]NEM-MARCKS-(151
175) and unlabeled MARCKS-(151
175)
and obtained essentially identical results, as expected if the NEM
label does not affect the binding.
MARCKS-(151
175) binds only weakly to PC vesicles (Fig. 2), so the
binding sites in PC/PIP2 LUVs should be PIP2.
If we assume that one MARCKS-(151
175) binds to one PIP2,
we get Equation 2, where Ka is the apparent
association constant for 1:1 binding, [P-PIP2]
is the molar concentration of the complex of peptide and
PIP2, [P] is the molar concentration of free
peptide in the bulk aqueous phase, [PIP2] is the molar
concentration of free PIP2, [P]tot
is the sum of bound and free peptide molar concentrations, and
[PIP2]tot is the sum of bound and free
PIP2 molar concentrations.
|
(Eq. 2)
|
These three equations were combined into a single quadratic
expression that predicts the 1:1 binding curve (shown in Fig. 3 with
Ka = 2 × 108
M
1, as derived from the 1%
PIP2 binding data in Fig. 2A).
Electrophoretic Mobility Measurements--
The electrophoretic
mobility (velocity/field) of MLVs was measured as described previously
(41) and used to calculate the zeta potential, the electrostatic
potential at the shear plane, which is located about 0.2 nm from the
surface (42). A mixture containing MARCKS-(151
175) and a low
concentration of MLVs was loaded into a glass electrophoresis tube that
had been pre-washed with MARCKS-(151
175) to minimize the loss of the
peptide. An electrical field was applied, and the electrophoretic
mobility of the vesicles, u, was measured directly using a
stopwatch and a microscope. The zeta potential was calculated using the
Helmholtz-Smoluchowski equation (42, 43).
|
(Eq. 3)
|
is the zeta potential of a vesicle, u
is the velocity of the vesicle in a unit electric field,
is the viscosity of the aqueous solution,
r is the dielectric constant of
the aqueous solution, and
0 is the
permittivity of free space. As discussed previously (42), the zeta
potential is proportional to the surface charge density and thus to the
number of charged peptides that absorb to the vesicles.
A control experiment showed that MARCKS-(151
175) has a similar effect
on the zeta potential of 98:2 PC/dipalmitoyl PI(4,5)P2 and
98:2 PC/native bovine brain PI(4,5)P2 MLVs (data not shown).
 |
RESULTS |
MARCKS-(151
175) Inhibits the PLC-catalyzed Hydrolysis of
PIP2 in a Monolayer--
Fig.
1 illustrates the effects of
MARCKS-(151
175) on the PLC-catalyzed hydrolysis of PIP2.
We deposited a mixture of lipids containing
[3H]PIP2 at the air-water surface; added
MARCKS-(151
175), PLC, and CaCl2 sequentially to the
subphase; collected samples of the subphase at the indicated times; and
measured the [3H]IP3 to determine the
percentage of [3H]PIP2 hydrolyzed as a
function of time (Fig. 1).

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Fig. 1.
MARCKS-(151 175)
inhibits the PLC-catalyzed hydrolysis of PIP2 in
monolayers. A, MARCKS-(151 175) (concentrations
indicated in the figure), PLC- 1 (0.1 nM),
and CaCl2 (free [Ca2+] 2 µM; t = 0) were added sequentially to the
subphase of a PC/PS/[3H]PIP2 (66.5:33:0.5)
monolayer and the percentage of PIP2 hydrolyzed was
measured at the times indicated. B, same experimental
protocol using 0.1 nM PLC- 1 instead of
PLC- 1. 10 100 nM MARCKS-(151 175) inhibits
the hydrolysis of PIP2 catalyzed by both PLC isoforms. The
data shown are representative of three sets of experiments. The lines
show linear fits to the data obtained in the first 6 min. The slopes of
the lines are the initial rates of hydrolysis of PIP2 (% PIP2 hydrolyzed/min) listed in Table I.
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In the absence of MARCKS-(151
175), PLC-
1 produces
rapid hydrolysis of PIP2 following addition of
CaCl2 to the subphase (free [Ca2+]
2 µM);
25% of the PIP2 in the monolayer is
hydrolyzed in 5 min (open circles in Fig. 1A).
