©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Membrane Association of the Myristoylated Alanine-rich C Kinase Substrate (MARCKS) Protein
MUTATIONAL ANALYSIS PROVIDES EVIDENCE FOR COMPLEX INTERACTIONS (*)

Sharon L. Swierczynski (§) , Perry J. Blackshear (¶)

From the (1) Howard Hughes Medical Institute, Durham, North Carolina 27710 and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent cellular substrate for protein kinase C, is associated with membranes in various cell types. MARCKS is myristoylated at its amino terminus; this modification is thought to play the major role in anchoring MARCKS to cellular membranes. Recent studies have suggested that the protein's basic phosphorylation site/calmodulin binding domain may also be involved in the membrane association of MARCKS through electrostatic interactions. The present studies used mutations in the primary structure of the protein to investigate the nature of the association between MARCKS and cell membranes. In chick embryo fibroblasts, activation of protein kinase C led to a decrease in MARCKS membrane association as determined by cell fractionation techniques. Cell-free assays revealed that nonmyristoylated MARCKS exhibited almost no affinity for fibroblast membranes, despite readily demonstrable binding of the wild-type protein. Similar experiments in which the four serines in the phosphorylation site domain were mutated to aspartic acids, mimicking phosphorylation, decreased, but did not eliminate, membrane binding when compared to either the wild-type protein or a comparable tetra-asparagine mutant. Addition of calmodulin in the presence of Ca also inhibited binding of the wild-type protein to membranes, presumably by neutralizing the phosphorylation site domain, or by physically interfering with its membrane association. Surprisingly, expression of a nonmyristoylatable mutant form of MARCKS in intact cells led to only a 46% decrease in its plasma membrane association, as determined by cell fractionation and immunoelectron microscopy. These results are consistent with a complex model of the interaction of MARCKS with cellular membranes, in which the myristoyl moiety, the positively charged phosphorylation site domain, and possibly other domains make independent contributions to membrane binding in intact cells.


INTRODUCTION

Phosphorylation of intracellular substrates by protein kinase C, the diacylglycerol-activated, Ca-dependent protein kinase, is the impetus for a wide range of cellular processes including differentiation, mitogenesis, and hormone secretion (1, 2) . Although the molecular events leading to activation of protein kinase C have been well-characterized, much less is understood about the role of its phosphorylated substrates in these cellular events.

One of the most prominent intracellular substrates for protein kinase C is the myristoylated alanine-rich C kinase substrate or MARCKS() protein (for reviews see Refs. 3 and 4). Although the precise function of this protein has yet to be defined, recent gene disruption studies have indicated that, at least in mice, MARCKS is essential for the normal development of the central nervous system and postnatal survival (5) . The protein is heat-stable, acidic, and characterized by anomalous migration on SDS-polyacrylamide gels (6, 7, 8, 9) . MARCKS contains three highly conserved domains: an amino-terminal myristoylation domain (10) , a region of conserved sequence at the single site of intron splicing (7, 9, 11) , and an internal phosphorylation site domain (PSD; Refs. 12 and 13).

This domain, in addition to containing the serines that are phosphorylated by protein kinase C (12, 13, 14) , also serves as the site of high affinity calmodulin binding (15, 16) . This region has also been shown to cross-link actin filaments in vitro(17) .

The myristoyl modification of MARCKS has been implicated in anchoring the protein to the plasma membrane through hydrophobic interactions (18-20). Although this myristoyl moiety appears to be required for maximal membrane association, other evidence suggests that the positively charged phosphorylation site/calmodulin binding domain (PSD) of the MARCKS protein may be involved in membrane interactions as well. For example, phosphorylation of MARCKS appears to result in its dissociation from cellular membranes in certain cell types (21, 22, 23, 24) . More recently, it has been shown that the MARCKS protein purified from bovine brain (25) , murine MARCKS expressed in a baculovirus system (26), and the MARCKS PSD peptide associate with synthetic lipid vesicles (27) , and that this association is markedly decreased following phosphorylation by protein kinase C (25, 26, 27) .

In the present studies, we have further investigated the potential role of the PSD in MARCKS association with cellular membranes using in vitro binding assays of in vitro synthesized MARCKS proteins mutated in both the myristoylation site and the PSD. In addition, we have examined the membrane association of the phosphorylated and the nonmyristoylated protein through subcellular fractionation and light and immunoelectron microscopy of intact cells. Our data indicate that myristoylation is required for MARCKS association with cellular membranes in a cell-free system, and that the PSD strengthens this interaction; somewhat surprisingly, however, the PSD and/or some other domain of the protein cause even the nonmyristoylated protein to associate with the plasma membrane in intact cells.


MATERIALS AND METHODS

Cells

Mouse LM/TK cells (7) and COS-P cells (28) (a generous gift from Dr. Bryan R. Cullen, Dept. of Immunology, Duke University) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Chick embryo fibroblast cells (CEF), prepared as described (29) , were maintained in minimal essential medium (MEM) supplemented with 5% (v/v) fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) tryptose phosphate.

Human 293 cells (American Type Culture Collection) and stable cell lines derived from them were maintained in MEM supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and, where indicated, 400 µg/ml Geneticin (Life Technologies, Inc.).

Subcellular Fractionation of CEF Cells

CEF cells were grown to confluence and serum-starved overnight in MEM containing 1% (w/v) bovine serum albumin (BSA, lyophilized and crystallized; Sigma), 10% tryptose phosphate, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were treated with 1.6 µM PMA in 0.01% (v/v) dimethyl sulfoxide, or the same concentration of dimethyl sulfoxide as a control, for the times indicated, washed three times with ice-cold phosphate-buffered saline (PBS), and scraped into a homogenization buffer containing 50 mM -glycerophosphate (pH 8.2), 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 10 mM benzamidine-HCl, 1 mM phenyl-methylsulfonyl fluoride, 2 µM pepstatin, 2 µM leupeptin, and 50 mM sodium fluoride. The cells were homogenized on ice with 25 strokes of a glass tissue homogenizer (Wheaton, Millville, NJ). The resulting homogenate was subjected to ultracentrifugation at 86,000 g for 57 min at 4 °C (TL 45 rotor, Beckman Instruments). The pellet fraction was resuspended by sonicating (Ultrasonics W-380, Farmingdale, NY, setting 10) for 15-20 s in an identical volume of homogenization buffer containing 1% (v/v) Triton X-100. The samples containing detergent were incubated on ice for 30 min and centrifuged at 13,440 g, and the resulting supernatant was taken as the particulate fraction. Soluble and particulate fractions were boiled for 10 min and centrifuged at 13,440 g for 15 min at 4 °C, and the supernatants containing the heat-stable proteins were combined with 1/5 volume of SDS sample buffer (1.5 M sucrose, 6% (w/v) SDS, 500 mM dithiothreitol, 60 mM EDTA, 0.006% (w/v) Pyronin Y), boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.

