From the Applied Biosystems Japan Ltd., 4-5-4 Hacchobori, Chuo-ku, Tokyo 104-0032, § Division of Food
Science and Biotechnology, Graduate School of Agriculture, Kyoto
University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, and
Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho,
Nada-ku, Kobe 657-8501, Japan
Received for publication, January 14, 2003, and in revised form, March 3, 2003
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ABSTRACT |
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Diacylglycerol kinase (DGK) and protein
kinase C (PKC) are two distinct enzyme families associated with
diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B,
and C1C) in the regulatory region. Although most PKC C1 domains
strongly bind phorbol esters, there has been no direct evidence that
DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich
sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGK Diacylglycerol kinase
(DGK)1 and protein kinase C
(PKC) both interact with the second messenger diacylglycerol (DG)
(1, 2). DGK phosphorylates DG to produce phosphatidic acid, whereas PKC
is allosterically activated by DG in the presence of
phosphatidylserine. Therefore, DGK may inhibit the activation of PKC by
attenuating DG levels, contributing to the regulation of intracellular
signal transduction.
To date, nine subtypes of mammalian DGKs have been cloned (3-15). All
DGK isozymes consist of a conserved catalytic domain and two or three
cysteine-rich C1 domains designated as C1A, C1B, and C1C (16). These
isozymes are classified into five classes according to the other
functional domains (Fig. 1). The class I
isozymes (DGK-C1A and DGK
-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGK
-C1A, human-DGK
-C1A, and human-DGK
-C1A gave Kd values of 3.6, 2.8, and 14.6 nM,
respectively, suggesting that DGK
and DGK
are new targets of
phorbol esters. An A12T mutation of human-DGK
-C1A enhanced the
affinity to bind PDBu, indicating that the
-hydroxyl group of
Thr-12 significantly contributes to the binding. The
Kd value for PDBu of FLAG-tagged whole rat-DGK
(4.4 nM) was nearly equal to that of rat-DGK
-C1A (3.6 nM). Moreover, 12-O-tetradecanoylphorbol
13-acetate induced the irreversible translocation of whole rat-DGK
and its C1B deletion mutant, not the C1A deletion mutant, from
the cytoplasm to the plasma membrane of CHO-K1 cells. These results
indicate that 12-O-tetradecanoylphorbol 13-acetate binds to
C1A of DGK
to cause its translocation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, -
, and -
) have calcium binding domains
(EF-hands). The class II isozymes (DGK
and -
) have a pleckstrin
homology domain at the N terminus, and their catalytic region is split into two domains unlike the other DGK isozymes. DGK
has a simple structure and is classified as a class III isozyme. The class IV
isozymes DGK
and
have a myristoylated alanine-rich C kinase substrate homology domain and four ankyrin repeats. DGK
, which has
three C1 domains unlike other DGK and PKC isozymes, is the only isozyme
in class V.
View larger version (32K):
[in a new window]
Fig. 1.
Structure and classification of mammalian DGK
and PKC isozymes. EF-hands and C2,
Ca2+-binding domain; C1A, C1B, C1C, and
C1, cysteine-rich domain (zinc-finger motif); PH,
pleckstrin homology domain; EPH, EPH C-terminal tail
homology domain; MARCKS, sequence homologous to the
myristoylated alanine-rich C kinase substrate phosphorylation site
domain.
The similarity between DGK and PKC isozymes in structure is in the
cysteine-rich C1 domains. Recent investigations using NMR spectroscopy
and x-ray crystallography have revealed the three-dimensional structure
of C1B domains of PKC, PKC
, and PKC
(17-20). Each PKC C1
domain has six conserved cysteines and two histidines in the typical
core structure
HX12CX2CX13-14CX2CX4HX2CX7C
(where X is any amino acid) that coordinates two atoms of
zinc in a tetrahedral geometry (21, 22). We previously synthesized the
50-mer core structure of the C1 domains of all PKC isozymes and showed
that most of the C1 domains of conventional and novel PKC isozymes strongly bind phorbol 12,13-dibutyrate (PDBu) with dissociation constants (Kd) in the nanomolar range (23, 24).