Similar results were reported previously (28, 29, 35). Addition of low
concentrations of MARCKS-(151
175) inhibits this hydrolysis; 50 nM MARCKS-(151
175) produces
50% inhibition, and 150 nM produces almost complete inhibition (decrease in initial
rate of hydrolysis or slope of line in Fig. 1A). (The
measurements were done at a surface pressure of 25 mN/m to minimize the
amount of PLC required. Control experiments at
= 30
35 mN/m,
at which the area of a lipid in a monolayer corresponds to that of a
lipid in a bilayer (see Footnote 2 in Ref. 28), produced qualitatively
similar results (data not shown) to the data shown in Fig.
1A.) Fig. 1B shows that MARCKS-(151
175) also
inhibits hydrolysis of PIP2 catalyzed by
PLC-
1, a different isoform of PLC; 10 nM
MARCKS-(151
175) produces
50% inhibition, and 50 nM
produces almost complete inhibition.
The monolayer results (Fig. 1) are consistent with those reported
previously using phospholipid vesicles if one notes that higher
concentrations of peptide were required to produce inhibition in the
vesicle experiments because most of the peptide was bound to the
vesicles (18). Specifically,
10 µM MARCKS-(151
175) produced 90% inhibition of PLC-catalyzed hydrolysis in vesicles containing
10 µM PIP2. As expected,
reducing the concentration of lipid decreased the concentration of
peptide required for inhibition in the vesicle experiments (18). In the
monolayer experiments shown in Fig. 1, there is much less
PIP2 than peptide, so most of the peptide is free in the
subphase. (If the PIP2 in a monolayer containing 0.5% of
this lipid were dispersed uniformly through the subphase, it would be
present at a concentration of
1 nM).
The simplest interpretation of the results illustrated in Fig. 1 is
that MARCKS-(151
175) decreases the rate of hydrolysis by binding to
PIP2 in the monolayer and competing with the catalytic domain of PLC for PIP2. The PLC assay system, however, is
complicated. We considered three other possible explanations for our
results. First, the inhibition could be due to the interaction of
MARCKS-(151
175) with PLC. However, we could obtain no evidence for
such an interaction using fluorescently labeled MARCKS and PLC (data
not shown). Second, the inhibition could be due to the neutralization
of the monovalent acidic lipids in the monolayer by MARCKS-(151
175);
the rate of the PLC-catalyzed hydrolysis of PIP2 decreases
as the mole fraction of monovalent acidic lipids decreases (28), and
addition of 50 nM MARCKS-(151
175) almost neutralizes the
2:1 PC/PS surface (44). However, we obtained similar results for both
PLC-
1 and PLC-
1 using PC/PIP2
(99.5:0.5) monolayers (Table I); these
monolayers are essentially electrically neutral prior to the addition
of MARCKS-(151
175). Thus, the inhibition is not due to a decrease in
the electrostatic surface potential of the monolayer. (We also observed
similar effects using PS/PIP2 (99.5:0.5) monolayers (Table I), so the inhibition does not depend strongly on the mole fraction of
monovalent acidic lipids in the monolayer.) Third, the inhibition could
be due to irreversible denaturation/complexation of PLC by a trace
contaminant in the peptide solution. This is unlikely because addition
of calmodulin reverses the inhibition induced by MARCKS-(151
175) in
the monolayer system (data not shown), as was observed previously in
the vesicle system (18).
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Table I
Effect of MARCKS-(151-175) on the PLC-catalyzed hydrolysis of
PIP2
MARCKS-(151-175), PLC- 1 (or PLC- 1), and
CaCl2 (free [Ca2+] 2 µM) were added
sequentially to the subphase of monolayers containing
[3H]PIP2, and the percentage of PIP2
hydrolyzed was measured as in Fig. 1. [Pep] indicates the
concentration of MARCKS-(151-175) added to the subphase of the
monolayer. % inhibition = 100% × (initial rate of hydrolysis
without peptide rate with peptide)/rate without peptide. This
was determined from experiments similar to those shown in Fig. 1. The
data are representative results from at least two sets of experiments.