Western Blot Analysis

Proteins from SDS-polyacrylamide gels were transferred to nitrocellulose with a pore size of 0.45 µm (Schleicher & Schuell) at 850 mA for 1 h at room temperature in 1 transfer buffer (25 mM Tris (pH 8.3), 192 mM glycine, 20% (v/v) methanol). The nitrocellulose filters containing immobilized proteins were then blocked in a solution of 3% (w/v) non-fat dry milk (Carnation) in TBS/T (10 mM Tris (8) , 154 mM NaCl, 0.3% (v/v) Tween 20) at room temperature for 1 h. Blots were then incubated with a polyclonal antibody to chicken MARCKS (18) at a 1:500 dilution in TBS/T for 2 h at room temperature. As a secondary antibody, I-Protein A (Amersham) was used at 0.2 mCi/ml in TBS/T for 1 h at room temperature for some experiments. The blots were exposed to a PhosphorImager screen (Molecular Dynamics) for analysis and quantitation. As a secondary antibody in other experiments, goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used at a 1:5000 dilution in TBS/T. Chemiluminescence (ECL kit, Amersham) was also used for detection of immunoreactive proteins in some experiments. Preparation of LM/TKMembranes-LM/TK cells (7) were plated in Petri dishes (100 mm) and grown to confluence. Cells from 5-6 plates were pooled and separated into cytosolic and membrane fractions as described previously (19, 30) except that the final membrane pellet was resuspended in 50 mM Tris (pH 7.4). The final protein concentration of the membrane suspension was determined by a dye binding assay (Bio-Rad).

In Vitro Transcription and Translation

Wild-type bovine MARCKS mRNA was prepared as described previously (19) . Mutations in the myristoylation and phosphorylation site domain (PSD) consensus sequences of the bovine MARCKS cDNA construct pBS80K1.2A (7) were generated by in vitro mutagenesis. Constructs were created in which the amino-terminal glycine was mutated to alanine or the four serines contained within the phosphorylation site domain (12) were mutated to aspartic acids (tetra-Asp) using the oligonucleotide-directed site mutagenesis kit from Amersham. The corresponding asparagine construct (tetra-Asn) was prepared using the Altered Sites in vitro mutagenesis kit (Promega). The mutant plasmids were linearized with EcoRI, and mRNA was synthesized using T3 RNA polymerase as described (Life Technologies, Inc.). The resulting mRNA was translated using a rabbit reticulocyte lysate system (Promega) for 1 h at 30 °C with 4 µg of RNA, 55 µM myristate, and 100 µCi of [S]cysteine (ICN or DuPont NEN) in a 100-µl reaction volume. These conditions have been shown to result in MARCKS that is completely myristoylated (19) . It should be emphasized that all of the expressed proteins contain the same number of cysteines; in addition, all constructs were translated in parallel using the identical reticulocyte lysate containing [S]cysteine of constant specific activity. Therefore, all expressed proteins should have the same specific radioactivity. To test this, we translated two of the constructs using the identical reticulocyte lysate, performed Western blot analysis on 20 µl of the lysate containing both expressed proteins, and quantitated MARCKS immunoreactivity by densitometry. We also measured the radioactivity of each expressed protein by scintillation counting. The resulting specific activities differed by only 16%, confirming that proteins expressed in parallel in this way have essentially identical specific radioactivities.

Membrane Binding Assays

To ensure that the membranes were not saturated in the binding reactions, a titration assay was performed with a constant amount of membrane protein (15 µg) and increasing amounts of reticulocyte lysate containing translated MARCKS protein. Binding for both the myristoylated and nonmyristoylated forms of MARCKS was linear between 1 and 10 µl of lysate added to the reaction (Fig. 2); binding was not saturated under these conditions (see below under ``Results''). In subsequent experiments, 3 µl of the reticulocyte lysate containing the in vitro-translated, [S]cysteine-labeled MARCKS protein was diluted to 50 µl with PBS and precleared of residual lysate material by ultracentrifugation at 107,000 g for 1 h at 4 °C (TLA 100.3 rotor, Beckman Instruments). For each binding assay, 50 µl of diluted, precentrifuged lysate was incubated with the indicated concentrations of membrane protein in PBS in a total volume of 100 µl. The reaction mixture was incubated at 25 °C for 20 min or varying times as indicated, then centrifuged at 107,000 g for 40 min. Under these conditions, binding was constant between 10 min and 1 h. The resulting membrane pellet was resuspended in 30 µl of SDS sample buffer, sonicated (Ultrasonics W-380, setting 10, 10 s), boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis. All gels were treated with Autofluor (National Diagnostics, Atlanta, GA), dried, and exposed to Kodak XAR film at -70 °C. Gel bands were excised, added to scintillation mixture (Ready-Safe, Beckman Instruments), and counted on an LS3801 -counter (Beckman Instruments).


Figure 2: Titration of myristoylated and nonmyristoylated MARCKS binding to LM/TK membranes. RNAs encoding wild-type and nonmyristoylated MARCKS were translated in the same reticulocyte lysate containing [S]cysteine of identical specific activity. Increasing amounts of the translated proteins were incubated with a constant amount (15 µg) of LM/TK membrane protein for 20 min at 25 °C. Following ultracentrifugation, the pellet fractions were subjected to SDS-PAGE. The gel bands were excised and subjected to scintillation counting. The resulting autoradiographs are shown in A and B. The graph in C plots the counts/min associated with the membrane fraction against the amount of lysate containing radioactive MARCKS added to a 100-µl reaction volume.



