From the
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
Phosphorylation of intracellular substrates by protein kinase C,
the diacylglycerol-activated, Ca
One of the most prominent intracellular
substrates for protein kinase C is the myristoylated alanine-rich C
kinase substrate or MARCKS
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.
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.).
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 [
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
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,
[
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
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
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.
We thank Dr. Samuel George for the purified chicken
calmodulin, Dr. Deborah Stumpo for the bovine WT, tetra-Asp, and
A
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
-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.
(
)
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).
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.
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/TK
Membranes-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.
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) .
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.
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) .
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).
/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.
(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.
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.
/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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.