From the
We reported previously that recombinant myristoylated,
alanine-rich protein kinase C substrate (MARCKS) expressed in
Escherichiacoli as well as MARCKS purified from rat
brain specifically bound to phosphatidylserine (PS) in a
calcium-independent manner and that the binding was regulated through
phosphorylation of MARCKS (Nakaoka, T., Kojima, N., Hamamoto, T.,
Kurosawa, N., Lee, Y. C., Kawasaki, H., Suzuki, K., and Tsuji, S.
(1993) J. Biochem. (Tokyo) 114, 449-452). In
this study, to identify the minimum PS-binding region of MARCKS and the
regulatory phosphorylation site, the binding of MARCKS to PS was
examined in deletion mutants producing glutathione
S-transferase (GST) fusion proteins. The mutant proteins
GST-6-180 and GST-127-160 had almost the same ability to
bind to immobilized PS as MARCKS purified from rat brain, whereas
GST-127-152 did not bind to it. In addition, the binding of
GST-6-156 to immobilized PS was 62% of that of GST-6-180,
but that of GST-6-152 was only 8% and that of GST-6-135 was
not detected. The effect of phosphorylation by protein kinase C was
examined in several mutants of GST-6-180 whose serine residues
were substituted with alanine. After phosphorylation, the mutants
GST-6-180[S156A and S163A],
GST-6-180[S156A], and GST-6-180[S163A]
did not bind to immobilized PS like native MARCKS and GST-6-180.
However, even after phosphorylation, GST-6-180[S152A]
and GST-6-180[S152A and S156A] could bind to
immobilized PS.
These results strongly suggest that MARCKS binds to
PS molecules in the inner leaflet of the plasma membrane through
residues 127-156, with residues 153-156 (FKKS) being
particularly important in the binding of MARCKS to PS, and that the
binding is regulated through the protein kinase C-catalyzed
phosphorylation of the serine at residue 152.
Protein kinase C (PKC)
PKC
(7) ,
synaptotagmin
(8) , and coagulation factor V
(9) have been
reported to bind specifically to phosphatidylserine (PS), and their
binding domains have been reported. The interaction of coagulation
factor V with the platelet surface is considered to involve binding to
PS. We reported previously that bacterially expressed full-length
recombinant MARCKS clearly binds to PS, just like MARCKS purified from
rat brain
(10) . The binding of MARCKS to PS is abolished on
PKC-catalyzed phosphorylation. This is consistent with the reversible
association of MARCKS with the plasma membrane. Thus, it is very
important to elucidate the physiological role of the PS binding of
MARCKS. We have investigated the structure of MARCKS for binding to PS
using a bacterial expression system. As a result, the
Ca
In the case of GST-6-180, the primer
GCGGTCTTGGATCCCTGTGCACCCA was annealed to the template DNA, and a new
BamHI site was created for ligation to pGEX-2T in frame at
Ser-6 of MARCKS. The resulting plasmid was further transfected into
cj236, and single-stranded DNA was prepared as a template DNA. Using
the primer GGCCTCGTCCTTGGCGATATCTCACTAGGCGCCTTCCGCCTC, a stop codon
(TAG) was introduced at residue 181 of MARCKS (the portion
corresponding to the stop codon is underlined).
For fusion to GST at
residues 46, 98, and 127, a new BamHI site was created by
site-directed mutagenesis using the following primers:
CTCGGCGGCGGCGGGGGATCCGTCCCCGTTCACTTT,
AGCCTCCTTGTCGGCGGATCCAGCGCCCGGCTCGGG, and
GTCCTCCGCCTTGGGGGATCCCGTGGAGGAGGCGGA, respectively. The authentic
threonine (residue 98) of rat MARCKS was substituted with serine for
ligation to the vector in GST-98-180. A new stop codon
(underlined) was introduced at residues 168, 161, 157, 153, and 136
using the following primers:
CTCAGCGCCCTCGCCGTTAACTCACTAGCTCTTCTTGAAGGAGAAGCCG,
CTTGCTCTTCTTGAAGTTAACTCACTAGCTCAGCTTGAAGGACTTCTT,
CTTGAAGGAGAAGCCGTTAACCTATCAGGACTTCTTGAAGGAAAAGCG,
GCCGCTCAGCTTGAAGTTAACCTATCAGGAA-AAGCGCTTCTTTTTTTT, and
CGGGGTCTCGCTGATATCTCACTAGGCCCCGTCCTC, respectively.