Membrane Binding Assay in the Presence of Calmodulin

Chicken calmodulin (31) (a generous gift from Dr. Sam George, Dept. of Medicine, Duke University) was dialyzed against PBS using a Centricon 10 microconcentrator (Amicon). The lysate (5 µl) containing [S]cysteine-labeled MARCKS was diluted 1:10 with PBS and boiled for 10 min prior to centrifugation in order to remove as many heat-labile proteins from the lysate as possible, particularly other calmodulin-binding proteins. A 50-µl aliquot of the resulting lysate was incubated for 10 min at room temperature in the presence of 50 µM CaCl, ±50 µM EGTA, 11.6 µM calmodulin, and PBS in a total volume of 95 µl. To this reaction mixture, 5 µg of membrane protein in 5 µl was added. The remainder of the assay was carried out as described above for membrane binding.

Two-dimensional Gel Electrophoresis

Samples for two-dimensional gel electrophoresis were prepared by translation of MARCKS mRNA in a rabbit reticulocyte lysate system as described above, with either 100 µCi of [S]cysteine or 200 µCi of [-P]ATP (ICN) in a 100-µl reaction volume. The proteins were precipitated with 25% (w/v) trichloroacetic acid, washed with acetone (-20 °C), and subjected to isoelectric focusing and SDS-polyacrylamide gel electrophoresis as described (32) .

Transient Transfection of COS-P Cells

COS-P cells were plated at a density of 8.7 10 cells per 60-mm plate in Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml streptomycin, and 100 µg/ml streptomycin and allowed to reattach overnight. The cells were transfected using the DEAE-dextran method (33) with 2-5 µg of pCMV/60K (wild-type chicken MARCKS) or pCMV/60K A/G (nonmyristoylatable MARCKS) cDNA (18) . Seventy-two hours after transfection, the cells were subjected to subcellular fractionation and Western blot analysis as described above for CEF cells.

Stable Transfection of 293 Cells

Human 293 cells were plated at a density of 2 10 cells per 100-mm plate in MEM containing 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin the night before transfection. The cells were transfected using the 4-h calcium phosphate method (34) with 10 µg of pCMV/60K (wild-type MARCKS) or pCMV/60K A/G (nonmyristoylatable MARCKS) cDNA and 1 µg of pSV2Neo cDNA per plate. Selection in media containing 400 µg/ml Geneticin (Life Technologies, Inc.) was begun 3 days after transfection. Cells were grown in selective media for 4 weeks until individual clones could be discerned. Thirty individual clones for both transfected constructs (pCMV/60K and pCMV/60K A/G) and 10 clones for pSV2Neo alone were then isolated with a pipette tip and transferred to 96-well plates. The cells were allowed to undergo several rounds of expansion and were consecutively transferred to 24-well plates, 12-well plates, 60-mm Petri dishes, and 100-mm Petri dishes. All clones were screened for expression of either wild-type or mutant (A/G) MARCKS by Western blot analysis as described above for CEF cells except that 1% (v/v) Triton X-100 was included in the initial homogenization buffer and sodium fluoride was excluded. For Western blot analysis, the clone lysates were matched for protein content using a dye binding assay as described above. Six clones expressing the highest levels of protein as determined by Western blot analysis were chosen for both wild-type and mutant MARCKS. As negative controls, three clones were selected which did not express either MARCKS construct. The cells from the stable lines were subjected to subcellular fractionation and Western blot analysis, as described above for CEF cells, and light and immunoelectron microscopy, as described below.

Immunostaining of Stable 293 Cell Lines Expressing Wild-type and Nonmyristoylatable MARCKS

Human 293 cells stably expressing wild-type and nonmyristoylatable MARCKS were plated at subconfluence on plastic Lab-Tek slides (1.8-cm, Nunc) and allowed to adhere overnight. Cells expressing aminoglycoside 3`-phosphotransferase (Neo) to confer resistance to Geneticin were plated at similar densities as controls. Cells were washed with PBS and fixed in 4% paraformaldehyde (w/v) in PBS for 5 min. Following fixation, the cells were washed with PBS three times, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and washed with PBS three more times. The Lab-Tek slides containing the fixed cells were blocked in a solution of 5% (v/v) normal goat serum and 0.5% (w/v) BSA (U. S. Biochemical Corp.) in PBS for 30 min at room temperature. Slides were then incubated with a polyclonal antibody to chicken MARCKS (18) at a 1:500 dilution, or preimmune serum at identical dilution, in blocking solution for 1 h at 37 °C. After the primary antibody incubation, slides were washed three times for 5 min in a solution containing 0.2% (v/v) Tween 20 and 0.5% BSA (w/v) in PBS. As a secondary antibody, goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (Organon Tecknika, Cappel Laboratories) was used at a 1:1000 dilution in blocking solution for 1 h at 37 °C in the dark. The slides were then washed three times for 5 min in Tween/BSA solution and covered with a glass coverslip using a 0.25% (w/v) solution of Airvol-205 (Air Products and Chemicals, Inc., Allentown, PA) mounting medium in PBS. Cells were viewed using a Zeiss Axiophot fluorescent microscope and photographed with Kodak T-MAX 400 film using a 30-s exposure.

Electron Microscopic Analysis of Stable 293 Cell Lines

To prepare cell pellets for electron microscopy, two 100-mm dishes each of 293 cells stably expressing wild-type chicken MARCKS (WT60K), nonmyristoylatable MARCKS (60K A/G), and pSV2Neo (Neo) were grown to confluence. Cells were washed three times with 5 ml of cold PBS and treated with 4 ml of a 50 µg/ml solution of Proteinase K (Boehringer Mannheim GmbH, Mannheim, Germany) in PBS. After the cells lifted, the resulting cell suspension was centrifuged for 3 min at 200 g. Cell pellets were resuspended in 1.5 ml of a 40 µg/ml solution of phenylmethylsulfonyl fluoride in PBS and centrifuged at 1000 g for 5 min at 4 °C. The supernatant was removed and replaced with 0.5 ml of a 4% paraformaldehyde solution in 200 mM PIPES (pH 7.0) for 1 h at room temperature. The 4% paraformaldehyde solution was carefully aspirated and replaced with 8% paraformaldehyde in PIPES buffer. The cell pellets were frozen in liquid nitrogen following treatment with 2.1 M sucrose in PBS, and ultrathin sections were prepared using a Reichert Ultracut E with an FC4E cryo attachment. The sections were transferred to a grid using a drop of 2.3 M sucrose in PBS. The grids were then blocked by floating them on a solution of 5% (v/v) fetal calf serum in PBS for 30-60 min at room temperature. The grids were treated with a polyclonal antibody to chicken MARCKS (18) at a 1:25 dilution, or preimmune serum at the same dilution, in the fetal calf serum solution for 45-60 min at room temperature. Following five washes in PBS, the grids were floated on 5 µl of Protein A Gold (35) (approximately 9 nm diameter; 1:12) in PBS/BSA for 30-45 min. The grids were then washed with PBS six times for 5 min and with distilled water four times over 5 min. The grids were mounted in 0.3% uranyl acetate, 2% methylcellulose and dried. A Phillips EM 300 electron microscope was used for all examinations and photography.


