(Received for publication, September 5, 1995; and in revised form, October 9, 1995)
From the
A monoclonal anti-idiotypic antibody, Id8F7, previously shown to
bind to a phosphatidylserine (PS)-specific binding site on protein
kinase C (PKC) has been used to identify a 12-amino acid consensus
sequence shared by PKC and phosphatidylserine decarboxylase (PSD). The
14-amino acid synthetic peptide derived from the corresponding region
of PSD (amino acids 351-364 of the enzyme from Chinese hamster
ovary cells) bound effectively and specifically to PS, and that derived
from rat PKC (amino acids 227-240) bound weakly but
specifically to PS. Analysis of binding of Id8F7 to various synthetic
peptides revealed that the consensus sequence motif,
FXFXLKXXXKXR, is responsible for
the interaction with both Id8F7 and PS. The results suggest that the
conserved amino acid residues represent a basic structural motif for
the specific interaction with PS, and the corresponding regions of PKC
and PSD form the PS-specific binding sites of these enzymes.
Although it is generally accepted that phospholipids in
membranes are essential for the catalytic activity of many
membrane-bound enzymes, it is still unclear how phospholipids regulate
the activity of the enzymes. Phosphatidylserine (PS) ()in
membranes is known to be an essential cofactor for the activation of
protein kinase C (PKC) (1, 2, 3) and blood
coagulation(4, 5, 6) . Recent analyses have
shown that it regulates the activity of various enzymes such as c-Raf-1
protein kinase(7) , nitric oxide synthase(8) ,
Na
/K
-ATPase(9) , Dynamin
GTPase(10) , and diacylglycerol kinase (11) and acts as
a ligand in recognition of apoptotic cells(12, 13) .
PKC is a family of phospholipid-dependent kinases, and its enzymatic
activity is allosterically regulated by
1,2-diacyl-sn-glycero-3-phospho-L-serine
(PS)(14, 15) . Analyses of the interaction of
conventional PKC (cPKC) with membrane lipids support a two-step model
for the activation of cPKC, which includes initial binding to membranes
that does not require PS since cPKC binds various acidic phospholipids
in a Ca
-dependent manner and allosteric activation by
specific interaction with PS and diacylglycerol(15) . The
initial binding of cPKC to membranes is believed to be mediated by the
Ca
-dependent phospholipid-binding (CaLB) domain
(amino acids 186-233 of PKC
), a sequence motif that is also
found in other cytosolic proteins such as cytosolic phospholipase
A
, GTPase-activating protein, and
synaptotagmin(16, 17) . However, it has been difficult
to validate the presence of the PS-specific binding site on PKC, since
the enzyme interacts with multiple phospholipid molecules during
activation. Our previous studies showed that the binding to PKC of the
monoclonal anti-idiotypic antibody Id8F7 raised against the combining
site of the PS-specific antibody is inhibited by PS but not by other
phospholipids including the synthetic PS analogue,
1,2-diacyl-sn-glycero-3-phospho-D-serine (18, 19) . The binding of Id8F7 to PKC is
significantly enhanced by the presence of diacylglycerol and is
independent of the presence of Ca
(19) . These
observations indicate that Id8F7 binds to the PS-specific binding site,
which is involved in the allosteric activation of PKC. In this study,
we first mapped the Id8F7 binding site within the C2 region of rat
PKC
, and then we identified a 12-amino acid peptide motif
responsible for the specific interaction with PS.
The
transformant cells were cultured at 37 °C and were induced by
addition of isopropyl--D-thiogalactopyranoside to the
final concentration of 0.5 mM. The cells were harvested by
centrifugation after 3 h of incubation, and the cell pellets were
resuspended in hypotonic buffer (HB; 20 mM Tris, 5 mM EDTA, 5 mM EGTA, 20 µg/ml leupeptin, 50 µg/ml
phenylmethylsulfonyl fluoride, 10 mM 2-mercaptoethanol, pH
7.5). PKC1-173 and PKC161-373 were purified by consecutive
washing with HB containing 1% Triton X-100 and 6 and 8 M urea.
The final precipitates were dissolved in HB containing 6 M guanidine. PKC161-220 and PKC220-296 were resuspended
in Tris-buffered saline (10 mM Tris, 150 mM NaCl, pH
7.4) containing 50 µg/ml phenylmethylsulfonyl fluoride and purified
to homogeneity by affinity chromatography with amylose-resin (New
England Biolabs). Expressed PKC fragments were at least 95% pure as
judged by SDS-polyacrylamide gel electrophoresis. To confirm the amino
acid sequences of the recombinant fragments, N-terminal amino acid
sequences of each polypeptide were identified by amino acid sequencer
(Applied Biosystems amino acid sequencer model 477A) after
purification.