The core structure of DGK C1 domains,
HX10-12CX2-6CX9-19CX2CX4HX2-4CX5-10C,
is slightly different from that of PKC C1 domains, but the six
cysteines and two histidines are precisely conserved. In particular,
DGK-C1A, DGK
-C1A, DGK
-C1A, DGK
-C1B, DGK
-C1A, and
DGK
-C1C have the same core structure as the PKC C1 domains (Fig. 2)
and are deduced to show a significant phorbol ester binding affinity.
However, there have been no reports that DGK isozymes bind phorbol
esters. Ahmed et al. (25) reported that human-DGK
expressed in Escherichia coli or in COS-7 cells did not bind
PDBu. Sakane et al. (26) also detected no specific PDBu
binding in either rat-DGK
or human-DGK
expressed in COS-7 cells.
On the other hand, a recent investigation by Shirai et al.
(27) clearly showed that DGK
expressed in Chinese hamster ovary-K1
(CHO-K1) cells was translocated from the cytoplasm to the plasma
membrane irreversibly following 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. They also suggested that the C1A domain of
DGK
is responsible for this translocation based on the point mutation of each C1 domain. These results prompted us to examine the
phorbol ester binding ability of each DGK
C1 domain, and we have
reported recently (28) that rat-DGK
binds PDBu with high affinity as
a preliminary communication. Further detailed studies on the C1 domains
of several DGK isozymes other than those of rat-DGK
have been
carried out along with the C1A peptide of human-DGK
which Sakane
et al. (26) employed. These additional data have been
compiled to give this full report.
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EXPERIMENTAL PROCEDURES |
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General--
The following spectroscopic and analytical
instruments were used: matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF-MS), Applied Biosystems
Voyager-DETM STR (20 kV); peptide synthesizer,
PioneerTM peptide synthesizer model 9030 (Applied
Biosystems); and HPLC, Waters model 600E with model 2487UV detector.
MALDI-TOF-MS measurements were carried out as follows; each peptide
dissolved in 0.1% trifluoroacetic acid aqueous solution (50 pmol/µl)
was mixed with saturated -cyano-4-hydroxycinnamic acid in 50%
CH3CN containing 0.1% trifluoroacetic acid at a ratio of
1:1. One microliter of the resultant solution was subjected to the
measurement. Angiotensin I and ACTH-(7-38) were used as external
references. HPLC was carried out on a YMC-packed SH-342-5 (ODS, 20 mm
inner diameter × 150 mm) column (Yamamura Chemical Laboratory)
for preparative purposes. [3H]PDBu (17.0 Ci/mmol) was
purchased from PerkinElmer Life Science. COS-7 cells were obtained from
the Riken Cell Bank (Tsukuba, Japan). Unless otherwise noted, reagents
were purchased from Sigma, Allexis, Wako Pure Chemical Industries, or
Nacalai Tesque.
Synthesis of the C1 Peptides of DGK Isozymes-- The 49-52-mer peptides corresponding to the cysteine-rich sequences of DGK isozyme C1 domains (Table I and Fig. 2) were synthesized in a stepwise fashion on 0.1 mmol of preloaded Fmoc-Gly-PEG-PS resin (Applied Biosystems) by PioneerTM using the Fmoc method as reported previously (23, 24). The coupling reaction was carried out using each Fmoc amino acid (0.4 mmol), N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (0.4 mmol) (29), and N,N-diisopropylethylamine (0.8 mmol) in N,N-dimethylformamide for 30 min (flow rate, 30 ml/min). After completion of the chain assembly, each peptide resin was cleaved, and the resultant crude peptide was precipitated by diethyl ether. The crude peptide was purified by gel filtration, followed by HPLC as reported previously (23, 24). Lyophilization gave a corresponding pure C1 peptide, the purity of which was confirmed by HPLC (>98%). Each purified peptide exhibited satisfactory mass spectrometric data. The yields and mass data of the C1 peptides synthesized in this study are summarized in Table I.
