(Received for publication, October 1, 1996, and in revised form, January 14, 1997)
From the A cDNA encoded a 462-amino acid protein,
which showed CDP-diacylglycerol synthase (CDS) activity was cloned for
the first time as the vertebrate enzyme molecule from rat brain
cDNA library. The deduced molecular mass of this rat CDS was 53 kDa, and putative primary structure included several possible membrane-
spanning regions. At the amino acid sequence level, rat CDS shared
55.5%, 31.7%, and 20.9% identity with already known
Drosophila, Saccharomyces cerevisiae, and
Escherichia coli CDS, respectively. This rat CDS preferred
1-stearoyl-2-arachidonoyl phosphatidic acid as a substrate, and its
activity was strongly inhibited by phosphatidylglycerol 4,5-bisphosphate. By immunoblotting analysis of COS cells overexpressed with the epitope-tagged for rat CDS, a 60-kDa band was detected. By
epitope-tag immunocytochemistry, the CDS protein was mainly localized
in close association with the membrane of the endoplasmic reticulum of
the transfected cells. The intense mRNA expression of CDS was
localized in the cerebellar Purkinje cells, the pineal body, and the
inner segment of photoreceptor cells. Additionally, very intense
expression was detected in postmitotic spermatocytes and
spermatids.
Phosphoinositide cycle mediates one of the intracellular signal
transduction pathways in eukaryotic cells and produces a class of
second messengers that are involved in cell growth (1, 2), differentiation (3, 4), the action of hormones and neurotransmitters (5), and sensory perception (6-8). Triggering of the cell surface
receptors, such as G-protein-coupled receptors and tyrosine kinase
receptors, initiates the cycle by activating phospholipase C
(PLC),1 resulting in the hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PIP2) into two
second messengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate
(IP3). DAG is subsequently phosphorylated by DAG kinase to
synthesize phosphatidic acid (PA), a presumed novel messenger (9-11).
In this cycle, CDP-diacylglycerol synthase (CDS) (CTP-phosphatidate
cytidylyltransferase) catalyzes the converting process from PA to
CDP-DAG, the precursor to phosphatidylinositol (PI), which is
phosphorylated to synthesize PIP2 eventually.
The importance of CDS in the signal transduction has been strengthened
in a recent study by Wu et al. (12), showing that overexpression of a photoreceptor cell-specific isoform of CDS in
Drosophila increases the amplitude of the light response.
Its mutants (cds mutants) cannot sustain a light-activated
current and undergo light-dependent retinal degeneration,
which can be suppressed by a mutation in PLC. The difference in
phototransduction mechanism between vertebrate and invertebrate is well
known, such as the primary second messenger role of cGMP in vertebrate
photoreceptors versus that of IP3 in
invertebrate. However, recent investigations have shown unique
distribution of phosphoinositide-specific PLC (13-15), protein kinase
C (16, 17), and IP3 receptor (18) in vertebrate rod outer
segment, and that light may enhance the activity of PLC (19-22) and
protein kinase C (23), and phosphoinositide synthesis (24). In
addition, it has been reported that cytoplasmic Ca2+
concentration mediates light adaptation in vertebrate photoreceptors (25, 26). Thus it is suggested that in vertebrates as well as
invertebrates the phosphoinositide cycle may play a role in the
phototransduction signaling. It is therefore possible that CDS is also
important in the signal transduction mechanism of vertebrate retina and
neural cells.
As a step toward understanding the possible functional significance of
this enzyme in vertebrate cellular signaling, the present study was
attempted to perform molecular cloning of a CDS molecule from rat brain
and to clarify its enzymatic feature and tissue and cell localization.
Our result shows that a newly identified CDS prefers
arachidonate-containing PA as a substrate, suggesting strongly a role
for CDS in the phosphoinositide synthesis. It is also shown that
the activity of CDS is inhibited by PIP2, suggesting that
polyphosphoinositides regulate their own synthesis through CDS
activity. In addition, we show the detailed localization at mRNA
level in the retina and brain, but unexpectedly the highest expression
of its mRNA is detected in the testis.