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Experiments measuring the effects of other molecules on the
PLC-catalyzed hydrolysis of PIP2 support our interpretation
that MARCKS-(151
175) inhibits the reaction by binding to
PIP2. Neomycin, a molecule that forms a 1:1 complex with
PIP2 with an equilibrium dissociation constant in the 1
10
µM range (45), also inhibits the PLC-catalyzed hydrolysis
of PIP2; 2 µM neomycin produces a significant
inhibition, and 20 µM neomycin produces
50%
inhibition of the PLC-
1-catalyzed hydrolysis of
PIP2 (data not shown). Conversely, simple polybasic
peptides such as pentalysine and heptalysine, which do not bind with
high affinity to PIP2 (46), do not inhibit the
PLC-catalyzed hydrolysis of PIP2. For example, 10 µM heptalysine, which binds sufficiently strongly to a
2:1 PC/PS membrane to reduce the zeta potential
50% from 
50 to
30 mV (20), has little effect on the hydrolysis of PIP2
under conditions similar to those described in Fig. 1 (data not shown).
MARCKS-(151
175) Binds Strongly to Vesicles Containing
PIP2--
To test more directly our hypothesis that
MARCKS-(151
175) binds to PIP2, we used both a
centrifugation technique (Fig.
2A) and a fluorescence
technique (Fig. 2B) to measure the binding of
MARCKS-(151
175) to vesicles containing PIP2. Fig.
2A illustrates that [3H]NEM-MARCKS-(151
175)
binds strongly to PC/PIP2 vesicles. We reported previously
that incorporation of 1% PIP2 into PC vesicles enhances
the binding of [3H]NEM-MARCKS-(151
175) to vesicles
104-fold (25). Fig. 2A extends the data to show
the effect of lower mole fractions of PIP2 on the binding.
[3H]NEM-MARCKS-(151
175) binds weakly to PC vesicles
(K
102
M
1), which can be explained by
the hydrophobic insertion of the 5 Phe residues into the bilayer (25).
The molar partition coefficient (Equation 1) increases as the mole
fraction of PIP2 in the membrane increases:
K
104, 105, and
106 M
1 for
PC/PIP2 LUVs containing 0.01%, 0.1%, and 1%
PIP2, respectively. In other words, a solution containing
10
8 M PIP2 in the
form of PC/PIP2 vesicles binds 50% of
[3H]NEM-MARCKS-(151
175). In Fig. 2B, we use
a fluorescence technique to measure the binding of
acrylodan-MARCKS-(151
175) to PC/PIP2 vesicles. The
acrylodan probe is polarity-sensitive; when it binds to a lipid
bilayer, its fluorescence signal increases and is blue-shifted. Fig.
2B shows that acrylodan-MARCKS-(151
175) binds to
PC/PIP2 vesicles containing 0.1% and 1% PIP2
with K
105 and 106
M
1, respectively, results
comparable to those shown in panel A. Thus,
measurements using both the centrifugation and the fluorescence techniques show that MARCKS-(151
175) binds strongly to
PC/PIP2 vesicles.

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Fig. 2.
MARCKS-(151 175) binds
strongly to PC/PIP2 vesicles. A, the
binding of [3H]NEM-MARCKS-(151 175) to sucrose-loaded
PC/PIP2 LUVs as measured by the centrifugation technique.
The percentage of peptide bound is plotted as a function of the
accessible lipid concentration (PC+PIP2). The
binding measurements were conducted with PC/PIP2 LUVs
containing 1% ( ), 0.1% ( ), 0.01% ( ), and 0% ( )
PIP2. The curves are the least square fits of Equation 1 to
the data. The molar partition coefficient, K, deduced from
these fits is the reciprocal of the lipid concentration that binds 50%
MARCKS-(151 175). The molar partition coefficients are 2 × 106 M 1, 8 × 104 M 1, 6 × 103 M 1, and 3 × 102 M 1for
PC/PIP2 vesicles containing 1%, 0.1%, 0.01%, and 0%
PIP2, respectively. B, the binding of
acrylodan-MARCKS-(151 175) to PC/PIP2 LUVs as measured by
the fluorescence technique. The molar partition coefficients are 5 × 105 M 1 and 4 × 104 M 1, for
PC/PIP2 LUVs containing 1% ( ) and 0.1% ( )
PIP2, respectively. The binding to PC vesicles is too weak
to measure accurately with the fluorescence technique; the curve is
drawn with K = 3 × 101
M 1.
