(Received for publication, September 7, 1995; and in revised form, October 26, 1995)
From the
The myristoylated alanine-rich C kinase substrate (MARCKS) is a
major cellular substrate of protein kinase C. Its concentration in
cells is important for the normal development of the central nervous
system, and perhaps other physiological processes. We found that MARCKS
concentrations in cells were regulated in part by a specific
proteolytic cleavage; this resulted in two fragments, each representing
about half of the intact protein, that co-existed with MARCKS in cells
and tissues. These fragments were present in significant concentrations
in quiescent fibroblasts; they disappeared, and the amount of intact
MARCKS increased, within 15 s of activation of protein kinase C by
serum. In vitro experiments demonstrated that phosphorylated
MARCKS was a poor substrate for a protease activity present in cell
extracts, whereas dephosphorylated MARCKS was a good substrate. Both
the protease activity and the specific MARCKS cleavage products were
essentially absent in brain, but present in many other cells and
tissues. The protease activity, which had the characteristics of a
cysteine protease, cleaved MARCKS between Asn and
Glu
of the bovine sequence, three amino acids to the
amino-terminal side of the MARCKS phosphorylation site domain. These
studies demonstrate that MARCKS is subjected to specific cleavage by a
cellular protease, in a manner dependent on the phosphorylation state
of the substrate. This represents a novel means of regulating cellular
MARCKS concentrations; these data also raise the interesting
possibility that MARCKS is involved in regulating the activity of this
novel cellular protease.
The myristoylated alanine-rich C kinase substrate, or MARCKS ()protein, is a prominent cellular substrate for protein
kinase C (PKC)(1, 2) . MARCKS and its relative, the
MARCKS-related protein (also known as F52 or MacMARCKS) comprise a
family of heat-stable, acidic proteins that are characterized by
several common properties. These include three highly conserved regions
within the primary protein sequence: The amino-terminal sequence, which
is responsible for directing myristoylation; a short sequence of
identity to the mannose 6-phosphate/insulin-like growth factor II
receptor which surrounds the splice site of the single known intron;
and an internal highly basic domain of 25 amino acids containing the
PKC phosphorylation sites. This basic phosphorylation site domain (PSD)
is also the binding site for calmodulin; this occurs in a
calcium-dependent fashion and is inhibited by PKC
phosphorylation(3) . The PSD also has been implicated in
binding to cytoskeletal actin, again in a phosphorylation- and
calmodulin-dependent manner(4) . The deduced amino acid
sequences of MARCKS from various animal species predict proteins of
28-33 kDa; however, MARCKS migrates anomalously on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), at molecular
mass
60,000-87,000 depending on the species.
Although the precise cellular function of MARCKS remains obscure, recent data from gene targeting experiments in the mouse indicate that it is necessary for normal development of the central nervous system, as well as extrauterine life(5) . Abnormalities seen in the MARCKS-deficient pups include exencephaly, agenesis of the corpus callosum and other forebrain commissures, failure of hemisphere fusion, and lamination abnormalities of the cerebral cortex and retina. The molecular basis of these abnormalities is not known; however, heterozygous mice expressing 50% of wild-type MARCKS levels are phenotypically normal, suggesting that a certain threshold MARCKS concentration is necessary for these developmental processes to occur normally.
For these and other reasons, regulating MARCKS protein
concentrations in cells is important. In general, MARCKS protein
concentrations closely parallel its mRNA levels; these can in turn be
regulated at the level of gene transcription. For example, tumor
necrosis factor and lipopolysaccharide can cause dramatic
increases in the levels of MARCKS mRNA and protein in neutrophils,
macrophages, and related cells(6, 7, 8) .
Decreases in MARCKS mRNA and protein have been seen in fibroblasts
transformed by a variety of oncogenes or by chemical
carcinogenesis(9, 10, 11, 12, 13) .
At the post-transcriptional level, MARCKS mRNA and protein are
decreased in Swiss 3T3 cells in response to several hours of exposure
to phorbol esters, bombesin, platelet-derived growth factor, and agents
that elevate cAMP(14, 15) . In a different study,
Lindner et al.(16) reported rapid decreases in MARCKS
protein levels after short-term (less than 15 min) exposure to phorbol
12-myristate 13-acetate (PMA).
