Enzymatic and Cellular Characterization of a Catalytic Fragment of CTP:Phosphocholine Cytidylyltransferase α

Abstract

To probe the mechanism of lipid activation of CTP:phosphocholine cytidylyltransferase (CCTα), we have characterized a catalytic fragment of the enzyme that lacks the membrane-binding segment. The kinetic properties of the purified fragment, CCTα236, were characterized, as well as the effects of expressing the fragment in cultured cells. CCTα236 was truncated after residue 236, which corresponds to the end of the highly conserved catalytic domain. The activity of purified CCTα236 was independent of lipids and about 50-fold higher than the activity of wild-type CCTα assayed in the absence of lipids, supporting a model in which the membrane-binding segment functions as an inhibitor of the catalytic domain. Thek cat/K m values for CCTα236 were only slightly lower than those for lipid-activated CCTα. The importance of the membrane-binding segment in vivo was tested by expression of CCTα236 in CHO58 cells, a cell line that is temperature-sensitive for growth and CCTα activity. Expression of wild-type CCTα in these cells complemented the defective growth phenotype when the cells were cultured in complete or delipidated fetal bovine serum. Expression of CCTα236, however, did not complement the growth phenotype in the absence of serum lipids. These cells were capable of making phosphatidylcholine in the delipidated medium, so the inability of the cells to grow was not due to defective phosphatidylcholine synthesis. Supplementation of the delipidated medium with an unsaturated fatty acid allowed growth of CHO58 cells expressing CCTα236. These results indicate that the membrane-binding segment of CCTα has an important role in cellular lipid metabolism. To probe the mechanism of lipid activation of CTP:phosphocholine cytidylyltransferase (CCTα), we have characterized a catalytic fragment of the enzyme that lacks the membrane-binding segment. The kinetic properties of the purified fragment, CCTα236, were characterized, as well as the effects of expressing the fragment in cultured cells. CCTα236 was truncated after residue 236, which corresponds to the end of the highly conserved catalytic domain. The activity of purified CCTα236 was independent of lipids and about 50-fold higher than the activity of wild-type CCTα assayed in the absence of lipids, supporting a model in which the membrane-binding segment functions as an inhibitor of the catalytic domain. Thek cat/K m values for CCTα236 were only slightly lower than those for lipid-activated CCTα. The importance of the membrane-binding segment in vivo was tested by expression of CCTα236 in CHO58 cells, a cell line that is temperature-sensitive for growth and CCTα activity. Expression of wild-type CCTα in these cells complemented the defective growth phenotype when the cells were cultured in complete or delipidated fetal bovine serum. Expression of CCTα236, however, did not complement the growth phenotype in the absence of serum lipids. These cells were capable of making phosphatidylcholine in the delipidated medium, so the inability of the cells to grow was not due to defective phosphatidylcholine synthesis. Supplementation of the delipidated medium with an unsaturated fatty acid allowed growth of CHO58 cells expressing CCTα236. These results indicate that the membrane-binding segment of CCTα has an important role in cellular lipid metabolism. CTP:phosphocholine cytidylyltransferase (CCT) 1The abbreviation used is: CCT, CTP:phosphocholine cytidylyltransferase; PC, phosphatidylcholine; FBS, fetal bovine serum; M/L, membrane/lipid; GCT, CTP:glycerol-3-phosphate cytidylyltransferase. is a critical participant in the CDP-choline pathway, catalyzing the synthesis of CDP-choline for the biosynthesis of phosphatidylcholine (PC), a major component of eukaryotic cell membranes (1Cornell R.B. Adv. Lipobiol. 1996; 1A: 1-38Google Scholar, 2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar). CCT is rate-limiting for the CDP-choline pathway and is extensively regulated at the enzymatic level. Mammalian CCT is present as both soluble and membrane-associated forms. Activation of CCT often occurs simultaneously with the translocation of the enzyme from a soluble form to membrane-associated form, resulting in an increase in the rate of PC synthesis. Consistent with in vivo membrane activation, the soluble form of CCT is activated by the addition of certain lipids in vitro. There are two isoforms of mammalian CCT; the most extensively studied, CCTα, is nuclear and apparently ubiquitously