From the
To investigate the role of phosphorylation and dephosphorylation
in modulating the activity and location of CTP:phosphocholine
cytidylyltransferase, we used site-directed mutagenesis to construct
four mutant forms of cytidylyltransferase. These forms were 5SP CTP:phosphocholine cytidylyltransferase catalyzes the conversion
of phosphocholine to CDP-choline, an important regulatory step for
phosphatidylcholine biosynthesis in mammalian cells(1) .
Cytidylyltransferase is mainly regulated at the post-transcriptional
level, although alterations in mRNA levels for cytidylyltransferase
have been reported(2, 3) . Cytidylyltransferase is a
nuclear protein that exists in both soluble and nuclear
envelope-associated forms (4, 5, 6) . In
normal cells most of the enzyme is in the soluble, relatively inactive
form. Activation of cytidylyltransferase is accompanied by conversion
to the particulate form (1) or, in some cells, to a soluble
lipoprotein complex(7, 8, 9) . Activation and
translocation of cytidylyltransferase to the membrane can be achieved
by treating cells with fatty acids (10, 11) or
phosphatidylcholine-specific phospholipase C(12, 13) ,
choline deficiency(14) , or supplementation with choline
analogs(15) . Soluble cytidylyltransferase is highly
phosphorylated(16) ; 15-16 Ser residues near the carboxyl
terminus have been determined to be the phosphorylation sites in
baculovirus-expressed cytidylyltransferase (17) . Activation
and translocation to the membrane are accompanied by extensive
dephosphorylation(18, 19) . Although it appears that
phosphorylation and dephosphorylation are key components in the
inactivation and activation of cytidylyltransferase, the precise
contribution of the phosphorylation state of cytidylyltransferase to
its activity and location are presently unknown. Treatment of CHO To
approach the role of protein phosphorylation in the function and
location of cytidylyltransferase, we have chosen to use site-directed
mutagenesis of the phosphorylation sites followed by expression of the
mutant enzymes in a heterologous cell. We previously found that a
suitable expression system is the strain 58 CHO cell line, in which
endogenous cytidylyltransferase is temperature-sensitive and is present
at very low levels even at the permissive
temperature(5, 20, 21) . We first mutated a
putative casein kinase II site, Ser
The strategy
for making the 16S The final PCR products were purified and subcloned into the BamHI and XbaI sites of M13mp19. The 0.6-kilobase
pair EcoRV-XbaI fragment of the COOH-terminal portion
of the cDNA (23) was sequenced to ensure that no undesired
mutation occurred during the multiple-round reactions. The EcoRV-XbaI portions were then subcloned into pCMV5CT,
from which the EcoRV-XbaI portion had been removed;
the resulting plasmids were pCMV5CT16S
For
measuring pools of phosphatidylcholine and aqueous choline metabolites,
cell were plated at 2
Figure 1:
Phosphorylation levels of wild type and
mutant forms of cytidylyltransferase. Cells were incubated with
The labeled
phosphopeptides in the partially phosphorylated cytidylyltransferase
mutants were analyzed by two-dimensional peptide mapping (Fig. 2). Even though 11 phosphorylation sites remain in the 5SP
Figure 2:
Phosphopeptide maps of
To study the effects of the
phosphorylation site mutations on the partitioning of
cytidylyltransferase between soluble and membrane fractions, the cell
lines expressing wild type and mutant cytidylyltransferase constructs
were harvested by digitonin extraction. Cytidylyltransferase activity
was determined in the presence of lipids (Table 3), and the mass
of cytidylyltransferase was determined by Western blotting (Fig. 3). As judged by the activity measurements, mutation of
Ser to Ala affected the distribution of cytidylyltransferase in a
cumulative manner (Table 3), with the amount of
membrane-associated enzyme increasing from 5% with the wild type to
about 20, 30, and 40% with 5SP
Figure 3:
Translocation of wild type and mutants in
response to oleate treatment. Cells were plated for 1 day and then
treated with 0.25 mM sodium oleate in Ham's F-12 medium
for 20 min. The cells were then washed and separated by digitonin
extraction into soluble (S) and particulate (P)
fractions of equal volumes. Samples were separated by
SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting;
equal volumes of all fractions were loaded, except for the use of 0.25
volume for 16S
Figure 4:
Cell growth at 40 °C. Cells were left
at 34 °C for 1 day after plating and then shifted to 40 °C for
up to 5 days. Viable cells were counted as described under
``Experimental Procedures.'' WT, wild type; CHO
58, untransfected strain 58 cells.