Total RNA was extracted
from adult Wistar rat brain by acid guanidinium
thiocyanate-phenol-chloroform extraction (27). Poly(A)+ RNA
was isolated by chromatography on an oligo(dT)-cellulose column.
First-strand cDNA was prepared using First-Strand cDNA Synthesis kit (Pharmacia Biotech Inc.).
Primers used for PCR were composed of two degenerate oligonucleotides,
which were made according to the amino acid sequences of conserved
regions between Drosophila CDS (12) and E. coli CDS (28): the regions corresponded to the amino acid sequences TW(E/Q)GFIGG (aa 276-283 for 5 A cDNA library of adult rat brain on
postnatal day 49 was constructed as described previously (29). Clones
(4 × 106) derived from the cDNA library were
screened by hybridization in the presence of 50% formamide at 42 °C
with the subcloned PCR fragments labeled with [32P]dCTP.
Washing conditions were carried out in 0.1 × SSC (SSC: 0.15 M NaCl, 0.015 M trisodium citrate), 0.1%
sodium dodecyl sulfate (SDS) at 42 °C. Among five clones isolated,
one clone (pCDS4) containing the longest cDNA insert (3.5 kb) was
selected for further sequence analysis on both strands as described
above.
A full-length cDNA (pCDS4)
for the newly cloned molecule was subcloned into the expression vector,
pSRE (pcDL-SR An epitope-tag composed of eight amino acids
(FLAG marker peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; Eastman Kodak
Corp.) was fused to the newly identified molecule by cloning the 24 base pairs of FLAG coding sequence up- or downstream to the coding region of the novel cDNA. The FLAG epitope-tagged molecules were expressed in COS-7 cells using the expression vector, pSRE, by the
DEAE-dextran method (30-32). Total lysate of the overexpressed COS-7
cells was prepared as described above. Total lysate boiled for 5 min in
Laemmli's sample buffer was subjected to 10% SDS-polyacrylamide gel
electrophoresis (35). The separated proteins were then
electrophoretically transferred to a nitrocellulose membrane (0.45-µm
pore size). After blocking the nonspecific binding sites in the 5%
skim milk (w/v) in phosphate-buffered saline, 0.01% Tween 20, the
membrane was incubated for 1 h at room temperature with antibody
against FLAG and then treated with peroxidase-conjugated anti-mouse IgG antibody for 1 h. The immunoreactive bands were detected using a
chemiluminescence detection kit (ECL Western blotting detection kit,
Amersham).
Transfected cells were fixed with 4% paraformaldehyde, 0.1 M sodium phosphate buffer (pH 7.2), 0.2% Triton X-100, or
fixed with the same fixative without the detergent and freeze-thawed after immersion in 40% sucrose. The cells were incubated with the
anti-FLAG antibody (Kodak) and sites of the antigen-antibody reaction
were visualized using the avidin-biotinylated peroxidase complex (ABC)
system (Vector Laboratories) with 3,3 Total RNAs were extracted from
adult rat brains, eyeballs, and several other tissues as described
above. Each of the total RNA samples (30 µg/lane) was denatured with
formamide and size-separated by formaline/agarose gel electrophoresis.