expressed (2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar). The recently discovered CCTβ is cytoplasmic and exhibits tissue-specific expression (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Mammalian CCTα contains several functional regions: an N-terminal nuclear localization signal, a central catalytic domain, a membrane/lipid (M/L) activation segment, and a C-terminal phosphorylation region (see Fig. 1). There is a high degree of sequence similarity within the catalytic domain of all known forms of CCT, with the yeast catalytic domain being 56% identical to that of mammalian CCTα, and the catalytic domains of CCTα and CCTβ being 90% identical. By contrast, there is much less conservation of primary structure in the N-terminal sequence or the C-terminal M/L and phosphorylation regions. Truncated forms of rat CCTα have been constructed to determine the regions responsible for lipid activation. Both full-length CCTα and truncated forms lacking the phosphorylation segment require lipids for full activity (4Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar, 5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Truncations lacking the M/L region, however, are not lipid-activated (4Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar, 5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and do not translate to membranes in the cell (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). It is notable that CHO58 cells that are temperature-sensitive for growth and CCTα activity can be complemented by exogenous expression of the truncated CCTα lacking the M/L region, which suggested that this segment is not important for growth under normal cell culture conditions (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These studies have contributed to the general understanding that the M/L region is truly responsible for membrane binding in the cell and lipid activation in vitro. On the other hand, two important questions regarding the M/L segment remain. First, what is the mechanism by which the M/L segment controls the activation of the enzyme by lipids? Second, what is the importance of the M/L segmentin vivo? The answer to the first question depends on an understanding of the level of activity remaining in the truncated enzyme lacking the M/L region, which has been a matter of some debate. The truncation expressed by Wang and Kent (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), CCTα236 (Fig. 1), is constitutively active as assessed by measuring enzyme activity in crude extracts and enzyme levels by quantitative immunoblotting. Slightly different forms expressed by Yanget al. (6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) or Cornell et al. (4Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar) are less than 10% as active as full-length, lipid-activated CCTα (7Feldman D.A. Weinhold P.A. J. Biol. Chem. 1987; 262: 9075-9081Abstract Full Text PDF PubMed Google Scholar). Thus the precise role of the M/L segment in enzyme activation is not clear. Wang and Kent (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) proposed that the M/L segment interacts with the catalytic domain to cause inhibition and that removal of the M/L segment by lipid binding results in activation of catalysis. Jackowski and co-workers (6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), however, proposed that lipids and the M/L domain are co-activators of the catalytic domain. In this paper we report the kinetic properties of purified CCTα236, showing that it is nearly as active as full-length CCTα and is indeed lipid-independent, supporting the model that the M/L segment is inhibitory. In addition, we report conditions under which the presence of the M/L segment in CCTα is necessary for cell growth. CTP, phosphocholine, protease inhibitors, lipids, CM-Sepharose, DEAE-Sepharose, and Sephacryl S-200-HR were obtained from Sigma. The cross-linkers, BS3 and Sulfo-MBS, were from Pierce. [3H]choline and [14C]phosphocholine were from Amersham Pharmacia Biotech. The Bac-to-Bac baculovirus expression system, Sf9 cells adapted for serum-free growth, Sf900II serum-free medium, and Ham's F-10 and F-12 media were from Life Technologies, Inc. Restriction endonucleases were from New England Biolabs. Fetal bovine serum was from BioWhittaker. Blue-Sepharose was from Amersham Pharmacia Biotech. A cDNA encoding rat liver CCTα (8MacDonald J.I. Kent C. Protein Expression Purif. 