Figure 5:
Incorporation of choline into lipids.
Cells were plated and left at 34 °C for 1 day. The cells were then
washed twice with calcium- and magnesium-free phosphate-buffered saline
and incubated with [
The levels of
aqueous choline metabolites were also determined at both 34 and 40
°C. As expected, the phosphocholine level was high and the
CDP-choline level was low for strain 58. The wild type and
phosphorylation mutants had lower phosphocholine levels and higher
CDP-choline levels than untransfected strain 58, which would be
expected from their abilities to complement the genetic defect. There
were, however, no consistent differences in metabolite levels between
cells expressing the wild type enzyme and those expressing the
phosphorylation site mutants (data not shown). Experiments described in this paper used site-directed
mutagenesis to determine the role of phosphorylation on the properties
of cytidylyltransferase. Mutations of as many as all 16 of the
phosphorylated residues did not dramatically alter enzymatic activity
as judged by assay in vitro in the presence of activating
lipids, suggesting that the COOH-terminal phospho-rylation region does
not interact closely with the more central catalytic core under these
conditions. The mutations do alter the mobility of the enzyme in SDS
gels, as would be expected from previous evidence showing that the
slower mobility of the soluble enzyme is due to
phosphorylation(18, 19) . Conversion of all 16 Ser
residues to Ala resulted in the same mobility as the fastest form of
wild type cytidylyltransferase. Conversion of the same residues to Glu
resulted in a slow moving enzyme, which is consistent with the mobility
differences being due to a high degree of negative charge in this
region. Previous studies in which the phosphorylation sites of
baculovirus-expressed cytidylyltransferase were determined by chemical
sequencing failed to detect any phosphorylated residues other than the
16 Ser residues near the COOH terminus(17) . Radiolabeling with The subcellular location of the
16S The amount of the 16S The fact that more of the 16S
Fig. 6depicts the state of cytidylyltransferase in its
various possible forms. Form I is soluble, relatively inactive, and
highly phosphorylated. Form IV is membrane-associated, active, and
extensively dephosphorylated. Both forms I and IV are well established
from many previous studies. The forms in columns II and III are
hypothetical intermediates in the transition between forms I and IV.
Forms IIa-IIc would be produced in one step from form I, and
forms IIIa-IIIc would be produced in one step from form IV. A in a circle and I in a square refer to
the relative state of activation in the cell (active and inactive,
respectively); all forms are proposed to be fully activable in
vitro. The existence of the intermediate forms that are connected
by arrows are supported by some evidence. Form IIa would
correspond to a situation in which activation of the enzyme is not
accompanied by dephosphorylation or appreciable translocation to
membranes, such as in phorbol ester-treated HeLa cells(16) .
Form IIa may be active in a soluble form because it is activated by
association with a lipoprotein complex(9) . Form IIb would
correspond to the 16S
Figure 6:
Possible
intermediate steps in activation, dephosphorylation, and association of
cytidylyltransferase with membranes. I, inactive; A,
active. The coils represents the membrane-binding
domains(32) , and the hatched bars represent the
membranes.
