©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lipid Activation of CTP:Phosphocholine Cytidylyltransferase Is Regulated by the Phosphorylated Carboxyl-terminal Domain (*)

Wannian Yang (1), Suzanne Jackowski (1) (2)(§)

From the (1)Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101 and the (2)Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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[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 Kvalues 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.


INTRODUCTION

CT()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.

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.


EXPERIMENTAL PROCEDURES

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[312-367] and CT[S315A] Mutants

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.

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.

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)] = nlog[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 Kvalues 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).


RESULTS AND DISCUSSION

Expression and Phosphorylation of CT, CT[S315A], and CT[312-367]

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] 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[312-367] by PtdCho/18:1 Vesicles

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] 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[312-367] by PtdCho/DAG Vesicles

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. 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.

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 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]



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM45737, Cancer Center (CORE) Support Grant CA 21765, National Research Service Award T32 CA09346 from the National Cancer Institute (to W. Y.), and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38101-0318. Tel.: 901-495-3494; Fax: 901-525-8025; E-mail: suzanne.jackowski@stjude.org.

The abbreviations used are: CT, CTP:phosphocholine cytidylyltransferase; PCR, polymerase chain reaction; PtdCho, phosphatidylcholine; 18:1, oleic acid; DAG, diacylglycerol; bp, base pair(s); bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.


ACKNOWLEDGEMENTS

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.


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