1993; 4: 1-7Crossref PubMed Scopus (32) Google Scholar) was placed in the baculovirus donor plasmid pFASTBAC1 using the BamHI and SphI sites of the multiple cloning site. To generate a cDNA encoding truncation mutant CCTα236 with appropriate restriction sites for the vector, oligonucleotide-directed polymerase chain reaction mutagenesis was used to introduce a stop codon following amino acid 236. The following oligonucleotides were utilized: 5′-oligo, 5′-CGCGGATCCAGATCTATGGATGCACAGAGTTCA-3′, and 3′-oligo, 5′-ACATGCATGCGGTACCTTAGTTGATAAAGCTGACATT-3′. Stocks of virus encoding wild-type CCTα and CCTα236 were prepared as described in the Bac-to-Bac Baculovirus expression system instruction manual. For protein production, 1-liter cultures of Sf9 cells at 1 × 106 cells/ml were infected at a multiplicity of infection of 2. Cells were harvested at 48 h post-infection for protein purification. Sf9 cells expressing wild-type CCTα were collected, lysed, and centrifuged as described (8MacDonald J.I. Kent C. Protein Expression Purif. 1993; 4: 1-7Crossref PubMed Scopus (32) Google Scholar). The 100,000 × g Sf9 cell supernatant was loaded directly onto a DEAE-Sepharose column equilibrated with buffer A (10 mm Tris-Cl, pH 7.5, 2 mm dithiothreitol, 1 mm EDTA) containing 150 mm NaCl. The column was washed with 10 column volumes of buffer A containing 150 mmNaCl and 1% Nonidet P-40 followed by 10 column volumes of buffer A containing 150 mm NaCl. The enzyme was eluted from the column with a 150–400 mm NaCl gradient in buffer A. The fractions constituting the peak of CCTα activity were pooled, diluted 5-fold in buffer A, and loaded onto a CM-Sepharose column equilibrated with buffer A containing 30 mm NaCl. The enzyme was eluted from the column with a gradient of NaCl from 30 to 150 mm, and the fractions containing the peak of the CCTα activity were pooled. The high speed Sf9 cell supernatant was loaded onto a CM-Sepharose column equilibrated with buffer A. The column was washed sequentially with 10 column volumes each of buffer A and buffer A containing 0.1 m NaCl. CCTα236 was eluted from the CM-Sepharose column with buffer A containing 0.2 mNaCl. The CM-Sepharose elution fraction was loaded onto a Blue-Sepharose 6 fast flow column. The column was washed with 10 column volumes of buffer A containing 0.2 m NaCl. CCTα236 was eluted from Blue-Sepharose with buffer A containing 0.5 mNaCl. Activity of purified enzymes was determined with a charcoal binding assay as described previously (9Wang Y. Kent C. J. Biol. Chem. 1995; 270: 17843-17849Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The kinetic parametersk cat and K m with respect to substrates CTP and phosphocholine were determined from secondary plots. Kinetic parameters were determined in the presence and absence of PC:oleate vesicles (10Weinhold P.A. Feldman D.A. Methods Enzymol. 1992; 209: 248-258Crossref PubMed Scopus (39) Google Scholar); the final concentration of each lipid was 100 μm. CHO58 cells expressing full-length CCTα or CCTα236 were obtained previously (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cell stocks were maintained at 34 °C in 5% CO2 in Ham's F-12 medium supplemented with 25 mm HEPES and10% fetal bovine serum (FBS). The stably transfected cell lines were maintained in the presence of 0.8 mg/ml G418 during one-third of the passages. For growth curves, cells were plated at 34 °C in Ham's F-12, 10% FBS at 50,000 cells/35-mm dish or 50,000 cells/well of a 6-well plate. After 24 h the medium was removed, cells were washed twice in Ca2+- and Mg2+-free phosphate-buffered saline, and then F-10 medium supplemented with 25 mm HEPES, 50 μg/ml gentamycin, and 10% FBS or 10% delipidated serum was The cells were then maintained at and cell was determined after by in with a were diluted the medium from a in For the cells were plated at dish in Ham's 10% FBS, and the medium as for growth Fetal bovine serum was delipidated by with and as described J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and then by The used is a of that described previously (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). 24 h after cells were washed twice with Ca2+- and Mg2+-free phosphate-buffered and Ham's 25 mm HEPES, 10% delipidated and 50 μg/ml Cells were then for 24 h at Cells were by at °C in of medium supplemented with 2 [3H]choline for the Cells were harvested by in Ca2+- and Mg2+-free phosphate-buffered and of at lipids were from 0.8 of the as described previously J. Biochem. PubMed Scopus Google Scholar). Protein concentration was determined by the assay of Biochem. PubMed Scopus Google Scholar) or et al. R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). we were in lipid activation of CCTα, we a for CCTα that not with lipids, as did the (7Feldman D.A. Weinhold P.A. J. Biol. Chem. 1987; 262: 9075-9081Abstract Full Text PDF PubMed Google Scholar). The on both DEAE-Sepharose and CM-Sepharose at pH the of in the enzyme is to it to to both at pH 7.5, most other The enzyme that was purified by this by of (Fig. The activity of the purified enzyme was to the activity of the enzyme purified by the (7Feldman D.A. Weinhold P.A. J. Biol. Chem. 1987; 262: 9075-9081Abstract Full Text PDF PubMed Google Scholar, J.I. Kent C. Protein Expression Purif. 