The RNAs were transferred and fixed to nylon membranes (Nytran,
Schleicher & Schuell) and hybridized with a probe corresponding to the
3 Fresh frozen blocks of
adult rat brains, eyeballs, and testes were sectioned at 10 or 25 µm
thickness on a cryostat. The sections were mounted on silane-coated
glass slides, fixed with 4% paraformaldehyde, 0.1 M sodium
phosphate buffer (pH 7.2), pretreated and hybridized as described
previously (29). Two kinds of probes were used in the experiment: a
cDNA probe corresponding to nucleotides 526-1311 labeled with
[ The nucleotide and deduced amino acid sequences of the composite
CDS cDNA are presented in Fig. 1. The putative
initiation codon was preceded by in-frame stop codons. The predicted
open reading frame encoded a protein of 462 amino acids with the
deduced molecular mass of 53 kDa. The deduced amino acid sequence of
this rat CDS shared 55.5%, 31.7% and 20.9% identity to that of
Drosophila (12), Saccharomyces cerevisiae (36),
and Escherichia coli (28), respectively (Fig.
2a). Near the carboxyl terminus, domains sharing more than 80% identity to each other were contained in all
molecules. Several protein kinase-recognition sequence motifs were
identified such as those recognized by myosin-I heavy chain kinase (aa
345-348), multifunctional
Ca2+/calmodulin-dependent protein kinase II (aa
52-57), cGMP-dependent protein kinase (aa 312-318),
proline-dependent protein kinase (aa 265-268 and
271-274), casein kinase I (aa 76-80), and glycogen synthase kinase 3 (aa 312-318) (37). Hydrophobicity analysis by the method of Kyte and
Doolittle (38) revealed that this novel molecule, like the other three
CDSs, was very hydrophobic and appeared to contain several possible
membrane-spanning regions, as judged by the fact that at least 19 sequential residues have an average hydrophilic value of greater than
+1.6 (Fig. 2b). The amino terminus of this molecule was
hydrophilic, similar to those of CDSs of Drosophila and
S. cerevisiae.
By measurement of the CDS activity of this novel molecule, the total
lysate from COS-7 cells transfected with the full length of the present
cDNA showed about 5-7 times higher activity toward PAs than the
control total lysate (e.g. 184 ± 9.3; 28 ± 1.5 pmol/min/mg total protein under the assay condition at 200 µM C18:0/C20:4-PA). The enzymatic activity of this
molecule was the highest toward 1-stearoyl-2-arachidonoyl-PA
(C18:0/20:4-PA) among the others, whereas little or no activity was
detected toward PAs containing saturated fatty acyl groups in both of
the sn-1 and -2 positions (di-C12:0-PA, di-C18:0-PA) (Fig.
3). From double-reciprocal (Lineweaver-Burk) plots,
values for the apparent Km toward C18:0/C20:4-PA, di-C18:1-PA, and PA from egg yolk lecithin were 102, 114, and 138 µM, respectively; and for the apparent
Vmax were 268, 259, and 198 pmol/min/mg total
protein, respectively. Values for apparent Vmax/Km (specificity
constant) calculated from Eadie-Hofstee plots were 2.51, 2.28, and
1.48, respectively. When the utilization of CTP and dCTP for the
activation of phospholipid intermediates was compared, dCTP was
incorporated into phospholipid precursors at almost the same rate as
CTP. In the analysis of inhibitory effects of PI, PIP, and
PIP2 on the CDS activity of the present molecule, the
activity decreased by 50% at 2 mol% PIP2 and by 80% at
10 mol% and more (Fig. 4), under the assay condition at 1000 µM C18:0/C20:4-PA (the present molecule showed the
maximum activity at 200-1000 µM C18:0/C20:4-PA). In
contrast, the activity decreased by at most 40% at 5-20 mol% PIP.
Addition of PI at various concentrations from 2 to 20 mol% produced
little effects on CDS activity. Similar results were obtained in the
experiments for C18:0/C20:4-PA and di-C18:1-PA at 100 or 500 µM (data not shown).
In the immunoblotting of epitope (FLAG)-tagged CDS with the antibody
against the FLAG tag, a single immunoreaction band was observed at size
of 60 kDa (Fig. 5).
In the immunocytochemistry (Fig. 6a) of the
transfected COS-7 cells with the antibody against FLAG tag,
immunoreactive cells accounted for approximately 20-40% of the total
cell population and they were randomly dispersed in each culture dish.