RESULTS

Effect of Protein Kinase C Activation on MARCKS Membrane Association in CEF Cells

It has been shown previously that phosphorylation of MARCKS can result in its dissociation from cellular membranes to supernatant fractions in certain cell types (21, 22, 23, 24) . To determine whether a similar phenomenon occurred in fibroblasts, confluent CEF cells were serum-starved overnight and then treated with either 1.6 µM PMA or control for 10 min. In preliminary experiments, involving time points from 5 to 45 min, we found that 10 min of incubation with PMA resulted in the lowest membrane/cytosol ratio (data not shown). Cells treated with PMA for 10 min exhibited a decreased membrane/cytosol ratio of MARCKS immunoreactivity, as determined from PhosphorImager analysis of the resulting Western blot (Fig. 1). This result was highly statistically significant (p < 0.005; n = 4); four similar experiments have yielded similar results. These results suggest that, in intact CEF as well as other cell types studied previously (21, 22, 23, 24) , the phosphorylation of MARCKS by protein kinase C results in at least a quantitative decrease in affinity for cellular particulate structures. These results led us to explore the possible independent contributions of both the myristoyl moiety and the phosphorylation site domain (PSD) to MARCKS binding to cellular membranes in a cell-free system. Binding of Myristoylated and Nonmyristoylated MARCKS to LM/TKCell Membranes-The cell-free binding studies used a modification of the LM/TK cell membrane assay described previously (19) . In order to ensure that our experiments were conducted in the linear range of binding with respect to translated MARCKS, we performed a titration using differing amounts of wild-type and nonmyristoylated [S]cysteine-labeled MARCKS with a constant amount (15 µg) of LM/TK membrane protein. The extent of binding was calculated by excising the bands containing the labeled MARCKS proteins from the gel after identification by fluorography and subjecting them to scintillation counting. For the wild-type protein, binding was linear between 1 and 10 µl of lysate containing translated MARCKS added per 100 µl assay volume (Fig. 2, A and C). The nonmyristoylated MARCKS protein bound much less avidly, but also in an apparently linear fashion with respect to the amount of translated protein (Fig. 2, B and C).


Figure 1: Effect of PMA on MARCKS membrane association in CEF cells. Chick embryo fibroblasts (CEF) were grown to confluence and serum-starved overnight. The cells were then treated with either PMA (1.6 µM in 0.01% dimethyl sulfoxide (DMSO)) or dimethyl sulfoxide alone for 10 min as described under ``Materials and Methods.'' The cells were then homogenized, and the homogenates were separated into membrane (M) and cytosolic (C) fractions by ultracentrifugation; the membranes were then resuspended in exactly the original volume of homogenization buffer. Equal volumes of these samples (approximately 5% of each fraction) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal antibody to chicken MARCKS. A representative Western blot is shown in B; blots of 4 such sets of samples were exposed to a PhosphorImager screen for quantitation. Shown in A are the mean ratios (± S.D.) of cytosol/membrane immunoreactivity (n = 4; p < 0.005 using Student's t test).



Absence of MARCKS Phosphorylation during in Vitro Translation

Since phosphorylation of MARCKS could result in a decreased affinity for membranes (25, 26, 27) , we investigated whether or not MARCKS was phosphorylated during normal translation reactions in the reticulocyte lysates. Wild-type bovine MARCKS mRNA was translated under normal conditions except that [-P]ATP was added; the reaction mixture was then subjected to two-dimensional gel electrophoresis and autoradiography. No phosphorylated protein was observed at the position to which the MARCKS protein characteristically migrates, even though many other lysate proteins were phosphorylated under these conditions (Fig. 3A). These results were identical with a control translation in which no mRNA was added to the translation reaction (Fig. 3B). When the translation was performed in the presence of [S]cysteine, a single spot corresponding to MARCKS at an approximate pI of 4.5 and an apparent M of 87,000 was observed (Fig. 3C). When MARCKS is phosphorylated, its two-dimensional gel pattern consists of an elongated spot containing multiple smaller spots (18, 32, 36) ; therefore, the observed single spot provides further evidence that MARCKS was not phosphorylated in the reticulocyte lysates under our usual translation conditions. Furthermore, the complete absence of the nonmyristoylated form, which migrates with a considerably lower apparent M (see below), confirms our earlier observation (19) that MARCKS is completely myristoylated under these reaction conditions.


Figure 3: Absence of MARCKS phosphorylation during in vitro translation. Wild-type bovine MARCKS mRNA was translated in the presence of [-P]ATP (A) or [S]cysteine (C). A control reaction (B) was performed in which no mRNA was added to the [-P]ATP-containing translation mixture. Reaction mixtures were subjected in parallel to two-dimensional gel electrophoresis and autoradiography. No P-labeled MARCKSwas present in A in the expected position shown in C.



Membrane Association of MARCKS Proteins Mutated in the Phosphorylation Site Domain

Because phosphorylation of MARCKS appears to decrease its association with membranes in certain cell types (21, 22, 23, 24) , in addition to CEF as described above, and because phosphorylated MARCKS exhibited markedly decreased association with synthetic membranes (25, 26, 27) , we examined the effect of mutating the phosphorylation site domain of MARCKS on membrane binding. Binding experiments performed with the positively charged phosphorylation site domain peptide indicated that this region of MARCKS may be involved in an electrostatic interaction with the membrane (27) . In addition, the phosphorylation site domain peptide exhibited decreased affinity to negatively charged synthetic membranes when the peptide was phosphorylated or the four serines were mutated to aspartic acids (27) . To further investigate the nature of this potential association using the intact protein, we first mutated the four serines in the phosphorylation site domain to aspartic acids. Since aspartic acid exhibits a negative charge at physiological pH, four of these residues could, in theory, mimic the fully phosphorylated protein (37) and potentially cause an electrostatic repulsion between MARCKS and the membrane. Given that the expressed proteins should have the same specific activities, the results were expressed as a percentage of the total amount of S-labeled MARCKS added to the assay to account for differences in translation efficiency. When binding assays were performed using the myristoylated tetra-Asp mutant, 6% of the total protein associated with the membranes, compared to 40% for the wild-type, tetra-Ser protein (Fig. 4). Similar results have been obtained in five similar experiments.