One means of controlling MARCKS protein levels is by specific proteolysis, perhaps regulated by some of the stimuli enumerated above. In the present study, we show that MARCKS can be cleaved into two major fragments that achieve significant concentrations in some cells and tissues. This cleavage is prevented by PKC-dependent phosphorylation of MARCKS. The putative protease involved is present in many cells and tissues, appears to be a cysteine protease, and cleaves MARCKS at a site between asparagine 147 and glutamate 148 in the bovine sequence, three amino acids amino-terminal to the PSD. These results indicate that MARCKS levels in cells can be controlled by a specific protease, whose proteolytic efficacy is dependent on the phosphorylation state of its substrate. They also raise the interesting possibility that MARCKS can physically associate with this uncharacterized protease, perhaps even regulating its activity toward other substrates.
For lysates from HFF cells that
had been boiled prior to clarification, further preclearing was
unnecessary. For boiled lysates from LM/TK cells and
non-boiled lysates from HFF cells, lysates were precleared by
incubation with a 1:100 dilution of nonimmune control antibody and
tumbling at 4 °C for 1 h. In the case of samples immunoprecipitated
with monoclonal antibodies, a secondary rabbit anti-mouse IgG antibody
(2 mg/ml; Pierce) was added at a 1:00 dilution followed by tumbling at
4 °C for 1 h. Protein A-Sepharose, 0.1 g, (Pharmacia Biotech Inc.,
Piscataway, NJ), washed and resuspended in 1 ml of lysis buffer, was
then added at a 1:10 dilution and samples were tumbled for 1 h at 4
°C. Protein A-Sepharose was sedimented by centrifugation at 6000
g for 3 min and the supernatants subjected to further
immunoprecipitation.
Samples that were precleared by boiling or by
treatment with nonimmune antibodies were subjected to
immunoprecipitation with 1:100 dilutions of the appropriate antibodies
and tumbled at 4 °C overnight. When competing peptides were used,
an equal volume of antibody and 5 mM solutions of the peptides
were mixed and incubated for 1 h at 4 °C prior to adding to
precleared lysates. In the case of immunoprecipitations with monoclonal
antibodies, lysates were treated with a rabbit anti-mouse secondary
antibody as described above. All samples were treated with a 1:10
dilution of protein A-Sepharose as described above. Protein A-Sepharose
was precipitated by centrifugation at 6000 g for 3 min
and the supernatants were discarded. The pellets were washed three
times with 0.5-1 ml of lysis buffer followed by centrifugation at
6000
g for 3 min. The final protein A pellet was
resuspended in 100 µl of SDS sample buffer containing 10% sucrose
(w/v), 100 mM dithiothreitol, 1.2% SDS (w/v), 12 mM EDTA, and 0.0012% (w/v) Pyronin Y and boiled for 3 min. The
immunoprecipitates were analyzed by electrophoresis on 10%
SDS-polyacrylamide gels. Gels were fixed in 40% methanol and 10% acetic
acid for 20 min, rinsed extensively with water for 20 min, and treated
with Autofluor (National Diagnostics, Atlanta, GA) for 20 min. Gels
were dried and exposed to Kodak X-Omat XAR film at -70 °C.
Human fibroblasts, serum starved and radiolabeled
overnight with L-[S]cysteine, were
stimulated with Me
SO or PMA for 10 min followed by
immunoprecipitation of cell lysates under low-stringency conditions. A
radiolabeled protein band corresponding to MARCKS was present in
lysates from both Me
SO and PMA-stimulated cells, and was
specifically immunoprecipitated by 2F12 and not by a control monoclonal
antibody, 6F6 (Fig. 1). In addition, 2F12 specifically
immunoprecipitated a
S-labeled protein of approximately 40
kDa (p40) that was present in lysates from Me
SO-treated
cells but absent in lysates from PMA-stimulated cells. In analogous
experiments using polyclonal antibodies generated against a peptide
representing the first 15 amino acids of MARCKS(22) , MARCKS
was detected in the immunoprecipitates from both PMA-stimulated and
control cells. However, in contrast to the data shown in Fig. 1using the carboxyl-terminal specific antibody, no band
migrating at 40 kDa was detected (data not shown).
