(Received for publication, July 3, 1995; and in revised form, September 18, 1995)
From the
MacMARCKS (also known as myristoylated alanine-rich C kinase
substrate (MARCKS)-related protein) is a member of the MARCKS family of
protein kinase C substrates, which binds
Ca/calmodulin in a phosphorylation-dependent manner.
Immunoprecipitation demonstrated that MacMARCKS is present in both PC12
cells and in neurons. Upon depolarization of PC12 cells with 60 mM KCl, MacMARCKS phosphorylation increased 4-fold over basal levels
in a Ca
-dependent manner. By immunofluorescence
microscopy, MacMARCKS was co-localized in PC12 cells to neurite tips
with the synaptic vesicle membrane protein synaptophysin and to
vesicles in the perinuclear region. Subcellular fractionation
demonstrated that MacMARCKS associates tightly with membranes in PC12
cells. In Percoll-purified rat cerebrocortical synaptosomes,
depolarization with 60 mM KCl in the presence of exogenous
Ca
transiently increased MacMARCKS phosphorylation,
whereas phorbol ester promoted a sustained increase in MacMARCKS
phosphorylation. Subcellular fractionation of rat brain indicated that
MacMARCKS was present in both soluble and particulate fractions;
particulate MacMARCKS was associated with both small vesicles and
highly purified synaptic vesicles. These results are consistent with a
role for MacMARCKS in integrating Ca
-calmodulin and
protein kinase C-dependent signals in the regulation of neurosecretion.
MacMARCKS (also known as MRP) ()is a member of the
myristoylated alanine-rich C kinase substrate (MARCKS) family of
protein kinase C (PKC) substrates(1, 2) . MacMARCKS
binds calmodulin in a Ca
-dependent manner, and this
binding is regulated by PKC-dependent phosphorylation of serine
residues within the calmodulin binding domain(3, 4) .
The amino acid sequence of the calmodulin-binding domain of MacMARCKS
is highly homologous to the calmodulin- and actin-binding domain of
MARCKS(1, 2) .
MacMARCKS cDNA has been cloned from
mouse macrophages(3) , mouse brain(5) , and rat kidney
epithelial cells(6) . Northern blot analysis of various tissues
indicates that MacMARCKS is widely expressed(7) . MacMARCKS is
highly enriched in bacterial lipopolysaccharide-stimulated
macrophages(3) , where it is associated with endocytic
compartments and the plasma membrane. ()However, the
cellular localization and the regulation of MacMARCKS phosphorylation
in other tissues remain to be established.
While investigating the potential role of MARCKS in neurosecretion, we identified a PKC substrate that had similar characteristics to MacMARCKS. We now report that MacMARCKS is phosphorylated in PC12 cells and, like MARCKS(8, 9) , is phosphorylated in rat brain synaptosomes upon depolarization and phorbol ester treatment. Within PC12 cells, MacMARCKS co-localizes with synaptophysin in distal neuronal processes and in perinuclear endosomes. It also associates with a number of membrane vesicular fractions purified from rat brain, including a synaptic vesicle fraction.
Synaptic fractions were prepared as described
previously (14) with minor modifications(15) . To
obtain S and P
, the S
fraction was
centrifuged at 165,000
g
for 2 h. The
eluate from the controlled pore glass (CPG) column was pooled into
three fractions (CPG1-CPG3). These fractions were kindly prepared
by Drs. P. S. McPherson and P. De Camilli.
Figure 1:
MacMARCKS
is expressed and phosphorylated in PC12 cells. A, PC12 cells
were labeled with [H]myristic acid, and MacMARCKS
was immunoprecipitated from the cell lysate. The lysate and
immunoprecipitate were resolved by 10% SDS-PAGE, and myristoylated
proteins were visualized by fluorography. Left lane, total
cell lysate; right lane, immunoprecipitated 48-kDa protein. B, lysates from PC12 cells were heat-treated and centrifuged,
and the heat stable proteins (supernatant) were subjected to Western
blotting with the anti-MacMARCKS antibody. C,
P-labeled PC12 cells were treated with 200 nM PMA
for 30 min, lysed, and immunoprecipitated with anti-MacMARCKS antibody.
