From the Departments of Biochemistry and
§ Virology and Molecular Biology, St. Jude Children's
Research Hospital, Memphis, Tennessee 38105 and the Departments of
Biochemistry and ¶ Pathology, University of Tennessee,
Memphis, Tennessee 38163
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ABSTRACT |
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CTP:phosphocholine cytidylyltransferase (CCT) is
a key regulator of phosphatidylcholine biosynthesis, and only a single
isoform of this enzyme, CCT, is known. We identified and sequenced a human cDNA that encoded a distinct CCT isoform, called CCT
, that is derived from a gene different from that encoding CCT
. CCT
transcripts were detected in human adult and fetal tissues, and very
high transcript levels were found in placenta and testis. CCT
and
CCT
proteins share highly related, but not identical, catalytic
domains followed by three amphipathic helical repeats. Like CCT
,
CCT
required the presence of lipid regulators for maximum catalytic
activity. The amino terminus of CCT
bears no resemblance to the
amino terminus of CCT
, and CCT
protein was localized to the
cytoplasm as detected by indirect immunofluorescent microscopy. Whereas
CCT
activity is regulated by reversible phosphorylation, CCT
lacks most of the corresponding carboxyl-terminal domain and contained
only 3 potential phosphorylation sites of the 16 identified in CCT
.
Transfection of COS-7 cells with a CCT
expression construct led to
the overexpression of CCT activity, the accumulation of cellular
CDP-choline, and enhanced radiolabeling of phosphatidylcholine. CCT
protein was posttranslationally modified in COS-7 cells, resulting in
slower migration during polyacrylamide gel electrophoresis. Expression
of CCT
/CCT
chimeric proteins showed that the amino-terminal portion of CCT
was required for posttranslational modification. These data demonstrate that a second, distinct CCT enzyme is expressed in human tissues and provides another mechanism by which cells regulate
phosphatidylcholine production.
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INTRODUCTION |
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CCT1 is a key enzyme in
the regulation of PtdCho biosynthesis, and the mechanisms that regulate
its expression, activity, and subcellular localization are the focus of
current research (for reviews, see Refs. 1 and 2). cDNAs that
encode CCT proteins have been identified and sequenced in rat (3),
hamster (4), mouse (5), and human (6), and there are only minor
differences among these mammalian cDNAs (see Ref. 6 for a
comparison). Their catalytic properties are thought to be essentially
identical, and CCT
can be divided into four distinct functional
domains (see Fig. 1). The amino-terminal domain between residues 1 and 71 contains a sequence that specifies the nuclear localization of the
protein between residues 2 and 28 (7, 8). The catalytic core extends
from residues 72 to 233. This region of the protein is conserved from
yeast to mammals and is responsible for substrate binding and
catalysis. In particular, the conserved HXGH motif is essential for
cytidylyltransferase activity (9, 10). The third domain, located
between residues 256 and 288, contains three 11-residue amphipathic
repeats that form
-helices following association with lipid
regulators and contribute to the reversible membrane association of the
enzyme (11-15). The binding of stimulatory lipids to this region
greatly enhances catalytic activity by lowering the
Km of the enzyme for CTP into the range
corresponding to cellular concentrations of the nucleotide (16). CCT
is also negatively regulated by lipids and is potently inhibited by
sphingosine (17), lysophosphatidylcholine (18), and antineoplastic
phospholipids (18, 19). The fourth domain of CCT
is the
carboxyl-terminal phosphorylation domain between residues 315 and 367. CCT
membrane association and activity are modulated by reversible
phosphorylation (20, 21), and all of the phosphorylation sites are
located in the carboxyl-terminal region (22). Phosphorylation
attenuates CCT
biochemical activity by interfering with lipid
stimulation (23), and unphosphorylated CCT
exhibits a greater degree
of membrane association in cells (20).