Figure 1:
Immunoprecipitation of
[S]cysteine-labeled proteins from
control and PMA-stimulated human fibroblasts. Human fibroblasts labeled
overnight with [
S]cysteine, followed by
stimulation for 10 min with 0.01% Me
SO (lanes 1 and 3) or PMA (1.6 µM in 0.01%
Me
SO; lanes 2 and 4) were subjected to
immunoprecipitation with control (6F6) or MARCKS carboxyl-terminal
specific (2F12) antibodies. Cell extracts were precleared with 6F6,
then immunoprecipitated with 6F6 (lanes 1 and 2) or
2F12 (lanes 3 and 4). Labeled proteins in
immunoprecipitates were analyzed by SDS-PAGE followed by fluorography.
The arrows indicate MARCKS (top) and p40 (bottom). The positions of protein molecular weight standards
are indicated.
Figure 2:
Evidence that p40 is a fragment of MARCKS. A, human fibroblasts were labeled overnight with either
[S]methionine (lanes 1-4) or
[
S]cysteine (lanes 5-8),
stimulated with Me
SO (lanes 1, 2, 5, and 6) or PMA (lanes 3, 4, 7, and 8), and then
subjected to immunoprecipitation with control (6F6; lanes 1, 3, 5, and 7) or anti-MARCKS (2F12; lanes 2, 4, 6, and 8) antibodies as described in the legend to Fig. 1. The arrows point to intact MARCKS (top) and p40 (bottom). B, human fibroblasts were labeled overnight
with [
S]cysteine and then were stimulated with
Me
SO (D) or PMA (P) as described in the
legend for Fig. 1. Extracts of the radiolabeled cells were
boiled prior to clarification, and the clarified supernatants were
subjected to immunoprecipitation with 2F12 (anti-MARCKS) monoclonal
antibodies. The arrows indicate intact MARCKS (top)
and p40 (bottom). C, control LM/TK
cells expressing vector alone (vector) and LM/TK
cells heterologously expressing bovine MARCKS (p80) were labeled
overnight with [
S]cysteine. Boiled and clarified
cell extracts from unstimulated cells were subjected to
immunoprecipitation with 2F12 monoclonal antibodies. Also shown is an
immunoprecipitation using 2F12 of boiled and clarified lysate from
unstimulated HFF. The arrows indicate intact MARCKS (top) and p40 (bottom).
To further confirm that p40 was a fragment of MARCKS or a
MARCKS-like protein we took advantage of the heat stability of MARCKS.
In the event that an associated protein was itself heat stable, it
seemed unlikely that the putative association of this protein with
MARCKS would withstand boiling. MARCKS-containing lysates from
[S]cysteine-labeled HFF were boiled for 10 min
followed by precipitation of the denatured proteins. MARCKS remains
soluble and immunoreactive following this procedure. Fig. 2B shows an autoradiograph of proteins present in immunoprecipitates
of boiled and cleared lysates. Boiling removed most of the nonspecific
bands detected in immunoprecipitates of non-boiled lysates. Full-length
MARCKS was readily detected in immunoprecipitates from
Me
SO- and PMA-treated cells. In addition, a
S-labeled protein migrating similarly to p40 was seen in
the immunoprecipitates of lysates from Me
SO-treated but not
PMA-treated cells. The presence of p40 in the immunoprecipitates of
boiled lysates supports the possibility that this protein is a fragment
of MARCKS or a MARCKS-like protein, rather than an associated protein.
To determine whether the presence of p40 in 2F12 immunoprecipitates
was dependent upon the presence of MARCKS in the lysates, cells lacking
MARCKS were tested for the presence of p40. LM/TK fibroblasts, a mouse cell line that lacks endogenous MARCKS, were
stably transfected with either vector alone or a plasmid expressing
full-length bovine MARCKS, which cross-reacts with the 2F12
antibody.
Immunoprecipitations using 2F12 resulted in the
presence of p40 only in cells expressing bovine or human MARCKS, and
not in cells lacking MARCKS (Fig. 2C). Taken together
with the previous data, these findings indicated that p40 was probably
a carboxyl-terminal fragment of MARCKS.