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Plasmid Construction for FLAG-tagged Rat-DGK--
The plasmid
for FLAG-tagged DGK
-(1-789) (FLAG-DGK
) was generated by PCR
using BS412 (rat-DGK
) as a template as reported previously (28). The
cDNA of DGK
-(1-789) was digested with MunI and
BamHI and then subcloned into the EcoRI and
BglII sites of pTB701-FL, a mammalian expression vector, to
express fusion protein with the N-terminal FLAG epitope.
Preparation of FLAG-DGK--
Transient transfection into
COS-7 cells was performed by electroporation as reported previously
(28). After transfection, the cells were cultured at 37 °C for
48 h and were harvested with phosphate-buffered saline(
),
followed by centrifugation at 600 × g for 5 min at
4 °C. The cells were resuspended in 300 µl of homogenization
buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 50 mM Tris/HCl, 20 µg/ml leupeptin,
1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100,
pH 7.4) and sonicated on ice. The homogenate was centrifuged at
10,000 × g for 30 min at 4 °C, and the resultant
supernatant was saved for the PDBu binding assay described below.
[3H]PDBu Binding Assay of DGK Isozyme C1 Peptides
and FLAG-DGK--
The PDBu binding assay was carried out using the
procedure of Sharkey and Blumberg (30) with slight modification (23, 24). The standard assay mixture (250 µl) in 1.5-ml Eppendorf tubes
contained 50 mM Tris maleate, pH 7.4, 50 µg/ml
1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phospho-L-serine (phosphatidylserine dioleoyl), 3 mg/ml bovine
-globulin,
[3H]PDBu (17.0 Ci/mmol), and each DGK
C1 peptide or
FLAG-DGK
. For determination of PDBu saturation curves for Scatchard
analysis, concentrations of free [3H]PDBu between 1 and
50 nM were used.
For chemically synthesized DGK C1 peptides, metal coordination was
carried out in a helium-purged distilled water solution (pH 5.5-6.0)
of each C1 peptide. Five mol eq of 10 mM ZnCl2
in helium-purged distilled water was added to the peptide solution, and
the solution (174 µM) was allowed to stand at 4 °C for
10 min. After 10 µl of the peptide solution was diluted with 990 µl
of helium-purged distilled water, the resultant solution (1.5-2.9 µl) was added to the standard assay mixture described above (247.1 µl), and the solution was incubated at 4 °C for 10 min. For
FLAG-DGK, 5 µl of the enzyme solution after homogenization was
similarly added and incubated at 30 °C for 20 min. To the tubes was
added 187 µl of 35% (w/w) poly(ethylene glycol) (average molecular
weight, 8000), and the mixture was vigorously stirred. The tubes were allowed to stand at 4 °C for 10 min and then centrifuged for 10 min
at 12,000 rpm in an Eppendorf microcentrifuge at 4 °C. A 50-µl aliquot of the supernatant of each tube was removed, and its
radioactivity was measured to determine the free [3H]PDBu
concentration. The remainder of the supernatant of each tube was
removed by aspiration. The tips of the tubes were cut off, and the
radioactivity in the pellets was measured to determine the bound
[3H]PDBu. Specific binding represents the difference
between the total and nonspecific binding. The nonspecific binding for
each tube was calculated from its measured free [3H]PDBu
concentration and its partition coefficient to the pellet (about
3%).
In competition experiments using rat-DGK-C1A, various concentrations
of an inhibitor in ethanol solution were added to the reaction mixture
mentioned above. The effective concentration of [3H]PDBu
and rat-DGK
-C1A was 20 and 5 nM, respectively. The final ethanol concentration of the mixture was less than 2%. Binding affinity was evaluated by the concentration required to cause 50%
inhibition of the specific [3H]PDBu binding,
IC50, that was calculated by a computer program (Statistical Analysis System) with a probability unit procedure (31).
The binding constant (Ki) was calculated from the
IC50 values and Kd for PDBu of
rat-DGK
-C1A by the method of Sharkey and Blumberg (30).
