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Originally published In Press as doi:10.1194/jlr.M600536-JLR200 on July 23, 2007

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Journal of Lipid Research, Vol. 48, 2172-2181, October 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

Angela Tie and Marica Bakovic1

Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Published, JLR Papers in Press, July 23, 2007.

1 To whom correspondence should be addressed. e-mail: mbakovic{at}uoguelph.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDP-ethanolamine (Kennedy) pathway. We have previously established that Pcyt2 is encoded by a single gene that can be alternatively spliced from an internal exon into two transcripts, designated Pcyt2{alpha} and Pcyt2ß. Little is currently known about the regulation of Pcyt2. Here, we functionally express both murine Pcyt2 (mPcyt2) transcripts and investigate the roles of the two proteins in the regulation of mPcyt2 activity. We demonstrate that the tagged and purified {alpha} and ß proteins differ significantly in their kinetic properties. The Km of mPcyt2{alpha} for phosphoethanolamine was 318.4 µM, compared with 140.3 µM for mPcyt2ß. The maximal velocities of the {alpha} and ß isoforms at saturating conditions for both substrates were 138.0 and 114.4 nmol/min/µmol enzyme, respectively. When phosphoethanolamine was used at a fixed concentration of 1 mM, the Km of mPcyt2{alpha} for CTP was 102.0 µM and that of mPcyt2ß was 84.09 µM. Using a combination of nondenaturing PAGE, gel filtration chromatography, and immunoprecipitation, we provide evidence that mPcyt2{alpha} and mPcyt2ß proteins can form both homodimeric and heterodimeric complexes. We show that alternative splicing of the mPcyt2 transcript is ubiquitous but could also be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2{alpha}/mPcyt2ß mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt2 activity is regulated.

Supplementary key words cytidylyltransferase • CDP-ethanolamine • phospholipids • dimerization

Abbreviations: Ni-NTA, nickel-nitrilotriacetic acid agarose; Pcyt2, CTP:phosphoethanolamine cytidylyltransferase; PE, phosphatidylethanolamine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the formation of CDP-ethanolamine from phosphoethanolamine and CTP in the rate-controlling reaction of the CDP-ethanolamine (Kennedy) pathway (1–3). This pathway is known as the sole route of de novo phosphatidylethanolamine (PE) synthesis in mammalian cells. PE is also produced by the mitochondrial decarboxylation of phosphatidylserine (4) and by base exchange with phosphatidylserine or phosphatidylcholine (5). PE is an important structural phospholipid in mammalian cell membranes, in part because of its tendency to form nonlamellar structures (6). These unique biophysical properties of PE confer a specialized role in membrane fusion-mediated events, including cytokinesis (7), endocytosis (8), and vesiculation (9). In autophagy, covalent linkage of PE to the C-terminal arginine of Apg8 is necessary for the association of this ubiquitin-like protein with the autophagosomal membrane (10). PE is a known reservoir of the potent biological mediator, arachidonic acid. The phospholipase A2, cPLA2{gamma}, is a microsomal enzyme that shows preferential activity toward arachidonic acid at the sn-2 position of PE (11). This selective arachidonic acid release is seen in response to agents of oxidative stress, such as H2O2 (11).

Specialized ether derivatives of PE known as plasmalogens constitute up to 18% of total phospholipid mass (12), with the highest content being found in brain myelin (13). The physiological functions of plasmalogens have not been well characterized, and their study is complicated by the multitude of metabolic abnormalities that accompany plasmalogen deficiency in severe inherited peroxisomal disorders such as Zellweger's syndrome (14). The importance of the CDP-ethanolamine pathway was highlighted by the observation that ethanolamine was used in the synthesis of both PE and plasmalogens, but no plasmalogens could be derived from products of phosphatidylserine decarboxylation (12).