We also wished to determine
if a corresponding amino-terminal fragment of MARCKS could be detected
whose formation was prevented by activation of PKC. Only three
cysteines are present in human MARCKS and these all reside very close
to the carboxyl terminus; therefore, an amino-terminal fragment would
not be detected following [S]cysteine labeling
of cells and immunoprecipitation with the amino-terminal specific
antibody. However, when the cells were radiolabeled with
[
C]alanine, full-length MARCKS as well as a
smaller protein were precipitated with the amino-terminal antibody (Fig. 3). This smaller protein or protein fragment was present
only in unstimulated cells and not in PMA-stimulated cells. This
smaller fragment migrated as a diffuse band of M
44,000. When similar immunoprecipitations were done in the
presence of competing peptide, both intact MARCKS and the entire
diffuse band were competed by the peptide (Fig. 3). In addition,
this same diffuse band was detected when HFF were labeled with
[
H]myristate and the lysates subjected to
immunoprecipitation with the amino-terminal MARCKS antibody (data not
shown). These data indicate that the diffuse band at M
44,000 is an amino-terminal fragment of MARCKS, whose
formation is also prevented by PMA-stimulation of the cells (Fig. 3). The sum of the M
of both the
amino-terminal and carboxyl-terminal fragments add up to the apparent M
of full-length MARCKS. Interestingly, both
fragments migrated anomalously on SDS-polyacrylamide gels, as does
full-length MARCKS.
Figure 3:
Immunoprecipitation of an amino-terminal
fragment of MARCKS. Human fibroblasts were labeled overnight with
[C]alanine and then stimulated with
Me
SO (DMSO) or PMA as described in the legend to Fig. 1. Detergent extracts of the radiolabeled cells were boiled
prior to clarification. The clarified supernatants were subjected to
immunoprecipitation with a polyclonal antibody generated to the
amino-terminal portion of MARCKS. Immunoprecipitations were performed
in the absence(-) or presence (+) of competing peptide.
Proteins present in immunoprecipitates were analyzed by SDS-PAGE
followed by fluorography. The arrow indicates MARCKS and the bracket indicates the amino-terminal fragment of
MARCKS.
Figure 4:
Serum stimulation results in the transient
disappearance of p40. Human fibroblasts were labeled overnight with
[S]cysteine followed by stimulation with fetal
calf serum for the indicated times. Boiled and clarified lysates were
subjected to immunoprecipitation with 2F12 and the immunoprecipitates
analyzed by SDS-PAGE and fluorography. Arrows indicate MARCKS (top) and p40 (bottom).
Figure 5:
p40 can be generated by incubation of cell
extracts without protease inhibitors or phosphatase inhibitors. Human
fibroblasts were labeled overnight with
[S]cysteine followed by stimulation with
Me
SO or PMA, as described in the legend to Fig. 1. A, cell extracts were prepared in the absence (lanes
1-3 and 7-9) or presence (lanes 4-6 and 10-12) of protease inhibitors and then
incubated at 30 °C for 0 (lanes 1, 4, 7, and 10),
10 (lanes 2, 5, 8, and 11), or 20 min (lanes 3,
6, 9, and 12). Following incubation, the extracts were
boiled, clarified, and then subjected to immunoprecipitation with 2F12. Lanes 1-6, extracts made from
Me
SO-stimulated cells. Lanes 7-12, extracts
made from PMA-stimulated cells. B, cell extracts were prepared
in the absence of protease inhibitors as described in A, and
in the presence or absence of NaF, and then subjected to incubation at
30 °C for 0 (lanes 1, 3, 5, and 7) or 30 min (lanes 2, 4, 6, and 8). Following incubation,
extracts were boiled, clarified, and then subjected to
immunoprecipitation with 2F12. Lanes 1-4, extracts made
from Me
SO-stimulated cells. Lanes 5-8,
extracts made from PMA-stimulated cells. Lanes 1, 2, 5, and 6, extracts prepared in the absence of NaF. Lanes 3, 4, 7, and 8, extracts prepared in the presence of NaF. In both A and B, the labeled proteins present in the
immunoprecipitates were analyzed by SDS-PAGE followed by fluorography. Arrows indicate MARCKS (top) and p40 (bottom).
The incomplete loss of full-length MARCKS in lysates from PMA-treated cells suggested that PKC activation partially protected MARCKS from proteolysis either through MARCKS phosphorylation or inhibition of the responsible protease. To determine which of these possibilities pertained, we prepared lysates without protease inhibitors but with sodium fluoride, a serine/threonine phosphatase inhibitor. As seen in Fig. 5B, incubation of lysates from PMA-stimulated cells in the presence of sodium fluoride inhibited the normal loss of MARCKS and generation of p40. Sodium fluoride had no effect on the generation of p40 in lysates from control cells. These data further support the possibility that a PKC-dependent phosphorylation event inhibits the generation of p40 and the concurrent decrease in MARCKS, but still do not distinguish between phosphorylation of MARCKS or inhibition of the responsible protease.
