©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of an Inhibitory Domain of CTP:Phosphocholine Cytidylyltransferase (*)

(Received for publication, April 18, 1995; and in revised form, June 14, 1995)

Yuli Wang Claudia Kent (§)

From theDepartment of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The function of the putative amphipathic helices between residues 236 and 314 of CTP:phosphocholine cytidylyltransferase was examined by constructing two truncation mutants; CT314 was missing the entire phosphorylation segment, whereas CT236 was missing both the region with the putative amphipathic helices and the phosphorylation segment. Stable cells lines expressing these truncation mutants in Chinese hamster ovary 58 cells were isolated and characterized. CT314 was predominantly soluble in control cells but became membrane-associated in cells treated with oleate, which also causes translocation of wild-type cytidylyltransferase. CT236 was found to be soluble both in control cells and in cells treated to cause translocation. These results strongly suggest that the membrane-binding site is located within residues 237-314. When assayed for activity in vitro, the mutant forms were catalytically active in the presence of exogenous lipids. CT236, moreover, was as active in the absence of lipids as in their presence, whereas CT314 required lipids for activity. The rate of phosphatidylcholine synthesis in cells expressing CT236 was considerably higher than in wild-type cells, consistent with the enzyme being constitutively active in the cells. These results indicate that residues 237-314 constitute an inhibitory segment; when this segment is removed from the catalytic domain by truncation or by binding to membranes, an inhibitory constraint is removed and cytidylyltransferase is activated.


INTRODUCTION

The major pathway for phosphatidylcholine biosynthesis in mammalian cells is the CDP choline pathway. The formation of the activated intermediate, CDP choline, is catalyzed by the second enzyme of the pathway, CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), which is a key regulatory enzyme for this pathway(1) . When cultured cells are subjected to choline starvation (2, 3) or various treatments that stimulate phosphatidylcholine synthesis(4, 5, 6, 7) , cytidylyltransferase changes from a relatively inactive, soluble form to an active, membrane-associated form. The soluble form is intranuclear, and the membrane form is associated with the nuclear envelope(8, 9, 10) . In some circumstances active cytidylyltransferase appears to be associated with a lipoprotein complex rather than a membrane(11, 12, 13) . When assayed in vitro, the soluble form requires lipids for activity, whereas the membrane form is usually quite active in the absence of exogenous lipids. Thus it appears that the soluble form is an inactive reservoir available to be activated when an increased requirement for phosphatidylcholine synthesis is sensed. The soluble form is highly phosphorylated and translocation of cytidylyltransferase to membranes is accompanied by extensive dephosphorylation(14, 15, 16, 17) . The phosphorylation state of cytidylyltransferase, however, does not appear to be a critical determinant of the distribution of the enzyme. (^1)

Sequences have been determined for several mammalian cytidylyltransferase cDNAs (18, 19, 20, 21) and the gene from Saccharomyces cerevisiae(22) . The mammalian protein has a molecular weight of about 42,000 and appears to be a dimer(13, 23) . A comparison of the mammalian and yeast sequences reveals a region from residues 79-209 that is highly conserved and is proposed to be the catalytic domain(21) . That this region is catalytic is supported by the similarity of this region to a small bacterial CTP:glycerol-3-phosphate cytidylyltransferase that is only as big as the putative catalytic domain of the eukaryotic cytidylyltransferases (24, 25, 26) . The phosphorylation domain extends from residue 315 to the COOH terminus(17) , and the nuclear localization sequence is near the NH(2) terminus (27) (see Fig.1).


Figure 1: Regions of cytidylyltransferase.



Between the phosphorylation region and the catalytic domain is a sequence of about 80 residues that is predicted to form amphipathic alpha-helices(21) . Cornell and co-workers proposed that this amphipathic helical region is a membrane-binding domain for cytidylyltransferase (21) . In support of this hypothesis, Craig et al., (28) showed by chymotryptic digestion that there is a correlation between the presence of the amphipathic helical region in a proteolytic fragment of cytidylyltransferase and the ability of the fragment to bind to lipids in vitro. The digested enzyme was not reported to be active, however, so it was not clear if the protease caused such a major structural change in the enzyme that a membrane-binding site distinct from the helical region might have been altered. Furthermore, it is not clear if binding to lipids in vitro occurs by the same mechanisms as binding to the nuclear envelope in vivo.

