From the
The role of the phosphorylated carboxyl-terminal domain of
CTP:phosphocholine cytidylyltransferase (CT) in the regulation of
enzyme activity was investigated by comparing the catalytic properties
of wild-type CT to two mutant proteins with altered carboxyl-terminal
phosphorylation domains. CT isolated from a baculovirus expression
system was extensively phosphorylated at multiple sites in the
carboxyl-terminal domain. The CT[S315A] mutant lacked a major
CT phosphorylation site, and the carboxyl-terminal deletion mutant,
CT[
CT
CT is
extensively phosphorylated at multiple sites in vivo, and a
detailed analysis of CT phosphorylation shows that these sites are
exclusively located in the carboxyl-terminal domain (residues
312-367)(21) . There is a correlation between the degree
of CT phosphorylation and the activity and membrane association of CT
in Chinese hamster ovary (22) and HeLa cells(23) . The
stimulation of PtdCho synthesis by exogenous 18:1 correlates with CT
dephosphorylation and enzyme association with the particulate
fraction(23) , whereas okadaic acid increases CT phosphorylation
and decreases membrane association(24) . The pattern of CT
phosphorylation in vivo is
complex(21, 22, 25, 26, 27) ,
and the negative correlation between CT phosphorylation and membrane
association is not always observed (27) indicating that
phosphorylation does not prevent membrane binding. Treatment of cells
with phorbol esters (28) increases PtdCho synthesis, whereas
treatment with cAMP analogs decreases PtdCho
synthesis(29, 30) . However, the phosphorylation state
of CT is not affected by these treatments (25, 31, 32) indicating that factors other than
phosphorylation regulate CT activity under these circumstances. Since
the cellular content of DAG increases in phorbol ester-treated cells
(33) and decreases in cAMP-treated cells(32) , the intracellular
concentration of DAG may be a determinant of CT activity.
The
biochemical consequences of CT phosphorylation are not clear. Sanghera
and Vance (34) report that dephosphorylation of purified CT
increases its catalytic activity whereas phosphorylation of CT with
protein kinase A decreases activity by 30%. The goal of the present
work is to define the biochemical basis for the modulation of CT
activity by the phosphorylated carboxyl-terminal domain.
The SacI/BamHI fragment of rat CT (0.57 kilobase)
was subcloned into pBS and used as the template for site-directed
mutagenesis. The mutant primer was designed on the basis of the
nucleotide sequence between bp 1038 to bp 1058 which contains the codon
for Ser-315 of CT. The primer, GGCCATCgcTCCCAAGCAGAG, substituted the
original AGT codon for Ser-315 with GCT and thus changed Ser-315 to
Ala-315. PCR site-directed mutagenesis used the mutant primer and the
M13 reverse primer to generate the first round PCR product. The first
round PCR product was then used together with the M13 forward primer to
generate the second round PCR product(37) . The second round PCR
product was precipitated with ethanol and digested by SacI/BamHI. The digested fragment (0.57 kilobase) was
ligated into the pBS plasmid, and the DNA sequence was confirmed. The
mutated SacI/BamHI fragment of CT was isolated and
co-ligated with digested pGEMEX-1 (SalI/BamHI), and
the SalI/SacI fragment was isolated from pCT-N/B.
This construct was denoted as pGEMEX-1CTS315A and contained the full
coding region of CT with a S315A point mutation. The NcoI/HindIII fragment of pGEMEX-1CTS315A was inserted
into pBlueBacIII. This construct was denoted as pCTS315A and used to
generate the baculovirus that expressed the CT[S315A] mutant
protein. The presence of the mutation and absence of other changes in
the nucleotide sequence were confirmed by DNA sequence analysis.
Sweitzer and Kent (26) reported that the original rat CT cDNA
was a cloning artifact containing 2 incorrect bases generating a G91S
and C114S mutant. These mutations were removed from our constructs by
replacing the EcoRI/StyI fragment (bp 189-783)
of the rat CT cDNA clone with the corresponding fragment of the murine
CT cDNA.
Our results provide a framework to interpret in vivo experiments that have investigated the relationship
between CT phosphorylation, membrane association, and PtdCho synthesis.
A model for the function of the phosphorylated CT carboxyl terminus
suggests that CT phosphorylation will interfere with CT association
with membrane bilayers, but will not prevent binding to membranes
containing high concentrations of activating lipids. Accordingly, some in vivo studies show a negative correlation between CT
phosphorylation and membrane association(22, 23) .
