©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Phosphatidylserine-binding Region of Rat MARCKS (Myristoylated, Alanine-rich Protein Kinase C Substrate)
ITS REGULATION THROUGH PHOSPHORYLATION OF SERINE 152 (*)

Takashi Nakaoka (1), Naoya Kojima (2), Teruhiko Ogita (1), Shuichi Tsuji (2)(§)

From the (1) Fourth Department of Internal Medicine, School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 112, Japan and the (2) Laboratory for Molecular Glycobiology, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-01, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We reported previously that recombinant myristoylated, alanine-rich protein kinase C substrate (MARCKS) expressed in Escherichiacoli as well as MARCKS purified from rat brain specifically bound to phosphatidylserine (PS) in a calcium-independent manner and that the binding was regulated through phosphorylation of MARCKS (Nakaoka, T., Kojima, N., Hamamoto, T., Kurosawa, N., Lee, Y. C., Kawasaki, H., Suzuki, K., and Tsuji, S. (1993) J. Biochem. (Tokyo) 114, 449-452). In this study, to identify the minimum PS-binding region of MARCKS and the regulatory phosphorylation site, the binding of MARCKS to PS was examined in deletion mutants producing glutathione S-transferase (GST) fusion proteins. The mutant proteins GST-6-180 and GST-127-160 had almost the same ability to bind to immobilized PS as MARCKS purified from rat brain, whereas GST-127-152 did not bind to it. In addition, the binding of GST-6-156 to immobilized PS was 62% of that of GST-6-180, but that of GST-6-152 was only 8% and that of GST-6-135 was not detected. The effect of phosphorylation by protein kinase C was examined in several mutants of GST-6-180 whose serine residues were substituted with alanine. After phosphorylation, the mutants GST-6-180[S156A and S163A], GST-6-180[S156A], and GST-6-180[S163A] did not bind to immobilized PS like native MARCKS and GST-6-180. However, even after phosphorylation, GST-6-180[S152A] and GST-6-180[S152A and S156A] could bind to immobilized PS.

These results strongly suggest that MARCKS binds to PS molecules in the inner leaflet of the plasma membrane through residues 127-156, with residues 153-156 (FKKS) being particularly important in the binding of MARCKS to PS, and that the binding is regulated through the protein kinase C-catalyzed phosphorylation of the serine at residue 152.


INTRODUCTION

Protein kinase C (PKC)() has been established to be the mainstay of a number of intracellular signaling systems, especially that concerned in Ca mobilization. Myristoylated, alanine-rich protein kinase C substrate (MARCKS) is a major substrate and is suggested to be involved in the cellular signaling pathway involving activation of PKC (1) . Although the physiological function of MARCKS has not yet been clearly elucidated, the association of MARCKS with the plasma membrane or cytoskeleton (2) has been suggested to be of importance regarding the function of MARCKS. MARCKS associates with the plasma membrane under regulation through PKC-catalyzed phosphorylation of MARCKS (3, 4) . It has been suggested that MARCKS is capable of associating with the plasma membrane through binding to phospholipids without interaction with membranous proteins (5, 6, 10) .

PKC (7) , synaptotagmin (8) , and coagulation factor V (9) have been reported to bind specifically to phosphatidylserine (PS), and their binding domains have been reported. The interaction of coagulation factor V with the platelet surface is considered to involve binding to PS. We reported previously that bacterially expressed full-length recombinant MARCKS clearly binds to PS, just like MARCKS purified from rat brain (10) . The binding of MARCKS to PS is abolished on PKC-catalyzed phosphorylation. This is consistent with the reversible association of MARCKS with the plasma membrane. Thus, it is very important to elucidate the physiological role of the PS binding of MARCKS. We have investigated the structure of MARCKS for binding to PS using a bacterial expression system. As a result, the Ca-independent PS-binding domain of rat MARCKS was identified within a region of 30 amino acid residues in length.