Figure 6:
Formation of p40 from in vitro translated wild-type and mutant MARCKS. A, in vitro translated wild-type and mutant MARCKS were incubated with lysis
buffer (lanes 1 and 4) or detergent lysates from
human fibroblasts which had been treated with MeSO (lanes 2 and 5) or PMA (lanes 3 and 6). Lanes 1-3, in vitro translated
wild-type MARCKS. Lanes 4-6, in vitro translated mutant nonmyristoylated MARCKS. Radiolabeled proteins
were analyzed by SDS-PAGE followed by fluorography. The arrows indicate myristoylated (top) and nonmyristoylated MARCKS (second from top) and p40 (bottom). B,
translated wild-type and mutant MARCKS were incubated with lysis buffer (lanes 1, 4, 7, and 10) or extracts from human
fibroblasts which had been treated with Me
SO (lanes 2,
5, 8, and 11) or PMA (lanes 3, 6, 9, and 12). Lanes 1-3, MARCKS (tetra-Asp). Lanes
4-6, MARCKS (tetra-Asn). Lanes 7-9, MARCKS
(tetra-Gly). Lanes 10-12, MARCKS (tetra-Ala). The
radiolabeled proteins were analyzed by SDS-PAGE followed by
fluorography. Arrows indicate MARCKS (top) and p40 (bottom).
As an additional test of whether the activity responsible for cleaving MARCKS might itself be regulated by PKC-dependent phosphorylation, lysates were prepared from control or PMA-stimulated cells in the presence or absence of NaF, and then assayed for their ability to cleave in vitro translated MARCKS. NaF had no effect on the ability of the cell lysates to cleave MARCKS (data not shown). Taken together, the data suggest that the protease activity is not regulated by PKC-dependent phosphorylation.
Figure 7: The generation of p40 from MARCKS is inhibited by cysteine protease inhibitors. A, radiolabeled in vitro translated nonmyristoylated MARCKS was incubated at 30 °C for 30 min with extracts from human fibroblasts in the absence or presence of specific protease inhibitors. Lane 1, no inhibitors; 2, 200 nM aprotinin; 3, 1 mM benzamidine-HCl; 4, 2 µM leupeptin; 5, 1 µM pepstatin; 6, 574 µM phenylmethylsulfonyl fluoride. Radiolabeled proteins were analyzed by SDS-PAGE followed by fluorography. Arrows indicate MARCKS (top) and p40 (bottom). B, radiolabeled in vitro translated nonmyristoylated MARCKS was incubated at 30 °C for 30 min with extracts from human fibroblasts in the presence of increasing amounts of leupetin (lane 1, 0.01 µM; 2, 0.05 µM; 3, 0.1 µM; 4, 0.5 µM; 5, 1 µM; 6, 2 µM) or antipain (7, 17 nM; 8, 170 nM; 9, 1.7 µM). Radiolabeled proteins were analyzed by SDS-PAGE followed by fluorography. Arrows indicate MARCKS (top) and p40 (bottom).
To support the idea that this activity was due to a cysteine protease, the concentration dependences of leupeptin and another cysteine protease inhibitor, antipain, were tested (Fig. 7B). Both of these protease inhibitors inhibited the p40 generating activity at concentrations compatible with the inhibition of a cysteine protease.
Figure 8: p40 is present in rapidly frozen bovine tissues. One hundred µg (brain and spleen) or 400 µg (liver and kidney) of protein were separated by SDS-PAGE followed by transfer to nitrocellulose. The filters were probed with 2F12 monoclonal antibody followed by horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were visualized by enhanced chemiluminescence. Arrows indicate MARCKS (top) and p40 (bottom).
To establish the authenticity of p40 in adult
bovine tissues, similar immunoblots were analyzed with a control
monoclonal antibody; in addition, the 2F12 monoclonal was tested in the
presence of a specific competing peptide and a nonspecific peptide. As
stated above, the control antibody did not detect either full-length
MARCKS or p40 in HFF extracts, and only the specific peptide competed
with 2F12 in the detection of full-length MARCKS and p40. Finally, bona
fide p40 that was immunoprecipitated from
[S]cysteine-labeled HFF was included in the gels
as a positive control. These experiments confirmed the authenticity of
p40 in bovine tissue and showed that p40 in bovine tissues comigrates
with p40 from immunoprecipitates of HFF (data not shown).