Although CTP:phosphocholine cytidylyltransferase (Pcyt1), the analogous enzyme of the CDP-choline pathway, has been studied extensively (15, 16), considerably less is known about Pcyt2. Unlike Pcyt1, the Pcyt2 protein lacks sufficient hydrophobic domains to span a membrane (17), and lipid association is not required for the activation of Pcyt2 (18). Pcyt2 protein has been purified from rat liver (5, 18, 19), and several of its properties indicate a dimeric structure, with two phosphoethanolamine binding sites within each monomer unit (5, 19). The yeast gene (17) and human (20) and rat (21) cDNAs have been cloned and characterized. We reported on the genomic organization and differential splicing of murine Pcyt2 (mPcyt2), designating two mRNA transcripts ({alpha} and ß). The resulting proteins differ by an 18 amino acid peptide sequence encoded by exon 7 of the Pcyt2 gene and present only in the {alpha} form. We found that an identical "exon-skipping" mechanism is conserved in a wide range of species, suggesting that both transcripts have physiological relevance (22). Nevertheless, information regarding the expression, interaction, and function of the two distinct proteins is lacking. Here, we report the expression and purification of epitope-tagged mPcyt2{alpha} and mPcyt2ß proteins that differ in catalytic activity and kinetic parameters. We show variations in the tissue expression pattern of the two isoforms and present the first evidence that mPcyt2{alpha} and mPcyt2ß are both functional proteins that can form products of homodimeric and heterodimeric association.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction and expression of epitope-tagged mPcyt2{alpha} and mPcyt2ß cDNAs
Plasmids containing the mPcyt2{alpha} and mPcyt2ß cDNAs (GenBank accession numbers BC003473 and BU557590) were purchased from the American Type Culture Collection. The inserts corresponding to each cDNA coding region were subcloned into the pcDNA4/myc-His vector (Invitrogen), which adds the myc epitope and polyhistidine tags to the C terminus of the expressed protein. In brief, the cDNA coding regions of mPcyt2{alpha} and mPcyt2ß were amplified using PCR primers designed to add the EcoRV and XhoI restriction sites to the 5' and 3' ends of the cDNAs, respectively. The translation termination codons were deleted so that the myc and Hisx6 tags would be added onto the mPcyt2 proteins. Amplified cDNAs were subcloned into the pGEM-T Easy vector (Promega), then removed by restriction digestion with EcoRV and XhoI and subcloned into the pcDNA4/myc-His vector. These two constructs are referred to as mPcyt2{alpha}-myc-His and mPcyt2ß-myc-His. The mPcyt2{alpha} cDNA was also cloned into the pcDNA3.1/V5-His vector (Invitrogen), which adds the C-terminal V5 epitope and polyhistidine tags. The cDNA inserts in all final constructs were sequenced at the Guelph Molecular Supercenter to ensure that coding errors had not occurred. COS-7 cells were grown in DMEM with 10% fetal bovine serum. For enzyme activity and Western blotting, cells in 60 mm dishes were transfected with 5 µg of empty vector, mPcyt2{alpha}-myc-His, or mPcyt2ß-myc-His constructs using 20 µl of Lipofectamine (Invitrogen). Forty-eight hours after transfection, the cells were harvested in lysis buffer [25 mM Tris-phosphate, pH 7.8, 15% glycerol, 1% Triton X-100, 8 mM MgCl2, 1 mM dithiothrietol, 10 µg/ml each leupetin, aprotinin, and soybean trypsin inhibitor, 0.5 mM PMSF, 1 µg/ml pepstatin A, 0.01% Nonidet P-40, and 0.2% phosphatase inhibitor cocktail II (Sigma)].

Enzyme activity assays
Total cell lysates were prepared by harvesting cells in lysis buffer as described above. After centrifugation at 13,000 g and 4°C to pellet cell debris, the supernatants were assayed for mPcyt2 activity as described (23), except that commercially available [14C]phosphoethanolamine (American Radiolabeled Chemicals) was used at a final specific activity of 2 µCi/µmol. The final concentrations of phosphoethanolamine and CTP were 1 and 2 mM, respectively. All reactions were incubated at 37°C for 15 min followed by boiling for 2 min to inactivate the enzyme. CDP-ethanolamine and phosphoethanolamine standards were added to the samples and resolved on Silica Gel G plates in a solvent system of methanol/0.5% NaCl/ammonia (50:50:5). Individual spots were visualized using 2% ninhydrin in 95% ethanol, then scraped and subjected to liquid scintillation analysis. Three measurements were taken from each of three independent transfections.