In the present manuscript we have expressed and characterized truncated forms of cytidylyltransferase that are missing either the phosphorylation region (CT314, Fig.1) or both the phosphorylation region and the amphipathic helical region (CT236). The truncated enzymes were examined for activity in vitro, subcellular location, ability to translocate, and ability to complement the phenotypes of a cell line in which the endogenous cytidylyltransferase is temperature-sensitive.


EXPERIMENTAL PROCEDURES

Materials

Sodium oleate, phosphate-free Dulbecco's modified Eagle's medium, and Ham's F-12 medium were purchased from Sigma. P(i) was obtained from ICN, and [methyl-^14C]choline chloride and [methyl-^3H]choline chloride were from Amersham. Lipofectin reagent, Opti-MEM, Protein A-agarose, and G418 were from Life Technologies, Inc. LK6D silica gel 60A plates were from Whatman. The ECL immunodetection kit was from Amersham. The pCMV5 vector was obtained from Dr. David Russell. Phospholipase C from Clostridium perfringens was prepared through the QAE-Sepharose step as described(29) .

Construction of Truncation Mutants

The truncation mutants were constructed by PCR (^2)with primers that substituted stop codons for the codons encoding residue 237 (CT236) or 315 (CT314). The template was pCMV5CT.^1 For both mutants, 5`-CGCGGATCCAGATCTATGGATGCACAGAGTTCA-3` was the NH(2)-terminal primer, corresponding to the amino terminus of cytidylyltransferase. The COOH-terminal primer for CT236 was 5`-TCCCCGGGTCTAGATTAGTTGATAAAGCTGACATT-3` and for CT314 was 5`-TCCCCGGGTCTAGATTAGATGGCCTGCAGCATCCG-3`. The PCR program was 92 °C for 1 min, 52 °C for 1 min, and 72 °C for 1.5 min for 20 cycles. The PCR products were purified by electrophoresis on a 1% agarose gel and subcloned into the BglII-XbaI sites of the pCMV5 vector to give pCMV5CT236` and pCMV5CT314`. To ensure that no undesired mutations were obtained during the PCR reactions, the EcoRV-XbaI fragments of pCMV5CT236` and pCMV5CT314` were isolated and subcloned into pCMV5CT, in which the EcoRV-XbaI fragments were removed. The EcoRV-XbaI regions of the resulting pCMV5CT236 and pCMV5CT314 were sequenced. The plasmids were purified by ion exchange chromatography using QIAGEN Maxi-kit as recommended by the manufacturer.

Cell Culture

CHO 58 cells (30, 31) were cultured with Ham's F-12 medium plus 10% fetal bovine serum at 34 °C with 5% CO(2). Stable 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 10^6 cells/60-mm dish for 1 day at 34 °C before experiments unless otherwise specified.

Stable Transfections

Plasmid DNA of pCMV5CT236 and pCMV5CT314 was transfected into CHO 58 cells by the Lipofectin method as recommended by the manufacturer. The cells were plated at 2 10^5 cells/60-mm dish. One day after plating, the cells were washed with CMF-PBS twice, and the 1.5 ml Lipofectin/DNA mix was added to the dish. The Lipofectin/DNA mix was prepared by mixing 10 µg of DNA in 0.75 ml of Opti-MEM with 30 µg of Lipofectin in 0.75 ml of Opti-MEM and incubating for 10 min at room temperature before addition to cells. The transfection was stopped after 10 h by adding an equal volume of Ham's F-12 medium supplemented with 20% fetal bovine serum. The cells were allowed to grow for 2 days and then were split into four 150-mm dishes in normal culture medium plus 0.8 mg/ml G418 and further grown for about 2 weeks. Individual colonies were picked and screened by immunofluorescence for cytidylyltransferase expression(27) .

Labeling of Cytidylyltransferase in Vivo

For labeling, cells in a 60-mm culture dish were labeled with 0.2 mCi of P(i) in 1.5 ml of phosphate-free Dulbecco's modified Eagle's medium. Cytidylyltransferase was immunoprecipitated, immunoblotted, and analyzed as described(16) .

Incorporation of [^3H]Choline

Cells were washed twice with CMF-PBS and incubated with 1.5 ml of culture medium containing 2 µCi/ml [^3H]choline for the indicated time at 40 °C. The cells were then washed twice with CMF-PBS and harvested by scraping into 1.0 ml of H(2)O at 0 °C. Lipids were extracted from 0.8 ml of the extract as described(32) .