In other
experiments, extracts from mouse tissues were analyzed for their
ability to generate p40 from in vitro translated MARCKS.
Spleen, liver, and kidney all contained readily detectable amounts of
this activity. This activity is similar to the activity present in HFF
in that it is inhibited by leupeptin. In addition, like the protease
present in HFF cells, the protease present in mouse tissues will cleave
the SerAsn mutant MARCKS protein, but will not cleave the
Ser
Asp mutant protein. Finally, the mouse protease will also not
cleave wild-type MARCKS previously subjected to phosphorylation with
PKM (data not shown). Taken together these data demonstrate that both
the protease activity and the resulting proteolytic fragment (p40) are
present in both tissue culture cells and intact tissues.
Figure 9: Alignment of MARCKS protein sequences from various species surrounding the proteolytic cleavage site. The amino acids surrounding the cleavage site of bovine MARCKS and immediately upstream of the PSD are shown. The corresponding sequences from other species are aligned with the bovine sequence. The numbers correspond to the bovine sequence. The arrow indicates the site of cleavage.
The importance of the PSD in regulating this interaction was further analyzed by testing the ability of synthetic peptides representing the PSD to inhibit the MARCKS protease. Wild-type peptide or peptides containing all four serines mutated to alanine or glycine partially inhibited this activity when added at concentrations of 100 µM to in vitro translated MARCKS. In contrast, similar concentrations of the peptide containing aspartic acid residues in place of the serines demonstrated no inhibitory effect on this activity, thus supporting the importance of the charged state of the PSD in regulating interactions with this protease (data not shown).
The alignment of MARCKS protein sequences in this region from
different animal species is shown in Fig. 9. The sequence is
identical among the species shown from Glu in the bovine
sequence through the PSD. The human and bovine sequences, as well as
that of Xenopus laevis, (
)are identical at the
cleavage site. However, the chicken, mouse, and rat sequences contain a
serine in place of asparagine at the amino side of the cleavage site.
Serine and asparagine are similar in that they are both polar,
uncharged amino acids. It is therefore possible that they could
substitute for each other at this cleavage site. The presence of the
proteolytic activity in mouse tissues supports the possibility that
mouse MARCKS is also a substrate for this activity. However, mutational
analysis of the cleavage site will be necessary to establish whether
Ser-Glu as well as Asn-Glu are recognized by this cellular protease.
This study shows that, in some cells and tissues, intact MARCKS co-exists with reasonably high concentrations of two cleavage products, each comprising about half of the intact protein sequence. The concentration of p40 ranges from undetectable in bovine brain to several times the full-length MARCKS concentration in bovine spleen, as determined by Western blotting. These cleavage products are formed by the action of a widely distributed, still uncharacterized cellular protease, apparently of the cysteine protease class. The rate of MARCKS cleavage by this protease activity is dependent upon its phosphorylation state, that is, phosphorylated MARCKS is a poor substrate for the protease whereas dephosphorylated MARCKS is a good substrate. This finding implied that the proteolytic cleavage site was at or near the PSD; direct amino acid sequencing of one of the cleavage products revealed a cleavage site between asparagine 147 and glutamate 148 of the bovine sequence, only three amino acids amino-terminal of the PSD. These data thus provide evidence for a novel means by which PKC can regulate MARCKS concentrations in the cell. They also suggest that MARCKS interacts physically with the responsible protease, and that the PSD represents an important component of the interaction.
That phosphorylation of a protein can affect its susceptibility to
proteolytic cleavage has been described previously. For example, Elvira et al.(25) demonstrated that phosphorylation of
connexin-32 by PKC, but not by the cAMP-dependent protein kinase,
protected the protein from calpain-mediated proteolysis. Chen and
Stracher (26) showed, both in intact cells and in a cell-free
system, that actin-binding protein of human platelets in its
phosphorylated form was a poorer substrate for calpain-mediated
proteolysis than its dephosphorylated form. Finally, protein kinase
A-directed phosphorylation of the microtubule-associated proteins MAP-2
and tau protected these two proteins from calpain-mediated proteolysis,
at least in a cell-free assay(27, 28) .
Phosphorylation may also make a protein more susceptible to proteolytic
cleavage. For example, Warrener and Petryshn (29) demonstrated
that phosphorylated nucleolin was more susceptible to a copurifying
protease than the dephosphorylated form. Another example is IB,
the inhibitor of the transcription factor NF-
B(30) .
