(Received for publication, July 27, 1994; and in revised form, October 19, 1994)
From the
CTP:phosphocholine cytidylyltransferase (CT) is a major
regulatory enzyme in phosphatidylcholine synthesis in mammalian cells.
CT is found in both soluble and particulate forms, both of which are
nuclear. We report here the identification of a 21-residue sequence at
the amino terminus of CT, KVNSRKRRKEVPGPNGATEED
, which was sufficient to
direct
-galactosidase into the cell nucleus. Further deletions
from either end of this sequence greatly reduced the nuclear
localization of
-galactosidase. Deletions of amino acids within
the nuclear localization signal or of the entire signal disrupted CT
nuclear localization, but CT was not completely excluded from the
nucleus. Clones of stable transfectants of the nuclear localization
signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells,
which is temperature-sensitive for growth and CT activity, were
isolated and characterized. The deletion mutants were active under the
same conditions as the wild-type enzyme. Despite the difference in
subcellular location from wild-type CT, the nuclear localization
mutants were fully able to complement the CT-deficient cell line CHO 58
for both growth and choline incorporation into phosphatidylcholine at
the nonpermissive temperature. The mobility of the mutant enzymes on
SDS gels was altered relative to the mobility of wild-type CT; however,
the extent of phosphorylation of the mutant enzymes was decreased only
slightly. Thus, the distribution of CT in both cytoplasm and nucleus,
rather than exclusively nucleus, has little effect on the ability of CT
to function in growing CHO cells.
Phosphatidylcholine (PC) ()is the principal
phospholipid of mammalian cell membranes. It is also an abundant
component of pulmonary surfactant and serum lipoproteins. In addition
to these major structural roles, PC hydrolysis products, including
diacylglycerol, phosphatidate, and arachidonate have been identified as
lipid second messengers in signal transduction(1, 2) .
CTP:phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme
for phosphatidylcholine synthesis in mammalian cells(3) . CT is
an ambiquitous protein existing in both soluble and particulate forms.
The particulate form is generally considered to be the more active form
while the soluble enzyme appears to serve as a relatively inactive
reservoir in vivo. The soluble CT can be converted into the
more active, particulate form under a variety of conditions, including
treatment of CHO-K1 cells with phospholipase C (4, 5) and stimulation of Hela cells with
oleate(6) . Moreover, soluble CT is highly phosphorylated while
the particulate enzyme is less phosphorylated(4, 6) .
Thus, translocation and activation of CT are accompanied by
dephosphorylation(4, 6) . Recently, Jackowski (7) has shown that phosphorylation of CT is regulated during
the cell cycle. To understand the mechanism of CT regulation, it is
important to understand the subcellular localization of both the
soluble and particulate forms of CT and the means by which CT is
transported to its locations. Early cell fractionation experiments
suggested that the membranous form of CT is associated with the
endoplasmic reticulum (8, 9) or Golgi
apparatus(10, 11) , and it has long been assumed that
the soluble form is cytoplasmic. Using immunofluorescence staining as
well as biochemical studies, however, we found that soluble CT is
nuclear (12) . Translocation of CT from the nuclear matrix to
the nuclear envelope was observed upon activation by treatment with
oleate or phospholipase C(5, 12, 13) . This
is a surprising observation because the enzyme catalyzing the final
step in the CDP-choline pathway, choline phosphotransferase, is in the
endoplasmic reticulum(14) , as are many other enzymes of lipid
biosynthesis. It is not obvious why an enzyme regulating
phosphatidylcholine biosynthesis should be located in the nucleus. In
order to study the physiological function of the nuclear localization
of CT, we have identified the nuclear localization signal of CT by
deletion analysis as well as by constructing chimeric fusions of CT
fragments to
-galactosidase. Stably transfected cells expressing
mutant forms of CT deficient in the nuclear localization signal were
also isolated and characterized.