EXPERIMENTAL PROCEDURES

Materials

PS, phosphatidylcholine (PC), phosphatidylinositol (PI), phorbol 12-myristate 13-acetate, and cholesterol were obtained from Sigma. Other chemicals were purchased from the following sources: [1,2-H]cholesterol and ENHANCE spray from DuPont NEN; bacterial expression vector pGEX-2T, goat anti-glutathione S-transferase antibody, protein A-Sepharose 4B, and glutathione-Sepharose 4B from Pharmacia Biotech Inc.; nylon sheets (Immobilon) from Millipore; 3,3`,5,5`-tetramethylbenzidine dihydrochloride and goat anti-rabbit IgG peroxidase-conjugated antibody from Cappel; and swine anti-goat IgG peroxidase-conjugated antibody from Tago Inc. Bacterial strain BL21(DE3)pLysS and bacterial expression vector pET-8c were obtained from the RIKEN GENE BANK. PKC was prepared from rat brain as described (10). Rabbit anti-MARCKS antiserum was prepared by immunizing a rabbit with 300 µg of purified rat MARCKS using the standard procedure. The resulting anti-MARCKS antiserum stained one band by immunostaining of a rat brain homogenate with a Konica Immunostain HRP kit.

Assay for Binding of Liposomes to MARCKS (Liposome Binding Assay)

Unilamellar liposomes labeled with [1,2-H]cholesterol were prepared as described previously (10). In the case of PC liposomes, lyso-PC was included at a PC/lyso-PC ratio of 7:3. Rat MARCKS was purified to a homogeneous state as judged by protein staining of SDS-polyacrylamide gel. To examine the binding of liposomes to MARCKS, the indicated amounts of the purified protein were adsorbed to the wells of a 96-well flat-bottom assay plate (Pro-bind, Falcon) for 16 h at 4 °C. After being blocked with 4% skim milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, 150 mM NaCl, pH 7.5), 50-µl aliquots of TBS, 2 mM EGTA containing labeled phospholipid liposomes (10 dpm/ml) were distributed in the wells, followed by incubation for 16 h with slow shaking at room temperature. After unbound liposomes had been removed by washing three times with TBS, 1 mM EGTA, the bound liposomes were collected in 2-propanol/hexane/HO (50:25:20) and then counted in triplicate using a scintillation counter.

Assay for Binding of Fusion Proteins to Immobilized Phospholipid (Protein Binding Assay)

Phospholipid (0.03-2.0 µg) in 50 µl of 50% aqueous ethanol was adsorbed to each well of a 96-well flat-bottom assay plate for 8 h at 37 °C. After being blocked with 4% skim milk in TBS, the wells were overlaid with fusion proteins in 50 µl of TBS, 2 mM EGTA containing 1% skim milk, followed by incubation for 12 h at 4 °C. After washing out the unbound proteins, the bound fusion proteins were measured as the absorbance at 650 nm of 3,3`,5,5`-tetramethylbenzidine dihydrochloride using anti-MARCKS antiserum or anti-GST antibody and corresponding peroxidase-conjugated secondary antibodies. The binding of fusion proteins to a PS-coated plate reached a plateau after incubation for >8 h at 37 °C. The amount of proteins bound to a PS-coated plate after incubation for 12 h at 4 °C was comparable to that after incubation for 8 h at 37 °C.

Construction of MARCKS Mutants

A 4.4-kilobase pair clone was obtained from a rat brain cDNA library as rat MARCKS cDNA (10) . An XhoII fragment (1.1 kilobase pairs) cloned into pBluescript (pBSK), which contained all the coding region of rat MARCKS (11) , was restricted with NotI, and then the resulting fragment (0.6 kilobase pair), which contained the start codon, was cloned into pBSK. The resulting plasmid, NotI SK, was transfected into bacterial strain cj236, and single-stranded DNA was prepared as a mutagenesis template. To construct a plasmid to express a fusion protein of GST and MARCKS, a new BamHI site and a new stop codon were introduced into a plasmid, which was cloned into pBSK by site-directed mutagenesis using Mutagene kits (Bio-Rad)

In the case of GST-6-180, the primer GCGGTCTTGGATCCCTGTGCACCCA was annealed to the template DNA, and a new BamHI site was created for ligation to pGEX-2T in frame at Ser-6 of MARCKS. The resulting plasmid was further transfected into cj236, and single-stranded DNA was prepared as a template DNA. Using the primer GGCCTCGTCCTTGGCGATATCTCACTAGGCGCCTTCCGCCTC, a stop codon (TAG) was introduced at residue 181 of MARCKS (the portion corresponding to the stop codon is underlined).