CCT has been localized using cellular in situ methods to
the nucleus in Chinese hamster ovary cells (7, 8), but in rat
hepatocytes, the protein has been detected in both the nuclear and
cytoplasmic compartments (24). CCT
has also been identified in
association with Golgi membranes (25, 26), endoplasmic reticulum, and
transport vesicles (27) using biochemical methods. There is only one
isoform of CCT expressed in yeast (28), and only one isoform of CCT
(CCT
) has been identified, purified, or cloned from mammalian
sources (2). The existence of a conditionally lethal Chinese hamster
ovary cell mutant with a temperature-sensitive defect in CCT
activity (29) also suggested that there was only a single CCT isoform
in mammalian cells. A single genetic locus for CCT
was identified on
mouse chromosome 16 (5), and the murine CCT
gene has been cloned
(30). In this work, we identify a unique, second human CCT isoform,
called CCT
. CCT
catalyzes the same enzymatic reaction as CCT
and requires the presence of lipids for full activity. However, CCT
lacks the nuclear targeting sequence and the phosphorylation domain of
CCT
, suggesting that CCT
is distinct from CCT
with regard to
its subcellular localization and regulation.
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EXPERIMENTAL PROCEDURES |
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Materials-- Sources of supplies were as follows: American Radiolabel Company, Inc., phospho[methyl-14C]choline (specific activity, 55 mCi/mmol) and [methyl-3H]choline (specific activity, 85 mCi/mmol); Amersham Pharmacia Biotech [35S]methionine (specific activity, >1000 Ci/mmol); CLONTECH, human multiple tissue Northern blots; Life Technologies, Inc., LipofectAMINE reagent; Promega, restriction endonucleases and other molecular biology reagents; Invitrogen, pcDNA3 plasmid; FMC Corp., Sea-Kem, molecular biology grade agarose; American Type Culture Collection, cDNA clone AA382871; Sigma, CTP and buffers; Avanti Polar Lipids, PtdCho and fatty acids; Analtech, thin-layer chromatography plates. All other supplies were reagent grade or better.
Anti-CCTIsolation of the CCT cDNA--
Human Genome Systems
identified and provided a clone that exhibited significant sequence
similarity to CCT
. The protein expressed from this cDNA,
however, did not exhibit significant CCT catalytic activity. Sequence
information from this clone was used to search the public expressed
sequence tagged data base. We identified a clone (GenBankTM
accession no. AA382871) that contained 40 bp of related sequence at the
5' end. We purchased this clone from American Type Culture Collection
and sequenced the cDNA on both strands using primers that flanked
the multiple cloning sites and internal primers that were synthesized
to ensure a complete read on both strands. The cDNA contained a
single open reading frame we called CCT
. A 1.3-kb BamHI-XhoI fragment was excised and subcloned
into the mammalian expression vector, pcDNA3, to generate plasmid
pPJ34, which expressed CCT
from the constitutive cytomegalovirus
promoter.
Construction of Plasmids for Expression of CCT Chimeras and
CCT Amino-terminal Truncation--
Rodent CCT
(pWYCT) and human
CCT
(pPJ34) cDNAs cloned into pcDNA3 were digested with
SspI. The pcDNA3 vector has an SspI site
distanced approximately 1 kb from the 5'-end of the T7 promoter. The
CCT
cDNA has an SspI site at nucleotide 260, whereas
CCT
has an SspI site at nucleotide 311. The fragments
that contained either CCT
or CCT
sequence plus vector sequence
were purified. The 1.3-kb fragment generated from CCT
cDNA that
encoded the amino terminus was ligated to the 5-kb fragment of pWYCT to
generate pCCT
/CCT
, and the 1.3-kb fragment from the CCT
cDNA encoding the amino terminus was ligated to the 5-kb fragment
of pPJ34 to generate pCCT
/CCT
. The resulting plasmids were
checked for correct orientation with the polymerase chain reaction
using the T7 and SP6 primers of pcDNA3.
CCT Assay-- CCT activity was determined essentially as described previously (31). The standard assay contained 64 µM lipid activator (PtdCho:oleic acid, 1/1), 4 mM CTP, 10 mM MgCl2, 150 mM bis-Tris-HCl, pH 6.5, 1 mM phospho[14C]choline (specific activity, 4.5 mCi/mmol), in a final volume of 50 µl. The reaction mixtures were incubated at 37 °C for 10 min. The reaction was stopped by the addition of 5 µl of 0.5 M Na3EDTA, and the tubes were vortexed and placed on ice. Next, 40 µl of each sample was spotted on preadsorbent Silica Gel G thin layer plates, which were developed in 2% ammonium hydroxide/95% ethanol (1:1, v/v). CDP-[14C]choline was identified by co-migration with a standard, scraped from the plate, and quantitated by liquid scintillation counting. Protein was determined according to the Bradford method (32).