For the pulse-chase analysis, cells in 60-mm dishes were washed with CMF-PBS twice and then incubated with 1.5 ml of Ham's F-12 medium containing 5% fetal bovine serum plus 5 µCi/ml [^3H]choline for 30 min at 34 °C. The labeled medium was then replaced with Ham's F-12 medium plus 5% fetal bovine serum and incubated at 40 °C for the indicated time. Cells were harvested as described above for continuous labeling. The lipid fraction was dried and counted to determine label in choline-containing lipids. The aqueous phase was dried under air overnight, and choline metabolites were separated on silica gel 60A thin layer plates as described(33) .

Protein Determinations

The total protein concentration in cell extracts was determined by the Bradford method with bovine serum albumin as standard(34) . To quantitate the amount of cytidylyltransferase protein, cell extracts were separated by SDS gel electrophoresis, immunoblotted with NH(2)-terminal antibody (8) , and visualized by chemiluminescence with the ECL method. The amount of cytidylyltransferase protein was quantitated with either a Bio-Rad Molecular Imager or a Pharmacia LKB Ultrascan XL densitometer.

Cytidylyltransferase Assay

The activity of cytidylyltransferase was assayed as described(35) . For lipid activation, assays were performed in a sonicated mixture of 0.2 mM oleate plus phosphatidylcholine (molar ratio, 1:1). For measuring activity in immunoprecipitates, cells in 150-mm dishes were harvested in 1.0 ml of digitonin and immunoprecipitated as described(16) .


RESULTS

Characterization of Truncation Mutants

To determine the effects of the truncations on the properties of cytidylyltransferase, cDNAs for the mutants and wild-type cytidylyltransferase were stably transfected into CHO 58 cells, a cell line in which the endogenous cytidylyltransferase is temperature-sensitive and present in low levels even at the permissive temperature(20, 31, 36) . G418-resistant clones were screened for expression of cytidylyltransferase by immunofluorescence(27) . Cytidylyltransferase was nuclear in all clones examined. Western blot analysis showed that the truncated cytidylyltransferase forms had a faster mobility than wild-type cytidylyltransferase, as expected (Fig.2). To confirm that the COOH-terminal truncations of cytidylyltransferase did not create new phosphorylation sites, cytidylyltransferase was immunoprecipitated from P-labeled cells. As expected, wild-type cytidylyltransferase was highly phosphorylated, whereas the two truncation mutants were not labeled at all (Fig.2).


Figure 2: Mobility and phosphorylation of wild-type cytidylyltransferase and truncation mutants. Cells were labeled at 34 °C, immunoprecipitated, electrophoresed, and blotted as described under ``Experimental Procedures.'' A, Western blot; B, autoradiogram of the blot. Lanes 1, wild-type cytidylyltransferase; lanes 2, CT314; lanes 3, CT236.



Because the extensive truncations of cytidylyltransferase might affect the catalytic activity of the enzyme, it was important to determine if the truncated forms were as active as the wild-type enzyme. Enzymatic activity in crude extracts was measured in the presence of activating lipids, and the amount of cytidylyltransferase protein was quantitated from Western blots (Table1). In the presence of the activating lipids, each mutant was quite active; there was no significant difference in mutant and wild-type activity.



Subcellular Location of Truncation Mutants

The distribution of wild-type and mutant cytidylyltransferase activities between soluble and membrane-associated forms was determined (Table2). The differences in the specific activities of the three cytidylyltransferase forms reflect the different expression levels of the transfectants. In untreated cells, wild-type cytidylyltransferase was mostly soluble, as expected, as was CT236. The majority of CT314 was also soluble, although more CT314 than wild type was membrane-associated, presumably because CT314 resembles the dephosphorylated form of cytidylyltransferase, which has an increased affinity for membranes.^1 When cells were treated with oleate, extensive translocation was observed with the wild-type enzyme and CT314. Very little translocation was observed with mutant CT236, indicating that this mutant remains soluble even when wild type and CT314 translocated. Furthermore, CT236 remained soluble when treated with phospholipase C, a treatment previously shown to cause translocation of cytidylyltransferase(4, 5) , even though wild type and CT314 both translocated when treated with phospholipase C (data not shown). Because the difference between CT236 and CT314 is the amphipathic helical region, this evidence strongly supports the concept that this is the membrane-binding region in vivo.