Figure 4: Effect of tetra-Asp, tetra-Asn, A/G, and A/G-D/S mutations on MARCKS binding to LM/TK membranes. The four serines in the wild-type phosphorylation site domain (wild-type or TS) of bovine MARCKS were changed to either aspartic acids (tetra-Asp or TD) or asparagines (tetra-Asn or TN), and the amino-terminal glycine was changed to alanine (A/G or A/G) using oligonucleotide-directed mutagenesis. A double mutant (A/G-tetra-Asp or A/G-TD) in which the four serines in the PSD were changed to aspartic acids and the amino-terminal glycine was changed to alanine was also created. All mRNAs were translated in parallel in the presence of identical specific activities of [S]cysteine. Binding reactions were performed in parallel as described under ``Materials and Methods'' using 15 µg of LM/TK membrane protein. The resulting autoradiographs are shown in A. T represents 33% of the total MARCKS radioactivity added to the reaction; in addition, background samples (B) containing no membranes were included for each protein. The histogram (B) shows the mean ± S.D. (n = 4) percentage of the total added radioactive MARCKS bound to the membranes as calculated by counting gel slices containing radioactive MARCKS shown in A. Background counts (lanes marked B in A) were subtracted from the reported values.



If the decrease in membrane association of the tetra-Asp protein was due primarily to its electrostatic properties, mutating the four serines to asparagines should have little effect on binding when compared to the wild-type protein. Asparagine is similar in structure to aspartic acid; however, it is neutral at physiological pH. When the four serines were mutated to asparagines, 27% of the total protein counts were associated with the membranes, as compared with 6% for the tetra-Asp protein (Fig. 4). Similar marked decreases in binding of the tetra-Asp mutant compared to the tetra-Asn mutant were observed in four additional experiments. When the amino-terminal glycine was mutated to alanine, resulting in a nonmyristoylated but otherwise wild-type protein, a small amount of binding could be detected on the autoradiograph (Fig. 4A); however, scintillation counting of the bands revealed that only 0.3% of the total protein associated with membranes under these reaction conditions (Fig. 4). In these studies, sedimentation of [S]cysteine in lysates prepared without MARCKS mRNA, indicating nonspecific membrane trapping, was approximately 0.2-0.5% of total. The double mutant, nonmyristoylatable and tetra-Asp protein, also did not exhibit binding above background by either autoradiography (Fig. 4A) or scintillation counting (Fig. 4B). These results suggest that myristoylation is required for MARCKS to associate significantly with membranes in this in vitro system and that the PSD also may be involved.

Effect of Calmodulin on Membrane Association of MARCKS

As an additional test of the importance of the phosphorylation site domain to membrane binding of the MARCKS protein, we performed binding experiments in the presence of exogenous calmodulin, ± Ca. Because the phosphorylation and calmodulin binding domains of MARCKS are identical (15) , it seemed likely that binding of acidic calmodulin (pI 3.9-4.3) to the basic PSD/calmodulin binding domain (pI 12.2) could neutralize the positive charges of this domain and decrease association of MARCKS with the membranes. Calmodulin might also physically interfere with the interaction between the PSD/calmodulin binding domain and the membranes. In addition, very recent experiments have shown that murine MARCKS expressed in a baculovirus system exhibited a decrease in affinity for negatively charged synthetic membranes in the presence of calmodulin (26) . In the present study, the addition of calmodulin (11.6 µM) in the presence of Ca resulted in a 48% decrease in MARCKS binding compared to a control, in which 50 µM EGTA was included in the reaction to chelate Ca ions and prevent formation of Ca/calmodulin (Fig. 5A). MARCKS is known to require Ca for its interaction with calmodulin (15) . The decrease in binding due to calmodulin was statistically significant (Fig. 5C; p < 0.0005). Parallel experiments without added calmodulin showed no change in binding upon addition of EGTA (Fig. 5, B and C). When identical experiments were performed with the tetra-Asp mutant, which binds calmodulin poorly (38) , no significant change in binding was observed in the presence or absence of EGTA in the assay mixture (data not shown).


Figure 5: Effect of calmodulin on MARCKS binding to LM/TK membranes. Wild-type bovine MARCKS mRNA was translated in the presence of [S]cysteine. Binding reactions were performed as described using 5 µg of LM/TK membrane protein in the presence (A) or absence (B) of 11.6 µM calmodulin. All reactions contained CaCl (50 µM); in addition, EGTA at a concentration of 50 µM was either included (+) or excluded (-). Shown are the resulting autoradiographs of the radioactive MARCKS contained in the pellet fractions and a histogram (C) of the total mean ± S.D. (n = 3) counts/min represented by radioactive MARCKS in the gel slices. The two means in the calmodulin group were significantly different (p < 0.0005 using Student's t test). Background counts, i.e. counts/min from samples incubated in the absence of membranes, were not subtracted in this experiment.



Effect of Lack of Myristoylation on Membrane Association of MARCKS in Intact Cells

Given the very low level of binding of the nonmyristoylated protein to fibroblast membranes in our cell-free assay, we wished to determine whether myristoylation of the protein was necessary for MARCKS to associate with membranes in intact cells or whether the PSD or other parts of the protein could confer some membrane association in the absence of the myristoyl moiety. To do this, we expressed the wild-type chicken protein and its nonmyristoylatable counterpart (glycine to alanine mutation) in COS and 293 cells, taking advantage of the fact that the chicken protein migrates on SDS-PAGE at a considerably lower apparent molecular weight than the endogenous monkey (COS) or human (293) protein (39) .