1993; 4: 1-7Crossref PubMed Scopus (32) Google Scholar) in a of the truncation mutant CCTα236 to on CM-Sepharose as well as Blue-Sepharose The enzyme was as assessed by (Fig. CCTα236 did not to DEAE-Sepharose at pH 7.5, that the C-terminal in CCTα are at that pH and with the The of the purified forms of CCTα to be activated by lipids was determined by in the presence and absence of vesicles of (10Weinhold P.A. Feldman D.A. Methods Enzymol. 1992; 209: 248-258Crossref PubMed Scopus (39) Google Scholar). The activity of wild-type CCTα, purified in the absence of lipids, is to the addition of lipid (Fig. the of CCTα is activated In contrast, the activity of CCTα236 was not by the addition of the activity of CCTα236 with and lipid was with lipid-activated wild-type CCTα (Fig. The kinetic parameters of purified wild-type CCTα and CCTα236 were in the presence and absence of 100 PC:oleate a concentration at which CCTα is Thek cat for wild-type CCTα in the absence of lipid was less than that for wild-type CCTα with 100 lipid Thek cat values for CCTα236 were independent of the presence of lipid and were only about lower than that for wild-type CCTα in the presence of The K for phosphocholine were for both enzyme forms in the presence and absence of lipids. The K m values for CTP were higher for CCTα236 than for wild-type CCTα. The catalytic cat/K for CCTα236 in the absence of lipid were as high as those for wild-type CCTα in the presence of lipids. In contrast, cat/K m values for CCTα236 were higher than those for wild-type CCTα in the absence of lipids The that the activity of CCTα236 is so much than the activity of wild-type CCTα in the absence of lipids the model in which the M/L region is to parameters of CCTα and cat/K for cat and K m are the for independent in a for cat and K m are the for independent CCTα is known to be a P.A. Feldman D.A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), the at the have not been the M/L region were important in the of CCTα, then CCTα236 be to be The structure of CCTα236 was assessed by using the BS3 and BS3 is for two functional an and Both wild-type CCTα and CCTα236 were by BS3 and (Fig. The major form for both to be CCTα to form high CCTα236 only the The structure of CCTα236 was by on Sephacryl The truncated enzyme eluted the protein bovine serum and The was and the of a of CCTα236 is both and that CCTα236 is a and the that the of CCTα in the catalytic domain and not the C-terminal it is that the M/L region of CCTα has a on enzyme the of this domain in the cell to Expression of CCTα236 in the CHO58 cell which is temperature-sensitive for growth and CCTα activity J. Biol. Chem. Full Text PDF PubMed Google Scholar), the temperature-sensitive growth (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). that the M/L region is not for lipid and cell we have recently discovered conditions under which expression of CCTα236 is not to complement the CHO58 CHO58 cells stably transfected with CCTα236 were to medium containing delipidated the cells not to grow at °C not these cells not grow in delipidated serum at a that is for the growth of CHO58 in complete serum or delipidated serum (Fig. Thus the inability of cells expressing CCTα236 to grow in delipidated medium is a CHO58 cells expressing wild-type CCTα were capable of growth at °C in complete or delipidated serum (Fig. To determine a lipid the of CHO58 cells expressing CCTα236 to a of different lipids were to the delipidated The unsaturated fatty acid was capable of supporting growth at the rate as in complete serum (Fig. growth was at from to 100 unsaturated fatty and growth at The fatty and at not growth. The of not growth, and the addition of and to the medium did not growth than These results indicate that expression of CCTα236 CHO58 cells unsaturated fatty acid previously that CHO58 cells expressing CCTα236 in complete serum were active at making PC than cells expressing wild-type CCTα (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), which is with the CCTα236 being constitutively To the of these cells to PC was defective in