The Effector Domain of Myristoylated Alanine-rich C Kinase Substrate Binds Strongly to Phosphatidylinositol 4,5-Bisphosphate*

Jiyao Wang, Anna Arbuzova, Gyöngyi Hangyás-Mihályné, and Stuart McLaughlinDagger

From the Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661

Received for publication, September 12, 2000, and in revised form, October 24, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151-175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187-26193). We report here that adding 10-100 nM MARCKS-(151-175) to a subphase containing either PLC-delta or -beta inhibits hydrolysis of PIP2 in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP2. Two direct binding measurements, based on centrifugation and fluorescence, show that approx 10 nM PIP2, in the form of vesicles containing 0.01%, 0.1%, or 1% PIP2, binds 50% of MARCKS-(151-175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151-175) forms an electroneutral complex with approx 4 PIP2. MARCKS-(151-175) binds equally well to PI(4,5)P2 and PI(3,4)P2. Local electrostatic interactions of PIP2 with MARCKS-(151-175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mM decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP2 in the plasma membrane, and release the bound PIP2 upon interaction with Ca2+/calmodulin or phosphorylation by protein kinase C.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Although phosphatidylinositol 4,5-bisphosphate (PIP2)1 constitutes only approx 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 approx 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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-alpha -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-alpha -dioleoyl-phosphatidylcholine, [inositol-2-3H]-L-alpha -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-delta 1 expressed in Escherichia coli, a generous gift from Dr. Mario J. Rebecchi, was purified as described elsewhere (22). Recombinant PLC-beta 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 approx 1 min. After 3 min, we added PLC-delta 1 or PLC-beta 1 (final concentration approx  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+ approx  2 µM, as measured using a Ca2+ electrode in separate experiments. A free Ca2+ concentration of 2 µM produces significant activation of both PLC-delta and -beta 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 pi  approx  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-delta and -beta 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, pi , 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-delta 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-delta 1 and PLC-beta 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 (approx  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.
<FR><NU>[P]<SUB>m</SUB></NU><DE>[P]<SUB><UP>tot</UP></SUB></DE></FR>=<FR><NU>K[L]</NU><DE>1+K[L]</DE></FR> (Eq. 1)

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 approx 370 nm and an emission peak at approx 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 approx 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.
P+<UP>PIP</UP><SUB>2</SUB> <LIM><OP><ARROW>⇄</ARROW></OP><UL>K<SUB>a</SUB></UL></LIM> P-<UP>PIP</UP><SUB>2</SUB>

K<SUB>a</SUB>=<FR><NU>[P-<UP>PIP</UP><SUB>2</SUB>]</NU><DE>[P][<UP>PIP</UP><SUB>2</SUB>]</DE></FR> (Eq. 2)

[P]<SUB><UP>tot</UP></SUB>=[P]+[P-<UP>PIP</UP><SUB>2</SUB>]

[<UP>PIP</UP><SUB>2</SUB>]<SUB><UP>tot</UP></SUB>=[<UP>PIP</UP><SUB>2</SUB>]+[P-<UP>PIP</UP><SUB>2</SUB>]
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).


&zgr;=u&eegr;/(ϵ<SUB>r</SUB>ϵ<SUB>0</SUB>) (Eq. 3)
zeta  is the zeta potential of a vesicle, u is the velocity of the vesicle in a unit electric field, eta  is the viscosity of the aqueous solution, varepsilon r is the dielectric constant of the aqueous solution, and varepsilon 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).


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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-delta 1 (0.1 nM), and CaCl2 (free [Ca2+approx  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-beta 1 instead of PLC-delta 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.

In the absence of MARCKS-(151-175), PLC-delta 1 produces rapid hydrolysis of PIP2 following addition of CaCl2 to the subphase (free [Ca2+approx  2 µM); approx 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 approx 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 pi  = 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-beta 1, a different isoform of PLC; 10 nM MARCKS-(151-175) produces approx 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, approx 10 µM MARCKS-(151-175) produced 90% inhibition of PLC-catalyzed hydrolysis in vesicles containing approx 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 approx 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-delta 1 and PLC-beta 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-delta 1 (or PLC-beta 1), and CaCl2 (free [Ca2+approx  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.

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 approx 50% inhibition of the PLC-delta 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 approx 50% from approx -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 approx  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 approx  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 approx 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 approx  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% (open circle ), 0.1% (), 0.01% (triangle ), and 0% (down-triangle) 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% (open circle ) 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.

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 approx 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, approx 50 nM MARCKS-(151-175) should be required to decrease the percentage of peptide bound from approx 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 (open circle ) 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.

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 approx 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 approx 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 (triangle ), 98:2 PC/PI(4,5)P2 (), and 98:2 PC/PI(3,4)P2 (black-down-triangle ) vesicles, but does not affect the zeta potential of PC (open circle ) vesicles. B, MARCKS-(151-175) has little effect on the zeta potential of MLVs containing monovalent acidic lipids: 97:3 PC/PS (triangle ), 94:6 PC/PS (), 94:6 PC/PI (black-down-triangle ) or PC only (open circle ). 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.

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 approx +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 approx -3 valent or three approx -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 approx 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 approx 100-fold for PC/PIP2 vesicles containing 1% ( and open circle ) and 0.1% (black-triangle and triangle ) PIP2. The filled and open symbols show the data obtained with 2 nM MARCKS-(151-175) and 5 nM MARCKS-(151-175), respectively.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 approx 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, approx 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-delta 1, two other well characterized molecules that bind PIP2 with high affinity. Addition of approx 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, approx 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.

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).


    ACKNOWLEDGEMENTS

We thank Mario J. Rebecchi, Srinivas Pentyala, and Edward Tall for a gift of PLC-delta 1 (supported by National Institutes of Health Grant GM43422 to M. J. Rebecchi), Suzanne Scarlata for a gift of PLC-beta 1, and Andrew Morris for a gift of PIP2. We thank Erwin London and David Cafiso for helpful discussions.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM24971 and National Science Foundation Grant MCB9729538 (to S. M.).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.

Dagger To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, NY 11794-8661. Tel.: 631-444-3615; Fax: 631-444-3432; E-mail: smcl@epo.som.sunysb.edu.

Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M008355200

2 D. Cafiso, personal communication.


    ABBREVIATIONS

The abbreviations used are: PIP2 or PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; MARCKS, myristoylated alanine-rich protein kinase C substrate; MARCKS-(151-175), peptide corresponding to residues 151-175 of bovine MARCKS; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PI(4)P, phosphatidylinositol 4-phosphate; NEM, N-ethylmaleimide; IP3, inositol 1,4,5-trisphosphate; PLC, phosphoinositide-specific phospholipase C; PH, pleckstrin homology; PKC, protein kinase C; K, molar partition coefficient (Equation 1); Ka, apparent association constant for 1:1 binding (Equation 2); LUV, large unilamellar vesicle; MLV, multilamellar vesicle; CD, circular dichroism; EPR, electron paramagnetic resonance; MOPS, 4-morpholinepropanesulfonic acid; N, newton(s).


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RESULTS
DISCUSSION
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