For construction of CT-lacZ fusions, the E. coli lacZ gene, starting from codon 7,
was produced by PCR amplification with pcD-SR
296-HMGal (kindly
provided by Dr. Robert D. Simoni) as template and
5`-GAAGATCTGCCATGGATATCAAGCTTGAGCTCTGCAGAATTCCACTGGCCGTCGTTTTA-3` and
5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. The PCR product was
subcloned into the BglII/XbaI site of the pCMV4
vector to produce pCMV4-lacZ. The CT portion of different
CT
-lacZ fusions were produced by PCR with the
NH
-terminal primer 5`-CGCGGATCCAGATCTATGGATGCACAGAGTTCA3`
and different COOH-terminal primers followed by a HindIII
restriction site. The primers were as follows:
pCMV4CT
-lacZ:
5`-CGGGATCCAAGCTTAGGGCCAGGTACCTCTT-3`;
pCMV4CT
-lacZ:
5`-CCGGATCCAAGCTTTGTTGCTCCATTAGGGC-3`;
pCMVCT
-lacZ:
5`-CCGGATCCAAGCTTATCTTCCTCTGTTGCT-3`;
pCMV4CT
-lacZ: 5`CGGGATCCAAGCTTGGAAGGAATTCCATC-3`;
pCMV4CT
-lacZ: 5`-CCCAAGCTTCCTGCAGGCTTCTTCCA-3`;
pCMV4CT
-lacZ:
5`-CGGGATCCAAGCTTCCCTGCCGAAGAGTAG-3`;
pCMV4CT
-lacZ:
5`-CGGGATCCAAGCTTCACAAATTCTTTCGACT-3`; pCMV4CT-lacZ:
5`-AGCGAGGATGAAGAGGACGAGCAGAAGCTTATCAGCGAGGAGGACCTCTAGAGTCGACTCGAG-3`.
The PCR products were then digested with BglII and HindIII and subcloned into the BglII/HindIII
site of pCMV4-lacZ in frame to produce
pCMV4CT
-lacZ, where
is the last codon of
the CT fragment at each construct. The junction of each
CT
-lacZ fusion was sequenced.
Plasmid
pCMV4CTN1-lacZ was obtained by PCR
amplification with pCMV4CT
N1 as template and
5`-CGCGGATCCAGATCTATGGATGCACAGAGTTCA-3` and
5`-CGGGATCCAAGCTTGGAAGGAATTCCATC-3` as primers. The PCR product was
then subcloned into the BglII/HindIII site of
pCMV4lacZ to produce
pCMV4CT
N1-lacZ.
pCMV4CT
2-11-lacZ was produced by PCR using
pCMV4CT
-lacZ as template and
5`-CGCGGATCCAGATCTATGAGGAGGAGGAGGAAAGAGG-3` and
5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. pCMV4N1-lacZ was
obtained by PCR with pCMV4-lacZ as template and
5`-CGCGGATCCAGATCTATGAGGAAGAGGAGGAAAGATATCAAGCTTGAGCT-3` and
5`-GCTCTAGATTATTATTTTTGACACC-3` as primers.
pCMV4CT
2-7-lacZ was produced by PCR with
pCMV4CT
-lacZ as template and
5`-GGAATTCAGATCTATGAAAGTCAATTCAAGGAAG-3` and
5`-GCTCTAGATTATTATTTTTGACACC-3` as primers. The three previous PCR
products were subcloned into the BglII/XbaI sites of
pCMV4 to produce pCMV4CT
2-11-lacZ,
pCMV4N1-lacZ, and pCMV4CT
2-7-lacZ,
respectively. The CT portions of all of these constructs were sequenced
to confirm that there were no undesired mutations. All the plasmids
were purified using QIAGEN plasmid purification kit and transfected
into CHO 58 cells.
To prepare the rabbit anti-rat
liver CT antibody, CT was purified as described (21) from
insect cells infected for 72 h with a baculovirus clone containing the
cDNA for rat liver CT, and a polyclonal antibody to the recombinant CT
was raised in rabbits. To obtain CT-specific antibodies from the serum
of immunized rabbits we first isolated the IgG fraction by passage of
the serum over protein A-agarose. Immune serum was added to protein
A-agarose which had previously been equilibrated in 0.15 M phosphate containing 0.9% NaCl and 0.3% bovine serum albumin, pH
8.2 and incubated with gentle rocking for 30-60 min at room
temperature. The slurry was then poured into a column (1 5 cm)
and washed with approximately 10 volumes of phosphate buffer.
Antibodies were eluted with 5 ml of 0.1 M glycine-HCl, pH 2.3
into 3 ml of 3.0 M Tris-HCl containing 0.1% bovine serum
albumin, pH 8.6. The IgG fraction was then dialyzed overnight against 4
liters of PBS, lyophilized, and resuspended in 1.0 ml of deionized
water.