Radiolabeling with [14C]ethanolamine
COS-7 cells were grown on six-well plates and transfected with 2.5 µg of plasmid DNA (empty vector, mPcyt2{alpha}-myc-His, or mPcyt2ß-myc-His) using 10 µl of Lipofectamine. Forty-six hours after transfection, the cells were preconditioned for 2 h in DMEM containing 50 µM ethanolamine, followed by 30 min of labeling with [14C]ethanolamine (0.2 µCi/well) in the presence of 50 µM unlabeled ethanolamine. Cells were then washed twice in ice-cold PBS-A and harvested by trypsinization. The lipid and water-soluble fractions were separated by the method of Bligh and Dyer (24), and the radiolabeled ethanolamine, phosphoethanolamine, and CDP-ethanolamine in the methanol-water phase were separated and analyzed as described above. Three experiments were performed in triplicate. Total ethanolamine incorporation into PE was determined by evaporation of the entire chloroform phase under nitrogen, followed by the separation of PE from lyso-PE in a TLC system of chloroform-methanol-ammonia (65:35:5). Spots corresponding to PE and lyso-PE standards were scraped and subjected to liquid scintillation analysis. Because the contribution of lyso-PE was negligible, only PE was considered in the analysis. PE analysis was performed twice in duplicate.

Purification of expressed mPcyt2{alpha} and mPcyt2ß proteins
COS-7 cells in 100 mm dishes were transfected with 16 µg of either mPcyt2{alpha}-myc-His or mPcyt2ß-myc-His using 20 µl of Lipofectamine (Invitrogen). After 48 h, cells were harvested by trypsinization and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, and 1% Triton X-100, pH 8.0) with protease and phosphatase inhibitor cocktails (Sigma). Cells were lysed by rocking at 4°C for 10 min and centrifuged at 4°C and 13,000 g for 10 min. For every 1 ml of lysis buffer used, 50 µl of Ni-NTA Superflow nickel-charged resin (Qiagen) was added to the supernatant (cleared lysate) and allowed to bind with rocking at 4°C for 90 min. The sample mixture was loaded onto a chromatography column, and the unbound fraction was allowed to flow through by gravity. The resin was washed four times with 50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH 8.0). Bound protein was eluted multiple times with 100 µl of elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole, pH 8.0). The cleared lysate, unbound fraction, washes, and eluted fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Eluted fractions containing histidine-tagged protein were pooled and dialyzed overnight against 500 volumes of 20 mM Tris-HCl, 0.5 mM EDTA, 2 mM dithioerythritol, and 5% (v/v) ethylene glycol. SDS-PAGE and Coomassie Brilliant Blue staining were used again to compare the purities of the final protein preparations.

Substrate kinetic studies with purified mPcyt2{alpha} and mPcyt2ß protein
Before the kinetic studies, multiple time courses were carried out at three different protein concentrations to ensure that CDP-ethanolamine formation was linearly related to protein amount. In addition, the molar concentration of mPcyt2{alpha} was determined using spectrophotometry, and Western blotting with the anti-myc antibody followed by densitometry (data not shown) was used to equalize the molar amounts of each enzyme used in the experiments. Enzyme activity assays were carried out as described (23), with CTP at a fixed concentration of 2 mM and unlabeled phosphoethanolamine varying from 31.25 µM to 1.0 mM. [14C]phosphoethanolamine was used at a final specific activity of 0.2 µCi/µmol. All reactions were incubated at 37°C for 18 min, followed by inactivation by boiling for 2 min. Thin-layer chromatography and liquid scintillation analysis were performed as described above. Experimental data were plotted as reaction velocity (V) versus phosphoethanolamine concentration ([P-etn]) using GraphPad Prism version 4.00 for Windows (GraphPad Software), and Km (µM) and Vmax (nmol/min/µmol enzyme) were calculated after fitting the experimental data into a hyperbolic form of the Michaelis-Menten equation. Each experimental point on the curve represents the mean ± SD of six replicates from two independent experiments.