Western blots of soluble and particulate fractions confirmed that the distribution of cytidylyltransferase protein followed that of cytidylyltransferase activity (Fig.3). Identical distributions in control and oleate-treated cells were observed by Western blotting for two different clonal isolates of each truncation mutant, indicating that the distributions in Table2were a property of the truncated enzyme and not an artifact of the specific clone (data not shown).


Figure 3: Subcellular distribution of cytidylyltransferase protein. Cells were treated in the absence (A) or the presence (B) of oleate at 40 °C and harvested as described in Table1. Identical volumes of the cell extracts were subjected to electrophoresis. This experiment was performed twice with the clonal isolate used here and once with a different clonal isolate for each mutant. Similar results were also obtained with a transient transfection. From the left the lanes correspond to wild-type soluble, wild-type particulate, CT314 soluble, CT314 particulate, CT236 soluble, and CT236 particulate.



CT236 Is Constitutively Active in Vitro

Because the absence of the putative amphipathic helix largely precludes membrane binding in the cell by CT236, it was of interest to know if the truncated enzyme was still lipid-activated. The ratio of activity in the presence of lipids to activity in the absence of lipids for the wild-type and truncated enzymes was determined (Table3). Assays were carried out with crude cell extracts and cytosols, as well as with enzyme purified by immunoprecipitation. Although the wild-type enzyme and CT314 required lipids for full activation, CT236 was as active in the absence of lipids as in the presence of lipids. Because the truncated enzymes were essentially fully active in the presence of exogenous lipids (Table1), CT236 must be constitutively activated. These results indicate that the membrane-binding domain is actually inhibitory to wild-type cytidylyltransferase.



CT236 Is Active in Vivo

If CT236 is active when assayed in the absence of lipids, then it might be more active than the soluble form of wild-type cytidylyltransferase in vivo. To determine if CT236 were more active in vivo, a pulse-chase experiment was performed to compare rates of incorporation of [^3H]choline into choline-labeled metabolites in cells expressing wild-type cytidylyltransferase and the truncation mutants (Fig.4, A-E). The rate of incorporation into phosphatidylcholine was appreciably higher in cells expressing CT236 than in cells expressing wild type or CT314. The ratio of labeled CDP choline to phosphocholine was much higher at all time points for CT236, indicative of the increased rate of the cytidylyltransferase reaction. Furthermore, there was a considerably higher rate of glycerophosphocholine labeling in CT236, suggestive of increased turnover of phosphatidylcholine. An increase rate of phosphatidylcholine synthesis in cells expressing CT236 was also observed in an experiment in which cells were continuously labeled with [^3H]choline (Fig.4F).


Figure 4: Incorporation of [^3H]choline into cells expressing wild-type and mutant cytidylyltransferases. A-E, pulse-chase protocol. Cells were labeled with 5 µCi/ml [^3H]choline for 30 min at 34 °C and then incubated with unlabeled medium at 40 °C for the indicated time. , CHO 58; , wild type; , CT314; , CT236. A, phosphocholine; B, CDP choline; C, phosphatidylcholine; D, glycerophosphocholine; E, the ratio of CDP choline to phosphocholine. F, a continuous labeling experiment. Cells were labeled with 2 µCi/ml [^3H]choline for at 40 °C for the indicated times. The amount of label incorporated into phosphatidylcholine is shown. The pulse-chase experiment was performed once; the continuous incorporation experiment was performed four times with similar results.



Complementation of Strain 58 for Growth

The CHO 58 cell line is temperature-sensitive for growth, and expression of wild-type rat liver cytidylyltransferase can rescue the phenotype for growth as well as for phosphatidylcholine synthesis(20) . The cell lines of CHO 58 stably expressing the truncation mutants were tested for their abilities to grow at 40 °C (Fig.5). Both truncation mutants were fully capable of supporting growth at 40 °C.


Figure 5: Cell growth at 40 °C. Stable cell lines expressing wild-type or mutant cytidylyltransferases were plated at 2 10^4 and incubated at 34 °C for 1 day and then shifted to 40 °C for the indicated time. Viable cells were determined by trypan blue exclusion. Two dishes were counted each day. Symbols are as in Fig.4.




DISCUSSION

These studies have characterized two truncation mutants of cytidylyltransferase in which the enzyme was shortened by either 53 or 131 residues, a reduction in length of 14 or 36%, respectively. The catalytic activities of these truncation mutants were quite similar to that of wild-type cytidylyltransferase, however. This supports the concept that the catalytic region is truly a structurally distinct domain that can exist by itself as a catalytically active unit. The fact that these mutants were active also indicated that the truncations did not cause a global structural alteration in enzyme structure and allowed us to use the truncation mutants to investigate the functions of the regions that were deleted.