The fact that
membrane-associated 16S
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
AP, in which five of the seven Ser-Pro sequences were converted to
Ala-Pro; 7SP
AP, in which all of the seven Ser-Pro sequences
converted to Ala-Pro; 16S
A, in which all sixteen Ser residues
that can be phosphorylated in wild type cytidylyltransferase were
converted to Ala; and 16S
E, in which all sixteen Ser residues
were converted to Glu. The mutant enzymes were expressed in the strain
58 Chinese hamster ovary cell line, which is temperature-sensitive for
growth and cytidylyltransferase activity. All mutant enzyme forms were
enzymatically as active as the wild type when assayed under optimal
conditions. In untreated cells, more of the Ser
Ala mutants were
membrane-associated than in cells expressing wild type enzyme,
consistent with the phosphorylation state of the enzyme affecting its
affinity for membranes. About half of the 16S
A mutant remained
soluble, however, indicating that dephosphorylation alone does not
trigger membrane association. Although the amount of
membrane-associated enzyme in the 16S
A mutant was about 10-fold
greater than that of wild type, phosphatidylcholine synthesis was
increased by only about 75%, suggesting that membrane association does
not necessarily cause full activation. All mutant forms, including the
16S
E mutant, translocated to the particulate fraction upon
oleate treatment, indicating that a high negative charge in the
phosphorylation region does not preclude association of
cytidylyltransferase with membranes. All mutant enzymes were able to
support growth of strain 58 at 40 °C, and the rate of
phosphatidylcholine synthesis was not greatly altered in the cell lines
expressing mutant cytidylyltransferase forms. These results are
consistent with a role for phosphorylation in the equilibrium
distribution of cytidylyltransferase but suggest that changes in enzyme
activity and location are not triggered exclusively by changes in the
phosphorylation state.
(
)cells with okadaic acid, an inhibitor of protein
phosphatase 1 and 2A, prevents dephosphorylation and translocation of
cytidylyltransferase in response to phospholipase C, indicating that
protein dephosphorylation is a necessary component of
activation(18) . It is not clear, however, whether the protein
that must be dephosphorylated is cytidylyltransferase or another
protein involved in the transduction of the signal from exogenous
phospholipase C to cytidylyltransferase. Okadaic acid does not block
translocation and dephosphorylation of cytidylyltransferase in HeLa
cells stimulated with oleate(19) , suggesting either that the
target of okadaic acid in the CHO cell system is a protein distinct
from cytidylyltransferase or that different protein phosphatases
activate cytidylyltransferase in the different cell systems.
, to Ala and found
that this change did not affect the activity of cytidylyltransferase,
its ability to translocate upon stimulation of cells expressing the
mutant, or its ability to support growth of the transfected cells at 40
°C(22) . Subsequent analysis of additional mutants in which
only a single site was mutated also revealed little, if any, change in
cytidylyltransferase function or location.
(
)In
the present study, we characterized mutants in which 5-16
phosphorylation sites were altered. We report that, although these
modifications dramatically altered the phosphorylation state of
cytidylyltransferase and in some cases modified its location, the
activation state of the enzyme was not appreciably altered.
Materials
Sodium oleate, Ham's F-12
medium, and phosphate-free Dulbecco's modified Eagle's
medium were from Sigma. P
(400-800
mCi/ml) was from ICN. [methyl-
H]Choline
chloride and [methyl-
C]choline chloride
were from Amersham Corp. Protein A agarose, Lipofectin reagent, and
G418 were from Life Technologies, Inc. QIAGEN supplied a plasmid
purification kit. The pCMV5 vector was a gift from Dr. David Russell,
University of Texas. All other reagents were from previously described
sources(17, 19) .
Construction of Phosphorylation Site Mutants
To
make the 5SP AP and 7SP
AP mutants, the HindIII-XbaI fragment of cytidylyltransferase from
pCMV4RCCT (22) was transferred into the pAlter vector of the
Promega Altered Sites mutagenesis system. Site-directed mutagenesis was
carried out to construct a 4SP
AP mutant; the oligonucleotides
were 5`-ATGAGTAGGGGCGCTGCTGGGAGCCTGCTTGGA for S319A,S323A and
5`-TGCTGGGGCGGAAGATGGGGCAGTCTTGC for S343A,S347A. The HindIII-XbaI fragment was then transferred to
M13mp19, and the Amersham in vitro mutagenesis kit was used to
convert Ser
to Ala with the oligonucleotide
5`-GCTTGGGAGCGATGGCCT to give 5SP
AP. The 7SP
AP mutation
was then constructed with 5SP
AP as template and
5`-GGAGGGGGCGGGGGCGCGCTCAT as the mutagenic oligonucleotide to
introduce S329A and S331A. The resulting mutant constructs were
sequenced entirely to ensure that only the desired mutants were
obtained. The HindIII-XbaI fragments were then cloned
into the HindIII-XbaI site of pCMV5.