Isolation of CCT from Endogenous Lipids--
CCT
was
isolated from COS-7 cells 48 h after transfection with plasmid
pPJ34. Cells were washed with phosphate-buffered saline and harvested
by centrifugation, and the pellet was lysed by incubation in lysis
buffer (10 mM NaCl, 1 mM EDTA, 2 mM
dithiothreitol, 1 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 50 mM
NaF, 100 µM Na3VO4, 10 mM HEPES, pH 7.4) for 1 h on ice. The cells were
disrupted by sonication, and the particulate matter was removed by
centrifugation. The supernatant was loaded onto a 0.5-ml DEAE-Sepharose
column and the column was washed with 1.5 ml of each of the following
in succession: lysis buffer, lysis buffer plus 1% Nonidet P-40, lysis
buffer, lysis buffer plus 0.25 M NaCl, lysis buffer plus
0.5 M NaCl, and lysis buffer plus 1.0 M NaCl.
The eluant was collected in 0.5-ml fractions, and CCT activity was
located in the 0.25 M NaCl wash. This procedure is essentially the same as described in previous papers (16, 23). CCT
activity that was eluted from the column could only be detected in the
presence of added lipid activators.
Transfection Experiments-- COS-7 cells were grown in 100-mm dishes to 80% confluency in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% glutamine. Transfections using LipofectAMINE reagent were performed according to the manufacturer's instructions. Briefly, 10 µg of plasmid and 60 ml of LipofectAMINE reagent were separately diluted into 0.8 ml of serum-free medium. The two solutions were combined and incubated at 25 °C for 45 min. Next, 6.4 ml of serum-free medium was added to each tube, and the diluted solution was overlaid onto the COS-7 cells that had been previously rinsed with serum-free medium. The cells and reagents were incubated at 37 °C for 5 h, and then 8 ml of growth medium containing twice the normal amount of serum was added. The medium was replaced 24 h after the start of the transfection procedure, and the cells were incubated for an additional 24 h at 37 °C and then harvested for analysis.
Metabolic Labeling--
COS-7 cells were transfected with
vectors expressing either CCT, CCT
, or a control. The total
plasmid amount in each of the transfections was 10 µg. At 24 h
after transfection, the medium was changed, and the cells were labeled
for an additional 24 h with
[methyl-3H]choline (3 µCi/ml). Cells were
washed three times with 10 ml of phosphate-buffered saline and
harvested in 10 ml of the same buffer, and the cell pellets were
extracted using 720 µl of chloroform/methanol/concentrated HCl
(1:2:0.02, v/v). Next, 240 µl of chloroform and 240 µl of water
were added, and following vortex mixing, the phases were separated by
centrifugation. The radioactivity in the soluble and phospholipid
phases was quantitated. Samples of the soluble phase were separated on
Silica Gel G thin layers developed with 2% ammonium hydroxide/95%
ethanol (1:1, v/v), and the organic phase was analyzed on Silica Gel 60 thin layers developed with chloroform/methanol/ammonium hydroxide
(60:35:8, v/v). About 90% of the labeled material in the organic phase
was PtdCho under these labeling conditions. Choline-derived metabolites
were identified by co-migration with standards.
Northern Blots--
Three multiple human tissue Northern blots
were purchased from CLONTECH and were hybridized
and washed according to the manufacturer's instructions. The blots
were first hybridized with 32P-labeled probe prepared from
a 1.3-kb BamHI-XhoI fragment that covered the
entire CCT cDNA. The blots were then stripped and hybridized
with a 32P-labeled probe prepared from the 582-bp
SacI fragment of the human phosphatidylinositol synthase
(pis1) cDNA (33). The blots were stripped again and
hybridized with a 32P-labeled probe prepared from a 320-bp
PstI-ApaI fragment representing the 3' region of
the CCT
cDNA. This area of the CCT
cDNA did not share any
sequences in common with CCT
.