I
B in its unphosphorylated state binds to NF-
B and keeps it
inactive and in the cytoplasm. Upon cellular stimulation, including PKC
activation, I
B is phosphorylated and proteolyzed, releasing
NF-
B and permitting its movement to the nucleus where it is active
as a transcription factor(31, 32, 33) .
The turnover of MARCKS with respect to PKC activation has been addressed previously. Brooks et al.(14, 15) demonstrated in Swiss 3T3 cells that long exposure (>5 h) to phorbol ester and bombesin, both of which activate PKC in these cells, resulted in down-regulation of MARCKS mRNA and protein levels. At times less than 5 h, a modest increase in MARCKS protein levels was observed, compatible with the present findings. However, these authors also noted that the down-regulation of MARCKS occurred following activation of protein kinase A, implying a completely different mechanism. In another study, Lindner et al.(16) described a rapid decrease in MARCKS protein levels in Swiss 3T3 cells that occurred within 15 min of PMA treatment. We cannot account for the discrepancies between their data and those of Brooks et al.(14, 15) , or between their data and those reported here, other than that human fibroblasts were used in the present studies rather than Swiss 3T3 cells.
Our data
show that when equal amounts of trichloroacetic acid precipitable
counts were immunoprecipitated from boiled lysates from
MeSO- or PMA-stimulated cells (see for example, Fig. 2B, Fig. 3, and Fig. 4, and data not
shown), the amount of full-length MARCKS in the immunoprecipitates was
always slightly greater in lysates from PMA-stimulated cells compared
to control lysates. This observation is consistent with phosphorylation
of MARCKS preventing its ongoing cleavage, so that the level of
full-length MARCKS is greater in stimulated cells than in unstimulated
cells. The question remains, what happens to the p40 and its
amino-terminal counterpart that are present in the cells prior to PKC
activation and disappear within 15 s of PKC activation? There are at
least three potential explanations for the rapid disappearance of these
fragments. First, activation of PKC might cause the tight association
of these fragments with an Nonidet P-40-insoluble cytoskeletal
fraction. To address this possibility, cell lysates were prepared and
centrifuged without boiling. The insoluble material was then
solubilized in 0.5 M NaCl, followed by dilution to salt and
detergent conditions appropriate for immunoprecipitation. Neither
MARCKS nor p40 was detected in the immunoprecipitates from this
fraction, from either control or PMA-stimulated cells (data not shown).
Second, activation of PKC might stimulate the secretion of these
fragments. To address this, media from
[
S]cysteine-labeled HFF stimulated with
Me
SO or PMA were subjected to immunoprecipitation. Antibody
2F12 detected a secreted protein that comigrated with p40 and was
competed for antibody binding by the epitope peptide. However, this
protein does not appear to be a fragment of MARCKS because: 1) its
presence in the medium was not increased by PMA treatment of the cells,
and 2) it was also detected in medium from cells lacking MARCKS.
Finally, p40 and its amino-terminal counterpart might be subjected to
rapid proteolysis following PKC activation. We addressed this issue by
analyzing lysates prepared from cells stimulated for less than 1 min
with PMA or serum, in which lysates from both
[
S]cysteine- and
[
H]alanine- labeled cells were immunoprecipitated
with 2F12 or the amino-terminal-specific antibody. Even when the
immunoprecipitates were analyzed on 15% acrylamide gels, no lower
molecular weight immunoreactive fragments were detected. However, it is
possible that smaller fragments would remain undetected if rapid
proteolysis were occurring following PKC activation. We favor, but
cannot prove, this final explanation for the rapid disappearance of p40
and the amino-terminal fragment that occurs following PKC activation.
To address this question, it will be necessary to perform studies in
which p40 is expressed in cells totally lacking MARCKS, or lacking the
forms that interact with the 2F12 antibody; the fate of p40 in response
to PMA can then be studied independently of the rate of its formation
from intact MARCKS.
GAP-43 (also known as B-50 and neuromodulin), a
neuron-specific protein that is highly enriched in growth cones and
nerve terminals, resembles MARCKS in that it is a heat-stable substrate
for PKC, it is fatty acylated, and it binds calmodulin in a
PKC-regulated fashion(34) . Coggins and Zwiers (35) have described the susceptibility of this protein to
specific proteolytic cleavage at or around the single PKC target
serine, Ser. They demonstrated that GAP-43, in either its
phosphorylated or dephosphorylated form, can be cleaved by
-chymotrypsin, and this suggested that this cleavage occurred at
Phe
. They also demonstrated the existence of a protease
that co-purifies with GAP-43, which presumably cleaves the protein at
Ser
. In a previous paper (36) they suggested that
the co-purifying protease was sensitive to cysteine-type inhibitors.