Construct of Plasmids Encoding Domain Deleted Mutants of
DGK--
Domain deleted mutants of DGK
were produced using an
ExSite PCR-based Site-directed Mutagenesis kit (Stratagene, La Jolla, CA). The plasmid (BS465) encoding rat-DGK
(27) with an
XhoI site in the 5' terminus and a SmaI site in
the 3' terminus was used as a template. The sense and antisense primers
for producing a C1A-deleted mutant (
C1A-DGK
) were
5'-GTGAAAACATACTCCAAAGCCAAAAGG-3' and 5'-GCGTCCATCCCCCTTGGAG-3',
respectively. The primers for a mutant DGK
lacking the C1B region
(
C1B-DGK
) were 5'-GATGGTGGGGAGCTCAAAGAC-3' and
5'-CTGCATCACCTCACCGCTC-3'. The sequence was confirmed by verifying sequences. Each C1A- or C1B-deleted mutant of DGK
cDNA was
subcloned into SalI and SmaI sites in pEGFPC1.
Observation of Translocation of the GFP Fusion Proteins-- Plasmids (~5.5 µg) were transfected into 1.0 × 106 cells by lipofection using FuGENE 6 transfection reagent (Roche Applied Science), according to the manufacturer's standard protocol. After being cultured at 37 °C for 16-24 h, the cells were spread onto glass bottom culture dishes (MatTek Corp., Ashland, MA). Experiments were performed 16-48 h after the transfection.
The culture medium was replaced with HEPES buffer composed of 135 mM NaCl, 5.4 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, 5 mM HEPES, and 10 mM glucose at pH 7.3 (Ringer's solution). Translocation of the GFP fusion protein was
triggered by a direct application of a 10 times higher concentration of
TPA into the Ringer's solution to obtain the appropriate final
concentration. The fluorescence of the fusion protein was monitored
with a confocal laser scanning fluorescent microscope (LSM 410 invert,
Carl Zeiss, Jena, Germany) at 488 nm argon excitation using a 515-535
nm bandpass barrier filter. All experiments were performed at
37 °C.
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RESULTS |
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Synthesis, Folding, and PDBu Binding of C1 Peptides of DGK
Isozymes--
DGK isozymes have cysteine-rich C1 domains quite similar
to those of PKC isozymes (1, 2). The zinc finger-like sequences of the
C1 domains of conventional and novel PKC isozymes specifically bind
phorbol esters (16, 23, 24). Although DGK binds zinc like PKC isozymes
(25), there has been no direct evidence that phorbol esters bind to
DGK. We carefully analyzed the cysteine-rich sequences of the C1
domains of all DGK isozymes on the basis of the 20 residues (Fig.
2) critical to PDBu binding (16, 24, 32).
DGK-C1A, DGK
-C1A, DGK
-C1A, DGK
-C1B, DGK
-C1A, and
DGK
-C1C have the same core structure as the PKC C1 domains, where
six cysteines and two histidines are conserved in the pattern
HX12CX2CX13-14CX2CX4HX2CX7C. Table II summarizes the
percentages of sequence homology of all DGK C1 domains to the 20 residues. Human-DGK
-C1A, rat-DGK
-C1A, and human-DGK
-C1A have
perfect sequence homology to all conventional and novel PKC isozyme C1
domains. The C1 domains, which show more than 50% sequence homology,
are rat-DGK
-C1A, human-DGK
-C1A, rat-DGK
-C1A, human-DGK
-C1A,
human-DGK
-C1A, human-DGK
-C1B, hamster-DGK
-C1A,
hamster-DGK
-C1B, human-DGK
-C1A, and human-DGK
-C1C that might
bind PDBu. We have thus synthesized these cysteine-rich sequences of
~50 amino acid residues with a PioneerTM peptide
synthesizer using HATU as an activator for Fmoc chemistry by a method
reported previously (23) (Table I). To prevent racemization and
oxidation during the synthesis, the C terminus of each peptide was
extended from the final cysteine to a glycine. These peptides exhibited
satisfactory mass spectrometric data (Table I), and their purity was
confirmed by HPLC analysis (>98%).