Immunoblots--
Cell lysates (50 µg of protein) were
separated by SDS-gel electrophoresis on 12% polyacrylamide gels and
transferred by electroblotting onto nitrocellulose membranes.
Immunoblotting was performed by incubation of the membranes with either
purified anti-CCT (1:200 dilution) or purified anti-CCT
(1:200
dilution) as the primary antibody. The Amersham Pharmacia Biotech ECL
Western blotting reagents and protocol were used to identify the
immunoreactive proteins.
Immunofluorescence Microscopy--
HeLa cells grown on
coverslips were fixed with 3.7% paraformaldehyde, permeabilized with
cold acetone, and processed as described (34). Affinity-purified
anti-CCT primary antibody was diluted in 0.15 M NaCl, 10 mM Tris-HCl, pH 8.0. The cells were incubated with
anti-CCT
antibodies at increasing dilutions followed by fluorescein-conjugated secondary antibodies. The coverslips were mounted with p-phenyldiamine, the cells viewed in a Zeiss
IM-35 microscope equipped with fluorescence optics, and photographs were made on Kodak Tri-X pan film. Controls from which the primary antibody was excluded showed no significant fluorescence. Preincubation of the primary antibody with the peptide did not yield significant fluorescence. Two other controls assured selective labeling of the
nuclear and cytoplasmic compartments. Both a nucleolar marker (anti-p120 antibodies, Becton Dickinson) and a cytoplasmic
(cytoskeletal) marker (anti-vimentin antibodies, Boehringer Mannheim)
were used to label the cells to confirm appropriate staining of
cellular compartments.
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RESULTS |
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Identification of the CCT Clone--
A BLAST search of the
proprietary human expressed sequence tagged data base of Human Genome
Sciences revealed the existence of a cDNA with considerable
similarity to mammalian CCTs. However, the protein expressed by this
cDNA was not catalytically active, indicating that it did not
contain a complete CCT coding sequence. Sequence information from this
clone was used to search the public expressed sequence tagged data
base. We identified a second clone (GenBankTM accession no.
AA382871) isolated from a human testis library that contained a 140-bp
related sequence at the 5' end. This clone, called CCT
, was
purchased from American Type Culture Collection. Both cDNA strands
were sequenced, and the clone contained the entire CCT
coding
sequence. The cDNA sequence of human CCT
was compared with the
cDNA sequence of human CCT
(see Fig. 2). The analysis of the
sequence (see below) indicated that the CCT
cDNA encoded a new
CCT isoform.
Similarities and Differences between the Predicted Protein
Sequences and the cDNAs of CCT and CCT
--
The predicted
amino acid sequences of human CCT
and CCT
are compared in Fig.
1. The catalytic cores of human CCT
and CCT
are nearly identical and extend from amino acids 72 through
233. The catalytic core in CCT
has 64% identity with the equivalent yeast CCT domain that is located between amino acids 99 and 260 of the
yeast protein (28). Three of the amino acids in CCT
that are
different from CCT
(N120K, V136L, and R162K) are identical to the
yeast CCT sequence. Three other amino acids that are different in
CCT
compared with CCT
(E126D, D134E, and E160K) are identical to
the residues found in the catalytic core of the yeast MUQ1 sequence,
which has been identified as phosphoethanolamine cytidylyltransferase (36). Also, the catalytic domains of human phosphoethanolamine cytidylyltransferase are highly related to the analogous domains in
CCT
and CCT
(37). These sequence similarities strongly suggested
that the CCT
cDNA encoded a protein with cytidylyltransferase activity.
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Pattern of CCT mRNA Expression--
The relative abundance
of CCT
mRNA expression in a wide variety of human tissues was
addressed by Northern blot analysis (Fig.
3). The blots were probed with the human
phosphatidylinositol synthase (pis1) cDNA as a loading
control. This enzyme in phosphatidylinositol biosynthesis is a
"housekeeping" protein that is expressed at a relatively uniform
level in human tissues (33). The blots were then probed with both the
entire CCT
cDNA (Fig. 3) and with a 32P-labeled
fragment from the 3' untranslated region of the CCT
cDNA. The
pattern of expression was the same with both probes (not shown). Two
sizes of CCT
transcripts were detected. The largest CCT
mRNA
(~6.5 kb) was most abundant in brain, ovary, testis, and all fetal
tissues examined. The second class of CCT
mRNAs were found
between 1.1 and 1.9 kb. The 1.1-kb mRNA was detected in placenta,
which was the most abundant source for CCT
mRNA in our survey.