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Incorporation of PIP2 into PC/PS vesicles also increases
the binding of MARCKS-(151
175) to these vesicles; the molar partition coefficient for 10:1 PC/PS vesicles, 6 × 103
M
1 (25), increases
100-fold to
5 × 105 M
1
(data not shown) when the vesicles contain 1% PIP2 (93:6:1
PC/PS/PIP2; the mole fraction of PS was reduced to keep the
electrostatic surface potential constant).
MARCKS-(151
175) Binds to More than One
PIP2--
Fig. 3 illustrates
that, as the concentration of MARCKS-(151
175) increases, the
percentage of MARCKS-(151
175) bound to PC/PIP2 vesicles
decreases (open circles). The curve illustrates the
predicted effect assuming the peptide forms a 1:1 complex with
PIP2; for example, increasing the concentration of
MARCKS-(151
175) from 2 nM to 10 nM should
have little effect on the fraction of peptide bound because the
accessible concentration of PIP2 = 30 nM. If only 1:1 complexes were formed,
50 nM MARCKS-(151
175)
should be required to decrease the percentage of peptide bound from
80% to 50%, but Fig. 3 shows that only 10 nM peptide
produces this reduction. These data strongly suggest that one
MARCKS-(151
175) binds to n (n > 1)
PIP2. It is difficult, however, to deduce with any
confidence the stoichiometry of the peptide/lipid interaction from
these data because the theoretical expressions are complicated and
model-dependent (47).

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Fig. 3.
Effect of
[MARCKS-(151 175)] on binding to
PC/PIP2 vesicles. The percentage of peptide bound to
PC/PIP2 (99.7:0.3) LUVs ( ) determined with different
concentrations of [3H]NEM-MARCKS-(151 175) and a
constant concentration of lipid (10,000 nM accessible lipid
or 30 nM accessible PIP2). The curve shows the
prediction of Equation 2, which assumes a 1:1
MARCKS-(151 175):PIP2 complex. The data for [peptide] 10 nM lie significantly below this curve, suggesting the
peptide binds more than one PIP2.
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Although both electrostatic repulsion between the negatively charged
PIP2 molecules and entropy effects make it unlikely that n-mers of PIP2 exist in the absence of peptide,
we did two control experiments to investigate this possibility. First,
addition of 0.1 mM EDTA to the bathing solution did not
change the binding of [3H]NEM-MARCKS-(151
175) to
PC/PIP2 (99.9:0.1) LUVs (data, similar to Fig.
2A, not shown), which suggests that trace concentrations of
divalent ions are not producing n-mers of PIP2. Second,
replacing 20% of PC by
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine did not change the binding of [3H]NEM-MARCKS-(151
175)
to PC/PIP2 (99:1) LUVs; similarly, changing the lipid
composition from PC/PIP2 (99.9:0.1) to
PC/PE/cholesterol/PIP2 (39.9:30:30:0.1) did not affect the
binding (data, similar to Fig. 2A, not shown). Thus, the
arachidonic acid chain at the 2-position on PIP2 is
unlikely to produce n-mers.
The simplest interpretation of the results shown in Fig. 3 is that one
peptide binds sequentially to n PIP2 monomers to
form a complex. Experiments with PC/NBD-PIP2 (99.9:0.1)
vesicles show addition of MARCKS-(151
175) quenches the fluorescence,
consistent with the peptide binding to several NBD-PIP2 and
inducing a local demixing (data not shown).
Electrophoretic Mobility Measurements Show MARCKS-(151
175) Binds
Equally Well to PI(4,5)P2 and
PI(3,4)P2--
We measured the electrophoretic mobility
(velocity/field) of PC/PIP2 MLVs and calculated the zeta
potential (potential 0.2 nm from surface) using Equation 3. The zeta
potential is proportional to the surface charge density (42) and thus
can be used to monitor the binding of positively charged
MARCKS-(151
175) to negatively charged PC/PIP2 MLVs. Fig.