A and 16S
E mutants was to use
multiple-round PCR to make a mutated fragment that became longer at the
COOH terminus with each round; the COOH-terminal primer for the second
through the fifth rounds overlapped with the COOH-terminal primer from
the previous round. The first round introduced mutations at Ser
residues 315, 319, 321, 322, and 323; the second round introduced
mutations at Ser residues 329, 331, and 333; the third round introduced
mutations at Ser residues 339, 343, 345, 346, and 347; the fourth round
introduced mutations at Ser residues 350 and 352; and the fifth round
introduced the mutation at Ser residue 362. The template for the PCR
was pCMV5CT, which was constructed by subcloning the HindIII-XbaI fragment of cytidylyltransferase from
pCMV4RCCT into the pCMV5 HindIII-XbaI site. The PCR
program was 92 °C for 1 min, 52 °C for 1 min, and 72 °C for
2 min for 20 cycles for each round. The NH
-terminal primer
for all rounds, corresponding to the first 16 base pairs of the coding
region for cytidylyltransferase, was
5`-CGCGGATCCAGATCTATGGGATGCACAGAGTTCA. For the first round, wild type
rat liver cytidylyltransferase cDNA in plasmid pCMV5CT was used as
template; the COOH-terminal primers were
5`-GAGTAGGGGCGGCGGCGGGAGCCTGCTTGGGAGCGATGGCCTGCAGCAT for 16S
A
and 5`-ATGAGTAGGTTCCTCTTCGGGTTCCTGCTTGGGTTCGATGGCCTGCAGCAT for 16S
E. The PCR product from the first round was separated from wild
type template by agarose gel electrophoresis and used as the second
round PCR template. For the second round the COOH-terminal primers were
5`-GGCCACCGAAAGGCGGGGGCGGGGGCGCGCTCATGAGTAGGGGCGGCGG for 16S
A
and 5`-GAAGGGCCACCGAAATTCAGGCTCGGGTTCGCGCTCATGAGTAGGTTCCTC for 16S
E. The template for the third, fourth, and fifth rounds were the
PCR products from the previous rounds. The COOH-terminal primers for
the third round were
5`-TGGGGCGGCAGCTGGGGCAGTCTTGCCAGCGAAGGGCCACCGAAAGGCG for 16S
A
and 5`-TTCTGCTGGTTCCTCTTCTGGTTCAGTCTTGCCTTCGAAGGGCCACCGAAA for 16S
E. The COOH-terminal primers for the fourth round were
5`-CACAGGTCACAGCCTTGCACCTAGCGAGAGCTGCTGGGGCGGCAGCTGG for 16S
A
and 5`-GATGTCACAGGTCACAGCCTTGCACCTTTCGAGTTCTTGCTGGTTCCTC for 15S
E. The COOH-terminal primers for the fifth round were
5`-TGCTCTAGATTAGTCCTCTTCATCCTCGGCGATGTCACAGGTCACAGCCT for 16S
A
and 5`-TGCTCTAGATTAGTCCTCTTCATCCTCTTCGATGTCACAGGTCAC for 16S
E.
A and pCMV5CT16S
E. All of the constructed plasmids were purified with the QIAGEN
Maxi-kit.
Cell Culture
CHO strain 58 cells were cultured
with Ham's F-12 medium plus 10% fetal bovine serum at 34 °C
with 5% CO. Stably transfected cell lines were cultured
with the same medium for experiments and with the addition of 20 mM HEPES and 0.8 mg/ml G418 for regular maintenance. Cells were
plated at 1
10
cells/60-mm dish for 1 day at 34
°C before experiments unless otherwise specified.