The immunoprecipitates were then subjected to two-dimensional
polyacrylamide gel electrophoresis.
Figure 2:
MacMARCKS is phosphorylated upon
depolarization of PC12 cells. A, PC12 cells radiolabeled with P
were either not treated (lane 1),
exposed to PMA (200 nM, 30 min, lane 2), or subjected
to depolarization (60 mM KCl for 1 min) either in the absence (lane 3) or presence (lane 4) of 1.5 mM extracellular Ca
. PC12 cells were solubilized in
lysis buffer and immunoprecipitated with anti-MacMARCKS antibody.
Immunoprecipitates were resolved by 10% SDS-PAGE and visualized by
autoradiography. B, PC12 cells were prelabeled with
[
H]myristic acid, treated with control buffer (C), 60 mM KCl (K
), or 200
nM PMA and fractionated to isolate particulate (P)
and cytosol (S) fractions as described under
``Experimental Procedures.'' The fractions were
immunoprecipitated with anti-MacMARCKS antibody, and MacMARCKS was
visualized by fluorography after 10% SDS-PAGE. C, PC12 cells
were radiolabeled with
P
and treated and
fractionated as described in B. Immunoprecipitated MacMARCKS
was visualized by autoradiography after 10%
SDS-PAGE.
Since the related PKC substrate, MARCKS, is released from
membranes upon phosphorylation(13, 16) , we examined
whether phosphorylation similarly displaces MacMARCKS from membranes.
In both unstimulated cells and in cells depolarized with elevated KCl,
greater than 95% of [H]myristoylated MacMARCKS
was associated with the membrane fraction (Fig. 2B).
Similarly, most of the phosphorylated MacMARCKS was associated with the
membrane fraction of control or depolarized PC12 cells (Fig. 2C), although the stoichiometry of
phosphorylation of the cytosolic protein appears higher than its
membrane-bound counterpart (Fig. 2, B and C,
and data not shown). PMA treatment resulted in the release of
approximately 20% of [
H]myristoylated MacMARCKS
from the membrane. The stoichiometry of PMA-induced phosphorylation of
the cytosolic form was also greater than that of the membrane bound
protein (Fig. 2, B and C, and data not shown).
Figure 3: MacMARCKS and synaptophysin co-localize in NGF-differentiated PC12 cells. A, differential interference contrast image; B, MacMARCKS immunoreactivity. MacMARCKS immunoreactivity is present in the tips of neurite processes, and in the perinulear region in a punctate manner. C and D, confocal immunofluorescence microscopy demonstrates the overlapping distribution of MacMARCKS (C) and synaptophysin (D) in PC12 cells. Arrows indicate neuronal processes where MacMARCKS and synaptophysin immunoreactivity are abundant.
Figure 4:
PKC agonists or depolarization induce the
phosphorylation of MacMARCKS in rat brain synaptosomes. A,
Percoll-purified synaptosomes were radiolabeled with P
and incubated with control buffer (lane
1), depolarized with 40 mM KCl for 30 s (lane
2), or treated with 300 nM PDBu for 1 min (lane
3). The reactions were terminated by the addition of cold lysis
buffer, and MacMARCKS was visualized by autoradiography following
immunoprecipitation and 10% SDS-PAGE. B, time course of PDBu
and depolarization-induced MacMARCKS phosphorylation.
P
-Labeled synaptosomes were exposed to 300
nM PDBu (circles) or 40 mM KCl (squares) for the indicated times, and MacMARCKS
phosphorylation was quantitated after immunoprecipitation and 10%
SDS-PAGE using a Molecular Dynamics PhosphorImager. The experiments
were done in triplicate. Mean and standard deviations are
indicated.
Figure 5: Subcellular distribution of MacMARCKS, MARCKS, synaptophysin, and synapsin 1. Proteins from rat brain subcellular fractions were fractionated by 10% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with polyclonal antibodies against MacMARCKS, MARCKS synapsin 1, or synaptophysin as indicated. H, Homogenate; P, pellet; S, supernatant; LP, lysed pellet; LS, lysed supernatant; CPG, controlled pore glass.