To identify the PS-specific binding site on PKC, we mapped
the Id8F7 binding site using recombinant and synthetic PKC
fragments. The fragments covering the C1 region (amino acids
1-173, PKC1-173) and the C2 region (amino acids
161-373, PKC161-373) of PKC
were expressed (Fig. 1A) (20) and examined for reactivity with
Id8F7. Id8F7 bound effectively to PKC161-373 but not to
PKC1-173 (Fig. 1B). Partial proteolytic digestion
of PKC161-373 with endoproteinase Lys-C followed by separation
and sequence analysis of the Id8F7-reactive peptides resulted in the
identification of a single reactive peptide with the amino acid
sequence, ATLNPVWNETFVFNLKPGDVER- - (single-letter amino
acid code, from amino acid 217). The analysis using the endoproteinase
Asp-N identified two Id8F7-reactive peptide fragments,
DPYVKLKLIPDPRN- - - (from amino acid 193) and
DPRNLTKQKTDTVKA- - - - (from amino acid 203).
These results suggest that the Id8F7 binding site is either located in
the CaLB domain (amino acids 186-242 of rat PKC
) (17) or spanned to amino acids outside the CaLB domain. We
expressed the fragments covering amino acids 161-220
(PKC161-220) and 220-296 (PKC220-296) in the C2
region (Fig. 1A) and showed that Id8F7 binds
specifically to PKC220-296 (Fig. 1B). To study
the functional role of this fragment, we examined the binding of the
fragments to various phospholipids. PKC220-296 bound effectively
to PS and showed some cross-reaction with phosphatidylinositol.
PKC161-220 did not show any significant binding to the
phospholipids (Fig. 2). These observations indicate that the
PS-specific binding site recognized by Id8F7 is located in amino acids
220-296 in the C2 region of PKC
.
Figure 1:
Binding of
anti-idiotypic monoclonal antibody Id8F7 to PKC fragments. A, schematic structure of rat brain PKC
showing the four
conserved regions (C1-C4) and the PKC fragments expressed in E. coli. B, the binding of Id8F7 to PKC
and PKC
fragments was examined by ELISA. PKC
(10 ng/well) or PKC fragments
(50 ng/well) were coated onto the microtiter plate, and binding of
Id8F7 (5 µg/ml) was determined using biotinylated anti-mouse IgM
and peroxidase-conjugated streptavidin. Bars represent the
mean values of three different experiments.
Figure 2: Binding of PKC recombinant fragments to PS. The binding of PKC161-220 and PKC220-296 to various phospholipids was tested by ELISA. The wells of the microtiter plate were coated with 50 pmol/well of the phospholipids and incubated with 5 µg/ml of the PKC fragments. The binding of the fragments to plate-coated phospholipids was determined by rabbit anti-maltose binding protein IgG and peroxidase-conjugated anti-rabbit IgG. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; CL, cardiolipin.
We performed
computer-aided similarity searching (SDC-GENETYX program, Software
Development, Tokyo) for amino acid sequences in PKC220-296 and
PSD from Chinese hamster ovary (CHO) cell(23) , the only
example of the enzyme that has been shown to specifically recognize the
molecular structure of PS(23, 24) . The analysis
revealed significant homology between the primary sequences of PKC
(amino acids 227-238) and PSD (amino acids 351-362) (Fig. 3A). The consensus sequence is conserved among the
cPKC family from different species(1) . We synthesized various
peptides derived from the consensus sequence of PKCs and PSD to
determine whether the consensus sequence is responsible for the
interaction with Id8F7. Id8F7 bound effectively to the synthetic
peptides derived from both PKC and PSD (Fig. 3B). Deletion
of the FXF residue greatly reduced reactivity, and replacement
of the consensus lysine or arginine residues with alanine also reduced
reactivity (Fig. 3B). The results indicate that Id8F7
recognizes the peptide motif with the consensus
FXFXLKXXXKXR amino acid sequence.
Figure 3: Identification of Id8F7-binding peptide sequence on PKC and PSD. A, comparison of amino acid sequences shared by rat PKC family and PSD from CHO cells. Conserved amino acid residues are indicated by boldface letters, with residue numbers on the right. Searching for similar amino acid sequences was performed using the SDC-GENETYX program (Software Development, Tokyo). B, binding of Id8F7 to the synthetic peptides derived from the consensus sequence of PKC and PSD. The synthetic peptides (100 ng/well) were coated onto the microtiter plate by incubating for 12 h at 37 °C and incubated with Id8F7 (5 mg/ml). Binding of Id8F7 was determined using biotinylated anti-mouse IgM and peroxidase-conjugated streptavidin.