Furthermore, the addition of 18:1 to cells triggers CT
dephosphorylation concomitant with increased translocation to the
membrane (23) in contrast to the action of okadaic acid which
increases CT phosphorylation and decreases membrane
association(24) . However, there is also evidence that
membrane-associated CT is not completely
dephosphorylated(17, 22, 23, 27) . These
experiments rely on the addition of pharmacological amounts of potent
lipid regulators to cells to modify CT phosphorylation and/or membrane
composition. The model predicts that the effects of phosphorylation on
CT activity are not absolute, but rather represent a more subtle fine
tuning of enzyme activity. CT phosphorylation will attenuate enzyme
activity only under conditions where the concentrations of lipid
regulators are low, a situation that may more accurately reflect
conditions normally found in vivo.
Our experiments do not
indicate whether one carboxyl-terminal phosphorylation site is more
important than another in regulating lipid association. A detailed
analysis of CT phosphorylation in Sf9 cells shows that virtually all
Ser residues in the carboxyl-terminal domain are phosphorylated to some
degree and that the phosphorylation of CT in mammalian cells is at
least as complex(21) . One possibility is that each
phosphorylation site in the carboxyl-terminal domain makes a
contribution to decreasing the affinity for lipid activators and
promoting negative cooperativity. This hypothesis suggests that the
carboxyl-terminal domain is a combinatorial switch capable of receiving
regulatory signals from a number of different kinases, the sum of which
combine to interfere with the stimulation of the enzyme by lipid
regulators. CT is ubiquitously expressed, and this hypothesis opens the
intriguing possibility that CT activity can be effectively regulated
not only by ubiquitously expressed protein kinases, but also by
tissue-specific protein kinases and/or by multiple kinases in the same
tissue whose activities are controlled by different physiological
stimuli. Although these kinases may phosphorylate different sites
within the carboxyl-terminal domain, each phosphorylation event would
contribute to negative cooperativity and lead to the down-regulation of
CT activity. Our analysis of the CT[S315A] mutant supports
this idea. The fact that K
We thank Dr. Charles O. Rock for his critical comments
on this research and Margarita Perez Pecha and Huong Nguyen for their
excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
312-367], was not phosphorylated. The higher
activities of CT[
312-367] and CT[S315A]
relative to CT were attributed to differences in the sensitivities of
the enzymes to lipid activators. The rank order of the apparent K
values for activation by either
phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol
was CT > CT[S315A] > CT[
312-367].
In addition, CT exhibited negative cooperativity in its activation by
phosphatidylcholine/oleic acid (n
= 0.64)
and phosphatidylcholine/diacylglycerol (n
=
0.74) vesicles, whereas CT[
312-367] and
CT[S315A] did not. These data support the concept that the
phosphorylation of the CT carboxyl-terminal domain interferes with the
activation of CT by lipid regulators.
(
)is considered a rate-controlling
enzyme in PtdCho biosynthesis (for reviews, see Refs. 1-3). CT
exhibits negligible activity in vitro in the absence of lipid
activators, and its stimulation by lipids is thought to contribute to
controlling the activity of the enzyme in vivo. CT activity is
stimulated by acidic lipids (such as 18:1) or DAG incorporated into
PtdCho bilayers(4, 5, 6) . Accordingly, the
pharmacological manipulation of cells by the addition of exogenous 18:1
or phosphatidylcholine-phospholipase C are both associated with
enhanced rates of PtdCho synthesis and increased association of CT with
cellular
membranes(7, 8, 9, 10, 11) . CT
activity and membrane association increase in cells deficient in PtdCho
correlating with the increased relative abundance of anionic
phospholipids(12, 13, 14, 15) . CT is
also potently inhibited by sphingosine(16) ,
lysophosphatidylcholine(17) , and antineoplastic
phospholipids(17) . The region between residues 251 and 286 of
the protein constitutes an amphipathic helical domain similar to
lipoprotein domains known to directly interact with
phospholipids(18) . Evidence obtained from limited chymotrypsin
proteolysis (19) and antibodies directed against the helical
region (20) are consistent with the idea that the helical domain is
responsible for the association of CT with lipid activators.
Materials
The rat CT cDNA was provided by Dr. R.