Substrate kinetics were then determined with CTP as the variable substrate and [P-etn] fixed at 250, 500, and 1,000 µM in turn. CTP concentration was varied from 3.125 to 1,000 µM in each experiment. The final specific activity of [14C]phosphoethanolamine used was 0.2, 0.4, or 0.8 µCi/µmol. Linear transformations of the data were carried out using GraphPad software to produce double reciprocal plots. All CTP experiments were performed twice in triplicate.

Electrophoresis, immunoblotting and gel filtration
For the identification of expressed mPcyt2{alpha} and mPcyt2ß proteins under denaturing conditions, 10–50 µg of total cell proteins was separated by 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with anti-His (Invitrogen) or anti-Pcyt2total (designed by us to recognize the epitope VTKAHHSSQEMSSEYRE, common to both isoforms). For native PAGE, total cell proteins (50 µg) were separated through a 4–20% acrylamide gradient and immunoprobed as described above for denaturing gels. Samples of purified mPcyt2{alpha} and mPcyt2ß were subjected to gel filtration chromatography on a BioGel P60 (Bio-Rad) column. The void volume of the column was first determined using BSA as a standard. Elution of mPcyt2 was monitored by Western blotting with anti-myc antibody (Invitrogen).

Immunoprecipitation
For coimmunoprecipitation studies, COS-7 cells were transfected with 4 µg each of mPcyt2{alpha}-V5-His and mPcyt2ß-myc-His per 60 mm dish. Cells were harvested in a lysis buffer, and 500 µg of total protein was precleared with 50 µl of protein G agarose (Sigma) and immunoprecipitated with a 1:100 dilution of anti-V5 (Invitrogen). The final pellet was resuspended in 50 µl of nonreducing sample buffer and boiled for 5 min. SDS-PAGE and Western blotting of total cell lysates, immunoprecipitation supernatant, and immunoprecipitation fractions were carried out using both anti-V5 and anti-myc (Invitrogen).

Tissue-specific expression of mPcyt2{alpha} and mPcyt2ß mRNA
We initially determined the total mRNA expression and enzyme activity of mPcyt2 (22). Here, the expression patterns of mPcyt2{alpha} and mPcyt2ß mRNAs were examined in various mouse tissues. The brain, adipose, heart, kidney, liver, and skeletal muscle were dissected from three adult female C57BL/6 mice, flash-frozen in liquid nitrogen, and stored at –80°C until RNA was extracted. Total RNA isolation and first-strand cDNA synthesis using TRIzol (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen) were carried out under standard conditions. To avoid complications arising from differing reaction efficiencies, a single primer set (F6, 5'-ggagatgtcctctgagtaccg-3'; R7, 5'-ggcaccagccacatagatgac-3') was chosen for simultaneous amplification of both isoforms. The region targeted by these primers includes the alternatively spliced exon 7, producing amplified fragments of 224 and 170 bp for mPcyt2{alpha} and mPcyt2ß, respectively. PCR amplification was carried out for 36 cycles with a 30 s annealing step at 62°C. A 450 bp fragment of GAPDH was used as an internal control and amplified for 22 cycles with primer annealing at 58°C. The primers used were GAPDH-FP (5'-tccaccaccctgttgctgta-3') and GAPDH-RP (5'-accacagtccatgccatcac-3'). The band densities of all DNA fragments were determined using the Scion Image densitometry program, and all ratios are expressed as means ± SD of three individual mice assayed in duplicate.