We have shown that the PKC substrate, MacMARCKS, is present
in the distal neuronal processes of PC12 cells, in rat brain
synaptosomes, and on small vesicles and synaptic vesicles isolated from
rat brain. The protein is phosphorylated when PC12 cells or rat brain
synaptosomes are stimulated with phorbol ester or depolarized with
elevated levels of KCl. Depolarization-induced phosphorylation of
MacMARCKS is dependent upon external Ca. MacMARCKS is
phosphorylated by PKC in vivo, since phosphopeptide maps
generated from the protein phosphorylated in PC12 cells are identical
to those generated from recombinant MacMARCKS phosphorylated in
vitro by purified PKC. The phosphopeptides correspond to the
underlined amino acids
(KKKKFS[1]FKKPFKLSGLS[2]FKR), and are found within
the effector domain of MacMARCKS, where both serines [1] and
[2] are phosphorylated by PKC(3) .
Considerable
evidence suggests that PKC has a role in neurosecretion (25, 26) . First, most conventional isozymes of PKC
are expressed at high levels in neurons and are present in synaptic
termini(25, 27) . Second, phorbol esters promote the
release of neurotransmitters, although the release is not restricted to
a specific transmitter (28, 29, 30, 31) . This effect is
stereospecific; only phorbol esters that activate PKC induce
neurotransmitter release(32) . Third, PKC inhibitors prevent
KCl-induced neurotransmitter release(25, 26) ,
although these data should be interpreted with caution because of poor
specificity of the inhibitors. Fourth, Ca and
diacylglycerol, which activate PKC, are elevated in active presynaptic
termini(33) . Although there is much evidence that PKC is
involved in neurotransmitter release, the mechanism by which PKC exerts
this effect is unknown. Most recent investigations of the role of PKC
in neurosecretion have concentrated on two PKC substrates found in
nerve termini, MARCKS(8, 34) , and neuromodulin (also
known as GAP-43 or B-50)(35, 36) . The current study
implicates a third PKC substrate, MacMARCKS.
MARCKS and MacMARCKS
are related proteins that share a number of features. Both are
elongated, rod-shaped molecules with a similar domain structure. They
have a myristoylated N terminus, a highly conserved MH2 domain, and a
basic effector domain that contains the PKC phosphorylation sites and
which binds calmodulin in a Ca-dependent
manner(3) . MARCKS binds and cross-links actin in a
phosphorylation-regulated manner(37) , and preliminary data
suggest that the effector domain of MacMARCKS also binds actin. (
)The function of MacMARCKS in the presynaptic terminal is
not yet clear, but it is likely to be involved with the rearrangement
of actin during neurosecretion and/or membrane retrieval. By analogy
with the synapsins (38) , it may have a role in reversibly
tethering synaptic vesicles to actin filaments. Alternatively,
MacMARCKS might regulate actin structure at the presynaptic terminal,
thereby affecting access of synaptic vesicles to the plasma membrane. A
possible role for MacMARCKS in membrane retrieval at the presynaptic
junction is supported by its behavior in macrophages, where it is known
to associate with phagosomes and endosomes.
PKC-dependent phosphorylation prevents calmodulin binding to
MacMARCKS(3, 4) , neuromodulin
(GAP-43)(39, 40) , and
MARCKS(8, 41) . It has been proposed that both
neuromodulin and MARCKS act as regulated calmodulin
stores(39, 2) , and it is possible that MacMARCKS has
a similar function. This is an attractive hypothesis since
PKC-dependent phosphorylation has been shown to mobilize calmodulin, an
event that activates calmodulin-dependent protein kinases implicated in
neurosecretion(42) . However, neuromodulin (GAP-43) would be
more suitable as a Ca sink than MARCKS or MacMARCKS,
since it binds calmodulin in a Ca
-independent manner (43) . Since MARCKS, MacMARCKS, and the calmodulin-dependent
protein-kinases would compete for calmodulin as the concentration of
intracellular Ca
increases, calmodulin availability
and hence Ca
/calmodulin-dependent protein kinase
activity could be regulated subtly by the phosphorylation of MARCKS and
MacMARCKS.