(Received for publication, July 19, 1995)
From the
The biochemical mechanism for the regulation of enzyme activity
by lipid modulators and the role of the amphipathic -helical
domain of CTP:phosphocholine cytidylyltransferase (CT) was investigated
by analyzing the kinetic properties of the wild-type protein and two
truncation mutants isolated from a baculovirus expression system. The
CT[
312-367] mutant protein lacked the
carboxyl-terminal phosphorylation domain and retained high catalytic
activity along with both positive and negative regulation by lipid
modulators. The CT[
257-367] deletion removed in
addition the region containing three consecutive amphipathic
-helical repeats. The CT[
257-367] mutant
protein exhibited a significantly lower specific activity compared to
CT or CT[
312-367] when expressed in either insect
or mammalian cells; however, CT[
257-367] activity
was refractory to either stimulation or inhibition by lipid regulators.
Lipid activators accelerated CT activity by decreasing the K
for CTP from 24.7 mM in their
absence to 0.7 mM in their presence. The K
for phosphocholine was not affected by lipid activators. The
activity of CT[
257-367] was comparable to the
activity of wild-type CT in the absence of lipid activators and the CTP K
for CT[
257-367]
was 13.9 mM. The enzymatic properties of the
CT[
231-367] mutant were comparable to those
exhibited by the CT[257-367] mutant indicating that
removal of residues 231 through 257 did not have any additional
influence on the lipid regulation of the enzyme. Thus, the region
between residues 257 and 312 was required to confer lipid regulation on
CT, and the association of activating lipids with this region of the
protein stimulated catalysis by increasing the affinity of the enzyme
for CTP.
CT ()is considered a key rate-controlling step in the
biosynthesis of PtdCho, a major phospholipid component of mammalian
membranes, and modulation of CT function by lipid regulators is an
important element in the control of enzyme activity (for reviews, see (1, 2, 3) ). Purified CT has essentially no
activity in the absence of lipids(4, 5) , and its
activity is revealed by the addition of anionic lipids (such as oleic
acid or phosphatidylglycerol) or neutral activators (such as
diacylglycerol) presented in PtdCho
vesicles(4, 5, 6, 7) . The
significance of lipid regulation of CT to the control of PtdCho
biosynthesis is supported by the stimulation of the pathway following
the treatment of cells with oleic acid (8, 9) or the
generation of diacylglycerol by the addition of exogenous
PtdCho-specific phospholipase C(10, 11, 12) .
The stimulation of PtdCho synthesis by these treatments correlates with
the translocation of CT to cellular membranes and the dephosphorylation
of the
enzyme(9, 10, 11, 12, 13, 14) .
The correlation between cellular diacylglycerol (15, 16, 17) or PtdCho (18, 19, 20) content with the rate of PtdCho
synthesis and CT membrane translocation provides compelling support for
the physiological importance of CT-lipid interactions. CT is also
negatively regulated by associations with lipids. CT activity in
vitro is inhibited by sphingosine(21) ,
lysoPtdCho(22) , and the antineoplastic lysoPtdCho analog,
ET-18-OCH
(22) . Treatment of cells with either
lysoPtdCho or ET-18-OCH
inhibits PtdCho synthesis and
triggers the accumulation of phosphocholine indicating that CT is a
target for these compounds in vivo(22) . The
observation that the inhibition of CT activity by these three lipids is
competitive with respect to the PtdCho/oleic acid activator suggests
that both positive and negative lipid regulators bind to the same site
on the enzyme(21, 22) .
CT can be divided into
several discrete functional domains (Fig. 1). The focus of this
work is the region between residues 228 and 315 which is predicted to
be primarily -helical with subdomains that exhibit significant
amphipathic character(23) . This helical domain is highly
conserved in all sequenced mammalian CT
proteins(23, 24, 25, 26) , although
the yeast CT sequence does not contain a homologous
domain(27) . The helical region contains a positively charged
cluster that is immediately followed by a series of three, 11-residue
repeats that are strongly predicted to form an amphipathic
-helix(23) . The predicted
-helix is broken at
residues 294-297, but this interruption is followed by another
predicted
-helix through residue 315 that also has amphipathic
character(23) . Protease protection experiments (28) implicate the
-helical region between residues 236
and 293 as responsible for the interaction of CT with phospholipid
bilayers. Also, antibodies directed against residues 247 to 257
interfere with CT membrane association (29) supporting the idea
that this region of CT is involved in lipid-protein interactions.