The immnoreactivity was localized widely throughout the cells in forms
of delicate networks composed of fine dots with a tendency of
condensation in one pole of juxtanuclear cytoplasm, and they were also
deposited along the nuclear rim. In immunoelectron microscopy (Fig.
6b), the immunoreactivity was detected along the membranes
of endoplasmic reticulum, vesicles and vacuoles, and nuclear envelopes.
No immunoreactivity was detected in any cells when the transfection was
made of the cDNA without the FLAG tag.
In Northern blot analysis (Fig. 7) of adult rat on
postnatal day 49, the hybridization bands were detected very strongly
in testis at sizes of 3.5 and 2.2 kb, suggesting a possibility of alternative splicing. The hybridization band was strongly detected in
eyeball and brain at a size of 3.5 kb. Among other tissues, a weak band
of 3.5 kb was detected in kidney, small intestine, and placenta,
whereas a faint band of 3.5 kb was seen in thymus and lung after long
exposure. Any hybridization bands could not be detected in heart,
liver, and ovary.
By in situ hybridization histochemical analysis of the adult
brain (Fig. 8), the most intense expression
signals were detected in the cerebellar Purkinje cells and intense
expression was found in the pineal body. Moderate to low expression was
seen in layers II-VI of the cerebral cortex, in the hippocampal
pyramidal cell layer and subiculum, and the olfactory mitral cells. Low
expression was seen in almost all neurons in the fore-, mid- and
hindbrains with much lower expression in the caudate putamen. Low
expression was also seen in the choroid plexuses. No significant
expression was detected in the white matter including the corpus
callosum and cerebellar medulla or in the glia limitans. In the retina, positive expression signals were confined to the inner segment of the
photoreceptor cells (Fig. 9).
In the adult testis, the hybridization signals were densely
deposited in most of the adluminal compartment of the seminiferous tubule composed of postmitotic spermatocytes and spermatids, while no
significant signals were detected in the apical compartment composed of
spermatozoa or in the basal compartment composed of proliferating
spermatogonia. No significant signals were found in the interstitial
cell clusters (Fig. 10).
In the control experiment in which several random sections of brain,
retina, and testis were hybridized with the labeled oligonucleotide probe in the presence of 100-fold excess amounts of the unlabeled probe, no significant expression signals were detected in any regions
of the tissue sections.
We report here for the first time the molecular cloning of
vertebrate CDS and clarify its primary structure, enzymatic features, and localization in detail. The primary structure of this rat CDS
shared 55.5%, 31.7%, and 20.9% identity to that of
Drosophila (12), S. cerevisiae (36), and E. coli (28), respectively (Fig. 2a). The high
hydrophobicity and the inclusion of several putative membrane-spanning
regions in the primary structure of the present molecule and the
localization associated with the endoplasmic reticulum and nuclear
envelopes are well consistent with available biochemical data of the
mammalian CDS so far reported (34, 39-41). The deduced molecular size
of 53 kDa is smaller than the size of 60 kDa detected by epitope-tagged
immunoblotting. This discrepancy may be ascribed to the
posttranslational modification of CDS in vivo. From the
sequence, it is clear that there are several potential serine/threonine
phosphorylation sites, suggesting the possibility that phosphorylation
by serine/threonine kinases may be involved in regulation of this
enzyme activity.
The present study shows the substrate specificity of rat CDS toward
1-stearoyl-2-arachidonoyl PA (C18:0/C20:4-PA) among several PAs (Fig.
3). In mammalian cells, CDP-DAG is used to produce
phosphatidylinositol, phosphatidylglycerol, and cardiolipin, and
CDP-DAG is therefore an important regulatory branching point in the
phospholipid metabolism. Since the PI predominantly consist of the
1-stearoyl-2-arachidonoyl species (42), the present substrate
specificity of rat CDS suggests strongly that this enzyme molecule
selectively participates in the phosphoinositide cycle, but not in the
synthesis of phosphatidylglycerol and cardiolipin.