In the point
mutation experiment, the authentic serine of the phosphorylation site
was substituted with alanine by site-directed mutagenesis using the
following primers (portions of nucleic acids for point mutations are
underlined): S152A, GAAGGACTTCTTGAACGCAAAGCGCTTCTTTT; S156A,
GCTCAGCTTGAACGCCTTCTTGAAGGAAAA; S163A, CTTGCTCTTCTTGAACGCGAAGCCGCTCAG;
S152A and S156A, GAAGCCGCTCAGCTTGAACGCCTTCTTGAACGCAAAGCGCTTCTTTT; and
S156A and S163A,
CTTCTTGCTCTTCTTGAACGCGAAGCCGCTCAGCTTGAACGCCTTCTTGAAGGAAAAGCG.
In the
case of ligation to pET-8c, all the mutants were ligated, using the
NcoI site (5`-end) and the blunt-ended BamHI site
(3`-end) of pET-8c, by introducing a new NcoI site by
site-directed mutagenesis. The structures of the mutants were confirmed
by DNA sequencing with a DNA sequencer (Pharmacia Biotech Inc.).
Fragments derived from corresponding plasmids were isolated from
agarose gel pieces and ligated into the BamHI site (5`-end)
and the SmaI site (3`-end) of pGEX-2T. Positive clones were
selected by restriction analysis and DNA sequencing.
For purification, the GST fusion
protein was adsorbed to glutathione-Sepharose 4B by the batch method
according to the manufacturer's instructions and then eluted with
10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0,
after washing five times with TBS containing 2 mM EGTA. The
eluted fraction was gel-filtrated on a Sephadex G-25 column in TBS and
then subjected to the binding assay.
The binding
of GST-6-180 to PS was linear within the range of 0.03-0.12
µg of immobilized PS, and the protein bound to PS at >0.3 µg
reached a plateau (Fig. 4). Therefore, the binding assay was
performed with 0.08 µg of immobilized PS. The binding of GST fusion
MARCKS mutants to immobilized PS is summarized in Fig. 2. The
binding of GST-46-180, GST-98-180, and GST-127-180
was the same as that of GST-6-180, indicating that residues
6-126 of rat MARCKS are not involved in the binding to PS. We
next examined whether or not the phosphorylation site domain (residues
145-169) was involved in the binding to PS. The binding of
GST-6-167, GST-6-160, and GST-6-156 to PS was 94, 71,
and 62% of that of GST-6-180, whereas the binding of
GST-6-152 was greatly reduced compared with that of
GST-6-180 and that of GST-6-135 was negligible
(Fig. 5). In addition, GST-127-160 bound to PS as
GST-6-160 did, whereas GST-127-152 did not (Fig. 2).
These results indicate that residues 127-156, particularly
residues 153-156, are closely involved in the binding to PS.
The results presented here for the GST fusion protein
clearly indicate that rat MARCKS interacts with PS mainly via a region
within residues 127-156. Furthermore, residues 153-156
(FKKS) of MARCKS are very important for the PS binding of MARCKS since
the binding of GST-6-152 to PS decreased by >80% of that of
GST-6-156. On the other hand, Taniguchi and Manenti
(5) reported that a synthetic peptide corresponding to residues
145-169 of rat MARCKS interacts with phospholipids including PS.
Considering this result and the results presented here, residues
145-156 (KKKKKRFSFKKS) may largely contribute to the binding of
MARCKS to PS.
The binding domains of proteins for binding to PS,
such as the C2 domain of PKC
(14) and the C2 domain-like repeats
of synaptotagmin
(8) , have been reported. Like MARCKS,
coagulation factor V does not need Ca
Cosubstitution of serine at
both residues 156 and 163 with alanine did not affect regulation of the
binding of MARCKS to PS through PKC-catalyzed phosphorylation, but
substitution of serine at residue 152 with alanine clearly affected
this regulation. Therefore, phosphorylation of serine at residue 152,
which exists in the PS-binding region of MARCKS, is sufficient for
affecting the binding to PS. Phosphorylated GST-6-180[S152A
and S156A] bound to PS more than phosphorylated
GST-6-180[S152A], although
GST-6-180[S156A] did not bind to PS after
phosphorylation, indicating that phosphorylation of serine at residue
156, in addition to serine at residue 152, has some additional effect
on the binding to PS.