Figure 1:
Domain structure of CT. CT contains
four distinct domains. There is an amino-terminal nuclear localization
signal located between residues 8 and 28, a central yeast homology
domain that is thought to be the catalytic center of CT between
residues 72 and 235, an -helical domain between residues 228 to
312 that we have conceptually subdivided into a cluster of positively
charged residues (++) followed by three consecutive
11-residue repeats that are strongly predicted to form an amphipathic
-helix between residues 257 and 288, and a carboxyl-terminal
Ser/Pro-rich domain extending from residue 312 to 367 that contains all
of the CT phosphorylation sites. The helical region between 298 and 315
is also predicted to have a significant hydrophobic moment. The
CT[
312-367] truncation removes the
carboxyl-terminal phosphorylation domain, the
CT[
257-367] deletion removes the three 11-residue
amphipathic
-helical repeats as well as the region between
residues 294 and 312. The CT[
231-367] mutant
removes the entire
-helical domain and 4 residues that extend into
the yeast homology domain.
There are two possible mechanisms that could account for the
regulation of CT activity by lipids. First, the amphipathic
-helical domain may inhibit CT activity, and this inhibition might
be relieved by the binding of the amphipathic
-helix to
phospholipid vesicles. Second, the binding of lipid regulators to the
amphipathic
-helix may trigger a conformational change that
activates the enzyme. The goal of the present work is to verify that
the amphipathic helical domain is required to confer lipid regulation
to CT and to determine if the interaction between this domain and lipid
regulators is responsible for increasing the catalytic activity of the
protein or relieving the inhibition of the enzyme.
The
carboxyl-terminal deletion mutant pCTD312 was constructed starting with
a CT mutant containing an MscI site at bp 1040. This new
restriction site was generated by adding an additional A at the 3` end
by Taq DNA polymerase, which changed A to
T
, when PCR was performed using primer
5`-GGCCATCgcTCCCAAGCAGAG-3` to generate the CT[S315A] mutant
constructed in a previous study(31) . The sequence was
confirmed by DNA sequencing. The region between bp 1040 and bp1342 was
deleted by digestion of the resulting plasmid with MscI/NotI. The NotI site was filled with
Klenow fragment, and the plasmid 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.
Plasmid pCTD231 was constructed from the NcoI/SacI fragment of CT cDNA subcloned into pTrc99A (Pharmacia). The pTrc99ACTN/S containing the NcoI/SacI fragment was digested with SacI and BamHI, and the plasmid fragment was isolated by electrophoresis. The BamHI overhang was filled with Klenow fragment, and the plasmid religated. The insert contained amino acid residues 1-230 plus Ile-Leu and was subcloned into NcoI/HindIII sites of pBlueBacIII. The resulting construct was called pCTD231. The pCTD257 was constructed by PCR site-directed mutagenesis. The SacI/BamHI fragment of rat CT (0.57 kilobase) was subcloned into pBS and used as the template for the deletion primer which was designed based on the nucleotide sequence from bp 861 to bp 882. The primer, 5`-GAAAGATGTGtAGTAAAAGTCG-3`, substituted the original GAG codon for Glu-257 with TAG, thus changing Glu-257 to a stop codon. Mutagenesis was performed using the deletion primer and the M13 reverse primer to generate the first round PCR product and then using the first round PCR product and the M13 forward primer to generate the second round PCR product(32) . The second round PCR product was digested with SacI/BamHI, ligated into the pBS plasmid, and the DNA sequence was confirmed. The mutated fragment was isolated by digesting the mutated pBS construct with SacI/HindIII and co-ligated with the CT NcoI/SacI fragment into pBlueBacIII yielding pCTD257.
The flagCT construct was made by
co-ligation of the CT NcoI/BamHI fragment with the
flag-tag oligonucleotide, 5`-AGCTTATGGACTACAAGGACGACGATGACAAGGC-3`
((+)-strand) and 5`-CATGGCCTTGTCATCGTCGTCCTTGTAGTCCATA-3`
((-)-strand), into the HindIII/BamHI sites of
plasmid pcDNA3. The flagCT[312-367] construct was
made by replacing the Sse8387I/XhoI fragment in
flagCT with the corresponding fragment from pBSIIKSCTD312. The
pBSIIKSCTD312 was obtained by subcloning the insert of pCTD312 into the BamHI/HindIII sites of pBluescript II KS
(Stratagene). The flagCT[
257-367] construct was
made by replacing the Sse8387/BamHI fragment with the
corresponding fragment from pCTD257.
In all cases, the identities of the mutations were confirmed by DNA sequencing.
Figure 2: Expression and activity of CT and the three deletion mutants. CT and the truncation mutants were expressed in Sf9 cells by infection with recombinant baculoviruses. The proteins were delipidated and partially purified by DEAE-Sepharose chromatography. A, SDS-gel electrophoresis analysis of the protein preparations showing the purity and the apparent molecular weights of CT and the mutant proteins. B, specific activities of CT and the two mutant proteins were compared by assaying CDP-choline formation in the presence of 80 µM PtdCho/oleic acid as described under ``Experimental Procedures.'' Assays were linear with time and protein.