Testis also was an abundant source for CCT
transcripts, and two
mRNAs of 1.6 and 1.9 kb were detected in this tissue. Although it
is difficult to see in Fig. 3 due to the very high expression of CCT
in placenta and testis, CCT
mRNA species of either 1.1 or 1.9 kb
were faintly detected in all tissues examined. Thus, CCT
mRNA is
widely distributed in human tissues and expressed at very high levels
in testis and placenta.
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Substrate Specificity of CCT--
The similarity of the
catalytic core domains of CCT
and CCT
suggested that CCT
was a
phosphocholine cytidylyltransferase. This prediction was confirmed by
transfecting COS-7 cells with a CCT
expression construct (pPJ34) and
measuring the CCT enzymatic activity in cell lysates (Fig.
4A). The introduction of the
CCT
expression vector into the COS-7 cells led to a significant
(8-fold) increase in the CCT specific activity from 4.8 to 38.4 nmol/min/mg of protein in cell extracts (Fig. 4A). These
data establish that the CCT
cDNA encodes an active
CTP:phosphocholine cytidylyltransferase. Alternative substrates for
CCT
(phosphoethanolamine, glycerol 3-phosphate, phosphatidic acid,
and lysophosphatidic acid) were also screened. Substitution of these
compounds for phosphocholine in the biochemical assay did not yield
significant activity. Substitution of deoxyCTP for the CTP in the assay
at concentrations up to 10-fold higher also did not yield significant
activity.
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Effect of CCT Expression on PtdCho Metabolism--
COS-7 cells
were transfected with the CCT
expression plasmid and were labeled
with [3H]choline for 24 h to determine whether
CCT
functions as a CCT in vivo and whether the
overexpression of this isozyme effects the PtdCho biosynthetic pathway
(Fig. 4B). Although the cellular content of
3H-labeled choline and phosphocholine were the same in
control and CCT
-transfected cells, there was a 3.4-fold increase in
the CDP-choline pool from 1963 ± 13 to 6706 ± 847 cpm/mg.
This finding was consistent with the identification of CCT
as a
phosphocholine cytidylyltransferase and illustrated that overexpression
of the enzyme leads to increased accumulation of its product,
CDP-choline in vivo. There was also a 50% increase in the
incorporation of [3H]choline into PtdCho from
715,228 ± 28,317 to 1.06 × 106 ± 15,959 cpm/mg
in CCT
-transfected cells indicating that CCT
overexpression
accelerated PtdCho synthesis. The 5-fold increase in the amount of
glycerophosphocholine, a breakdown product of PtdCho, from 7027 ± 251 to 35,835 ± 7083 cpm/mg in the CCT
-expressing cells
indicated an acceleration of PtdCho turnover similar to that previously
observed with CCT
overexpression (39).
Lipid Stimulation of CCT Activity--
The similarities in the
amphipathic helical domains between CCT
and CCT
suggested that
lipids would stimulate CCT
activity in a similar manner to CCT
.
The addition of the PtdCho:oleic acid lipid activator mixture to crude
cell lysates did not enhance CCT
activity (not shown). However, the
lack of lipid regulation in cell lysates could be attributed to the
presence of endogenous lipid activators. This point was tested by
removing the endogenous lipid activators from the transfected COS-7
cell lysates by ion-exchange chromatography (16, 23) and determining
the ability of PtdCho:oleic acid vesicles to activate the enzyme (Fig.
4C). CCT
activity was not detected following the removal
of endogenous lipids, and it was potently stimulated by the addition of
PtdCho:oleic acid vesicles to the sample. There remain many kinetic
details related to the specificity of the lipid regulation of CCT
to
be investigated with purified CCT
, and there may be subtle
differences between the two proteins because the amphipathic helical
domains are not identical. Nonetheless, our experiments establish that
CCT
, like CCT
, is critically dependent on the presence of
stimulatory lipids for activity.