B. Cornell (18), the murine CT cDNA was isolated in our
laboratory(35) , and the CT recombinant baculovirus vectors were
constructed and expressed in Sf9 cells as described
previously(36) . [P]Orthophosphate
(carrier-free) and
phospho[methyl-
C]choline (specific
activity 55 mCi/mmol) were from DuPont NEN. Cell culture media were
obtained from Life Technologies, Inc., and thin layer plates were from
Analtech. Molecular biology reagents were obtained from Promega.
Construction of the CT[
The carboxyl-terminal deletion
mutant pCTD312 was constructed and a PCR mutant was used to introduce
an MscI site at bp 1040 of CT, and this mutant was subcloned
into pBlueBacIII. The region between bp 1040 and bp 1342 was deleted by
digestion of the resulting plasmid with MscI/NotI.
The NotI site was filled with Klenow fragment, and the plasmid
was religated. In this construct, the codons for Gln-312 through
Asp-367 were deleted, and the codons for five additional amino acid
residues (Leu-Gly-Pro-His-Ala) were added after the codon for Leu-311.
312-367] and
CT[S315A] Mutants
Delipidation of Sf9 Cell CT Lysate
Endogenous
lipids were removed from the CT preparation by DEAE-Sepharose
chromatography as described previously(17) .
CT Assay
The standard CT activity assay contained
the delipidated CT, CT[S315A], or
CT[312-367] fraction (0.4-2 µg of
protein) in a final volume of 40 µl containing 120 mM bis-Tris-HCl, pH 6.5, 1 mM
phospho[methyl-
C]choline (0.5 µCi),
2 mM CTP, 80 µM PtdCho/18:1 (1:1). The reaction
was for 10 min at 37 °C and was stopped by the addition of 5 µl
of 0.5 M EDTA and incubated on ice.
CDP-[
C]choline formation was determined by thin
layer chromatography(36) . Protein was determined according to
the method of Bradford(38) .
Kinetic Analysis
The degree of cooperative binding
of CT to PtdCho/18:1 or PtdCho/DAG vesicles was determined by analyzing
the data according to a transformation of the Hill equation: log
[(v/(V - v)]
= n
log[A] - logK,
where v is the CT catalytic rate, V
is
the CT maximum catalytic rate, n
is the Hill
coefficient constant, A is the concentration of lipid activator, and K is a constant(39) . Under these conditions, the
constant (K) approximates the K
value predicted by the Michaelis-Menten equation. Linear
correlation coefficients for the data used to determine the slope (n
) in the Hill plots exceeded 0.98 in all cases. V
values used in the Hill plots were determined
independently by double reciprocal plots using the data obtained with
40-120 µM PtdCho/oleate (1:1) or 1-32
µM PtdCho/DAG (3:1). The K
values for CTP or phosphocholine were obtained from the
Michaelis-Menten equation by a double reciprocal plot of 1/Vversus 1/S at 80 µM PtdCho/18:1 (1:1).
Expression and Phosphorylation of CT,
CT[S315A], and CT[
To
biochemically characterize CT and its carboxyl-terminal modifications,
these three proteins were expressed in Sf9 cells following infection
with recombinant baculoviruses(36) . The endogenous lipids were
removed, the proteins were partially purified by DEAE-Sepharose
chromatography, and the purity and relative levels of CT,
CT[S315A], and CT[312-367]
312-367] in these
preparations are shown in Fig. 1A. The CT band was
always less distinct than either the CT[S315A] or
CT[
312-367] band since two isoforms of wild-type
CT were present in Sf9 cells and the more extensively phosphorylated CT
isoform migrates slightly slower than the less extensively
phosphorylated form of CT(40) . The degree of CT,
CT[S315A], and CT[
312-367]
phosphorylation was determined by labeling the Sf9 cells with
[
P]orthophosphate and immunoprecipitating the CT
proteins from the cell lysates (Fig. 1B). CT and
CT[S315A] were extensively phosphorylated in this experiment,
whereas
P label was not incorporated into the
CT[
312-367] mutant. These data are consistent with
the results of MacDonald and Kent (21) who used analytical
techniques to demonstrate that the extensive phosphorylation of CT
expressed in Sf9 cells was localized to the carboxyl-terminal domain.
Therefore, in our experiments, a mixture of phosphorylated CT isoforms
are produced by Sf9 cells, and our kinetic analysis reflects the
average property of the mixture.
Figure 1:
Expression and phosphorylation of CT,
CT[S315A], and CT[312-367] in Sf9 cells.