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These peptides were folded using zinc chloride by a method reported
previously (23, 24) and subjected to a PDBu binding assay. Because our
recent investigation found that some C1 domain fragments of PKC
isozymes suffered from temperature-dependent inactivation
(24, 33), the incubation temperature of the binding assay was set at
4 °C for the DGK isozyme C1 peptides. The specific binding to PDBu
of the DGK C1 peptides is summarized in Fig.
3. Only the DGK-C1A and DGK
-C1A
peptides showed significant binding at 20 nM, whereas other
DGK C1 peptides with over 50% sequence homology to PKC C1 domains were
completely inactive even at 100 and 500 nM (data not
shown). Rat-DGK
-C1A and human-DGK
-C1A showed quite similar PDBu
binding although their sequences were different. In a control
experiment, rat-DGK
-C1B did not show any binding.
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Scatchard analyses of rat-DGK-C1A and human-DGK
-C1A for PDBu
binding gave Kd values of 3.6 ± 0.5 and
2.8 ± 0.1 nM with Bmax values
of 31.2 ± 4.5 and 44.1 ± 7.8%, respectively (Fig.
4, a and b). These
values are close to those of most PKC C1 peptides (0.45-7.4
nM) (24), suggesting that whole DGK
shows strong PDBu
binding affinity. The binding affinity of human-DGK
-C1A was also
great (Kd = 14.6 ± 1.7 nM;
Bmax = 42.9 ± 1.3%). However, the
Kd of rat-DGK
-C1A (202 ± 13 nM)
with a Bmax value of 8.3 ± 1.0% was
considerably larger than that of human-DGK
-C1A (Fig. 4, c
and d).
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The weak PDBu binding affinity of human-DGK-C1A
(Kd = 14.6 nM) compared with
human-DGK
-C1A (Kd = 2.8 nM) suggests
that the Ala-12 residue is responsible for the decrease in binding
affinity because the Thr-12 residue is strictly conserved in the
conventional and novel PKC isozymes that show strong PDBu binding (24).
The Thr-12 residue along with the Leu-21 and Gly-23 residues of
PKC
-C1B is shown to be involved in the phorbol ester binding in the
x-ray structure (20). To prove this, the A12T mutant of
human-DGK
-C1A was synthesized, and its PDBu binding affinity was
tested. The Kd value for PDBu of the A12T mutant was
4.9 ± 1.0 nM with a Bmax value
of 42.4 ± 2.8% (Fig. 4e), suggesting that the Thr-12
residue plays a significant role in the PDBu binding of DGK
-C1A.
PDBu Binding Affinity of Whole DGK--
FLAG-tagged whole
rat-DGK
(FLAG-DGK
) was expressed in COS-7 cells to investigate
whether whole rat-DGK
as well as rat-DGK
-C1A peptide binds PDBu.
After homogenization and centrifugation of the COS-7 cells, the
supernatant was saved for PDBu binding assay. Scatchard analysis of
FLAG-DGK
in the PDBu binding was performed with the incubation
temperature set at 30 °C as for whole PKC isozymes (24, 34). The
FLAG-DGK
solution (5 µl) showed remarkable specific PDBu binding
(24,600 ± 790 dpm) when 20 nM [3H]PDBu
and 50 µg/ml phosphatidylserine dioleoyl were employed. The
corresponding background binding because of endogenous PKC isozymes was
3320 ± 560 dpm. These data indicate that the background binding
does not significantly affect the Kd value of FLAG-DGK
. The Kd value for PDBu binding of
FLAG-DGK
was 4.4 ± 0.3 nM as shown in Fig.
4f. This is in good agreement with that of rat-DGK
-C1A
(3.6 nM), indicating that DGK
is a new receptor of
tumor-promoting phorbol esters.