delipidated of PC were 24 h after to delipidated medium, a at which the cells were as determined by in Fig. cells were capable of making PC in delipidated serum. The rate of PC synthesis was higher by a of in cells expressing CCTα236 than in cells expressing wild-type CCTα, as been when these cells were in complete serum. The higher rate of PC synthesis in cells expressing CCTα236 is with the M/L domain being in the can be for the role of lipid binding in by CCTα (Fig. In model A, the M/L region interacts with the catalytic domain in the absence of lipids, and this enzyme activity. of lipids to the M/L segment it to from the catalytic domain, and the inhibition is In model the catalytic domain in the enzyme not with the M/L the M/L region with lipids it then with the catalytic domain to catalysis. Both that CCTα236 have activity. A, however, that CCTα236 be as active as the wild-type enzyme in the presence of lipids, model that CCTα236 be as as the wild-type enzyme in the absence of lipids. The results in this that the activity of CCTα236 is indeed independent of lipids. CCTα236 is nearly as active as full-length CCTα in the presence of lipids and much active than full-length CCTα in the absence of lipids. The presence of the regions in the absence of lipid the by CCTα about These model A, in which the M/L region as an inhibitor in the absence of lipids. truncated forms of rat CCTα have been expressed with lower activity than CCTα236. of (4Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar) and (6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), were several that are highly conserved in all from to (2Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar). It is that these are important for or for enzyme In addition both these amino at which have activity. activity (6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), which the that of the region of the M/L to end truncation at on sequence conservation (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), of the CCTα that a the of the catalytic domain at this W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). that the catalytic domain truly end at residue 236. The role of lipid in the activation of CCTα has been to be due to both a of the K m for CTP as well as an increase in (6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). of kinetic parameters of wild-type CCTα only an increase cat in the presence of lipids, with in K m for CTP It is not there is a in these K It be that the kinetic m is a of rate that is a of binding J. in Protein New Scholar). It has been that the of an or inhibitor m is not as as a as the on m cat/K m Biochem. J. PubMed Scopus Google Scholar). It is that the for CCTα lipids by a of in the studies of et al. (6Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and cat/K m values for CCTα lipids in the present by a of these values are the that the of catalytic to K m m is the appropriate for the effects of lipids on activity. obtained by (Fig. and indicate that CCTα236 is a indicate that the responsible for in wild-type CCTα are within the catalytic domain or the CTP:glycerol-3-phosphate cytidylyltransferase a enzyme in primary sequence to the catalytic domain of CCT, is a Kent C. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), supporting the that in the catalytic domain are for have recently the structure of Y. S. S. C. and for The of at the of are or identical to the in CCTα, that the within the catalytic domain is a conserved of this cytidylyltransferase A role for the M/L or phosphorylation regions in a be not the role of the M/L region in in understanding of the of this enzyme in the It was that expression results that the regions of CCTα are not by cells, at under the cell culture conditions (5Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The present results however, that the presence of the M/L region can be critical under certain Expression of CCTα236 in CHO58 cells maintained in delipidated serum was the cells were with an unsaturated fatty The was a in that it at a for growth of CHO58 cells. The was not due to an inability of the cells to PC under these conditions (Fig. we do not the for the for an unsaturated fatty two to is that the cells are making so much PC that the responsible for making unsaturated fatty with the with the that much is and The other is that CCTα is in the of the in medium, and the M/L region a role in that The that CCTα has a role in lipid which nuclear The of a role for the M/L region in medium has an system in which that role can be for cell culture Y. for with serum and and for