4A illustrates that addition
of MARCKS-(151
175) changes the zeta potential of PC/PIP2
vesicles significantly. Specifically, 10
7
M MARCKS-(151
175) decreases the zeta potential of 98:2
PC/PIP2 MLVs from
15 mV to
8 mV (Fig. 4A,
open squares). If we assume that the peptide
forms electroneutral complexes with PIP2,
10
7 M MARCKS-(151
175) binds
50% of accessible PIP2 in PC/PIP2 MLVs. This agrees qualitatively with the results from the monolayer and
binding measurements:
10
8
10
7
M MARCKS-(151
175) inhibits significantly the hydrolysis
of PIP2 in monolayers (Fig. 1 and Table I) and
10
8 M PIP2 (in the
form of PC/PIP2 vesicles) binds
50% of
MARCKS-(151
175) in the direct binding experiments (Fig. 2). Fig.
4A also illustrates that MARCKS-(151
175) has a similar
effect on the zeta potential of MLVs formed from 98:2
PC/PI(4,5)P2 (open squares), and 98:2 PC/PI(3,4)P2 (filled triangles). This lack of
specificity between PI(3,4)P2 and PI(4,5)P2 is
expected if the relatively unstructured peptide binds mainly through
electrostatic interactions with the phosphoinositides.

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Fig. 4.
Effect of
MARCKS-(151 175) on zeta potential of
vesicles. A, MARCKS-(151 175) produces significant
changes in the zeta potentials of 99:1 PC/PI(4,5)P2 ( ),
98:2 PC/PI(4,5)P2 ( ), and 98:2 PC/PI(3,4)P2
( ) vesicles, but does not affect the zeta potential of PC ( )
vesicles. B, MARCKS-(151 175) has little effect on the zeta
potential of MLVs containing monovalent acidic lipids: 97:3 PC/PS
( ), 94:6 PC/PS ( ), 94:6 PC/PI ( ) or PC only ( ). The zeta
potential was calculated from the measured electrophoretic mobility of
MLVs using Equation 3. Each point shows the average of at least 20 measurements in two different experiments; the error bars
illustrate the standard deviations when they are larger than the size
of the symbols.
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Valence of PIP2--
A comparison of panels
A and B in Fig. 4 illustrates that, in the
absence of MARCKS-(151
175), MLVs containing 3% or 6% PS have
similar zeta potentials to MLVs containing 1% or 2% PIP2, respectively. These results suggest that, although the maximum valence
of PIP2 is
5, the effective valence of PIP2
is
3 at pH 7.0, in agreement with previous results (48). NMR
experiments show about 1 proton is bound to PIP2 at pH 7 (48, 49). The results in Fig. 4 suggest that a K+ is also
bound to PIP2 in 100 mM KCl to yield an
effective valence of
3. However, we do not know the valence of
PIP2 when it is bound to a basic peptide such as
MARCKS-(151
175); it could lose a bound proton or potassium ion.
Stoichiometry of Complex from Zeta Potential Data--
When the
[lipid]
[peptide], 10
8
M PIP2 binds 50% MARCKS-(151
175) (Fig. 2);
thus, 10
8 M MARCKS-(151
175)
should bind significantly to PIP2 when the [peptide]
[lipid]. Assuming MARCKS-(151
175) forms a 1:1 complex with
PIP2, the valence of a 1:1 peptide:PIP2 complex
would be
+10, and the zeta potential would be positive for
[peptide] > 10
8M. The data in Fig.
4A, however, show that the zeta potentials of 99:1
PC/PIP2 or 98:2 PC/PIP2 MLVs remain negative
after the addition of
10
8
10
5
M MARCKS-(151
175), providing additional evidence that one
MARCKS-(151
175) binds to several PIP2 to form an
electroneutral complex (i.e. one +13 valent
MARCKS-(151
175) combines with four 
3 valent or three 
4
valent PIP2). We stress that the results in Figs. 3 and 4
provide only indirect evidence about the stoichiometry of the complex;
the question needs to be addressed using more direct experimental approaches.