Our first experiments used transient transfection of these plasmids in COS cells. To determine whether or not the expressed proteins were myristoylated, we transfected cells with 5 µg of either wild-type or nonmyristoylatable MARCKS cDNAs using DEAE-dextran, grew the cells to confluence, homogenized the cells, and isolated particulate and cytosolic fractions by ultracentrifugation. The fractions were subjected to SDS-PAGE and Western blot analysis using the polyclonal antibody to chicken MARCKS (18) . The nonmyristoylatable mutant MARCKS was found almost exclusively in the cytosolic fraction (Fig. 6), suggesting that myristoylation was necessary for significant MARCKS membrane association in vivo. Wild-type myristoylated MARCKS was found in both the cytosolic and membrane fractions (Fig. 6). However, the cytosolic fraction from the cells transfected with the wild-type vector also contained a significant amount of nonmyristoylated MARCKS (Fig. 6). This identification was made based on differences in apparent molecular weight of the two forms of the protein; the nonmyristoylated protein routinely migrated to a lower apparent molecular weight than the myristoylated form on SDS-polyacrylamide gels (19) . In addition, when COS cells transfected with the wild-type chicken MARCKS cDNA were labeled with [H]myristate and subjected to immunoprecipitation, a single band corresponding to the top band in Fig. 6was detected on the resulting autoradiograph (data not shown).


Figure 6: Subcellular fractionation of chicken MARCKS expressed in COS-P cells. COS-P cells were transfected with 5 µg of either wild-type (WT60K) or nonmyristoylatable mutant (MUT60K) chicken MARCKS cDNA using DEAE-dextran. A mock transfection with no added cDNA was included as a control. The cells were homogenized, and the homogenates were separated into membrane (M) and cytosolic (C) fractions by ultracentrifugation. The membranes were then resuspended in the original volume of homogenization buffer, and then equal volumes (approximately 10% of each sample) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to chicken MARCKS. Shown is the resulting Western blot. Each pair of lanes represents results from a single plate of cells. The top arrow points to the fully myristoylated form of the protein; the bottom arrow points to the nonmyristoylated form.



Because constructs using the CMV promoter are expressed at high levels in COS cells, we postulated that overexpression of the wild-type MARCKS protein might overwhelm the capacity of the cells to myristoylate it cotranslationally. As a possible solution to this problem, we co-transfected a vector expressing the enzyme peptide N-myristoyltransferase (EC 2.3.1.97 (40) ) with the chicken MARCKS cDNA. Expression of additional N-myristoyltransferase had little effect on the levels of nonmyristoylated protein synthesized from the wild-type mRNA (data not shown). In case the cellular levels of myristate were limiting, we added additional myristate to the media for 72 h following transfection. The addition of the extra myristate decreased the proportion of the protein in the nonmyristoylated state; however, increasing ambient myristate concentrations to the point of cell toxicity did not result in the complete myristoylation of all newly synthesized wild-type MARCKS (Fig. 7).


Figure 7: Subcellular fractionation of chicken MARCKS expressed in COS-P cells incubated with exogenous myristate. COS-P cells were transfected with 2 µg of wild-type chicken MARCKS cDNA using DEAE-dextran. A mock transfection with no added cDNA was included as a control. After transfection, the cells were treated with 0-150 µM myristate, as indicated, and incubated in the presence of myristate for a further 72 h until they were homogenized. The resulting homogenates were matched for protein concentration and then separated into membrane (M) and cytosolic (C) fractions by ultracentrifugation. The membranes were then resuspended in the original volume of homogenization buffer, and then equal volumes (approximately 10% of each sample) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to chicken MARCKS. Shown is the resulting Western blot, which is overexposed to show minor species. The top arrow points to the fully myristoylated form; the bottom arrow points to the nonmyristoylated form of the protein.



In order to obtain cells in which expressed wild-type chicken MARCKS could be completely myristoylated, we created stable cell lines in 293 cells. Like COS cells, 293 cells were chosen for their ability to overexpress constructs cloned into the pCMV vector (41) . When stably expressed in 293 cells, the wild-type chicken MARCKS protein was completely myristoylated, as in normal CEF cells (Fig. 8). These data are representative of results from 10 other cell lines expressing wild-type protein. Upon fractionation of the cells by ultracentrifugation, the majority of the wild-type protein (82%) expressed in this cell type was membrane-associated. When cells expressing the nonmyristoylated protein were fractionated in the same way, a significant proportion (43%) of mutant MARCKS was found still to be associated with the membrane fraction (Fig. 8). This result suggested that, in contrast to the cell-free binding data, there was considerable membrane association of the nonmyristoylated protein in intact cells. These data suggested the rather surprising conclusion that additional domains of the MARCKS protein, possibly including the PSD, can cause the protein to associate with membranes in intact cells, even in the absence of protein myristoylation.


Figure 8: Subcellular fractionation of chicken MARCKS stably expressed in 293 cells. Human 293 cells were stably transfected with 10 µg of either wild-type (WT60K) or mutant (60K/A/G) chicken MARCKS cDNA and 1 µg of pSV2Neo cDNA (Neo) using calcium phosphate. The cells were homogenized and separated into membrane (M) and cytosolic (C) fractions by ultracentrifugation. Cells expressing only the Neo construct were included as a control. The membranes were then resuspended in the original volume of homogenization buffer, and then equal volumes (approximately 5% of each sample) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to chicken MARCKS. Shown is the resulting Western blot; the blot is overexposed to show the complete absence of nonmyristoylated MARCKS in the WT60K cells, although it is the only form expressed in the 60K/A/G cells. Note the complete absence of chicken MARCKS immunoreactivity in the cells transfected with Neo alone.



To confirm that the nonmyristoylated protein could associate with membranes in intact cells, we performed immunolocalization experiments of the wild-type and nonmyristoylatable mutant protein in 293 cells at the light and electron microscopic levels. At the light level, cells expressing the wild-type chicken protein exhibited a more speckled pattern of immunoreactivity when compared to the cells expressing nonmyristoylated MARCKS (Fig. 9). Although the staining of the cells expressing the mutant protein was more diffuse, there were also scattered, bright areas of staining that appeared to be at the plasma membrane (Fig. 9c). Electron microscopic studies showed that the anti-chicken antibodies did not cross-react with any endogenous human proteins in the cells expressing Neo alone (Fig. 10A). Wild-type MARCKS was localized both to the plasma membrane and to the membranes of large vesicular structures within the cell (Fig. 10B), as shown previously in CEF.() In addition, there was a near absence of gold particles in the cytoplasm of the cell (Fig. 10B). By contrast, gold particles were found in the cytoplasm in the 293 cells expressing the nonmyristoylatable MARCKS mutant (Fig. 10C). However, there was also considerable association of the nonmyristoylated protein with the plasma membrane and with the membranes of cytoplasmic vesicles (Fig. 10C). Similar results were observed in many similar sections.