Figure 3:
The activity of
CT[257-367] was not stimulated by PtdCho/oleic
acid vesicles. The ability of the potent lipid activator mixture
(PtdCho/oleic acid) to stimulate the activity of CT,
CT[
312-367], and CT[
257-367]
was determined as described under ``Experimental
Procedures.'' The maximum activities for the protein preparations
used in this experiment were: CT, 305 nmol/min/mg;
CT[
312-367], 628 nmol/min/mg; and
CT[
257-367], 22.7
nmol/min/mg.
Figure 4:
Effect of ET-18-OCH on the
activities of CT, CT[
312-367], and
CT[
257-367]. The three proteins were assayed in
the presence of 80 µM PtdCho/oleic acid and the indicated
concentration of ET-18-OCH
as described under
``Experimental Procedures.'' The maximum activities for the
protein preparations used in this experiment were: CT, 420 nmol/min/mg;
CT[
312-367], 680 nmol/min/mg; and
CT[
257-367], 17.8
nmol/min/mg.
Figure 5:
Expression of flagCT,
flagCT[312-367], and
flagCT[
257-367] in COS7 cells. COS7 cells were
transfected with plasmids directed to express epitope-tagged proteins
from the cytomegalovirus promoter as described under
``Experimental Procedures.'' At 48 h after transfection, the
cells were harvested. A, a sample of the cell lysate (100
µg of protein) was separated by SDS-gel electrophoresis, the
proteins were transferred to nitrocellulose membrane, and the levels of
flagCT, flagCT[
312-367], and
flagCT[
257-367] were determined by immunoblotting
with the M2 monoclonal antibody to the flag-tag epitope. B,
cell lysates were assayed for CT activity to determine the specific
activity of CT in the transfected cell populations as described under
``Experimental Procedures.'' Assays were linear with time and
protein.
Figure 6:
Phosphocholine kinetic constants for CT in
the presence and absence of lipid activators. The K for phosphocholine was determined for CT in the presence of
PtdCho/oleic acid vesicles and 2 mM CTP (A) or in the
absence of lipid activators in the presence of 32 mM CTP (B). Enzyme assays were performed as described under
``Experimental Procedures.''
A different result was obtained
when the CTP K was determined in the presence and
absence of PtdCho/oleic acid activator (Fig. 7). The presence of
activating lipids significantly increased the affinity of CT for CTP (Fig. 7, A and B). In the presence of
PtdCho/oleic acid, the CTP K
was 0.7 mM which increased to 24.7 mM (35-fold) in the absence of
lipids. The V
in the presence of lipids was
6.3-fold higher than in the absence of lipids (Fig. 7, A and B), indicating that the 700-1400-fold increase in CT
specific activity by PtdCho/oleic acid when measured at 2 mM CTP was due almost entirely to the ability of lipid activators to
increase the affinity of CT for CTP. The CTP K
values for CT and CT[
312-367] in the
presence of lipid activators were similar to those reported for
purified CT by ourselves and other
investigators(31, 37) , and, in both cases, the CTP
kinetics were a close match to the Michaelis-Menten equation showing
little cooperativity, with Hill coefficients (n
)
close to 1.0 (Table 1). The CTP K
calculated
for CT[
257-367] was 13.2 mM (Table 1). This value was considerably higher than the
values for the CT and CT[
312-367] in the presence
of lipid activators, but was close to the K
determined for CT and CT[
312-367] in the
absence of lipid activators. These data indicated that the PtdCho/oleic
acid stimulates CT activity by lowering the CTP K
by 20-30 fold and that the CT[
257-367]
truncation mutant cannot undergo this allosteric transition because it
lacks the lipid interaction domain.
Figure 7:
Kinetic constants for CTP in the presence
and absence of lipid activators. The K for CTP was determined for CT in the presence of
PtdCho/oleic acid and 1 mM phosphocholine (A) or in
the absence of lipid activator and 1 mM phosphocholine (B). Assays were performed using the indicated concentrations
of CTP as described under ``Experimental
Procedures.''
These data predicted that there
would be little difference between the activity of CT and
CT[257-367] when assayed in the absence of lipid
activators. Therefore, we compared the activities of these two proteins
in the absence of lipids as a function of CTP concentration and found
that indeed the two proteins possessed almost identical activities
under these assay conditions (Fig. 8). In both cases, the
enzymes required high concentrations of CTP for maximal activity. Thus,
the presence of the amphipathic
-helical domain did not inhibit CT
activity in the absence of lipids, but rather was required along with
lipid activators to lower the K
for CTP.
Figure 8:
A comparison of the specific activity of
CT and CT[257-3367] as a function of CTP
concentration and in the absence of lipid activators. The activities of
CT and CT[
257-367] as a function of CTP
concentration were compared in the absence of lipid activators and in
the presence of 1 mM phosphocholine using the CT assay
described under ``Experimental
Procedures.''