The present study also clarifies the marked inhibition of rat CDS
activity in vitro by PIP2 (Fig. 4), a presumed
end product of the phosphoinositide cycle. This represents another new
example of the potential for acidic phospholipids including
phosphoinositides to modulate the in vitro activities of
several membrane enzymes related to the signal transduction.
PI-4-phosphate 5-kinase appears to be inhibited by PIP2 and
be activated by PA (43-45), casein kinase I appears to be inhibited by
PIP2 (46), phosphoinositide-specific PLC- The PIP2-induced inhibition has recently been demonstrated
to occur on arachidonoyl-DAG kinase, which catalyzes the synthesis of
PA from sn-1-acyl-2-arachidonoyl-DAG selectively by
phosphorylation (51). Considering the fact that DAG kinase and CDS are
sequentially involved in the phosphoinositide resynthesis, the finding
that both arachidonoyl-specific DAG and CDS are inhibited by
PIP2 strongly suggests that PIP2 is synthesized
on demand as opposed to being stored, and the conversion process from
DAG to CDP-DAG through PA, the initial steps of resynthesis of
phosphoinositides, is tightly regulated in the
phosphoinositide-mediated signaling cascades. In addition, similar to
the CDS molecule, the arachidonoyl-DAG kinase has been shown to be
expressed also highly in the testis as well as the brain and to be an
integral membrane protein (52). It is thus suggested that these two
arachidonoyl-specific enzyme molecules are located in the same
subcellular membranes and are regulated by the lipid microenvironment
within the membranes in a paired fashion in the two consecutive
reactions of the phosphoinositide cycle. The detailed cellular and
subcellular localization of the arachidonoyl-DAG kinase in the testis
as well as the brain and retina, and its comparison with that of this
CDS may be informative in this regard. The functional significance of
the most intensive expression of rat CDS mRNA in testis, especially
in postmitotic spermatocytes and spermatids remains to be
elucidated.
The localization of this CDS mRNA in the inner segment of rat
photoreceptor cells (Fig. 9) corresponds well to the presence of the
Drosophila homologue in the photoreceptor cells, suggesting some similar functional significance between the two homologous molecules of far remote animal species. As cited in the Introduction, it has clearly been demonstrated that phosphoinositide-specific PLC
isoforms cloned from mammalian retina are distributed in the photoreceptor cells (13-15) and that light induces the decrease in
PIP2 and increase in IP3 in the photoreceptor
cells (19-22), and light also enhanced synthesis of PI by activation
of phosphoinositide cycle in these cells (24). The existence of
IP3 receptor in rod outer segment has also been shown (18),
and Ca2+-mediated light adaptation has been demonstrated in
vertebrate photoreceptor cells (25, 26). In addition, protein kinase C,
an enzyme activated by DAG, is reportedly present in the rod outer
segment (16, 17), and its activity appears to be stimulated by light
(23). Protein kinase C is known to phosphorylate several rod outer
segment proteins, including rhodopsin (53) and phosphodiesterase The molecular identification of the first mammalian CDS completed by
the present study would accelerate further elucidation of the
functional significance of this enzyme in the phosphoinositide-related signal pathway.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000517[GenBank]. We thank Dr. Hiroshi Munakata (Department of
Biochemistry, Tohoku University School of Medicine) for helpful advice
and suggestions for this work.
Department of Anatomy,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
Polymerase Chain Reaction (PCR)
primer) and IPGHGGI(M/L) (aa 391-398 for 3
primer) (amino acid numbers represent those of
Drosophila CDS). The sequences for the primers were designed
according to the mammalian codon usage:
GGAATTCAC(C/A)TGG(G/C)A(G/A)GG(C/A)TT(C/T)AT(C/T)GG(C/A/G)GG for 5
primer and
CGGGATCCA(T/G)(G/A)AT(G/ T)CC(G/T)CC(G/A)TG(G/T)CC(G/A)GG(G/A)AT for 3
primer. The 5
ends of 5
and 3
primers were designed to
contain an EcoRI site and a BamHI site,
respectively, for subsequent cleavage of subcloned cDNA fragments.