It is apparent that N-terminal myristoylation
(18) is not necessary for binding to PS because GST fusion
mutants bind to PS. The myristoyl moiety of proteins is not considered
to serve as a hydrophobic anchor because many myristoylated proteins
are found in the cytosol, in contrast to palmitoylated proteins, which
are exclusively localized in the membrane
(19) . Although the
role of the myristoyl moiety has not yet been established, many
myristoylated proteins exist as components of intracellular signaling
systems. The myristoyl moiety and PS-binding domain identified in this
study must regulate the localization of MARCKS cooperatively. In this
connection, it is interesting that demyristoyl activity of MARCKS
exists in the synaptic fraction to regulate the function of
MARCKS
(20) .
The linking of the plasma membrane to the
cytoskeleton is suggested to be an important feature of MARCKS. The
ability of MARCKS to bind to PS may assure the association of MARCKS
with the plasma membrane directly. It may be consistent with the
reversible association of MARCKS with the plasma membrane that the
binding of MARCKS to PS is abolished on PKC-catalyzed phosphorylation.
On the other hand, it was reported that the phosphorylation and
translocation of MARCKS are not parallel in the N1E-115 neuroblastoma
cell line
(21) . The interaction of MARCKS with calmodulin
(22) or actin may largely contribute to its function. It is also
probable that PS acts as a cofactor when MARCKS interacts with other
proteins, as in the case of PKC. The expression of MARCKS mutants, such
as with substitution of serine at residue 152 with alanine, in
eucaryotic cells may allow elucidation of whether the PS binding
property is involved in the membrane association or the interaction of
MARCKS with other molecules.
The binding of MARCKS purified from rat brain and GST fusion
proteins expressed in E. coli. to 0.08 µg of phospholipid
was measured by the protein binding assay using anti-MARCKS and
peroxidase-conjugated goat anti-rabbit IgG antibodies as described
under ``Experimental Procedures.'' Data are expressed in
relation to the binding to PS taken as 100%.
We are grateful to Dr. Masato Umeda (Faculty of
Pharmaceutical Science, University of Tokyo) for helpful discussion.
(
)
has been
established to be the mainstay of a number of intracellular signaling
systems, especially that concerned in Ca
mobilization. Myristoylated, alanine-rich protein kinase C
substrate (MARCKS) is a major substrate and is suggested to be involved
in the cellular signaling pathway involving activation of
PKC
(1) . Although the physiological function of MARCKS has not
yet been clearly elucidated, the association of MARCKS with the plasma
membrane or cytoskeleton
(2) has been suggested to be of
importance regarding the function of MARCKS. MARCKS associates with the
plasma membrane under regulation through PKC-catalyzed phosphorylation
of MARCKS
(3, 4) . It has been suggested that MARCKS is
capable of associating with the plasma membrane through binding to
phospholipids without interaction with membranous
proteins
(5, 6, 10) .
-independent PS-binding domain of rat MARCKS was
identified within a region of 30 amino acid residues in length.
Materials
PS, phosphatidylcholine (PC),
phosphatidylinositol (PI), phorbol 12-myristate 13-acetate, and
cholesterol were obtained from Sigma. Other chemicals were purchased
from the following sources: [1,2-H]cholesterol
and EN
HANCE spray from DuPont NEN; bacterial expression
vector pGEX-2T, goat anti-glutathione S-transferase antibody,
protein A-Sepharose 4B, and glutathione-Sepharose 4B from Pharmacia
Biotech Inc.; nylon sheets (Immobilon
) from Millipore;
3,3`,5,5`-tetramethylbenzidine dihydrochloride and goat anti-rabbit IgG
peroxidase-conjugated antibody from Cappel; and swine anti-goat IgG
peroxidase-conjugated antibody from Tago Inc. Bacterial strain
BL21(DE3)pLysS and bacterial expression vector pET-8c were obtained
from the RIKEN GENE BANK. PKC was prepared from rat brain as described
(10). Rabbit anti-MARCKS antiserum was prepared by immunizing a rabbit
with 300 µg of purified rat MARCKS using the standard procedure.
The resulting anti-MARCKS antiserum stained one band by immunostaining
of a rat brain homogenate with a Konica Immunostain HRP kit.