MARCKS-(151
175) Binds Only Weakly to Vesicles Containing Small
Fractions of Other Acidic Lipids--
Addition of MARCKS-(151
175) to
6% PS or 6% PI vesicles has little effect on the zeta potential of
the vesicles (Fig. 4B). The peptide exerts an intermediate
effect on the zeta potential of PC/PI(4)P vesicles;
10
7 M MARCKS-(151
175) changes
the zeta potential of 4% PI(4)P vesicles from
18 mV to
15 mV (data
not shown).
Although MARCKS-(151
175) binds strongly to vesicles containing high
mole fraction of monovalent acidic lipids (e.g. PS and PG)
due to nonspecific diffuse double layer effects, it binds only weakly
to vesicles containing a low mole fraction (<10%) of monovalent
acidic lipids (Fig. 4B and Ref. 25). Thus, the strong
binding of MARCKS-(151
175) to PC/PIP2 vesicles apparent from the data in Figs. 2 and 4A is not due to the average
electrostatic potential (zeta potential) of the vesicles.
Local Electrostatic Effects Are Important--
We measured the
effect of ionic strength on the binding of MARCKS-(151
175) to
PIP2. Fig. 5 shows that
increasing [KCl] from 100 mM to 500 mM
decreases the peptide's molar partition coefficient
100-fold for
both 99:1 PC/PIP2 and 99.9:0.1 PC/PIP2
vesicles; the average electrostatic potential at the surface of these
vesicles is very small or negligible and cannot drive the strong
binding of MARCKS-(151
175) to PIP2. Specifically, the
zeta potential (average potential 0.2 nm from surface) is only
8 mV
(Fig. 4) or
1 mV for 1% PIP2 or 0.1% PIP2
vesicles, respectively. Note that the molar partition coefficient does
not change when the peptide concentration is increased from 2 nM (Fig. 5, filled symbols) to 5 nM
(Fig. 5, open symbols). This observation that the molar partition coefficient is independent of peptide concentration when the
[lipid]
[peptide] (see Equation 1), provides an important control against several potential artifacts (e.g. the
binding sites are not saturated).

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Fig. 5.
The binding of
MARCKS-(151 175) to PC/PIP2 LUVs
depends on the salt concentration. The molar partition
coefficients (K) deduced from binding measurements like
those described for Fig. 2A are plotted versus
the salt concentration of the bathing solution. Increasing the salt
concentration 5-fold decreases the binding 100-fold for PC/PIP2
vesicles containing 1% ( and ) and 0.1% ( and )
PIP2. The filled and open symbols
show the data obtained with 2 nM MARCKS-(151 175) and 5 nM MARCKS-(151 175), respectively.
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Three observations suggest that the binding of MARCKS-(151
175) to
PIP2 depends on a local electrostatic
interaction. First, peptide binding decreases as the salt concentration
increases (Fig. 5). Second, MARCKS-(151
175) binds with similar
affinity to PI(4,5)P2 and PI(3,4)P2 (Fig.
4A). Third, MARCKS-(151
175) binding to the
phosphoinositides (PI, PI(4)P, and PIP2) increases with
lipid charge (Fig. 4). The local positive electrostatic potential adjacent to a MARCKS-(151
175) peptide adsorbed to a bilayer
containing 30% monovalent acidic lipid is illustrated in Fig. 3 of
Ref. 17.
Structure of MARCKS-(151
175) Bound to PC/PIP2
Vesicles--
Both CD and EPR measurements indicate that
MARCKS-(151
175) is in an extended nonhelical form when it is bound to
PC/PS vesicles (50). Our CD measurements suggest that MARCKS-(151
175)
also is in an extended form when it is bound to PC/PIP2
vesicles (data not shown). Thus, the picture (17) of an extended
MARCKS-(151
175) with its aromatics penetrating the polar head group
region derived previously from CD, EPR (50), and monolayer measurements
(25) is probably also valid for the interaction of MARCKS-(151
175) with PC/PIP2 vesicles.