Sf9 cells were infected with recombinant baculoviruses expressing
either CT, CT[S315A], or CT[
312-367],
and cells were harvested 48 h after infection. A, cells were
lysed, and endogenous lipids were removed by ion exchange
chromatography as described under ``Experimental
Procedures.'' The column fractions containing either the CT,
CT[S315A], or CT[
312-367] used in the
biochemical assays reported in this paper were subjected to SDS-gel
electrophoresis and stained with Coomassie Brilliant Blue. B,
Sf9 cells were infected with recombinant baculoviruses expressing
either CT, CT[S315A], or CT[
312-367]. At
48 h after infection, the cells were labeled with
[
P]orthophosphate and incubated for an
additional 24 h. The cells were then lysed and immunoprecipitated with
anti-CT polyclonal antibody (40). The immune complexes were separated
by SDS-gel electrophoresis, and location of the radiolabeled proteins
on the gel was determined by autoradiography.
CTP and Phosphocholine Kinetic Constants
The
kinetic substrate affinities calculated for CT, CT[S315A],
and CT[312-367] were similar (). The
apparent CTP K
calculated from
double-reciprocal plots was 0.3 mM for CT, 0.2 mM for
CT[S315A], and 0.5 mM for
CT[
312-367]. The apparent phosphocholine K
was 0.7 mM for CT, 0.8 mM for CT[S315A], and 0.5 mM for
CT[
312-367]. These data indicated that there was
little difference between wild-type, CT[S315A], and truncated
CT with respect to the interaction between the substrates and the
enzymes.
Differences in the Regulation of CT and
CT[
The
regulation of CT activity by lipid activators was examined following
the removal of endogenous lipids from the protein preparation by ion
exchange chromatography. CT and CT[312-367] by PtdCho/18:1 Vesicles
312-367]
exhibited essentially no activity following removal of the endogenous
lipids. The most potent lipid activators of CT are anionic compounds
typified by 18:1. Both enzymes were significantly stimulated by the
presence of PtdCho/18:1 vesicles (Fig. 2A), and the
lipid activation kinetics were analyzed by double reciprocal plots (Fig. 2B). There was a significant discrepancy between
the activities of the two proteins at PtdCho/18:1 concentrations above
4 µM (Fig. 2A), indicating that CT became
refractory to activation at higher lipid concentrations compared to
CT[
312-367]. Consistent with this interpretation,
the double-reciprocal plots were not linear (Fig. 2B)
indicating cooperative interactions between lipid activators and the
enzymes. Analysis of the data using the Hill equation (Fig. 2C) indicated that there were two factors that
contributed to the enhanced activity of
CT[
312-367] compared to CT. First, the K
values for PtdCho/18:1 vesicles
calculated from the Hill plots were 51 µM for CT and 17
µM for CT[
312-367] ()
illustrating that the truncated enzyme was more sensitive to lipid
activators. Second, CT exhibited negative cooperativity (n
= 0.64), whereas the Hill coefficient (n
= 1.11) for
CT[
312-367] did not indicate negative
cooperativity.
Figure 2:
Regulation of CT and
CT[312-367] by lipid activators. A,
activation of CT and CT[
312-367] by PtdCho/18:1
vesicles. B, analysis of the kinetics of CT and
CT[
312-367] activation by PtdCho/18:1 vesicles by
double reciprocal plots. C, the kinetic data in B were plotted according to the Hill equation to determine the
degree of cooperativity exhibited by the activation of CT and
CT[
312-367] by PtdCho/18:1 vesicles. D,
activation of CT and CT[
312-367] by PtdCho/DAG
vesicles. E, analysis of the kinetics of CT and
CT[
312-367] activation by PtdCho/DAG vesicles by
double reciprocal plots. F, the kinetic data plotted according
to the Hill equation for cooperative activation of CT and
CT[
312-367] by PtdCho/DAG
vesicles.
Differences in the Regulation of CT and
CT[
CT is
also activated by the nonionic lipid, DAG, incorporated into PtdCho
vesicles(4, 5, 6) , and CT activity in vivo may be regulated by the levels of DAG in the
membrane(24, 32, 33) . Therefore, we performed
the same kinetic analysis comparing the stimulation of CT and
CT[312-367] by PtdCho/DAG Vesicles
312-367] by PtdCho/DAG vesicles. Both CT and
CT[
312-367] were activated by PtdCho/DAG (Fig. 2D), and, as in the case with PtdCho/18:1,
CT[
312-367] was stimulated to a greater extent at
lower PtdCho/DAG concentrations. Analysis of the activation curves by
double reciprocal plots (Fig. 2E) indicated that CT
activation by PtdCho/DAG exhibited negative cooperativity, whereas
CT[
312-367] activation appeared to more closely
obey Michaelis-Menten kinetics. The Hill plots (Fig. 2F)
showed that the Hill coefficient for CT was 0.76, whereas
CT[
312-367] had a Hill coefficient of 0.93.