Expression and Modification of CCT Protein--
The predicted
molecular size of the CCT
protein was confirmed by transcription and
translation of the CCT
cDNA in vitro using a
reticulocyte lysate (Fig. 5). The
expressed proteins were radiolabeled with
[35S]methionine, and the products were separated by
SDS-polyacrylamide gel electrophoresis on 12% gels. A protein of an
apparent size of 35 kDa was identified in reactions using CCT
cDNA as template and was consistent with the predicted size of 36.3 kDa for CCT
protein. As a control, CCT
was expressed in the
transcription/translation system, and the expected 42-kDa protein was
detected (Fig. 5). The CCT
expression plasmid was transfected into
COS-7 cells, and cell lysates were analyzed for expression of CCT
protein by immunoblotting with an antibody raised against amino acids 27-39 of the CCT
polypeptide sequence (Fig. 5). Two forms of CCT
were detected following expression in COS-7 cells. The less abundant
species migrated at the same apparent size as the protein made in
vitro (CCT
), and there was a second, slower migrating form
(CCT
M). The location of both CCT
M and
CCT
at approximately the same position on the gel was not due to
cross-reactivity of the two affinity-purified antibodies. The
specificities of the anti-CCT
and anti-CCT
amino-terminal
antibodies were clearly demonstrated in the same experiment (Fig. 5).
The larger apparent size of CCT
M suggests that a
significant portion of the expressed protein is modified
posttranslationally.
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Regulation of CCT Activity--
CCT
activity was examined
in vitro to determine whether the NH2-terminal
sequence and modification of the protein influenced expression and/or
catalytic activity. COS-7 cells were transfected with the plasmids
encoding CCT
, CCT
, the chimeric CCT proteins (CCT
/CCT
and
CCT
/CCT
), and the NH2-terminal truncated
CCT
[
1-26]. The CCT protein species were expressed at
approximately equivalent levels based on the immunoblots (Fig. 6). The
CCT protein species in the cell lysates were assayed in the presence of
excess stimulatory lipids to evaluate their relative activities.
Although cells overexpressing (CCT
plus CCT
M) had
higher activity (76 nmol/min/mg) than the endogenous control activity
(8 nmol/min/mg), [CCT
plus CCT
M] was less active
than CCT
and its multiply phosphorylated species (1105 nmol/min/mg)
(Fig. 7). Substitution of the
NH2-terminal domain of CCT
onto CCT
enhanced
biochemical activity (215 nmol/min/mg), whereas the amino-terminal
domain of CCT
dramatically reduced the activity of CCT
(91 nmol/min/mg). These data suggested that the protein modification
directed by the amino terminus of CCT
attenuates biochemical
activity. In support of this hypothesis, truncation of the first 26 amino acids of CCT
elevated activity almost 10-fold (686 nmol/min/mg), supporting the idea that the NH2 terminus
plays a role in the cellular regulation of CCT
activity.
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Cellular Localization of CCT--
The amino terminus of CCT
bears little resemblance to the amino terminus of CCT
, which harbors
a nuclear localization sequence. CCT
is reported to be predominately
an intranuclear protein based on indirect immunofluorescence using an
antibody raised against an amino-terminal peptide of CCT
(7, 8). The
lack of a nuclear localization motif in CCT
suggested that it would
not be found in the cell nucleus. This hypothesis was tested by
evaluating the distribution of CCT
in human HeLa cells using the
anti-CCT
peptide antibody and indirect immunofluorescence microscopy
(Fig. 8). The affinity-purified peptide
antibody detected CCT
protein in the cytoplasm and did not detect
the protein in the nucleus, even at the lowest antibody dilutions. The
cytoplasmic staining appeared diffuse but was higher than the apparent
background staining of the nucleus. The background signal associated
with the cell nucleus/nucleolus was due to reaction with the
fluorescein-conjugated secondary antibodies used in the assay (Fig.
8B). Monoclonal anti-p120 antibody positively identified the
nucleolus in these cells (Fig. 8C), and anti-vimentin
monoclonal antibody, which signaled the cytoskeleton, was used as a
cytoplasmic marker (Fig. 8D). These data confirm that CCT
is localized primarily outside of the nucleus.