For fusion to GST at residues 46, 98, and 127, a new BamHI site was created by site-directed mutagenesis using the following primers: CTCGGCGGCGGCGGGGGATCCGTCCCCGTTCACTTT, AGCCTCCTTGTCGGCGGATCCAGCGCCCGGCTCGGG, and GTCCTCCGCCTTGGGGGATCCCGTGGAGGAGGCGGA, respectively. The authentic threonine (residue 98) of rat MARCKS was substituted with serine for ligation to the vector in GST-98-180. A new stop codon (underlined) was introduced at residues 168, 161, 157, 153, and 136 using the following primers: CTCAGCGCCCTCGCCGTTAACTCACTAGCTCTTCTTGAAGGAGAAGCCG, CTTGCTCTTCTTGAAGTTAACTCACTAGCTCAGCTTGAAGGACTTCTT, CTTGAAGGAGAAGCCGTTAACCTATCAGGACTTCTTGAAGGAAAAGCG, GCCGCTCAGCTTGAAGTTAACCTATCAGGAA-AAGCGCTTCTTTTTTTT, and CGGGGTCTCGCTGATATCTCACTAGGCCCCGTCCTC, respectively.

In the point mutation experiment, the authentic serine of the phosphorylation site was substituted with alanine by site-directed mutagenesis using the following primers (portions of nucleic acids for point mutations are underlined): S152A, GAAGGACTTCTTGAACGCAAAGCGCTTCTTTT; S156A, GCTCAGCTTGAACGCCTTCTTGAAGGAAAA; S163A, CTTGCTCTTCTTGAACGCGAAGCCGCTCAG; S152A and S156A, GAAGCCGCTCAGCTTGAACGCCTTCTTGAACGCAAAGCGCTTCTTTT; and S156A and S163A, CTTCTTGCTCTTCTTGAACGCGAAGCCGCTCAGCTTGAACGCCTTCTTGAAGGAAAAGCG.

In the case of ligation to pET-8c, all the mutants were ligated, using the NcoI site (5`-end) and the blunt-ended BamHI site (3`-end) of pET-8c, by introducing a new NcoI site by site-directed mutagenesis. The structures of the mutants were confirmed by DNA sequencing with a DNA sequencer (Pharmacia Biotech Inc.). Fragments derived from corresponding plasmids were isolated from agarose gel pieces and ligated into the BamHI site (5`-end) and the SmaI site (3`-end) of pGEX-2T. Positive clones were selected by restriction analysis and DNA sequencing.

Expression and Purification of the Bacterially Expressed Protein

BL21(DE3)pLysS transformed with the recombinant clone in pGEX-2T was cultured in M9H broth (12) containing 30 µg/ml ampicillin and 30 µg/ml chloramphenicol. Following the addition of isopropyl-1-thio--D-galactopyranoside to 0.4 mM at the mid-growth stage, the cells were further cultured for 4 h at 30 °C. After harvesting, Escherichia coli cells suspended in 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride were sonicated for 30 s twice at 4 °C. The lysate was centrifuged at 10,000 g for 15 min at 4 °C, and the resulting supernatant was collected as the soluble fraction.

For purification, the GST fusion protein was adsorbed to glutathione-Sepharose 4B by the batch method according to the manufacturer's instructions and then eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, after washing five times with TBS containing 2 mM EGTA. The eluted fraction was gel-filtrated on a Sephadex G-25 column in TBS and then subjected to the binding assay.

Phosphorylation with PKC

Mutant proteins were phosphorylated by incubation with PKC in 20 mM Tris-HCl, pH 7.4, 10 mM MgCl, 2 mM CaCl, 5 mM NaF, 0.1 mM NaVO, 1 µM phorbol 12-myristate 13-acetate, and 0.5 mM ATP for 30 min at 37 °C following the addition of EGTA (final concentration, 10 mM). The binding of phosphorylated proteins to PS was compared with that of mutant proteins incubated with PKC without ATP in the presence of 10 mM EGTA instead of MgCl.

RESULTS

MARCKS Recognizes PS Molecules

Using a liposome blotting method, we showed previously that PS liposomes bind to MARCKS (10) . To determine how the phospholipid composition affects the binding, we analyzed the binding of phospholipid/cholesterol liposomes with different PS/PC ratios to immobilized MARCKS by the liposome binding assay (Fig. 1). In this assay, the binding of PC liposomes (100% PC as phospholipid) to MARCKS was 5% of that of PS liposomes (100% PS as phospholipid). As the ratio of PS to PC decreased, the binding of liposomes to MARCKS also decreased, indicating that MARCKS recognizes PS on the liposomes.