Stable Transfection
Wild type and mutant
constructs of cytidylyltransferase were transfected into CHO 58 cells
with Lipofectin as recommended by the manufacturer. Cells were plated
at 2 10
cells/60-mm dish, cultured for 1 day, and
then incubated with Lipofectin and the DNA overnight. The transfection
was stopped by the addition of medium containing 20% fetal bovine
serum, after which the cells were incubated in normal medium for 2
days. The cells were then trypsinized and subcultured in four 150-mm
dishes with 0.8 mg/ml G418. Single colonies were picked, replated, and
screened by indirect immunofluorescence (19) for expression of
cytidylyltransferase.
Cell Labeling
For measuring the rate of choline
incorporation into phosphatidylcholine, cells were plated at 1
10
cells/60-mm dish at 34 °C for 1 day. The cells were
then washed twice with calcium- and magnesium-free phosphate-buffered
saline and incubated in 1.5 ml of culture medium containing 2
µCi/ml [
H]choline for 0.5-2 h at either
34 or 40 °C. The cells were then washed twice with calcium- and
magnesium-free phosphate-buffered saline and harvested by scraping into
1.0 ml of H
O at 0 °C. Lipids were extracted from 0.8 ml
of the cell extract by the Bligh-Dyer method(24) . The
chloroform phase containing the lipids was dried and counted.
10
cells/60-mm dish and
incubated at 34 °C for 1 day. The cells were then washed twice with
calcium- and magnesium-free phosphate-buffered saline, fed with culture
medium containing 0.5 µCi/ml [
C]choline, and
incubated at 34 °C for 1 more day. Maintaining the cells in the
labeled medium, two dishes of each cell line were then kept at 34
°C for an additional day, while two other dishes were incubated at
40 °C for the additional day. The cells were then washed twice with
calcium- and magnesium-free phosphate-buffered saline and harvested by
scraping into 1 ml of H
O. Lipids and aqueous metabolites
were separated by Bligh-Dyer extraction, and the lipid phase was dried
and counted. Under these conditions 90% of the lipid-associated label
was in phosphatidylcholine. The aqueous phase was dried, dissolved in
60 µl of methanol:H
O (1:1), and chromatographed on a
silica gel G thin layer plate in methanol:5% NaCl:ammonia (50:50:1).
The plates were dried and exposed to a PhosphorImager screen, and
metabolites were quantitated in a Molecular Dynamics PhosphorImager.
Labeling and Immunoprecipitation of
Cytidylyltransferase
Labeling, immunoprecipitation, and
detection of cytidylyltransferase by Western blotting and
autoradiography were performed as described(19) .
Two-dimensional peptide mapping was performed as
described(17) ; the electrophoresis buffer was 1% ammonium
bicarbonate.Immunoblots
Western blots were performed as
described (19) except that a 1:6000 dilution of amino-terminal
antiserum was used as the first antibody.Growth Curve
Cells were plated at 2.3
10
cells/60-mm dish and incubated for 1 day at 34 °C.
The cells were then incubated at 40 °C for up to 5 days. Viable
cell counts were determined by counting cells excluding trypan blue.
Two dishes of each cell line were counted each day.
Enzymatic Assay
Cells were fractionated into
soluble and particulate fractions by use of digitonin(19) .
Cytidylyltransferase activity was determined with previously described
conditions for enzyme incubation (25) and binding of the
product to charcoal(6) .Oleate Treatment
Cells were treated with 0.25
mM sodium oleate in F12 medium without serum for 20 min at 34
°C. Under these conditions the cells were viable as determined by
the exclusion of trypan blue, and the translocation of
cytidylyltransferase was reversible by transfer to fresh medium without
oleate.Protein Assay
Protein was determined by the
Bradford method (26) .
Construction of Phosphorylation Site Mutants
In
order to test the effect of phosphorylation on the properties of
cytidylyltransferase, we introduced mutations in phosphorylation sites
by site-directed mutagenesis as described under ``Experimental
Procedures.'' The sequences encoded by the wild type and mutant
constructs are listed in Table 1. In the 5SP AP mutant,
the Ser residues followed by Pro that are found in the repeated SPSSSP
sequence plus an additional Ser followed by Pro at position 315 were
changed to Ala. In the 7SP
AP mutant, the remaining two Ser
residues followed by Pro were similarly modified. All 16 Ser residues
from position 315 to the COOH terminus were mutated to Ala in 16S
A, so that the resulting mutant cytidylyltransferase could not
be phosphorylated. The same residues were changed to Glu in 16S
E in an attempt to mimic the negatively charged phosphorylated enzyme.