When the truncation mutants were expressed in stable cell lines, their subcellular distribution was altered compared with that of the wild type. The extent of membrane association was higher for mutant CT314 (about 20%) than for wild-type cytidylyltransferase (about 5%), consistent with the concept that the dephosphorylated enzyme has a higher affinity for membranes. This behavior has been previously observed with a cytidylyltransferase mutant in which all 16 phosphorylation sites were mutated from Ser to Ala.^1 Like the 16SA mutant, (^3)a high proportion CT314 was soluble even in control cells, further indicating that dephosphorylation per se does not trigger membrane binding.

The most striking feature of the subcellular location of the truncation mutants was the fact that CT236 was soluble, even in cells treated to cause the enzyme to translocate. The fact that this enzyme remains soluble but active provides very strong support for the hypothesis that the putative amphipathic helical structure that is predicted to be formed by residues 236-315 constitutes the membrane-binding domain of cytidylyltransferase(18) . It might be argued that the amphipathic helical region only affects membrane binding indirectly, for instance by influencing the region amino-terminal of the catalytic domain to form the actual membrane-binding domain. The fact that a synthetic peptide corresponding to residues 236-293 can bind to lipid vesicles that activate cytidylyltransferase but not those that do not activate, however, supports the hypothesis that the amphipathic helical region itself is truly the membrane-binding domain(37) . The involvement of amphipathic helices in membrane binding is a relatively new concept that is strongly supported by the crystal structure of prostaglandin H synthase, in which amphipathic helices are proposed to mediate membrane binding(38) .

Studies on the effect of lipids on the activities of the truncation mutants revealed that CT236 is not activated by lipids. In fact, CT236 is apparently constitutively active both in the cell as well as in enzymatic assays. Not only is the rate of phosphatidylcholine synthesis increased in cells expressing CT236, but the rate of production of glycerophosphocholine is also increased, a phenomenon that occurs in cells expressing high levels of cytidylyltransferase activity(39) . Thus the membrane-binding region serves as an autoinhibitory region. This would be consistent with the amphipathic helices interacting closely with the catalytic domain in order to inhibit activity. When the membrane-binding domain interacts with a suitably activating membrane surface, the helices would move away from the catalytic domain and inhibition would be released.

It is important to note that membrane binding does not necessarily trigger activation, however. For example, the amount of membrane-associated CT314 is considerably higher than wild type, yet the rate of phosphatidylcholine synthesis in cells expressing CT314 is the same as in cells expressing wild-type cytidylyltransferase. Likewise, the level of membrane-bound cytidylyltransferase with the 16SA mutations is about 10 times higher than wild type, yet the rate of phosphatidylcholine synthesis in the 16SA cells is less than 2-fold higher than wild type.^1 This suggests that an additional factor is necessary for full activation of membrane-associated cytidylyltransferase. Whether that factor is lipid or protein in nature remains to be determined.

Cytidylyltransferase undergoes changes in its phosphorylation state during the cell cycle; these changes correlate with altered rates of phosphatidylcholine synthesis(40) . It is therefore surprising that CT236 and CT314 are fully able to complement the growth phenotype of CHO 58 cells. The membrane-binding and phosphorylation segments, therefore, do not appear to be necessary for progression through the CHO cell cycle under standard culture conditions. It is difficult to believe that a third of the polypeptide chain of cytidylyltransferase is useless to the cells, however. Like the unexpected location of cytidylyltransferase in the nucleus, the seeming dispensability of the membrane-binding and phosphorylation segments presents an enigma that remains to be addressed in the future.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA64159. 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 should be addressed: Dept. of Biological Chemistry, University of Michigan Medical Center, 4417 Medical Science I, Box 0606, Ann Arbor, MI 48109.

^1
Wang, Y., and Kent, C. (1995) J. Biol. Chem.270, in press.

^2
The abbreviations used are: PCR, polymerase chain reaction; CMF-PBS, calcium- and magnesium-free phosphate-buffered saline; CHO, Chinese hamster ovary.

^3
16SA indicates a mutant in which 16 serines have been mutated to alanine.


ACKNOWLEDGEMENTS

We thank Jharna Saha for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.