Assay for Binding of Liposomes to MARCKS (Liposome
Binding Assay)
Unilamellar liposomes labeled with
[1,2-H]cholesterol were prepared as described
previously (10). In the case of PC liposomes, lyso-PC was included at a
PC/lyso-PC ratio of 7:3. Rat MARCKS was purified to a homogeneous state
as judged by protein staining of SDS-polyacrylamide gel. To examine the
binding of liposomes to MARCKS, the indicated amounts of the purified
protein were adsorbed to the wells of a 96-well flat-bottom assay plate
(Pro-bind, Falcon) for 16 h at 4 °C. After being blocked with 4%
skim milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, 150
mM NaCl, pH 7.5), 50-µl aliquots of TBS, 2 mM
EGTA containing labeled phospholipid liposomes (10
dpm/ml)
were distributed in the wells, followed by incubation for 16 h with
slow shaking at room temperature. After unbound liposomes had been
removed by washing three times with TBS, 1 mM EGTA, the bound
liposomes were collected in 2-propanol/hexane/H
O (50:25:20)
and then counted in triplicate using a scintillation counter.
Assay for Binding of Fusion Proteins to Immobilized
Phospholipid (Protein Binding Assay)
Phospholipid
(0.03-2.0 µg) in 50 µl of 50% aqueous ethanol was
adsorbed to each well of a 96-well flat-bottom assay plate for 8 h at
37 °C. After being blocked with 4% skim milk in TBS, the wells were
overlaid with fusion proteins in 50 µl of TBS, 2 mM EGTA
containing 1% skim milk, followed by incubation for 12 h at 4 °C.
After washing out the unbound proteins, the bound fusion proteins were
measured as the absorbance at 650 nm of 3,3`,5,5`-tetramethylbenzidine
dihydrochloride using anti-MARCKS antiserum or anti-GST antibody and
corresponding peroxidase-conjugated secondary antibodies. The binding
of fusion proteins to a PS-coated plate reached a plateau after
incubation for >8 h at 37 °C. The amount of proteins bound to a
PS-coated plate after incubation for 12 h at 4 °C was comparable to
that after incubation for 8 h at 37 °C.
Construction of MARCKS Mutants
A 4.4-kilobase pair
clone was obtained from a rat brain cDNA library as rat MARCKS
cDNA
(10) . An XhoII fragment (1.1 kilobase pairs)
cloned into pBluescript (pBSK), which contained all
the coding region of rat MARCKS
(11) , was restricted with
NotI, and then the resulting fragment (0.6 kilobase pair),
which contained the start codon, was cloned into pBSK
.
The resulting plasmid, NotI SK
, was
transfected into bacterial strain cj236, and single-stranded DNA was
prepared as a mutagenesis template. To construct a plasmid to express a
fusion protein of GST and MARCKS, a new BamHI site and a new
stop codon were introduced into a plasmid, which was cloned into
pBSK
by site-directed mutagenesis using Mutagene kits
(Bio-Rad)
Expression and Purification of the Bacterially Expressed
Protein
BL21(DE3)pLysS transformed with the recombinant clone in
pGEX-2T was cultured in M9H broth
(12) containing 30 µg/ml
ampicillin and 30 µg/ml chloramphenicol. Following the addition of
isopropyl-1-thio--D-galactopyranoside to 0.4 mM
at the mid-growth stage, the cells were further cultured for 4 h at 30
°C. After harvesting, Escherichia coli cells suspended in
50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1% Triton X-100, 2
mM phenylmethylsulfonyl fluoride were sonicated for 30 s twice
at 4 °C. The lysate was centrifuged at 10,000
g for 15 min at 4 °C, and the resulting supernatant was
collected as the soluble fraction.
Phosphorylation with PKC
Mutant proteins were
phosphorylated by incubation with PKC in 20 mM Tris-HCl, pH
7.4, 10 mM MgCl, 2 mM CaCl
, 5
mM NaF, 0.1 mM Na
VO
, 1
µM phorbol 12-myristate 13-acetate, and 0.5 mM
ATP for 30 min at 37 °C following the addition of EGTA (final
concentration, 10 mM). The binding of phosphorylated proteins
to PS was compared with that of mutant proteins incubated with PKC
without ATP in the presence of 10 mM EGTA instead of
MgCl
.