A CT affinity column was constructed by binding approximately 500 µg of purified recombinant CT to CNBr-activated Sepharose (Pharmacia Biotech Inc.), following the manufacturer's instructions. The column was stored in PBS containing 0.2% azide. The purified IgG fraction was incubated at room temperature for 2 h with gentle rocking with CT-Sepharose which had been previously equilibrated with phosphate buffer as above. The slurry was returned to the column and CT-specific antibody was eluted, dialyzed and lyophilized as described above. The CT-Sepharose was regenerated by washing with 2-3 volumes of 6 M guanidine HCl followed by 10 volumes of PBS containing 0.2% azide.
Mouse anti-E. coli -galactosidase monoclonal antibody was purchased from Promega
Biotech Inc. FITC-conjugated goat anti-rabbit IgG(H+L) antibody
and FITC-conjugated horse anti-mouse IgG(H+L) antibody were from
Vector Laboratories. Horseradish peroxidase-conjugated goat anti-rabbit
IgG(H+L) was from Life Technologies, Inc.
Immunoprecipitation was performed as previously described (6) except 5 µl of affinity-purified CT antibody were used as the first antibody. The immunoprecipitation sample were resolved on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and subjected to autoradiography. The relative amount of phosphorylation was quantitated with a PhosphorImager from Molecular Dynamics. The same samples were then probed with CT antibody as the first antibody and alkaline phosphatase-conjugated goat anti-rabbit IgG as the second antibody. After the alkaline phosphatase reaction was run, the membrane was made transparent with ethylene glycol and the relative amount of CT protein was quantitated with a Pharmacia LKB Ultrascan XL. The level of phosphorylation was then normalized to the amount of CT protein.
Figure 1:
Construction of two potential NLS
mutants. Sequence analysis of rat liver CT (4) revealed two
basic motifs of amino acids which could be potential nuclear
localization signals (black bars). The first one is close to
the NH terminus of CT(N1). The second one is in the
COOH-terminal one-third of CT(N2). Deletion mutants of either one or
both of the two putative nuclear localization signals were constructed
by site-directed mutagenesis and subcloned into mammalian expression
vector pCMV4.
Figure 2: Immunoblot of wild-type and deletion mutants of rat liver CT. Plasmids of wild-type and deletion mutants of CT were transfected into CHO 58 cells in a 60-mm tissue culture dish. Two days after the transfection was stopped, the cells were separated into soluble (S) and particulate (P) fractions in digitonin buffer. Equal amounts of protein were loaded on each lane for the immunoblot. A, 1:3000 dilution of affinity-purified N-antibody; B, 1:1000 dilution of affinity-purified CT-antibody. Lane 1, wild-type rat liver CT; lane 2, N1 deletion of rat liver CT; lane 3, N2 deletion of rat liver CT; lane 4, N1N2 double deletion of rat liver CT.
Figure 3:
Subcellular localizations of CT nuclear
localization signal mutants. CHO 58 cells were transfected with the CT
expression plasmids, and the location of the transiently expressed
proteins was determined by indirect immunofluorescence with N-antibody
for wild-type CT and CT-antibody for the deletion mutants of CT. A and B, wild-type rat liver CT (pCMV4CT). A is
stained with DAPI for DNA and B is immunofluorescence. C and E, N2 deletion of CT (pCMV4CTN2). C is
phase contrast and E is immunofluorescence; D,
immunofluorescence of N1 deletion of CT (pCMV4CT
N1); F,
Immunofluorescence of N1N2 double deletion
(pCMV4CT
N1N2).
Figure 4:
Representative subcellular locations of
CT-lacZ fusions. Cells were transfected with
pCMV4-lacZ (A and B); pCMV4CT-lacZ (C); pCMV4CT
-lacZ (D);
pCMV4CT
N1-lacZ (E);
pCMV4CT
-lacZ (F);
pCMV4CT
2-7-lacZ (G);
pCMV4CT
-lacZ (H); pCMV4N1-lacZ (I).
Figure 5:
Summary of subcellular locations of
-galactosidase protein fused to CT. CHO 58 cells were transfected
with indicated pCMV4CT
-lacZ plasmid. Two days
after the transfection was stopped, cells were fixed with 3%
formaldehyde followed by methanol:acetone (1:1). Subcellular locations
were detected with monoclonal antibody to
-galactosidase (1:200)
and FITC-conjugated rabbit anti-mouse second antibody. Open
bar, CT; slashed bar, lacZ (not proportional to
scale); black bar, N1.