PCR amplification was performed by using the first-strand cDNA and
Ampli-Taq DNA polymerase according to following schedule:
94 °C for 30 s, 55 °C for 45 s, and 72 °C for 2 min,
for 30 cycles, followed by further incubation at 72 °C for 7 min.
Subcloned cDNA fragments were sequenced by the model 373 DNA
autosequencer (Applied Biosystems).
296, Refs. 30 and 31). The constructs or vector alone
as a control was transfected into COS-7 cells by DEAE-dextran method
(32). After incubation for 3 days in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum, transfected cells were
harvested and lysed by sonication in lysis buffer (31). After removal
of undisrupted cells by centrifugation (550 × g, 10 min, at 4 °C), resulting supernatant was termed a total lysate.
Protein concentrations were determined by the method of Lowry et
al. (33), with bovine serum albumin as a standard. CDS activity
was measured by the mixed-micelle assay methods of Wu et al.
(12) and Sparrow and Raetz (34) with some modifications. The reaction
mixture (50 µl) contained 0.1 M Tris-HCl (pH 7.5), 0.2 M KCl, 1 mg/ml bovine serum albumin, 5 mM
(0.3%) Triton X-100, 0.25 mM dithiothreitol, 10 mM MgCl2, 0-1000 µM PA, 1 mM [
-32P]CTP (105 cpm/nmol).
In some experiments, [
-32P]dCTP was used instead of
[
-32P]CTP. PAs used as substrates for CDS are composed
of PA from egg yolk lecithin:
1,2-dilauroyl-sn-glycero-3-phosphate (di-C12:0-PA) as a
short chain PA, and 1,2-distearoyl-sn-glycero-3-phosphate (di-C18:0-PA), 1,2-dioleoyl-sn-glycero-3-phosphate
(di-C18:1-PA), and
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate
(C18:0/C20:4-PA) as long chain PAs. To analyze the sensitivity of CDS
activity to phosphoinositide, PI, PIP (phosphatidylinositol
4-monophosphate), and PIP2 (Sigma) were
added to the reaction mixture at concentration of 2, 5, 10, and 20 mol%. The reaction was continued for 10 min at 30 °C and was
stopped with 100 µl of 1 N HCl. A two-phase system was
created by adding 250 µl of chloroform/methanol (1:1, v/v), and the
mixtures were thoroughly vortexed and then centrifuged. The lower
organic phase was washed with 100 µl of chloroform, 1 N
HCl (1:1, v/v) and was analyzed by thin layer chromatography which was
developed in a solution containing chloroform/methanol/water/acetic acid (50:28:8:4, v/v/v/v).
1,2-Diacyl-sn-glycero-3-diphosphocytidine (CDP-DAG) from egg
yolk lecithin (Sigma) was used as a marker. The bands
of CDP-DAG detected by autoradiography was scraped with a sharp spatula
and collected for liquid scintillation counting.
-diaminobenzidine tetrahydrochloride as a substrate. Some of the freeze-thawed specimens, after immunoreaction, were postfixed with 1% OsO4, treated
with 0.5% uranyl acetate, and embedded in Epon 812. Ultrathin sections were examined under electron microscope.
non-coding sequences (nucleotides 1423-1875) labeled with
[32P]dCTP. Conditions for hybridization and washing were
performed as described previously (29).
-35S]dATP by nick translation and a probe composed of
45-mer antisense oligonucleotides complementary to a part of 3
non-coding sequences (nucleotides 1423-1467) labeled with
[
-35S]dATP by terminal deoxynucleotidyl transferase.