RESULTS
MARCKS Recognizes PS Molecules
Using a liposome
blotting method, we showed previously that PS liposomes bind to
MARCKS
(10) . To determine how the phospholipid composition
affects the binding, we analyzed the binding of
phospholipid/cholesterol liposomes with different PS/PC ratios to
immobilized MARCKS by the liposome binding assay (Fig. 1). In
this assay, the binding of PC liposomes (100% PC as phospholipid) to
MARCKS was 5% of that of PS liposomes (100% PS as phospholipid). As the
ratio of PS to PC decreased, the binding of liposomes to MARCKS also
decreased, indicating that MARCKS recognizes PS on the liposomes.
Figure 1:
Phospholipid composition affects the
binding of liposomes to MARCKS. H-Labeled
phospholipid/cholesterol liposomes (1:1 mol/mol) were prepared. The
ratio of PS to PC (mole/mole) was 1:0 (closedcircles), 1:1 (opencircles), 1:3
(opensquares), and 0:1 (closedsquares). The binding of each type of liposome to MARCKS
was measured as described under ``Experimental
Procedures.''
To
quantify the specificity of the binding of MARCKS to phospholipid, the
binding of MARCKS to immobilized phospholipid was determined (protein
binding assay). MARCKS bound to immobilized PS, but the binding of
MARCKS to immobilized PI was only 5% of that to PS and that to PC was
not detected ().
Binding of Recombinant MARCKS to PS
As previously
reported, bacterially expressed full-length MARCKS, as well as MARCKS
purified from rat brain, binds to PS
(10) . To investigate
further the PS-binding domain of MARCKS, several deletion mutants were
expressed as GST fusion proteins, and PS binding was determined by the
protein binding assay (Fig. 2). The purity of the fusion proteins
was checked by SDS-PAGE (Fig. 3). The exact migration position of
fusion proteins on SDS-PAGE is hard to estimate from their molecular
mass because native MARCKS is anomalously retarded from the position
estimated from its molecular mass on SDS-PAGE. GST-6-180,
GST-46-180, GST-6-152, and GST-6-135 migrated as
single bands on SDS-PAGE. GST-98-180 and GST-127-180 were
doublets on SDS-PAGE. Although proteolysis may be involved in these
cases, these proteins were used for the binding assay without further
purification.
Figure 2:
Construction of mutants. The structure of
rat MARCKS, composed of 309 amino acids, is schematically represented.
The phosphorylation site domain (residues 145-169; hatchedbox), myristoylation site (Myr), and
phosphorylation site (P) are indicated. The mutants of MARCKS
expressed as GST fusion proteins are designated according to the amino
acid sequence of MARCKS, with the GST portion denoted by a filledbox. The binding of each protein to 0.08 µg of
immobilized PS, as determined by the protein binding assay, was
compared. Data are expressed in relation to the binding of
GST-6-180 taken as 100%.
Figure 3:
Purified mutants of MARCKS. The GST fusion
proteins, which were purified with glutathione-Sepharose 4B, were
subjected to SDS-PAGE on a 5-20% gel. The gels were stained with
Coomassie Brilliant Blue R-250 and then dried. GST-6-180,
GST-46-180, GST-98-180, and GST-127-180 migrated as
66-, 56-, 43-, and 31-kDa bands, respectively. GST-6-152 migrated
indistinguishably from GST-6-180, whereas GST-6-135
migrated as a 48-kDa band. The molecular mass standards are indicated
on the left.
PS binding of the protein was not affected by fusion
with GST since both GST-6-309 and GST-6-180 specifically
bound to PS, but not to PI and PC, like MARCKS purified from rat brain
(). On the other hand, the control GST did not bind to
immobilized PS. GST-6-180 bound to PS at a molecular ratio of
1:200, which is the same as in the case of native MARCKS. Thus,
residues 181-309 are not involved in PS binding.
Figure 4:
Characterization of the binding of
GST-6-180 to PS. The binding of GST-6-180 to increasing
amounts of immobilized PS was measured in triplicate by the protein
binding assay as described under ``Experimental Procedures.''
Bound proteins were measured as the absorbance at 650 nm of
3,3`,5,5`-tetramethylbenzidine dihydrochloride using goat anti-GST
antibodies and peroxidase-conjugated secondary
antibodies.