We then began deletion from the NH terminus
using the first 28 residues of CT as the starting point to determine
which residues within this portion of the sequence are sufficient for
nuclear targeting. Deletion of residues 2 though 7 had no significant
effect on nuclear localization (Fig. 4G). Deletion of
residues 2 though 11 eliminated the ability of the CT sequence to
direct
-galactosidase to the nucleus (Fig. 5). The smallest
segment of CT that is sufficient for nuclear targeting was thus
identified as
KVNSRKRRKEASSPNGATEED
. It is
interesting to note that although the deletion of the 5-residue N1
sequence did not exclude CT from the nucleus (Fig. 3), an N1
deletion in a CT fusion protein containing the first 32 residues of CT
resulted in the exclusion of
-galactosidase from the nucleus (Fig. 5E). The N1 sequence, therefore, is necessary,
but not sufficient, for targeting
-galactosidase to the nucleus.
Figure 6:
Confocal indirect immunofluorescence of
CTN1 and CT
2-32 clones. Cells were fixed and detected the
same as in Fig. 4except 1:100 dilution of CT-antibody was used
as first antibody. Immunofluoresence was then detected with a confocal
microscope with a 0.5-nm slice at about 12-nm thickness from the bottom
of the cells. CT
N1 clone 3-5 (A); CT
N1 clone 28-3 (B); CT
2-32 clone 3-5 (C); CT
2-32 clone
10-3 (D).
CHO
58, the host cell line used for these transfections, is both deficient
in and temperature sensitive for CT(15) . The cells can grow at
34 °C but not at 40 °C, and expression of wild-type rat liver
CT in these cells allows them to grow at 40 °C(18) . Growth
rates at 40 °C of CHO 58 cells expressing CTN1 or CT
2-32
were no different from those of cells expressing wild-type CT,
indicating that the mutant CTs were fully capable of complementing the
mutant phenotype.
Figure 7:
Choline incorporation into lipids. Cells
were plated at 4 10
cells per 60-mm dish and grown
for 1 day at 34 °C and then 40 °C for 24 h. Cells were then
labeled with 1 µCi/ml
[methyl-
H]choline for 0.5-2 h.
Lipids were extracted by the Bligh-Dyer method. CHO 58 cells (open
circle), rat liver wild-type CT stable transfection (open
square), CT
N1 clone 3-5 (open diamond), CT
N1
clone 28-3 (closed square), CT
2-32 clone 3-5 (closed
circle), CT
2-32 clone 10-1 (closed
diamond).
Figure 8:
Immunoblot of stable transfection clones
of CTN1 and CT
2-32. Clones of stable transfection of
wild-type rat liver CT as well as CT
N1 and CT
2-32 were
isolated as described. Cells were plated at 4
10
cells/60-mm tissue culture dish and grown for 2 days at 34
°C. The cells were then washed twice with CMF-PBS and harvested in
digitonin buffer as soluble (S) and particulate (P)
fractions. Lane 1, wild-type rat liver CT; lane 2, N1
deletion of rat liver CT clone 3-5; lane 3, N1 deletion of rat
liver CT clone 28-3; lane 4, CT
2-32 deletion of rat liver
CT clone 3-5; lane 5, CT
2-32 deletion of rat liver CT
clone 10-1. The particulate fraction of CT
2-32 deletion clone 10-1
(not shown) was the same as the particulate fractions in lanes
1-4.
To
analyze further the CT phosphorylation pattern, CT was
immunoprecipitated from cells labeled in vivo with P
. The CT protein levels were determined by
immunoblot analysis and phosphorylation levels were detected by
autoradiography (Fig. 9). Although there was much more
fast-migrating CT in the deletion mutants than in wild-type CT, the
extent of phosphorylation of the mutants was, on average, only 20%
lower than that of wild-type CT (Table 1). Furthermore, the
two-dimensional tryptic phosphopeptide maps of the mutants were similar
to that of the wild-type CT (not shown).
Figure 9:
Phosphorylation of rat liver wild-type and
nuclear localization signal deletion mutants of CT. Cells were labeled
and samples were immunoprecipitated as described under
``Experimental Procedures.'' The precipitated samples were
then separated by SDS-PAGE and subjected to immunoblotting with CT
antibody (A) and autoradiography (B). Lane
1, wild-type rat liver CT; lane 2, CT2-32 clone 3-5; lane 3, CT
2-32 clone 10-1; lane 4, CT
N1
clone 3-5; lane 5, CT
N1 clone
28-3.