To assess the functional role of the consensus sequence, we examined
the binding of each synthetic peptide to various phospholipids. In this
assay, each peptide was conjugated with alkaline phosphatase, and its
binding to plate-coated phospholipids was examined by ELISA. Among the
synthetic peptides examined, the peptide derived from PSD
(FNFRLKAGQKIRFG) bound effectively and specifically to PS, and the
peptide derived from PKC (FVFNLKPGDVERRL) bound weakly to PS (Fig. 4, A and B). The peptide derived from
PKC
(FRFQLKESDKDRRL) tends to adhere nonspecifically to the
plate-coated phospholipids, but it bound effectively to PS when the
lower concentration of phospholipids was used to coat the wells of
microtiter plates (Fig. 4C). No significant binding of
the other synthetic peptides to the phospholipids was observed (data
not shown). Since the binding of the peptides to PS correlates well
with their binding to Id8F7, it is likely that the conserved amino acid
residues, FXFXLKXXXKXR, in the consensus
sequence also play a dominant role in the interaction with PS.
Figure 4:
Specific binding of 14 amino acids
synthetic peptides to PS. Synthetic peptides, FNFRLKAGQKIRFGC from PSD (A) and FVFNLKPGDVERRLC from PKC (B), were
conjugated to alkaline phosphatase as described under
``Experimental Procedures.'' The alkaline
phosphatase-conjugated peptides (10 µg/ml) were incubated with the
plate-coated phospholipids (50 pmol/well for A and B and 5 pmol/well for C), and the binding of the peptides
was determined by the addition of 0.25 mM 4-methylumbelliferyl
phosphate substrate solution.
The
PSD, which catalyzes the conversion of PS to phosphatidylethanolamine,
has been isolated from CHO cell(23) , E.
coli(25) , and yeast(26, 27) . The
consensus sequence motif observed in the PSD of CHO cell is conserved
in the yeast PSD (amino acids 475-486, FKFDVRVGDKVK) but not in
the E. coli enzyme. PSD is a pyruvoyl-dependent enzyme, and
the post-translational cleavage of a proenzyme (-subunit) is
believed to form the pyruvoyl prosthetic group at the N terminus of the
subunit, which is composed of the C-terminal 32 amino acids of
the PSD of CHO cells and 38 amino acids of the yeast
enzyme(23, 26, 27) . It is noteworthy here
that the consensus sequences in yeast and mammalian PSD are located in
the
subunit of the enzyme, which are close to the catalytic
carbonyl group of the enzyme. Recently, Trotter et al. (28, 29) isolated the structural gene encoding the
second PSD gene (PSD2) from yeast. The PSD2 amino acid
sequence contains a region of about 40 residues from 534 to 577 with
high homology to the C2 regions of PKC (amino acids 211-254 of
rat PKC
) and the two C2 regions of synaptotagmin(29) .
Trotter et al.(29) suggested that this C2 homology
domain represents a phospholipid-binding motif of these proteins. The
present consensus sequence motif of rat PKC
(amino acids
227-238) resides in the C2 homology domain, and the similar
sequence motif is also identified in PSD2 (amino acids 561-572)
but not in synaptotagmins. Since PKC (14, 15) and PSD2 (29) were shown to interact specifically with PS but
synaptotagmin binds nonspecifically to various acidic phospholipids (16) , the consensus sequence between PKC and PSD,
FXFX(I/V/L)(K/R)XX(D/Q)K, is likely to
mediate the specific interaction of these proteins with PS.
The
present study provides the first structural information of polypeptides
responsible for the specific interaction with PS. Since the 14-amino
acid synthetic peptides derived from PS decarboxylase bound effectively
and specifically to PS, the corresponding region may form the substrate
recognition site of the enzyme. The results using the synthetic
peptides suggest that the conserved amino acid residues among PKC and
CHO cell PSD, FXFXLKXXXKXR, which
are recognized by the anti-idiotypic antibody, represent a basic
structural motif for the specific interaction with PS. Computer-aided
similarity searching showed that various cellular proteins have a
homologous sequence motif; typical examples are DNA topoisomerase
II (amino acids 1022-1033)(30, 31) ,
spectrin
(amino acids 1598-1609)(32) ,
calmodulin-sensitive adenylate cyclase (amino acids
148-159)(33) , complement receptor type II (amino acids
381-392)(34) , urinary fatty acid binding protein (amino
acids 73-86)(35) , Dengue virus non-structural protein
NS2A (amino acids 1332-1343)(36) , and RNA polymerase
subunit (amino acids 2108-2119)(37) . Among these
proteins, rat DNA topoisomerase II
isolated from mouse fibroblast
cell line FM3A and spectrin purified from human red blood cells were
recently shown to bind effectively to Id8F7. The binding of Id8F7 to
these proteins was specifically inhibited by the preincubation of the
proteins with PS, suggesting that these proteins have a specific
binding site for PS. (
)Although further investigations are
required to determine the structure and biological significance of the
consensus sequence motif, the motif may represent a putative
PS-specific binding motif commonly present in cellular proteins. The
anti-idiotypic antibody will serve as a useful tool for identifying
PS-binding proteins and locating PS-specific binding sites, which have
been difficult to determine by the conventional biochemical techniques.