After hybridization at 42 °C for 18 h, the slides were
sequentially rinsed twice in 2 × SSC, 0.1% Sarkosyl at 42 °C
for 15 min each, three times in 0.1 × SSC, 0.1% Sarkosyl at
42 °C for 40 min each, and dehydrated in 70% and 100% ethanol
containing 0.3 M ammonium acetate. The sections were
autoradiographed using NTB2 nuclear track emulsion (Kodak) for 15-60
days.
Fig. 1.
Nucleotide sequence of the composite cDNA
and the deduced primary structure of rat CDS. In-frame stop codons
in 5 non-coding region are underlined.
[View Larger Version of this Image (85K GIF file)]
Fig. 2.
a, identity of the amino acid sequence
among rat, Drosophila, S. cerevisiae, and
E. coli CDSs. Conserved residues are stippled. b, linear representation of molecular structure of the CDSs
described above. Hydrophilic regions, and possible membrane-spanning
regions are shown.
[View Larger Version of this Image (57K GIF file)]
Fig. 3.
CDS activity toward various PAs in total
lysates of COS-7 cells transfected with pSRE-rat CDS cDNA. The
inset shows double-reciprocal (Lineweaver-Burk) plots of PA
with different fatty acyl composition dependence. Values for the
apparent Km toward C18:0/C20:4-PA, di-C18:1-PA, and
PA from egg yolk lecithin were 102, 114, and 138 µM,
respectively; and for the apparent Vmax were
268, 259, and 198 pmol/min/mg total protein, respectively. (Values for
apparent Vmax/Km (specificity
constant) calculated from Eadie-Hofstee plots were 2.51, 2.28, and
1.48, respectively.) The CDS activity for control lysate is 28 ± 1.5 pmol/min/mg total protein toward C18:0/C20:4-PA under the assay
condition at 200 µM C18:0/C20:4-PA. Values shown are
means ± S.D. (n = 6).
[View Larger Version of this Image (41K GIF file)]
Fig. 4.
Effects of PIP2 (), PIP (
),
and PI (×) on rat CDS activity under the assay condition at 1000 µM C18:0/C20:4-PA. Each point represents the average
of three determinations performed in duplicate. The error ranges of all
data are within 10%. Similar result were obtained in the experiments
for C18:0/C20:4-PA and di-C18:1-PA at 100 or 500 µM (data
not shown).
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Immunoblot of total lysate extracted from the
cells overexpressed with pSRE vector only (lane 1), and
epitope-tagged rat CDS cDNA (lane 2).
Immunoreaction was performed using anti FLAG-tag antibody.
[View Larger Version of this Image (17K GIF file)]
Fig. 6.
Immunocytochemistry of the COS-7 cell
transfected with epitope-tagged rat CDS cDNA using anti FLAG-tag
antibody. a, light micrograph, bar = 10 µm. b, electron micrograph (original magnification,
×5000), bar = 500 nm. Arrows show typical
immunodeposits. N, nucleus; M, mitochondria;
PM, plasma membrane.
[View Larger Version of this Image (175K GIF file)]
Fig. 7.
Northern blot analysis of rat CDS mRNA in
various rat tissues on postnatal day 49. Size markers
(arrowhead) represent 28 and 18 S rRNAs.
[View Larger Version of this Image (35K GIF file)]
Fig. 8.
In situ hybridization of rat CDS
mRNA in adult rat brain. a, dark field micrograph of
parasagittal section through caudate putamen (CP).
OB, olfactory bulb; Co, cerebral cortex; H, hippocampal formation; Th, thalamus;
SC, superior colliculus; IC, inferior colliculus;
Pn, pontine nuclei; Cb, cerebellar cortex (bar = 2 mm). b, dark field micrograph of
sagittal section through the pineal body (P) (bar = 1 mm). c, bright field micrograph of higher magnification of
cerebellar cortex. Mo, molecular layer; Pk,
Purkinje cell; Gr, granular layer. (Hematoxylin stain, 25 µm thickness, autoradiographed for 60 days; bar = 50 µm.).