Figure 9: Immunofluorescence of 293 cells expressing chicken MARCKS. Human 293 cells expressing wild-type chicken MARCKS (a and b), nonmyristoylatable MARCKS (c and d), or Neo (e and f) were grown on plastic slides, fixed in 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. These are the same cell lines analyzed in Fig. 8. The cells were incubated with either a polyclonal antibody to chicken MARCKS (a, c, and e) or preimmune serum (b, d, and f). As a secondary antibody, goat anti-rabbit IgG conjugated to fluorescein isothiocyanate was used. See the text for further details.




Figure 10: Immunoelectron microscopic localization of normal and mutant chicken MARCKS in 293 cells. Human 293 cells expressing Neo alone (A), wild-type chicken MARCKS (B. WT), or the A/G nonmyristoylatable mutant MARCKS (C) were prepared for immunoelectron microscopy as described in the text, using an antiserum directed at chicken MARCKS. A demonstrates that there is no cross-reactivity between the antiserum and endogenous human MARCKS. The arrowheads indicate the plasma membrane. Bar = 0.2 µm.



These sections were also analyzed by counting the gold particles and classifying them according to their subcellular localization. For this analysis, the ``membrane'' was specified as the plasma membrane and the membranes of large, clear, cytoplasmic vesicles. Any other portion of the cell was labeled ``cytosolic.'' In six sections of cells expressing wild-type chicken MARCKS, 82 ± 3.4% (S.D.) of the gold particles (total of 1503 counted) were associated with the plasma or vesicular membrane. However, in four sections of cells expressing nonmyristoylatable chicken MARCKS, 44 ± 8.5% (S.D.) of the gold particles (total of 1051 counted) were associated with these membranes. These values are strikingly similar to those obtained in the subcellular fractionation experiments (82% membrane association for wild-type MARCKS and 43% for nonmyristoylatable MARCKS). Therefore, despite the complete absence of myristoylation, a large fraction (44%) of chicken MARCKS was apparently still associated with the plasma membrane and membranes presumably derived from it.


DISCUSSION

These studies were designed to further investigate the nature of MARCKS association with cellular membranes. Our results in a cell-free system and in intact CEF stimulated with PMA support a two-component model for MARCKS membrane association, involving independent contributions of both the myristoylated amino terminus and the phosphorylation site domain (PSD) of the protein (26, 27) . However, the data from 293 cells also indicate that, in contrast to the cell-free data, a substantial proportion (almost 44%) of nonmyristoylated MARCKS was still associated with the plasma membrane in intact cells. These findings are even more compelling because essentially identical results were achieved by cell fractionation methods followed by immunoblotting and by immunoelectron microscopy. In intact cells, therefore, myristoylation of the protein does not seem to be necessary for plasma membrane association of the protein, thus implicating other domains of the protein in this interaction.

These findings are in contrast to results obtained with many other myristoyl proteins. For example, subcellular fractionation experiments demonstrated that it was necessary for enzymatically active, immunoprecipitable pp60 to be myristoylated in order for it to associate with cellular membranes (42, 43) . More recently, the nonmyristoylated forms of several other myristoyl proteins were found almost exclusively in cytosolic fractions, using subcellular fractionation and Western blotting experiments similar to those described here. When the nonmyristoylatable forms of the human immunodeficiency virus (HIV) Nef protein (44) , endothelial nitric-oxide synthase (45) , and the subunits of several G proteins (46) were transfected into COS cells, association with membranes was essentially completely myristoylation-dependent. Similarly, nonmyristoylatable p56 expressed in NIH-3T3 cells was found almost exclusively in cytosolic fractions by Western blotting of immunoprecipitates (47) . When the analyses were performed by immunomicroscopic techniques rather than by subcellular fractionation, immunoelectron microscopy of thin sections of yeast cells expressing nonmyristoylated Pr55 protein from HIV revealed that the protein was not targeted to the membrane, in contrast to the membrane association of the wild-type protein (48) . Immunostaining of QT6 fibroblasts transfected with the nonmyristoylatable form of the 43-kDa postsynaptic protein (49) , and Ba/F3 lymphoblastoid cells transfected with the nonmyristoylatable form of Abl (50) , demonstrated that very little of the expressed proteins was found at the plasma membrane. Finally, in other cell-free studies, Ca-dependent binding of in vitro-translated hippocalcin to crude rat hippocampal membranes was essentially eliminated when the protein was not myristoylated (51) .

Our data suggest that the continued affinity of nonmyristoylated MARCKS for membranes in intact cells is due to membrane interactions with other domains of the protein. The domain most likely to be involved in this membrane association is the positively charged (pI 12.2) PSD. There is considerable experimental evidence in support of this conclusion. For example, [H]myristate-labeled MARCKS was found almost exclusively in the cytosolic compartment of neutrophils treated with PMA, as determined by subcellular fractionation (23) . Similarly, [H]myristate-labeled MARCKS was found in the supernatant of isolated macrophage membrane preparations phosphorylated in vitro by protein kinase C (22). MARCKS was also released into soluble fractions from depolarized P-labeled synaptosomes (21) . Phosphorylation of H-labeled MARCKS by PMA in C6 glioma cells resulted in an increase of MARCKS in soluble fractions (24) . Finally, we show here that immunoreactive MARCKS increased in cellular supernatant fractions from CEF treated for 10 min with PMA. All of these studies are consistent with a model in which the positively charged PSD, which contains 13 basic and no acidic residues within its 25-amino acid length, becomes avidly associated with negatively charged membrane lipids by electrostatic interactions. This association would be reversed by the negative charges introduced upon protein kinase C-dependent phosphorylation, at which time this domain can become phosphorylated to a maximum stoichiometry of 3 mol/mol (or 4 in some cases; reviewed in Ref. 4).

This model is supported by several types of data in cell-free systems. For example, binding of bovine brain MARCKS or recombinant mouse MARCKS to negatively charged synthetic lipid vesicles was inhibited by protein kinase C-dependent phosphorylation (25, 26) . In addition, studies with the PSD peptide showed that this peptide exhibited a decrease in affinity to negatively charged synthetic lipid vesicles, either upon phosphorylation with protein kinase C or when phosphorylation was mimicked by the substitution of aspartic acids for the four potentially phosphorylated serines (27) . Our present data also show that mutation of these same serines to aspartic acids markedly decreased association of intact MARCKS with cellular membranes in a cell-free system, when compared with either the wild-type protein or the corresponding tetra-Asn mutant protein. Finally, when calmodulin (pI 3.9-4.3) was allowed to bind to this same domain (15) , it also inhibited MARCKS association with cellular membranes, as seen here, or with synthetic lipid vesicles (26) , presumably either by neutralizing some of the positive charges in the PSD or by physically interfering with the ability of the PSD to come into contact with the lipid membranes.