Statistics
All statistical analyses were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software). To evaluate the differences in enzyme activities and metabolic labeling, the paired and unpaired Student's t-test, respectively, were used. To test for goodness of fit of the enzyme data to the Michaelis-Menten hyperbolic function, a least sum of squares test was used. For differences in mRNA expression between tissues, one-way ANOVA followed by Dunnett's multiple pairwise comparison was used. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpressed mPcyt2{alpha}-myc-His and mPcyt2ß-myc-His differ in activity
Results of the functional expression of myc-His-tagged mPcyt2{alpha} and mPcyt2ß are shown in Figs. 1 , 2 . Western blotting using the anti-histidine antibody showed that the mPcyt2ß protein had an apparent molecular mass of ~49 kDa, whereas mPcyt2{alpha} appeared slightly larger (Fig. 1A), indicative of the additional 18 amino acid peptide it contains (22). Western blotting with Pcyt2total antibody (Fig. 1B) was then used to confirm the identities of the expressed proteins. The total Pcyt2 activities per milligram of total cell protein in control cells (baseline) and mPcyt2{alpha} and mPcyt2ß overexpressors are depicted in Fig. 2A. Densitometry was performed on the Western blot shown in Fig. 1A, and the total Pcyt2 activity of the {alpha} and ß overexpressors was normalized for the amount of tagged mPcyt2 present, after subtraction of the baseline Pcyt2 activity in vector control cells. Figure 2B shows the specific activities of mPcyt2{alpha} and mPcyt2ß per milligram of total cell protein adjusted for mPcyt2 expression level. The adjusted mean activities of the {alpha} and ß isoforms were 9.883 ± 0.9084 and 2.375 ± 0.4867 nmol/min/mg, respectively, a significant 4-fold difference (P < 0.002).


Figure 1
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  		Fig. 1. A, B: Detection of mPcyt2{alpha}-myc-His and mPcyt2-ß-myc-His expressed in COS-7 cells. Cells in 60 mm dishes were transfected with 5 µg of empty vector or pcDNA4/myc-His containing either murine CTP:phosphoethanolamine cytidylyltransferase (mPcyt2) {alpha} or ß cDNA using 20 µl of Lipofectamine. Total cell lysates were prepared at 48 h after transfection, and proteins (10–50 µg) were separated through 12% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and immunoblotted using antibodies as indicated. Lane 1, vector control; lane 2, mPcyt2{alpha}-myc-His; lane 3, mPcyt2ß-myc-His.

 

Figure 2
Figure 2
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  		Fig. 2. A: In vitro activity of mPcyt2{alpha} and mPcyt2ß. COS-7 cells were transfected with 5 µg of empty vector or with pcDNA4/myc-His containing either mPcyt2{alpha} or mPcyt2ß cDNA. Cells were harvested after 48 h, and 13,000 g supernatants were assayed for mPcyt2 activity as described (15), except that commercially available [14C]phosphoethanolamine (American Radiolabeled Chemicals) was used at a final specific activity of 2 µCi/µmol. Results are means ± SD of three independent experiments expressed as nmol/min/mg total protein. * Significantly different from control (P < 0.001); ** significantly different from both control and Pcyt2{alpha} (P < 0.002). B: Total Pcyt2 activity of the {alpha} and ß overexpressors normalized for the amount of mPcyt2 present, as determined by densitometry, and after subtraction of baseline Pcyt2 activity. Results are means ± SD. The adjusted mean activities of the {alpha} and ß isoforms were 9.883 ± 0.9084 and 2.375 ± 0.4867 nmol/min/mg, respectively, a significant 4-fold difference (P < 0.002).