The results reported in this study identify residues
8-28 of CT as a nuclear localization signal. This sequence
represents a minimum sequence to direct a non-nuclear protein into the
nucleus. Deletion of several residues at either end of this sequence
greatly diminished nuclear targeting. Within this sequence is found a
more classic nuclear targeting sequence, RKRRK. Although
similar short, basic sequences are capable of directing some proteins
to the nucleus(26, 27) , the N1 sequence was not
sufficient for nuclear targeting of
-galactosidase.
Deletion of
the N1 sequence from a nuclear-targeted fusion protein rendered
-galactosidase completely cytoplasmic. In contrast, deletion of N1
or even the more extensive region of residues 2-32 from CT
rendered it both cytoplasmic and nuclear. There are several possible
reasons for the continued presence of the CT mutants in the nucleus.
First, CT might be interacting with another nuclear protein and could
be transported into the nucleus while attached to the other protein.
For example, deletion of the nuclear localization signal of the product
of the retinoblastoma gene does not exclude this nuclear protein from
the nucleus(30) . Additional mutations, however, in a region
involved in protein:protein interaction result in exclusion from the
nucleus (30) . It has been shown that CT interacts with a
110-kDa protein(31) , but the nature and the region of the
interaction are not clear. It is possible that CT is brought into the
nucleus though interaction with this or another protein. Second, CT
might have an additional nuclear localization signal. Polyoma large T
antigen (32) and yeast ribosomal protein L29 (33) each
have two nuclear localization signals. Mutation of either one
individually impairs but does not eliminate the ability of the protein
to enter the nucleus; mutations of both result in an exclusively
cytoplasmic location. While it is not likely that the N2 region serves
as an additional signal, there may be a sequence in CT that is not a
conventional, basic residue-rich nuclear localization sequence. Third,
rat liver CT expressed in the transfected cells may interact with the
endogenous CT in CHO 58 cells. CT is known to be a
dimer(34, 35) . The CT in CHO 58 cells is apparently
less stable than wild-type CT. At the permissive temperature (34
°C), the mutant cell lines contain approximately 5% of the CT
activity found in wild-type CHO K1 cells, while at the non-permissive
temperature (40 °C) the activity drops to less that
1%(36) . The decreased activity is accompanied by decreased
protein levels(18) . Expression of exogenous rat liver CT in
CHO 58 cells could result in heterodimer formation between rat liver CT
and CHO 58 CT. If rat liver CT could stabilize CHO 58 CT in the
heterodimer, then it is possible that the total CT level in the nucleus
could be substantial. An argument against this possibility, however, is
that immunoblot analysis of extracts from CT
2-32 cells did not
reveal a significant increase in endogenous CHO 58 CT levels.
The altered subcellular location of CT resulting from deletions of the nuclear targeting signal has no effect on the ability of exogenous CT to complement the defective CHO 58 CT. Since the product of the CT-catalyzed reaction, CDP-choline, is soluble, it should be able to diffuse to the site of choline phosphotransferase whether CT is nuclear or cytoplasmic. While the deletions of the nuclear localization signal had no apparent effect on phosphatidylcholine biosynthesis, they affected the mobility of CT on SDS gels as detected by Western blots. The faster mobility of CT in both mutants suggested a lower level of phosphorylation in the mutants, but a quantitative estimation of the degree of phosphorylation indicated only a 20% reduction. Furthermore, there was not a dramatic change in the pattern of phosphorylation as detected on two-dimensional phosphopeptide maps. Thus it appears that the altered location of CT in the mutants does not subject CT to abnormal phosphorylation or phosphatase activity.
While it is clear that a substantial change in the location of the enzyme has no dramatic effect on phosphatidylcholine biosynthesis, the apparent presence of some CT in the nucleus in the mutant cell lines precludes a definitive conclusion at this time as to the importance of CT location for phosphatidylcholine biosynthesis. In fact, given the low level of CT in CHO strain 58, which functions normally at 34 °C, it is likely that the level of nuclear CT in the nuclear localization mutants is sufficient to carry out any functions that must be performed in the nucleus. Further experiments to determine the necessity of the nuclear location of CT must await either the identification of the region of CT responsible for its continued nuclear location in the mutants or an alternative means of anchoring CT in the cytoplasm.