Figure 1: Phospholipid composition affects the binding of liposomes to MARCKS. H-Labeled phospholipid/cholesterol liposomes (1:1 mol/mol) were prepared. The ratio of PS to PC (mole/mole) was 1:0 (closedcircles), 1:1 (opencircles), 1:3 (opensquares), and 0:1 (closedsquares). The binding of each type of liposome to MARCKS was measured as described under ``Experimental Procedures.''



To quantify the specificity of the binding of MARCKS to phospholipid, the binding of MARCKS to immobilized phospholipid was determined (protein binding assay). MARCKS bound to immobilized PS, but the binding of MARCKS to immobilized PI was only 5% of that to PS and that to PC was not detected ().

Binding of Recombinant MARCKS to PS

As previously reported, bacterially expressed full-length MARCKS, as well as MARCKS purified from rat brain, binds to PS (10) . To investigate further the PS-binding domain of MARCKS, several deletion mutants were expressed as GST fusion proteins, and PS binding was determined by the protein binding assay (Fig. 2). The purity of the fusion proteins was checked by SDS-PAGE (Fig. 3). The exact migration position of fusion proteins on SDS-PAGE is hard to estimate from their molecular mass because native MARCKS is anomalously retarded from the position estimated from its molecular mass on SDS-PAGE. GST-6-180, GST-46-180, GST-6-152, and GST-6-135 migrated as single bands on SDS-PAGE. GST-98-180 and GST-127-180 were doublets on SDS-PAGE. Although proteolysis may be involved in these cases, these proteins were used for the binding assay without further purification.


Figure 2: Construction of mutants. The structure of rat MARCKS, composed of 309 amino acids, is schematically represented. The phosphorylation site domain (residues 145-169; hatchedbox), myristoylation site (Myr), and phosphorylation site (P) are indicated. The mutants of MARCKS expressed as GST fusion proteins are designated according to the amino acid sequence of MARCKS, with the GST portion denoted by a filledbox. The binding of each protein to 0.08 µg of immobilized PS, as determined by the protein binding assay, was compared. Data are expressed in relation to the binding of GST-6-180 taken as 100%.




Figure 3: Purified mutants of MARCKS. The GST fusion proteins, which were purified with glutathione-Sepharose 4B, were subjected to SDS-PAGE on a 5-20% gel. The gels were stained with Coomassie Brilliant Blue R-250 and then dried. GST-6-180, GST-46-180, GST-98-180, and GST-127-180 migrated as 66-, 56-, 43-, and 31-kDa bands, respectively. GST-6-152 migrated indistinguishably from GST-6-180, whereas GST-6-135 migrated as a 48-kDa band. The molecular mass standards are indicated on the left.



PS binding of the protein was not affected by fusion with GST since both GST-6-309 and GST-6-180 specifically bound to PS, but not to PI and PC, like MARCKS purified from rat brain (). On the other hand, the control GST did not bind to immobilized PS. GST-6-180 bound to PS at a molecular ratio of 1:200, which is the same as in the case of native MARCKS. Thus, residues 181-309 are not involved in PS binding.

The binding of GST-6-180 to PS was linear within the range of 0.03-0.12 µg of immobilized PS, and the protein bound to PS at >0.3 µg reached a plateau (Fig. 4). Therefore, the binding assay was performed with 0.08 µg of immobilized PS. The binding of GST fusion MARCKS mutants to immobilized PS is summarized in Fig. 2. The binding of GST-46-180, GST-98-180, and GST-127-180 was the same as that of GST-6-180, indicating that residues 6-126 of rat MARCKS are not involved in the binding to PS. We next examined whether or not the phosphorylation site domain (residues 145-169) was involved in the binding to PS. The binding of GST-6-167, GST-6-160, and GST-6-156 to PS was 94, 71, and 62% of that of GST-6-180, whereas the binding of GST-6-152 was greatly reduced compared with that of GST-6-180 and that of GST-6-135 was negligible (Fig. 5). In addition, GST-127-160 bound to PS as GST-6-160 did, whereas GST-127-152 did not (Fig. 2). These results indicate that residues 127-156, particularly residues 153-156, are closely involved in the binding to PS.