 
Overexpression of mPcyt2{alpha} and mPcyt2ß increases the synthesis of CDP-ethanolamine
Results of metabolic labeling experiments, in which [14C]ethanolamine was incorporated into the Kennedy pathway, are shown in Fig. 3 . COS-7 cells transfected with control vector, mPcyt2{alpha}-myc-His, or mPcyt2ß-myc-His were pulse-labeled for 30 min with [14C]ethanolamine, and the production of water-soluble PE precursors and PE were compared. Figure 3A, B show no significant differences in radiolabeled ethanolamine and phosphoethanolamine (the immediate precursor of CDP-ethanolamine and a substrate of mPcyt2). As expected from the data in Fig. 2, the expression of mPcyt2{alpha} and mPcyt2ß resulted in 4.2- and 2.5-fold increases in the labeling of their immediate product CDP-ethanolamine (Fig. 3C). The amount of CDP-ethanolamine formed in 30 min of labeling when mPcyt2{alpha} was overexpressed was 1.7 times that of cells expressing the ß form, agreeing closely with the in vitro activity data (Fig. 2). Lastly, overexpression of either form of mPcyt2 had no effect on the rate of PE formation (Fig. 3D). This was likely because the reaction catalyzed by ethanolamine phosphotransferase, the last enzymatic step in the Kennedy pathway, is limited by the supply of diacylglycerol (25).


Figure 3
Figure 3
Figure 3
Figure 3
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  		Fig. 3. Incorporation of [14C]ethanolamine (A) into phosphoethanolamine (B), CDP-ethanolamine (C), and phosphatidylethanolamine (PE) (D). COS-7 cells in 35 mm dishes were transfected with 2.5 µg of empty vector or with pcDNA4/myc-His containing either mPcyt2{alpha} or mPcyt2ß cDNA using 10 µl of Lipofectamine. Forty-six hours after transfection, cells were incubated for 2 h with 50 µM ethanolamine and for 30 min with [14C]ethanolamine (0.2 µCi/well) in the presence of 50 µM unlabeled ethanolamine. Lipid and water-soluble fractions were separated by the method of Bligh and Dyer (24), and the methanol-water phase-soluble metabolites were resolved on Silica Gel G plates in methanol/0.5% NaCl/ammonia (50:50:5). All metabolites were visualized with 2% ninhydrin. Individual spots were subjected to liquid scintillation analysis. Total PE was determined from the chloroform phase. Results for water-soluble intermediates are means ± SD of three experiments performed in triplicate. For PE, means are of two experiments in duplicate. * Significantly different from control (P < 0.0001); ** significantly different from both control and mPcyt2{alpha} (P < 0.002).

 
Purified mPcyt2{alpha} and mPcyt2ß differ in their kinetic properties
Figure 4 shows the relative purities of mPcyt2{alpha} and mPcyt2ß proteins. Multiple protein bands were apparent in the whole cell lysates (Fig. 4, lanes 1, 3). For each isoform, a single band was apparent (Fig. 4, lanes 2, 4) after purification and dialysis, as described in Materials and Methods. By comparing the activities per milligram of total cell protein in crude lysates with those of known quantities of pure protein, determined spectrophotometrically using a standard protein curve, we estimated a minimum 128-fold purification of the enzyme activity. Before kinetic experiments were performed, the activities of the final protein preparations were determined, and activity assays were performed at multiple time points with several different protein concentrations to establish the linearity of the reaction with protein amount and time. Figure 5A shows saturation curves after plotting of the reaction velocity in nmol/min/µmol enzyme as a function of [P-etn] for both mPcyt2{alpha} and mPcyt2ß. Curve-fitting of velocity data to the Michaelis-Menten equation indicated that the two Pcyt2 isoforms were functionally distinct proteins, with differences in both substrate affinity and the rate of catalytic turnover. The Km of mPcyt2{alpha} for phosphoethanolamine was 318.4 µM, compared with 140.3 µM for mPcyt2ß. The maximal velocities of the {alpha} and ß isoforms were 138 and 114.4 nmol/min/µmol enzyme, respectively. Figure 5B, C show the saturation curves obtained when CTP concentration was varied from 31.25 to 1,000 µM at several fixed concentrations of phosphoethanolamine. When 1 mM phosphoethanolamine was used, the Km of mPcyt2{alpha} for CTP was 102 µM and that of mPcyt2ß was 84.09 µM. Double reciprocal plots of the same data from Fig. 5B, C resulted in a series of lines intersecting at a common point (Fig. 5D, E), indicative of a sequential reaction mechanism.