Affinity of PKC Activators Other than PDBu to Bind
DGK-C1A--
The binding affinity of PKC activators other than PDBu
for rat-DGK
-C1A was evaluated by inhibition of the specific binding of [3H]PDBu. Dose-response curves were plotted for each
compound, and the concentration at which 50% of the specific
[3H]PDBu binding was inhibited (IC50) was
calculated. The binding constant (Ki) was calculated
from the Kd for PDBu of rat-DGK
-C1A by the method
of Sharkey and Blumberg (30). We determined the Ki
values of OAG, bryostatin 1, IL-V, and Ing-3-Bz. OAG is a
membrane-permeable analogue of natural diacylglycerols without tumor
promoting activity (35). Bryostatin 1 is a macrocyclic lactone with
potent antileukemic activity (36). IL-V (37-39) and Ing-3-Bz (40) are
naturally occurring tumor promoters. Table
III summarizes the binding data for these
compounds. The Ki values of PKC
and PKC
C1
peptides were also determined as references. Rat-DGK
-C1A as well as
PKC C1 peptides bound significantly OAG, bryostatin 1, IL-V, and
Ing-3-Bz. The ability of each ligand to bind rat-DGK
-C1A was similar
to that to bind PKC C1 peptides. It is noteworthy that DG can bind to rat-DGK
-C1A. This is the first evidence that DG specifically binds
to the C1 domain of DGK isozymes.
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Translocation of Rat-DGK to the Plasma Membrane with TPA
Treatment--
Because TPA induces an irreversible translocation of
conventional and novel PKC isozymes by binding to the C1 domains (41, 42), we examined the TPA-induced translocation of rat-DGK
with green
fluorescent protein (GFP-DGK
) in CHO-K1 cells. To clarify the role
of each C1 domain in the translocation, C1A or C1B deletion mutants of
DGK
fused with GFP (GFP-
C1A-DGK
and GFP-
C1B-DGK
) were
also analyzed. As shown in Fig. 5, the
fluorescence of GFP-DGK
, GFP-
C1A-DGK
, and GFP-
C1B-DGK
was observed throughout the cytoplasm and in the nucleus. Treatment
with 1 µM TPA induced an obvious translocation of
GFP-DGK
and GFP-
C1B-DGK
from the cytoplasm to the plasma
membrane (Fig. 5, top and bottom panels). This
translocation began within 30 s and remained for at least 60 min
after the TPA treatment. In contrast, GFP-
C1A-DGK
did not respond
to TPA (Fig. 5, middle panel).
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DISCUSSION |
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It has long been believed that the cysteine-rich C1 domains in DGK
isozymes do not bind phorbol esters (1, 2). However, there was
circumstantial evidence that DGK binds phorbol esters; TPA induced
the translocation of DGK
from the cytoplasm to the plasma membrane
of CHO-K1 cells (27). We have recently synthesized the cysteine-rich
sequences of all conventional and novel PKC isozyme C1 domains by the
solid-phase Fmoc strategy in high purity (23). These peptides folded by
zinc chloride showed strong PDBu binding in the presence of
phosphatidylserine with Kd values in the nanomolar
range comparable with the respective whole PKC isozymes (24). We
adopted this method to prove the phorbol ester binding ability of DGK
isozymes. Among the DGK C1 peptides with good sequence homology to the
PKC C1 domains, the DGK
-C1A peptides bound most strongly PDBu. There
was no significant difference in PDBu binding affinity between
rat-DGK
-C1A and human-DGK
-C1A (Fig. 4) although 6 amino acid
residues differ between them (Fig. 2). This remarkable binding was also
proved by using whole rat-DGK
; the Kd value for
PDBu binding of rat-DGK
-C1A (3.6 nM) was almost equal to
that of whole rat-DGK
(4.4 nM). This is the first
unambiguous evidence that DGK
is one of the specific receptors of
phorbol esters. The lack of PDBu binding by rat-DGK
-C1B clearly showed that the major binding site in whole rat-DGK
is the C1A domain. The TPA-induced translocation experiment using the C1A or C1B
deletion mutants of rat-DGK
also indicated that TPA binds directly
to the C1A domain of DGK
to cause its translocation from the
cytoplasm to the plasma membrane of CHO-K1 cells as observed for PKC
isozymes (41, 42). These results suggest that DGK
as well as PKC
isozymes should be taken into account to interpret the biological
phenomena evoked by phorbol esters. Furthermore, popular PKC activators
other than phorbol esters (bryostatin 1, IL-V, and Ing-3-Bz) bound to
rat-DGK
-C1A like to PKC isozymes (Table III), suggesting that DGK
also serves as the receptor of these PKC activators.