Figure 4: Characterization of the binding of GST-6-180 to PS. The binding of GST-6-180 to increasing amounts of immobilized PS was measured in triplicate by the protein binding assay as described under ``Experimental Procedures.'' Bound proteins were measured as the absorbance at 650 nm of 3,3`,5,5`-tetramethylbenzidine dihydrochloride using goat anti-GST antibodies and peroxidase-conjugated secondary antibodies.




Figure 5: PS binding of GST MARCKS mutants whose C terminus was deleted stepwise. GST fusion proteins, with almost the same absorbance as determined by enzyme-linked immunosorbent assay using anti-GST antibody, were overlaid on immobilized PS. Proteins bound to immobilized PS were measured by the protein binding assay as described for Fig. 4. Opencircles, GST-6-180; closedcircles, GST-6-167; closedtriangles, GST-6-160; closedsquares, GST-6-156; open triangles, GST-6-152; opensquares, GST-6-135.



Effect of Phosphorylation with PKC

Since the phosphorylation of MARCKS with PKC significantly decreased the binding to PS, as described (10) , we examined how phosphorylation with PKC affects the binding of MARCKS to PS using a GST fusion protein and mutant proteins whose serine residues were substituted with alanine residues. Phosphorylation of GST-6-180 clearly abolished the binding of the protein to PS. The possible phosphorylation sites of rat MARCKS for PKC are considered to be Ser-152, Ser-156, Ser-163, and Ser-167, of which Ser-152, Ser-156, and Ser-163 are conserved between species and were demonstrated to be phosphorylated by PKC in chick MARCKS (13) . Substitution of serine with alanine did not affect the binding to PS of these mutants (Fig. 6). The binding of GST-6-180[S156A], GST-6-180[S163A], and GST-6-180[S156A and S163A] to PS was almost completely abolished when the mutant proteins were phosphorylated with PKC, as in the case of native MARCKS and GST-6-180 (Fig. 6). On the other hand, the binding of phosphorylated GST-6-180[S152A] to PS decreased compared with that of the unphosphorylated form, but still occurred (42% of the unphosphorylated level). Phosphorylation of GST-6-180[S152A and S156A] decreased the binding to PS only slightly (74 ± 4% of the unphosphorylated level) (Fig. 6). Thus, PKC-catalyzed phosphorylation of Ser-152 is closely involved in regulation of the binding to PS.


Figure 6: Effect of the phosphorylation site on PS binding. The phosphorylation site of GST-6-180 was substituted as described under ``Experimental Procedures.'' The mutants, incubated with PKC and ATP (phosphorylated; shaded bars) or without ATP (unphosphorylated; open bars), were overlaid on immobilized PS. Proteins bound to 0.12 µg of immobilized PS were measured by the protein binding assay as described for Fig. 4.



DISCUSSION

We previously reported that MARCKS specifically binds to PS (10) . On the other hand, Taniguchi and Manenti (5) reported that MARCKS binds to other phospholipids as well as to PS. To clarify this difference, two assay systems for the binding of phospholipids and MARCKS were used to delineate the specificity of the binding in this study. With the liposome binding assay, the binding of PI liposomes to MARCKS was comparable to that of PS liposomes, and only weak binding of PC liposomes to MARCKS was detected. On the other hand, MARCKS clearly bound to only PS, i.e. not to PI or PC, when the binding of MARCKS to immobilized phospholipids was quantified using the protein binding assay. This apparent discrepancy may reflect the difference in the strength of the binding of MARCKS and phospholipid in the two assay systems. Therefore, MARCKS recognized PS much more strongly than other phospholipids such as PC and PI. It should be tested whether or not PS molecules among phospholipid really play a role in the association of MARCKS with the plasma membrane in vivo.

The results presented here for the GST fusion protein clearly indicate that rat MARCKS interacts with PS mainly via a region within residues 127-156. Furthermore, residues 153-156 (FKKS) of MARCKS are very important for the PS binding of MARCKS since the binding of GST-6-152 to PS decreased by >80% of that of GST-6-156. On the other hand, Taniguchi and Manenti (5) reported that a synthetic peptide corresponding to residues 145-169 of rat MARCKS interacts with phospholipids including PS. Considering this result and the results presented here, residues 145-156 (KKKKKRFSFKKS) may largely contribute to the binding of MARCKS to PS.