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DISCUSSION |
Previous work showed that both the MARCKS protein and a peptide
corresponding to its effector domain, MARCKS-(151
175), inhibit the
PLC-catalyzed hydrolysis of PIP2 in phospholipid vesicles containing physiological concentrations (33%) of the monovalent acidic
lipid PS; interaction of the peptide with Ca2+/calmodulin
or phosphorylation by PKC reverse the inhibition (18). The results we
report here show that this reversible inhibition is not an artifact
related to the peptide-induced aggregation of the vesicles (20);
MARCKS-(151
175) inhibits the PLC-catalyzed hydrolysis of
PIP2 in monolayers comprising a mixture of
PC/PS/PIP2 (Fig. 1). The simplest interpretation of these
results is that the effector domain of MARCKS binds to PIP2
with high affinity and competes successfully with the catalytic domain
of PLC for this lipid. Our observation that
100 nM
MARCKS-(151
175) produces 90% inhibition of PLC-catalyzed hydrolysis
of PIP2 (Fig. 1) thus provides information about the
affinity of the peptide for PIP2 presented in a
physiologically relevant PC/PS/PIP2 surface. This information is difficult to obtain by conventional binding techniques because the peptide also binds strongly to surfaces containing physiological concentrations of monovalent acidic lipids like PS
(25).
Direct binding measurements show that incorporating PIP2
into PC vesicles greatly enhances the binding of MARCKS-(151
175) to
the vesicles (Fig. 2). Specifically,
10
8
M PIP2, in form of PC/PIP2 vesicles
containing either 0.01%, 0.1%, or 1% PIP2, binds 50% of
MARCKS-(151
175). This strong binding probably involves formation of
an electroneutral complex consisting of one +13 valent
MARCKS-(151
175) and three or four PIP2 because peptide
binding does not produce charge reversal of vesicles in the
electrophoretic mobility experiments (Fig. 4A) and
competition measurements suggest the peptide binds to several
PIP2 (Fig. 3). Two experiments suggest that local,
nonspecific electrostatic interactions contribute strongly to the
binding; MARCKS-(151
175) exhibits no specificity for
PI(4,5)P2 versus PI(3,4)P2 (Fig.
4A), and increasing the salt concentration 5-fold decreases
the binding 100-fold (Fig. 5).
The interaction of PIP2 with MARCKS-(151
175) may be
compared with its interaction with neomycin and the PH domain of
PLC-
1, two other well characterized molecules that bind
PIP2 with high affinity. Addition of
10
5, 10
6, or
10
8 M PIP2 (in the
form of PC/PIP2 vesicles) binds 50% of neomycin (45), the
PH domain (51), or MARCKS effector domain (Fig. 2), respectively.
Neomycin and the PH domain form 1:1 complexes with PIP2
(45, 51-53) whereas MARCKS-(151
175) probably interacts with several
PIP2 (this report).
The binding of MARCKS-(151
175) to vesicles containing monovalent
acidic lipids such as PS involves three forces: a long range Coulomb
attraction, a short range Born/dehydration repulsion, and a short range
hydrophobic attraction of the aromatic residues for the polar head
group region of the bilayer (25). Although these three forces also must
be important in mediating the interaction of the peptide with membranes
containing PIP2, this binding is not yet well understood at
a molecular level. Additional experimental information is required to
understand the role of the 5 Phe and other specific residues in the
binding of MARCKS-(151
175) to PIP2. Ongoing NMR
experiments, EPR measurements with spin-labeled PIP2,2 and our
quenching measurements with fluorescent PIP2 should provide useful information. The direct binding measurements we report here,
however, are sufficient to show that the effector domain of MARCKS
binds to PIP2 with high affinity (Figs. 2
5) and
sequesters the PIP2 away from the catalytic domain of PLC
(Fig. 1). Extrapolating from our observations on the MARCKS protein and
effector domain peptide in model systems, we hypothesize that the
effector domain of MARCKS reversibly sequesters a significant fraction
of the PIP2 in the plasma membrane of cells.