Stable cell lines of strain 58 CHO cells transfected with each mutant
were isolated and screened with indirect immunofluorescence to monitor
purity of the clones. Cytidylyltransferase was nuclear in all clones
expressing either wild type cytidylyltransferase or the phosphorylation
site mutants.
To determine the level of phosphorylation of the
cytidylyltransferase mutants, the cells expressing the mutant enzymes
were labeled with P-Labeling of Phosphorylation Site
Mutants
P
, and the mutant enzymes
were immunoprecipitated and analyzed by Western blotting and
autoradiography (Fig. 1). As expected, there was no
phosphorylation detected in the mutants in which all 16 phosphorylation
sites were mutated either to Ala or Glu. The extents of phosphorylation
of 5SP
AP and 7 SP
AP were considerably decreased. The
low degree of phosphorylation in the latter two mutants suggested that
one or more Pro-directed protein kinases may be important in
determining whether or not other sites are modified.
P
, and cytidylyltransferase was
immunoprecipitated as described under ``Experimental
Procedures.'' The samples were then separated by
SDS-polyacrylamide gel electrophoresis and detected by immunoblotting (A), and the blot was subjected to autoradiography (B). Lane 1, 5SP
AP; lane 2, 7SP
AP; lane 3, 16S
E; lane 4, 16S
A; lane 5, wild type.
AP mutant, only two major phosphopeptides were observed for 5SP
AP. When the 5SP
AP peptides were mixed with those of the
wild type, the two major peptides from 5SP
AP did not coincide
with the major peptides of wild type cytidylyltransferase. Only one
major phosphorylated peptide was observed with 7SP
AP. From the
position of this peptide, it appears to correspond to the COOH-terminal
tryptic pepide, which contains a putative casein kinase II
site(22) . The minor phosphopeptide does not appear to coincide
with peptides from the wild type. Because of the low amount of label
associated with 7SP
AP, there was insufficient material to mix
with wild type peptides.
P-labeled cytidylyltransferase. Labeled samples were
immunoprecipitated and blotted as described for Fig. 1. After
the labeled cytidylyltransferase bands were detected by
autoradiography, the bands on the blots were excised, digested with
trypsin, and subjected to two-dimensional mapping as described under
``Experimental Procedures.'' A, wild type. B, 5SP
AP. C, a mixture of 5SP
AP and
wild type. D, 7SP-AP.
Activities and Subcellular Distributions of
Phosphorylation Site Mutants
To determine if the changes in
phosphorylation sites dramatically affected the catalytic ability of
cytidylyltransferase, soluble and particulate fractions were assayed
under optimal conditions in the presence of lipids, and the activity
was normalized to the amount of cytidylyltransferase protein in the
fraction as determined by immunoblotting (Table 2). Under these
conditions, the activities of the cytidylyltransferase mutants were
essentially the same as the activity of wild type cytidylyltransferase.
The mutant enzymes required lipids for maximal activity in the assay in vitro, as does the wild type enzyme; the extent of
activation of the mutant enzymes by lipids was the same as that of the
wild type enzyme (data not shown).
AP, 7SP
AP, and 16S
A, respectively. It is notable that removal of all the phosphorylation
sites, however, did not cause complete translocation to the membrane of
16S
A; about 60% of the 16S
A mutant enzyme remained
soluble. As expected, conversion of all the sites to Glu resulted in a
normal, soluble distribution in unstimulated cells. The Western blots
agreed with the activity measurements regarding the subcellular
distributions and expression levels of the mutants (Fig. 3). The
16S
E mutants had a considerably slower mobility than the 16S
A mutant. This decreased mobility is similar to the effect of
phosphorylation on protein mobility and is consistent with the higher
charge of this mutant cytidylyltransferase.
E S minus oleate and 16S
E S and P plus oleate and 0.5 volume for 16S
E P minus oleate. A, control cells. B,
oleate-treated cells. Lanes 1, wild type; lanes 2,
16S
A; lanes 3, 16S
E; lanes 4; 7SP
AP.