The binding domains of proteins for binding to PS, such as the C2 domain of PKC (14) and the C2 domain-like repeats of synaptotagmin (8) , have been reported. Like MARCKS, coagulation factor V does not need Ca for binding to PS-containing liposomes and two potentially different regions of factor V have been separately suggested to be the PS-binding domain: one is the region within the third A domain of the light chain, which contains two hydrophobic segments (15) , and the other is a region within the second C-type domain (16) . However, there is no homology between the PS-binding domain of MARCKS and those of other PS-binding proteins. Since the phosphatidylinositol bisphosphate-binding sequence of gelsolin was identified as only a 9-amino acid peptide consisting of a motif of basic amino acids (17) , the PS-binding domain of MARCKS seems to be rather similar in the case of phosphatidylinositol bisphosphate-binding proteins (gelsolin, gCap39, villin, profilactin, etc.). However, the PS-binding domain of MARCKS is considered to have a unique Ca-independent PS recognition motif because the binding of MARCKS to phosphatidylinositol bisphosphate was not detected.()

Cosubstitution of serine at both residues 156 and 163 with alanine did not affect regulation of the binding of MARCKS to PS through PKC-catalyzed phosphorylation, but substitution of serine at residue 152 with alanine clearly affected this regulation. Therefore, phosphorylation of serine at residue 152, which exists in the PS-binding region of MARCKS, is sufficient for affecting the binding to PS. Phosphorylated GST-6-180[S152A and S156A] bound to PS more than phosphorylated GST-6-180[S152A], although GST-6-180[S156A] did not bind to PS after phosphorylation, indicating that phosphorylation of serine at residue 156, in addition to serine at residue 152, has some additional effect on the binding to PS.

It is apparent that N-terminal myristoylation (18) is not necessary for binding to PS because GST fusion mutants bind to PS. The myristoyl moiety of proteins is not considered to serve as a hydrophobic anchor because many myristoylated proteins are found in the cytosol, in contrast to palmitoylated proteins, which are exclusively localized in the membrane (19) . Although the role of the myristoyl moiety has not yet been established, many myristoylated proteins exist as components of intracellular signaling systems. The myristoyl moiety and PS-binding domain identified in this study must regulate the localization of MARCKS cooperatively. In this connection, it is interesting that demyristoyl activity of MARCKS exists in the synaptic fraction to regulate the function of MARCKS (20) .

The linking of the plasma membrane to the cytoskeleton is suggested to be an important feature of MARCKS. The ability of MARCKS to bind to PS may assure the association of MARCKS with the plasma membrane directly. It may be consistent with the reversible association of MARCKS with the plasma membrane that the binding of MARCKS to PS is abolished on PKC-catalyzed phosphorylation. On the other hand, it was reported that the phosphorylation and translocation of MARCKS are not parallel in the N1E-115 neuroblastoma cell line (21) . The interaction of MARCKS with calmodulin (22) or actin may largely contribute to its function. It is also probable that PS acts as a cofactor when MARCKS interacts with other proteins, as in the case of PKC. The expression of MARCKS mutants, such as with substitution of serine at residue 152 with alanine, in eucaryotic cells may allow elucidation of whether the PS binding property is involved in the membrane association or the interaction of MARCKS with other molecules.

  
Table: Binding of MARCKS to phospholipid

The binding of MARCKS purified from rat brain and GST fusion proteins expressed in E. coli. to 0.08 µg of phospholipid was measured by the protein binding assay using anti-MARCKS and peroxidase-conjugated goat anti-rabbit IgG antibodies as described under ``Experimental Procedures.'' Data are expressed in relation to the binding to PS taken as 100%.



FOOTNOTES

*
This work was supported by Grants-in-aid for Scientific Research on Priority Areas 0625213 (to S. T.) and for Encouragement of Young Scientists 06770042 (to N. K.) from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Frontier Research Program, Inst. of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, Japan. Tel.: 81-48-462-1111 (ext. 6521); Fax: 81-48-462-4692.

The abbreviations used are: PKC, protein kinase C; MARCKS, myristoylated, alanine-rich protein kinase C substrate; PS, phosphatidylserine; PC, phosphatidylcholine; PI, phosphatidylinositol; TBS, Tris-buffered saline; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

T. Nakaoka, N. Kojima, T. Ogita, and S. Tsuji, unpublished observation.


ACKNOWLEDGEMENTS

We are grateful to Dr. Masato Umeda (Faculty of Pharmaceutical Science, University of Tokyo) for helpful discussion.


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