[View Larger Version of this Image (168K GIF file)]
Fig. 9.
Phase-contrast micrograph (a) and
dark field light micrograph (b) of rat CDS mRNA
expression in the adult retina by in situ hybridization.
GC, ganglion cell layer; IP, inner plexiform layer; IN, inner nuclear layer; OP, outer
plexiform layer; ON, outer nuclear layer; IS,
inner segment; OS, outer segment; PE, pigment
epithelium. Note significant expression for CDS mRNA in the inner
segment. (Hematoxylin stain, 25 µm thickness, autoradiographed for 60 days; bars = 50 µm.).
[View Larger Version of this Image (146K GIF file)]
Fig. 10.
Bright field light micrograph
(a) and dark field light micrograph (b) of rat
CDS mRNA expression in the seminiferous tubule of adult rat testis
by in situ hybridization. Note intense hybridization
signals in the zone of postmitotic spermatocytes (pSc) and
spermatids (St). Sg, spermatogonia;
Sz, spermatozoa; I, interstitium. (Hematoxylin
stain, 25 µm thickness, autoradiographed for 30 days;
bars = 200 µm.).
[View Larger Version of this Image (128K GIF file)]
1 appears to be
activated by PA (47), and several protein kinase C isoform are
activated by PIP2 (phosphatidylinositol 3,4-bisphosphate as
well as phosphatidylinositol 4,5-bisphosphate) and phosphatidylinositol 3,4,5-trisphosphate (48-50). This PIP2-induced enzymatic
inhibition and the substrate specificity of rat CDS suggest the
presence of a mechanism for PIP2 to regulate its own
synthesis through the phosphoinositide cycle by feedback.
(54). Therefore, despite the known difference in the primary second
messenger species, cGMP versus IP3,
between vertebrate and invertebrate phototransduction, our finding
implies the possibility that some additional roles for the
phototransduction are played by this CDS molecule through the
phosphoinositide cycle in rat photoreceptor cells. The intense gene
expression of this CDS in the pineal organ (Fig. 8b), a
phylogenically homologous organ to the retina, is in favor of this
possibility. The heterogeneous but wide expression localization of CDS
mRNA in the gray matter of the brain (Fig. 8a) is also
instructive for the functional significance of CDS in the neuronal
signal transduction. The intense expression of CDS mRNA in the
cerebellar Purkinje cells and moderate expression in the hippocampal
pyramidal cells are consistent with the gene expression pattern of the
receptor for IP3, one of the two second messengers produced
by phosphoinositide cycle (55).
*
This study was supported by Grants-in-aid for Scientific
Research 07457001, 07278203, and 08270205 (to H. K.) and 07780670 and
08780718 (to K. G.) from the Ministry of Education, Science and Culture
of Japan and by a grant from Nishijima Neurosurgery Hospital
Foundation, Numazu, Japan.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: Dept. of Anatomy,
Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai
980-77, Japan. Tel.: 81-22-717-8033; Fax: 81-22-717-8021.
1
The abbreviations used are: PLC, phospholipase
C; PIP, phosphatidylinositol 4-monophosphate; PIP2,
phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; DAG,
diacylglycerol; IP3, inositol 1,4,5-triphosphate; PA,
phosphatidic acid; CDS, CDP-diacylglycerol synthase; aa, amino acid(s);
PCR, polymerase chain reaction; kb, kilobase(s); di-C12:0-PA, 1,2-dilauroyl-sn-glycero-3-phosphate; di-C18:0-PA,
1,2-distearoyl-sn-glycero-3-phosphate; di-C18:1-PA,
1,2-dioleoyl-sn-glycero-3-phosphate; C18:0/C20:4-PA, 1-stearoyl-2-arachidonoyl-snglycero-3-phosphate.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.