Figure 5:
PS binding of GST MARCKS mutants whose C
terminus was deleted stepwise. GST fusion proteins, with almost the
same absorbance as determined by enzyme-linked immunosorbent assay
using anti-GST antibody, were overlaid on immobilized PS. Proteins
bound to immobilized PS were measured by the protein binding assay as
described for Fig. 4. Opencircles, GST-6-180;
closedcircles, GST-6-167; closedtriangles, GST-6-160; closedsquares, GST-6-156; open triangles,
GST-6-152; opensquares,
GST-6-135.
Effect of Phosphorylation with PKC
Since the
phosphorylation of MARCKS with PKC significantly decreased the binding
to PS, as described
(10) , we examined how phosphorylation with
PKC affects the binding of MARCKS to PS using a GST fusion protein and
mutant proteins whose serine residues were substituted with alanine
residues. Phosphorylation of GST-6-180 clearly abolished the
binding of the protein to PS. The possible phosphorylation sites of rat
MARCKS for PKC are considered to be Ser-152, Ser-156, Ser-163, and
Ser-167, of which Ser-152, Ser-156, and Ser-163 are conserved between
species and were demonstrated to be phosphorylated by PKC in chick
MARCKS
(13) . Substitution of serine with alanine did not affect
the binding to PS of these mutants (Fig. 6). The binding of
GST-6-180[S156A], GST-6-180[S163A], and
GST-6-180[S156A and S163A] to PS was almost completely
abolished when the mutant proteins were phosphorylated with PKC, as in
the case of native MARCKS and GST-6-180 (Fig. 6). On the
other hand, the binding of phosphorylated
GST-6-180[S152A] to PS decreased compared with that of
the unphosphorylated form, but still occurred (42% of the
unphosphorylated level). Phosphorylation of GST-6-180[S152A
and S156A] decreased the binding to PS only slightly (74 ±
4% of the unphosphorylated level) (Fig. 6). Thus, PKC-catalyzed
phosphorylation of Ser-152 is closely involved in regulation of the
binding to PS.
Figure 6:
Effect of the phosphorylation site on PS
binding. The phosphorylation site of GST-6-180 was substituted as
described under ``Experimental Procedures.'' The mutants,
incubated with PKC and ATP (phosphorylated; shaded bars) or
without ATP (unphosphorylated; open bars), were overlaid on
immobilized PS. Proteins bound to 0.12 µg of immobilized PS were
measured by the protein binding assay as described for Fig.
4.
DISCUSSION
We previously reported that MARCKS specifically binds to
PS
(10) . On the other hand, Taniguchi and Manenti
(5) reported that MARCKS binds to other phospholipids as well as
to PS. To clarify this difference, two assay systems for the binding of
phospholipids and MARCKS were used to delineate the specificity of the
binding in this study. With the liposome binding assay, the binding of
PI liposomes to MARCKS was comparable to that of PS liposomes, and only
weak binding of PC liposomes to MARCKS was detected. On the other hand,
MARCKS clearly bound to only PS, i.e. not to PI or PC, when
the binding of MARCKS to immobilized phospholipids was quantified using
the protein binding assay. This apparent discrepancy may reflect the
difference in the strength of the binding of MARCKS and phospholipid in
the two assay systems. Therefore, MARCKS recognized PS much more
strongly than other phospholipids such as PC and PI. It should be
tested whether or not PS molecules among phospholipid really play a
role in the association of MARCKS with the plasma membrane in
vivo.
for binding to
PS-containing liposomes and two potentially different regions of factor
V have been separately suggested to be the PS-binding domain: one is
the region within the third A domain of the light chain, which contains
two hydrophobic segments
(15) , and the other is a region within
the second C-type domain
(16) . However, there is no homology
between the PS-binding domain of MARCKS and those of other PS-binding
proteins. Since the phosphatidylinositol bisphosphate-binding sequence
of gelsolin was identified as only a 9-amino acid peptide consisting of
a motif of basic amino acids
(17) , the PS-binding domain of
MARCKS seems to be rather similar in the case of phosphatidylinositol
bisphosphate-binding proteins (gelsolin, gCap39, villin, profilactin,
etc.). However, the PS-binding domain of MARCKS is considered
to have a unique Ca
-independent PS recognition motif
because the binding of MARCKS to phosphatidylinositol bisphosphate was
not detected.
(
)
Table: Binding of MARCKS to phospholipid
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.