Translocation of Phosphorylation Mutants in Response to
Fatty Acid Treatment
Fatty acid treatment causes translocation
and activation of cytidylyltransferase in HeLa and other cultured cells (11, 27, 28) . CHO cells are much more
sensitive to oleate treatment than HeLa cells, but we determined that
cells remained viable when treated with 0.25 mM oleate for 20
min, which is sufficient for translocation of cytidylyltransferase. To
ask if the phosphorylation mutants could translocate in response to
cell stimulation, the cell lines were treated with oleate, and the
distributions were determined by activity measurements and Western
blots. Oleate stimulated translocation of 77% of wild type
cytidylyltransferase to the particulate fraction (Table 3), and
this correlated with a shift in mobility from multiple bands in the
soluble fraction to a faster migrating band in the particulate fraction (Fig. 3). All of the Ser to Ala mutants translocated to a
similar extent as the wild type enzyme. A considerable amount of the
16S E mutant also translocated in response to oleate. Although
the percentage of total 16S
E enzyme that translocated appeared
less than that in wild type, the actual amount of membrane-associated
enzyme in the 16S
E mutant was about three times higher than in
wild type due to the higher level of expression of this mutant. Thus,
the high negative charge in the phosphorylation region of the 16S
E mutant did not preclude its association with the membrane.
Abilities of Phosphorylation Mutants to Complement the
Defects in Strain 58
Because of its temperature-sensitive
cytidylyltransferase, the strain 58 cell line cannot grow or synthesize
phosphatidylcholine at 40 °C(20, 21) . Expression
of rat liver cytidylyltransferase in strain 58 cells, however, corrects
the defect and allows both phosphatidylcholine synthesis and growth at
40 °C (22) . All phosphorylation site mutants were capable
of supporting growth of strain 58 CHO cells at 40 °C (Fig. 4), consistent with the fact that these mutant enzymes
retain catalytic activity (Table 2). At either 34 °C or 40
°C, all cell lines expressing wild type or mutant
cytidylyltransferases incorporated radiolabeled choline into
phosphatidylcholine far better than did untransfected strain 58 control (Fig. 5). The rates of choline incorporation relative to wild
type were somewhat variable for most mutants; cells expressing 16S
A, however, always had a somewhat higher rate of choline
incorporation than the wild type and other mutants. The increased
choline incorporation in 16S
A, however, was far less than
expected, considering there was about 10-fold more membrane-associated
enzyme in these cells (Table 3). In a total of six labeling
experiments, including one pulse-chase protocol, the rate of
phosphatidylcholine synthesis was 74 ± 46% higher than in the
wild type. This suggests that cytidylyltransferase can be
membrane-associated and still be relatively inactive.
H]choline at 40 °C for
the indicated times. The cells were then harvested and extracted as
described under ``Experimental Procedures.'' WT,
wild type; CHO 58, untransfected strain 58 cells.
P of the 16S
A and 16S
E mutants confirmed
the protein chemistry, indicating that no other residues are
phosphorylated in these mammalian cells. Labeling of the mutants in
which Ser-Pro sites were modified to Ala-Pro sites resulted in rather
dramatic changes in labeling; loss of a third of the 16
phosphorylatable sites in the 5SP
AP mutant reduced labeling to
only two major tryptic peptides, and loss of only two additional sites
in the 7SP
AP mutant nearly eliminated labeling. These results
suggest that modification by a Ser-Pro-directed protein kinase may be
important for subsequent phosphorylation events by other protein
kinases. This is supported by the observations of
Jackowski(29) , who has shown that the phosphorylation state of
cytidylyltransferase varies with the cell cycle, in which several
Ser-Pro-directed kinases function.