If the PSD is indeed responsible for the continued membrane association of the nonmyristoylated protein in vivo, then the current data support a two-component model for MARCKS association with membranes, as proposed previously (26, 27) . According to this model (Fig. 11), MARCKS would be tethered to the plasma membrane by its myristoyl tail through hydrophobic interactions and by its PSD through electrostatic interactions. During acute elevations in intracellular calcium levels, MARCKS could associate with Ca/calmodulin through its PSD; this interaction would disrupt the electrostatic interaction between the PSD and the membrane, resulting in a decreased affinity of MARCKS for the membrane. Similarly, phosphorylation by protein kinase C would decrease the strength of the electrostatic interaction between the PSD and the membrane, again resulting in a decreased affinity of the whole protein for the membrane. Both interactions could be reversed rapidly, either by dissociation of calmodulin occurring concomitantly with decreasing intracellular [Ca] and/or by dephosphorylation of MARCKS by phosphatases 1 and 2a (52) . During all of these events, the protein still could be anchored to the membrane through its myristoyl tail. However, that phosphorylation decreased the overall affinity for the membrane is suggested by the subcellular fractionation experiments in which activation of protein kinase C leads to an increase in the cytosol/particulate ratio of the protein.


Figure 11: Two-component model for MARCKS membrane association. This model proposes that MARCKS associates with cellular membranes by its myristoyl tail through hydrophobic interactions and by its PSD through electrostatic interactions. Association of the PSD with calmodulin and/or phosphorylation by protein kinase C could disrupt the electrostatic interaction with the membrane; however, the protein could still be anchored to the membrane through its myristoyl tail.



One potential problem with this model is that, despite the relatively nonspecific nature of the proposed MARCKS:membrane interactions, i.e. hydrophobic and electrostatic, the protein somehow associates specifically with the plasma membrane and cytoplasmic vesicles presumably derived from it. A potential explanation for this finding comes from studies of Ras and related proteins, in which so-called polybasic domains (e.g. KKKKKK in wild-type Ki-ras (B)) are involved in specifically targeting these isoprenylated proteins to specific cellular locations. Ki-ras (B) is found at the plasma membrane (53, 54) ; in this case, the polybasic motif in combination with farnesylation has been described as conferring specific plasma membrane localization, by a still unknown mechanism (55) . Since the MARCKS PSD contains a similar polybasic motif (KKKKKR), it seems possible that it is conferring the same plasma membrane specificity to the MARCKS protein, even though the myristoyl group and the PSD are separated by 150 amino acids.

It remains possible, however, that the continued membrane association of nonmyristoylated MARCKS in intact cells is mediated by one or more domains distinct from the PSD and its polybasic motif. Besides the PSD and the myristoylation consensus sequence, the third major conserved domain is at the site of intron splicing, where eight consecutive amino acids are conserved with a similar small region in the cytoplasmic tail of the insulin-like growth factor II receptor. It is not obvious how these other conserved domains would confer membrane association; however, it seems likely from previous data (19) that a cytoplasmic-face protein receptor is not involved, as has been proposed for pp60(56, 57, 58, 59) . This does not exclude other types of potential protein-protein interactions occurring at the plasma membrane, e.g. between MARCKS and calmodulin (15, 16) or actin (17) . Ultimately, it should be possible to determine the other protein domains involved in plasma membrane association using expression of mutant MARCKS proteins in stable cell lines, as described here.

One final point is a cautionary note concerning the transient expression of myristoyl proteins in COS cells, using powerful vectors such as that used here which contain SV40 origins of replication (60) . We were somewhat surprised to find that a large proportion of wild-type MARCKS was not myristoylated when this cDNA was transiently transfected into COS cells. We attempted to increase the fraction of the protein that was myristoylated by decreasing the amount of cDNA in the transfections; increasing the concentration of myristate in the cell incubation medium; and cotransfecting a eukaryotic expression vector expressing N-myristoyltransferase. However, even when all three approaches were optimized, we could not obtain 100% myristoylation. It was important to achieve 100% myristoylation for a variety of reasons, particularly for the immunoelectron microscopy experiments. In the event, this was achieved readily by creating stable cell lines in 293 cells, even without employing the extra manipulations listed above. This situation may be unusual or even unique to MARCKS expression in COS cells, although we have observed a similar phenomenon when MARCKS was expressed in insect Sf9 cells using a baculovirus expression vector.It seems likely that, under certain circumstances, translation of an abundant message can overwhelm the ability of the cotranslational myristoylation machinery to keep up, perhaps resulting in the pool of nonmyristoylated MARCKS that has been noted in other studies (61, 62) . Whatever the mechanisms of this effect, it seems prudent to recommend that the extent of myristoylation of proteins be monitored when transient transfection experiments are performed.


FOOTNOTES

*
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.

§
Supported in part by the Howard Hughes Medical Institute Graduate Student's support fund.

Investigator of the Howard Hughes Medical Institute. To whom correspondence and reprint requests should be addressed: Box 3897, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-8760; Fax: 919-684-5458.

The following abbreviations are used: MARCKS, myristoylated alanine-rich C kinase substrate; PSD, phosphorylation site domain; MEM, minimal essential medium; CEF, chick embryo fibroblasts; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HIV, human immunodeficiency virus; CMV, cytomegalovirus; BSA, bovine serum albumin.

R. T. Sperling, D. J. Stumpo, J. K. Chuprun, and P. J. Blackshear, submitted for publication.

C. H. Cabell, G. M. Verghese, N. B. Rankl, D. J. Burns, and P. J. Blackshear, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Samuel George for the purified chicken calmodulin, Dr. Deborah Stumpo for the bovine WT, tetra-Asp, and A/G constructs, Dr. Jon Graff for the CMV/chicken MARCKS constructs and chicken antisera, Dr. Jeffrey Gordon for the N-myristoyltransferase cDNA, and Dr. Bryan Cullen for the COS-P cells. We also thank Susan Hester of the Duke Comprehensive Cancer Center Electron Microscopy Shared Resource for performing the electron microscopy and Drs. Terrence Oas and Patrick Casey for helpful discussions.


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