Biological Implications of MARCKS Effector Domain Binding to
PIP2--
Fig. 6 illustrates
what is known about the binding of MARCKS to membranes. Work from many
different laboratories has shown the MARCKS protein binds to
phospholipid vesicles
and by implication to plasma membranes in
cells
through hydrophobic interaction of its N-terminal myristate with
the interior of the bilayer and electrostatic interaction of its basic
effector domain (residues 151
175) with acidic lipids (Refs. 54
58;
reviewed in Refs. 16 and 17). Fig. 6 illustrates the 13 positively
charged residues (blue plus signs) and 5 aromatic residues
(green ovals) in the effector domain; much of what we know
about the interaction of the effector domain with membranes comes from
experiments with the MARCKS-(151
175) peptide, which appears to bind
in a similar manner to the effector region in the intact protein
(reviewed in Ref. 17). The regions of MARCKS flanking the effector
domain contain negatively charged residues (red minus signs)
and should be repelled from the negatively charged surface. What is new
is the recognition that the effector region can bind PIP2
(lipids with red circles in Fig. 6) with high affinity; in a
PC/PIP2 membrane,
4 PIP2/peptide are bound.
Biological membranes such as the plasma membrane also contain
monovalent acidic lipids that may affect the binding stoichiometry, so
the exact number of PIP2 bound is not known. It must be
significant, however, because both the peptide and protein inhibit
PLC-catalyzed hydrolysis of PIP2 on membranes that contain
33% monovalent acidic lipids (see Fig. 1 and Ref. 18).

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Fig. 6.
Schematic of MARCKS interacting with the
plasma membrane. MARCKS is shown with its N-terminal
myristate (orange) inserted into the lipid bilayer and its
effector domain (151 175) associated with the inner leaflet of the
plasma membrane. The 13 positively charged residues (blue
plus signs) of the effector domain interact electrostatically with
acidic lipids such as PS and PIP2. The lipids with
red head groups represent PIP2; lipids with
white head groups represent other phospholipids such as PS
and PC. The 5 Phe residues (green) of the effector domain
penetrate into the polar head group region. For residues 125 200,
blue plus signs indicate positively charged amino acids and
red minus signs indicate negatively charged amino acids.
Binding of Ca2+/calmodulin to the effector domain or
phosphorylation of 3 Ser residues within this domain by PKC produce
desorption of the effector domain from the membrane and concomitant
release of the bound PIP2.
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There are several corollaries to our hypothesis that MARCKS binds a
significant fraction of PIP2 in the plasma membrane and releases it upon interaction with Ca2+/calmodulin or
phosphorylation by PKC. First, if MARCKS is to bind a significant
fraction of the PIP2, it must be present in cells at
concentrations comparable to PIP2; it is in many cell types
(e.g. 10 µM in brain, Refs. 13 and 15).
Second, if reversible binding of PIP2 by MARCKS is
important for phosphoinositide function, overexpression of MARCKS
should stimulate increased production of PIP2 to maintain a
constant free concentration of PIP2 in the membrane; this
has been observed in some cells (59). Third, in cells where MARCKS is
distributed nonuniformly in the plasma membrane (presumably because of
protein-protein interactions, possibly with actin), PIP2
should be colocalized with MARCKS; in fibroblasts, both MARCKS and
PIP2 are concentrated in membrane ruffles (60
62). Fourth,
the mechanism by which MARCKS is targeted selectively to the plasma
membrane is unknown; the interaction of the effector domain with
PIP2 documented here could contribute to this targeting, as
could the less specific electrostatic targeting mechanism that Silvius
and co-workers (63) recently proposed with respect to the targeting of
Ki-Ras4B to the plasma membrane. Fifth, and perhaps most importantly,
local activation of PKC or calmodulin should produce a local increase
in the free concentration of PIP2 in the plasma membrane;
although this has been observed in model systems (see Fig. 3 in Ref.
18), it is not known if Ca2+/calmodulin and PKC can produce
the release of PIP2 bound by the effector domain of MARCKS
in biological cells. This putative function of MARCKS could be examined
in cells using green fluorescent protein-MARCKS (64) and different
fluorescent indicators for PIP2, such as fluorescently
labeled neomycin (65) and green fluorescent protein-PH (66-69).