E mutant in control cells did not differ from that of the
wild type enzyme, which is not surprising given that most of the wild
type enzyme in control cells is soluble and highly phosphorylated. On
the other hand, the subcellular location of all mutants in which Ser
residues were changed to Ala was modified considerably. The specific
activities of membrane-associated mutant enzymes assayed in vitro were 5-17-fold higher than that of wild type
cytidylyltransferase in control cells, suggesting that
dephosphorylation certainly promoted membrane association. The
membrane-associated enzymes were not fully activated, however, as
indicated by the lack of a dramatic increase in phosphatidylcholine
biosynthesis. It is not likely that the membrane-associated 16S
A enzyme was actually 10-fold more active but that the rate of
phosphatidylcholine degradation was also increased, because the
experiments represented in Fig. 5were measuring initial rates
of phosphatidylcholine biosynthesis. Furthermore, there was no increase
in glycerophosphocholine levels in the 16S
A cells, suggesting
that increased turnover did not occur.
A mutant that remained soluble ranged from about 40 to 60%, indicating
that a completely dephosphorylated cytidylyltransferase can remain
soluble. This suggests that dephosphorylation of cytidylyltransferase
does not cause it to associate with the membrane but rather influences
the extent of its association with the membrane. The lack of extensive
phosphorylation of soluble cytidylyltransferase has previously been
noted upon reversal of fatty acid treatment in HeLa cells(19) .
Within 1 or 2 min after removal of the fatty acid, cytidylyltransferase
becomes predominantly soluble but migrates with the fast mobility of
the membrane associated form, indicating that it is not highly
phosphorylated. It was not clear from those studies, however, if the
fast moving, soluble form is partially phosphorylated. In
choline-starved HepG2 cells, forms of cytidylyltransferase with
similarly low levels of phosphorylation are found in both soluble and
membrane fractions(14) .
A mutant became membrane-bound with oleate treatment lends
further support to the concept that a process distinct from
dephosphorylation is involved in causing the enzyme to associate with
membranes. In addition, the fact that the 16S
E mutant did not
seem impaired in its ability to interact with membranes indicates that
a high degree of negative charge in the phosphorylation region does not
preclude cytidylyltransferase from associating with membranes. Although
a Glu residue would not be as highly charged as phosphoserine, the
evidence implies that phosphorylation per se does not keep
cytidylyltransferase soluble. This is consistent with the observation
that, upon treatment of rat hepatocytes with fatty acids or
phospholipase C, phosphorylated cytidylyltransferase first becomes
membrane-associated and subsequently is dephosphorylated(30) .
A mutant in control cells where much of
the enzyme remains soluble. Whether such a form exists for wild type
cytidylyltransferase remains to be determined. The fact that the 16S
E mutant can readily associate with membranes in oleate-treated
cells supports the existence of form IIIa. Moreover, form IIIa is
strongly indicated by the translocation of phosphorylated
cytidylyltransferase in rat hepatocytes discussed above(30) .
Form IIIb, which is soluble, active, and relatively dephosphorylated,
is found in choline-starved HepG2 cells (14) and immediately
after removal of oleate from HeLa cells(19) . Form IIIc would
correspond to the 16S
A mutant that is membrane-associated but
relatively inactive in control cells. We are not aware of evidence
supporting form IIc, in which inactive, phosphorylated
cytidylyltransferase is membrane-associated, but such a form cannot yet
be ruled out. Although evidence suggests that these various forms can
exist, the preferred route of interconversion of forms I and IV is not
yet clear. Multiple pathways may operate in some circumstances. For
example, the route of formation for form IIIb in choline-starved cells
may be I
IIa
IIIa
IV
IIIb.
A is not fully activated suggests that
an additional component in the membrane is required to activate the
enzyme. The question as to what causes activation of
cytidylyltransferase in vivo is currently unresolved, but much
evidence supports the concept that a suitably altered change in
membrane lipid content can activate
cytidylyltransferase(1, 31) . What role, if any, does
phosphorylation play? It seems evident that phosphorylation and
dephosphorylation are not the triggers to inactivate and activate,
respectively, cytidylyltransferase. The phosphorylation state of the
enzyme, however, may be important for stabilizing it in either its
soluble or membrane-associated forms. This in turn would shift the
equilibrium between soluble and membrane forms, serving to promote the
continued activation or inactivation of the enzyme.
We thank Jharna Saha for technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.