Contrary to our results, Sakane et al. (26) did not detect
specific PDBu binding in whole human-DGK. In experiments using a
whole enzyme, there are several factors to attenuate the binding potency as follows: instability or low concentration of the enzyme, problem with the folding, and so on. The contribution of the molecular chaperon would be large because rat-DGK
expressed in the E. coli system did not show any detectable PDBu binding (data not
shown). Moreover, the quality of the commercially available
phosphatidylserine might be one of the reasons for the discrepancy
between Sakane et al. (26) and our group. We have found
recently that a synthetic phosphatidylserine such as phosphatidylserine
dioleoyl (Sigma) gives more reproducible PDBu binding data than natural
phosphatidylserine derived from the bovine
brain.2 We experienced that
some lots of natural phosphatidylserine did not work at all as a
cofactor in the PDBu binding assay using whole PKC isozymes. Long
storage or long purification process of natural phosphatidylserine
would result in oxidation to produce reactive oxygen species, which
might have oxidized the cysteine-rich sequences of the DGK
isozyme C1 domains to abolish their PDBu binding ability.
According to the sequence analysis, the DGK-C1A peptides as
well as the DGK
-C1A peptides have the highest sequence homology to
the PKC isozyme C1 peptides (Table II): 95% homology for
rat-DGK
-C1A and 100% homology for human-DGK
-C1A. Both DGK
-C1A
peptides showed significant PDBu binding as expected (Fig. 3). However,
the Kd values for rat-DGK
-C1A and
human-DGK
-C1A were considerably different. Human-DGK
-C1A showed
potent PDBu binding affinity (Kd = 14.6 nM), whereas the binding affinity of rat-DGK
-C1A was
very weak (Kd = 202 nM). The
Val-44 deletion in the rat-DGK
-C1A sequence is deduced to be
responsible for this weak binding affinity because the C-terminal
cysteine residue is one of the zinc coordination sites (20-22). After
the completion of this study, we reexamined the sequence of
rat-DGK
-C1A and found that rat-DGK
-C1A as well as
human-DGK
-C1A have a valine residue at position
44.3 These results indicate
that both rat-DGK
and human-DGK
are targets of tumor-promoting
phorbol esters. The Val-44 deletion in rat-DGK
proved to be reported
accidentally.4
The Kd value of human-DGK-C1A was ~5
times larger than that of human- and rat-DGK
-C1A peptides (Fig. 4),
suggesting that there are additional important residues other than the
20 residues (Fig. 2) that play a significant role in the PDBu binding. After careful comparison of the sequence of DGK
-C1A with DGK
-C1A, we focused on the Ala-12 residue which might decrease the PDBu binding
affinity of the DGK
-C1A peptides. The Thr-12 residue of PKC
-C1B
was deduced to contribute to the hydrogen bonding with phorbol
13-acetate in an x-ray analysis (20). Molecular modeling and
site-directed mutagenesis studies also suggest the involvement of the
-hydroxyl group of the Thr-12 residue in phorbol ester binding (43).
Moreover, the Thr-12 residue is strictly conserved in the conventional
and novel PKC isozymes that have potent PDBu binding ability (24).
These considerations led us to synthesize the A12T mutant of
human-DGK
-C1A. This mutant peptide showed a Kd
value for PDBu quite similar to that of the DGK
-C1A peptides (Fig.
4), indicating that the
-hydroxyl group of Thr-12 is involved in the
hydrogen bonding between DGK
-C1A and PDBu.