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DISCUSSION |
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The existence of a second isoform of CCT opens the door to a
series of experiments to determine the physiological function of
CCT. Our data indicate that the biochemical properties of the two
CCT isoforms are similar and the overexpression of either isoform is
capable of perturbing PtdCho biosynthesis and metabolism. However, the
distinct differences between the amino and carboxyl-terminal domains of
CCT
and CCT
indicate that the two isoforms likely have unique
regulatory properties. The two isoforms are clearly the products of
different genes and are differentially expressed in tissues and perhaps
also during development. Because CCT
is the only isoform detected in
the large volume of work in this area (1, 2), it is possible that
CCT
is expressed at lower levels or in a specific developmental
settings compared with than the more widely distributed CCT
.
Previous data may have to be reinterpreted in light of the existence of
CCT. For example, data on whole tissue CCT activity and distribution
will need to be reevaluated. The Northern blots cannot be used to
predict the relative levels of CCT
and CCT
proteins in particular
tissues. Total CCT specific activity is relatively low in most tissues,
illustrating that neither isoform is expressed at high levels. The
properties of CCT
when expressed in vivo and assayed
in vitro suggest that there are no significant differences
in biochemical characteristics that could distinguish between the two
isozymes in crude extracts. The general impression is that CCT
may
be more restrictive in its expression compared with CCT
because
CCT
has been the only mammalian isoform cloned to date. Nonetheless,
we think that these data will be important to gather because some of
the controversial issues in the CCT field may be explained by the
presence of two isoforms.
The CCT anti-peptide antibody used previously to identify its
nuclear localization (7, 8) does not cross-react with CCT
(Figs. 5
and 6). Houweling et al. (24) report that CCT is both a
nuclear and cytoplasmic protein in primary hepatocytes using a peptide
antibody raised against amino acids 164-176 of CCT
. This sequence
in the 164-176 region (DFVAHDDIPYSSA) is identical in human CCT
and
CCT
; therefore, antisera raised against this peptide would be
predicted to react with both CCT isoforms. We detect both CCT
and
CCT
transcripts in rodent
liver,2 and it will be very
interesting to determine whether the cytoplasmic CCT detected in
primary rodent hepatocytes can be attributed to the presence of CCT
in this tissue.
The co-migration of CCT and CCT
M on denaturing gels
and their similar reliance on lipid activators for biochemical activity makes it difficult to determine whether CCT
is a component of purified CCT preparations from mammalian sources. The regulation of
CCT
is governed by phosphorylation of its carboxyl terminus at
multiple sites, resulting in at least two species that migrate more
slowly on denaturing gels (Figs. 5 and 6 and Ref. 40). Phosphorylation
interferes with the stimulatory action of lipids on CCT
activity
(23), and there is some correlation with membrane dissociation of
CCT
in vivo (20), but the regulation by this mechanism is
not an absolute on/off switch (23, 35). On the other hand, the activity
of CCT
M, a protein with approximately the same molecular
weight as CCT
, is lower (Fig. 7) due to posttranslational modification that is dependent on the amino terminus. The nature of the
modification of CCT
protein and its role in the regulation of PtdCho
biosynthesis is currently under investigation.
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ACKNOWLEDGEMENTS |
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We thank Pam Jackson for her expert technical assistance, Wannian Yang for the construction of pWYCT, and Chuck Rock for his comments on the research. We thank Human Genome Sciences for providing us with information on the CCT-like cDNA contained in their library.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 45737, Cancer Center (CORE) Support Grant CA 21765, and the American and Lebanese Syrian Associated Charities.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF052510.
** To whom correspondence should be addressed: Biochemistry Department, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3494; Fax: 901-525-8025; E-mail: suzanne.jackowski{at}stjude.org.
1
The abbreviations used are: CCT,
CTP:phosphocholine cytidylyltransferase; PtdCho, phosphatidylcholine;
-isoform, the previously discovered CCT;
-isoform, the new CCT
described in this paper; kb, kilobase(s); bp, base pair(s).
2 A. Lykidis and S. Jackowski, unpublished observations.
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REFERENCES |
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