Our data reveal that the association of wild-type CT with
activating lipids accelerates catalysis by increasing the affinity of
the enzyme for CTP. This model asserts that the binding of activating
lipids to the amphipathic -helical domain induces a conformational
change that lowers the CTP K
. The increase in CT
specific activity triggered by activating lipids when assayed at CTP
concentrations around 1-2 mM is so large
(
1,000-fold) that most investigators report that CT has essentially
no activity when stripped of lipid
activators(4, 5, 22, 31) . However,
when delipidated CT is assayed at high CTP concentrations
(approximately 30 mM), the difference in the specific activity
in the presence and absence of lipid activators is reduced to
approximately 6-fold (Table 1, Fig. 3and Fig. 7).
Thus, the ability of lipid mixtures to induce a conformational change
that leads to higher affinity CTP binding to the enzyme is the primary
mechanism that accounts for the ability of activating lipids to
accelerate CT catalysis. Indeed, the activities of CT and the
CT[
257-367] mutant are nearly identical as a
function of the CTP concentration in the absence of lipid activators (Fig. 8) clearly showing that the binding of lipids to the
amphipathic
-helical domain is responsible for activating CT as
opposed to relieving the inhibition of the enzyme.
Our results show
that the region between residues 257 and 312 is necessary for the
regulation of CT activity by lipids. Although removal of this region in
CT[257-367] resulted in a 30-fold reduction in
specific activity of the expressed protein, significant residual
catalytic activity remained that was refractory to activation by lipid
regulators (Fig. 3). Our data also indicate that the region
between residues 257 and 312 of CT is involved in negative regulation
of CT activity by lipids. Craig et al.(28) report
that all of the chymotrypsin fragments bind to PtdCho/sphingosine
vesicles and suggest that CT interaction with this negative lipid
modulator is not mediated by the amphipathic
-helix, but rather by
the amino-terminal domain. However, our experiments with
ET-18-OCH
(Fig. 4) are consistent with the
hypothesis that negative lipid modulators associate with the same
domain of the protein as positive regulators. The idea that both
positive and negative lipid modulators bind to the same domain on the
protein is supported by the finding that inhibition by lipid effectors
(sphingosine, ET-18-OCH
, and lysoPtdCho) is competitive
with respect to lipid activator
concentration(21, 22) . Thus, the cumulative data
point to the three 11-residue repeats that are strongly predicted to
form an amphipathic
-helix as a region of CT that is essential for
all lipid-protein interactions. However, although our data show that
the region from residues 257 through 312, inclusive, is necessary for
lipid-protein interaction, the data do not prove that it is sufficient
and other regions of the protein may also participate in the regulation
of CT by lipids. The role of the region between residues 231 and 312
needs to be tested further by the construction and analysis of
additional carboxyl-terminal truncation mutants and internal deletion
mutants that specifically remove one or more of the
-helical
repeat motifs.
Modulation of the CTP K by lipid
regulators is an effective mechanism for controlling CT activity in
vivo. The average intracellular concentration of CTP in a variety
of cell types is 278 ± 242 µM (29 ± 19
µM for dCTP)(38) , which is close to the CTP K
found by us and others in vitro (Table 1)(31, 37) . These data indicate
that if CT is not associated with lipid activators in vivo,
the enzyme would be essentially inactive due to the high CTP K
(
25 mM). The fact that the in
vivo CTP concentration is close to the CTP K
for CT suggests that alterations in the intracellular CTP levels
could affect the rate of PtdCho synthesis. Indeed, the elevation of the
intracellular CTP concentration accelerates PtdCho synthesis in
neuronal cells(39, 40) supporting the idea that
modulation of intracellular CTP levels could participate in the
regulation of enzyme activity in vivo. Cytosolic CT
preparations have a CTP K
of 2 mM which
is lowered to 0.2 mM by the addition of lipid activators (41) indicating that ``soluble'' CT is
lipid-associated. Increased phosphorylation of the CT carboxyl-terminal
domain leads to reduced enzyme activity by interfering with the
association of activating lipids with the enzyme(31) . Thus, CT
phosphorylation fits into the regulatory scheme by interfering with the
ability of activating lipids to lower the CTP K
.
In summary, the large change in the CTP K
following lipid binding to the CT amphipathic
-helical
domain between residues 257 and 312 represents an extremely effective
and physiologically relevant on-off switch that governs CT catalysis
and hence the rate of PtdCho production.
Note Added in Proof-Our finding of a requirement for the residues between 257 and 312 for lipid regulation is consistent with the recent results of Wang and Kent(42) ; however, our conclusion that lipid binding triggers a conformational change that activates CT by increasing its affinity for CTP does not agree with their conclusion that lipid binding or carboxyl-terminal truncation activates CT by removing an inhibitory domain.