The potent PDBu binding ability of the DGK-C1A peptide
suggests that DGK
is also a new receptor of tumor-promoting phorbol esters. In fact, Caricasole et al. (15) have reported
recently that human-DGK
was redistributed from the cytoplasm to the
plasma membrane of HEK-293 cells upon TPA treatment. However, we could not confirm this translocation in our assay system using CHO-K1 cells
because almost all GFP-DGK
exists in the plasma membrane and not in
the cytoplasm (data not shown). Further investigations are necessary to
prove that DGK
is a receptor of phorbol esters.
DGK C1 peptides other than DGK-C1A and DGK
-C1A did not show any
significant PDBu binding (Fig. 3). What then is the intrinsic role of
the C1 domains of DGK isozymes? They might bind DG by analogy to PKC
isozymes. However, DG binding cannot be demonstrated directly using
radioisotope-labeled DG because DG is highly hydrophobic like TPA whose
specific binding to PKC isozymes cannot be proved by the usual method
(44). The sequence homology of the DGK C1B and C1C domains to the PKC
C1A and C1B domains is considerably lower than that of the DGK C1A
domains, but the DGK-C1B and C1C domains have the conserved sequence of
15 amino acid residues at their C terminus which is characteristic to
DGK isozymes. Because of this sequence, DGK-C1B and C1C domains are
called "extended cysteine-rich domains" (1, 2), and must be closely
related to the function of DGK isozymes. It has been shown recently (2, 45) that the extended cysteine-rich sequence of DGK
-C1C is necessary
for DGK activity. If DG binds to the C1B or C1C domains of DGK
isozymes, these C1 domains might assist DG to bind to the catalytic
domain for phosphorylation. To verify this hypothesis, it is
indispensable to develop a new ligand with moderate hydrophobicity that
shows strong affinity to bind these extended cysteine-rich domains.
Although OAG bound to the C1A domain of DGK
-C1A (Table III), this
binding would be related to the translocation of DGK
rather than to
the phosphorylation of DG.
Overall, we have identified DGK and DGK
as novel phorbol
ester-binding sites by the chemical synthesis of the cysteine-rich sequences of DGK isozyme C1 domains. These C1 peptide receptors obtained in high yield and high purity gave more reproducible binding
results than the whole enzymes prepared by DNA recombination techniques. The synthetic PKC C1 homology domain peptides are now
established as an effective screen for identifying new phorbol ester
receptors. They are also useful for identifying selective binding
agents for individual PKC and DGK isozymes.
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FOOTNOTES |
---|
* This work was supported in part by a Grant-in-aid for Scientific Research on Priority Areas (A) (2) 12045241 and 13024245 from the Ministry of Education, Science, Culture, and Sports of Japan (to K. I.) and a grant from Takeda Science Foundation (to K. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-6282; Fax: 81-75-753-6284; E-mail: irie@kais.kyoto-u.ac.jp.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M300400200
2 M. Shindo, K. Irie, A. Masuda, H. Ohigashi, Y. Shirai, K. Miyasaka, and N. Saito, unpublished results.
3 N. Saito, unpublished results.
4 K. Goto, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
DGK, diacylglycerol
kinase;
CHO, Chinese hamster ovary;
DG, diacylglycerol;
GFP, green
fluorescent protein;
HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium
hexafluorophosphate N-oxide;
IL-V, ()-indolactam-V;
Ing-3-Bz, ingenol 3-benzoate;
MALDI-TOF-MS, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry;
OAG, 1-oleoyl-2-acetyl-sn-glycerol;
PDBu, phorbol
12,13-dibutyrate;
phosphatidylserine dioleoyl, 1,2-di-(cis-9-octadecenoyl)-sn-glycero-3-phospho-L-serine;
PKC, protein kinase C;
TPA, 12-O-tetradecanoylphorbol
13-acetate;
HPLC, high pressure liquid chromatography;
Fmoc, N-(9-fluorenyl)methoxycarbonyl.
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