However, the most striking difference between the two enzymes was the
apparent K
for PtdCho/DAG activator (). The apparent K
for
CT[
312-367] was 6 µM, almost an order
of magnitude lower than the 45 µMK
for CT. Thus, the presence of the phosphorylated
carboxyl-terminal domain in CT reduced the affinity of the enzyme for
PtdCho/DAG activator and induced negative cooperative binding to
PtdCho/DAG, consistent with the conclusions drawn from the analysis of
enzyme regulation by PtdCho/18:1 (see above).
Effect of Eliminating a Major Phosphorylation Site on
Cooperative Activation
We attempted to dephosphorylate CT and
assess its kinetic properties. Although we were able to effectively
dephosphorylate CT using acid phosphatase(40) , the recovery of
CT activity from these reactions was always <2%, thus preventing the
further detailed analysis of enzyme kinetics. Other phosphatases were
less effective in dephosphorylating CT, and recovery of activity was
poor in all cases. Ser-315 is a major phosphorylation site in CT
expressed in Sf9 cells (21) and is the phosphorylation site
closest to the putative lipid binding helical domain(18) . To
test the importance of this particular site and to verify that negative
cooperativity was a property associated with the phosphorylation of the
carboxyl terminus, the CT[S315A] mutant was constructed,
expressed in Sf9 cells, and the kinetics of lipid activation were
measured. Although CT[S315A] was a phosphoprotein (Fig. 1), the kinetics of CT[S315A] activation by
PtdCho/18:1 was a good fit to the Michaelis-Menten equation and the
Hill plot showed reduced negative cooperativity compared to CT (Fig. 3A). The same kinetic analysis was applied to the
activation of CT[S315A] by PtdCho/DAG (Fig. 3B), and, again, CT[S315A] exhibited a
lower degree of negative cooperativity compared to CT. In both cases,
the Hill coefficient for CT[S315A] was intermediate between
CT and CT[312-367] indicating that phosphorylation
of the carboxyl-terminal domain was responsible for the negative
cooperativity associated with CT. Most importantly, the K
values for the lipid activation of
CT[S315A] were intermediate between the higher K
values exhibited by CT and the lower K
values exhibited by
CT[
312-367] (). Thus, the kinetic
constants of CT[S315A] were between those of CT and
CT[
312-367].
Figure 3:
Regulation of CT[S315A] activity
by lipid activators. Kinetic analysis was performed as described in
Fig. 2, and the Hill plots of the data on the activation of
CT[S315A] by PtdCho/18:1 (A) and PtdCho/DAG (B) vesicles are shown.
Conclusions
These data are consistent with the
idea that the phosphorylated carboxyl-terminal domain of CT modulates
enzyme activation by lipid regulators. CT and a truncated mutant
lacking the entire carboxyl-terminal domain both require specific
lipids for activity and have similar kinetic constants for the two
substrates, CTP and phosphocholine. However, there are significant
differences between the two proteins with respect to the kinetics of
lipid activation. The K values for the
two lipid activator mixtures are lower for
CT[
312-367] than for CT, and, in addition, CT
activation exhibits pronounced negative cooperativity with respect to
the two lipid regulators, whereas the CT[
312-367]
mutant does not. To verify that the effects on the K
and negative cooperativity arise from the phosphorylation of
the carboxyl-terminal domain, the phosphorylation state of CT was
modified by eliminating a major phosphorylation site. The
CT[S315A] mutant has affinities for lipid activator mixtures
that lie between CT and CT[
312-367] illustrating
that changes in the phosphorylation state can account for the
differences in CT regulation by lipid activators. Thus, our results
indicate that the phosphorylation of the carboxyl-terminal domain both
decreases the affinity of the enzyme for lipid regulators and induces
negative cooperativity.
values for
lipid activators of CT[S315A] lie between those for CT and
CT[
312-367] indicate that phosphorylation of
Ser-315 contributes to, but is not solely responsible for, the negative
regulation of CT activity.
Table: Kinetic affinity constants for CT,
CT[S315A], and CT[312-367]
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.