These authors also demonstrated that binding of calmodulin to the
unphosphorylated GAP-43 protects it from cleavage by the copurifying
protease(36) . To date, we have seen no effect of calmodulin to
inhibit the proteolytic cleavage of MARCKS. The facts that the
co-purifying protease cleaves GAP-43 in either its phosphorylated or
unphosphorylated forms, and that this is inhibited by calmodulin,
suggest a completely different level of regulation from that observed
with the MARCKS protease described here.
Recently, Allen and Aderem (37) described the PKC-mediated cycling of MARCKS between the plasma membrane and lysosomes in mouse fibroblasts. Studies from several laboratories have established that dephosphorylated MARCKS is associated with the plasma membrane through both the PSD and the myristoylated amino terminus(19, 38, 39, 40, 41, 42, 43) ; upon PKC-mediated phosphorylation, membrane association through the PSD is inhibited(42, 44, 45, 46, 47) . In this paper(37) , the authors demonstrate co-localization of phosphorylated MARCKS with a lysosome-specific marker following PMA stimulation of mouse embryo fibroblasts. Preliminary evaluation of the protease described here suggests that it is associated with the particulate fraction of the cell and is active at acidic pH, both characteristics of a lysosomal enzyme. However, our studies indicate that phosphorylated MARCKS is not a substrate for this protease. Therefore, movement of phosphorylated MARCKS to the lysosome may represent an alternative mechanism of MARCKS regulation to the proteolytic cleavage we describe here; alternatively, movement to the lysosomes may be followed first by dephosphorylation and then by the specific proteolytic cleavage described here.
The presence of a proteolytic activity in extracts of multiple tissues from mouse, human, and cow that is capable of generating p40 from MARCKS demonstrates that this protease is widely distributed. However, there are significant, tissue-specific differences in the apparent activity of this protease. For example, both the protease activity and p40 are essentially absent in brain, a tissue that expresses the highest levels of MARCKS(17, 24) . On the other hand, liver and kidney, which express low levels of MARCKS(17, 24) , express the protease and contain readily detectable amounts of p40. Whether the protease is involved in regulating MARCKS levels, or whether MARCKS is involved in regulating the activity of the protease, are questions that remain to be answered.
Preliminary characterization of the MARCKS protease suggests that it is a cysteine protease. One well known cysteine protease that is important in PKC regulation is calpain(48, 49, 50) . Calpain is a cytosolic protein that can be translocated to the membrane upon activation of PKC(51) . It has an absolute requirement for calcium and consists of two forms distinguished by their micromolar or millimolar calcium requirements. Calpain is also under the regulation of an endogenous protein inhibitor, calpastatin. In addition, many calpain substrates also appear to be calmodulin binding proteins(52) . For these reasons, we considered that calpain might be a good candidate for the MARCKS protease described here. However, this activity shows no calcium dependence, and thus is unlikely to be calpain. Further studies are in progress to characterize and identify this protease.
Amino-terminal sequencing of p40 from bovine spleen established the
site of cleavage to be between an asparagine and glutamate, three amino
acids upstream of the PSD. These residues are identical in the human
and Xenopus MARCKS sequence and contain a serine in place of
the asparagine in the rat, chicken, and mouse sequences. Although a
search of the literature did not reveal a previously identified
cellular cysteine protease with specificity for the dipeptide
Asn/Ser-Glu, there are a few examples of asparagine-specific proteases.
These include an asparagine-specific cysteine protease that is involved
in the post-translational modification of the plant storage protein,
glycinin. This protease recognizes an Asn-Gly linkage(53) . In
addition, the metalloproteinase stromelysin recognizes an Asn-Phe
linkage in pig cartilage proteoglycan. Human and rat proteoglycan also
contain this sequence, but the bovine sequence contains a serine in
place of the asparagine(54) . This suggests that, like the
protease described here, stromelysin may be able to accept an
AsnSer change and still cleave its substrate. Experiments are in
progress to analyze the ability of the MARCKS protease to recognize a
Ser-Glu site in place of the Asn-Glu site.