Figure 4
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  		Fig. 4. Relative purity of mPcyt2{alpha} and mPcyt2ß preparations. Ten 10 cm culture dishes of COS-7 cells were transfected with mPcyt2{alpha} or mPcyt2ß in pcDNA4/myc-His. Cells were harvested after 48 h, and cleared lysates were applied to a Ni-NTA Superflow (Qiagen) column. The column was washed four times, and histidine-tagged proteins were eluted in multiple fractions. Pure protein fractions were dialyzed against 500 volumes of dialysis buffer, as described in Materials and Methods, and the final preparations were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Lane 1, cleared lysate; lane 2, purified mPcyt2{alpha}; lane 3, cleared lysate; lane 4, purified mPcyt2ß.

 

Figure 5
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Figure 5
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  		Fig. 5. A: Substrate kinetics of mPcyt2{alpha} and mPcyt2ß with CTP at a fixed concentration of 2 mM and phosphoethanolamine concentration ([P-etn]) varying from 31.25 µM to 1.0 mM. [14C]phosphoethanolamine was used at a final specific activity of 0.2 µCi/µmol. B, C: CTP kinetics of mPcyt2{alpha} (B) and mPcyt2ß (C) with phosphoethanolamine at fixed concentrations of 250, 500, and 1,000 µM and CTP varying from 31.25 µM to 1.0 mM. [14C]phosphoethanolamine was used at a final specific activity of 0.2–0.8 µCi/µmol. D, E: Double reciprocal plots of the data in B, C. All reactions were incubated at 37°C for 18 min. Curve-fitting to the hyperbolic Michaelis-Menten equation and Lineweaver-Burk transformations of experimental data were done using GraphPad Prism software. All experiments were performed twice in triplicate. Results are means ± SD.

 
mPcyt2{alpha} and mPcyt2ß associate as homodimers and heterodimers
Using native gradient PAGE and the Pcyt2total antibody, we investigated the formation of higher order protein complexes by mPcyt2{alpha} and mPcyt2ß expressed individually and together in COS-7 cells. As shown in Fig. 6A (lane 2), myc-His-tagged mPcyt2{alpha} appears as a single band of ~108 kDa, which represents the {alpha}/{alpha} homodimer. Under the same conditions, mPcyt2ß (Fig. 6A, lane 3) appears as two bands, the weaker of the two at ~110 kDa and the stronger one at ~90 kDa. Based on the estimated monomeric size of ~50 kDa (Fig. 1), these results show that both isoforms associate as homodimers and that mPcyt2ß has at least two ß/ß mobility variants, the cause of which is yet unknown. When both isoforms were expressed together (Fig. 6A, lane 4), complexes that electrophoretically reassembled ß/ß homodimers were apparent, although the presence of {alpha}/{alpha} homodimers and {alpha}/ß heterodimers could not be ruled out.


Figure 6
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  		Fig. 6. A: Nondenaturing gradient PAGE of mPcyt2 dimers. mPcyt2{alpha} and mPcyt2ß were expressed separately and together in COS-7 cells. Whole cell lysates were subjected to nondenaturing PAGE in a 4–20% gradient, transferred to polyvinylidene difluoride membranes, and immunoblotted with antibody to total Pcyt2. Lane 1, vector control cells; lane 2, cells overexpressing mPcyt2{alpha}-myc-His; lane 3, cells overexpressing mPcyt2ß-myc-His; lane 4, cells overexpressing both mPcyt2{alpha}-V5-His and mPcyt2ß-myc-His. B, C: Western blot with anti-myc antibody after gel filtration chromatography of mPcyt2{alpha} (B) and mPcyt2ß (C) dimers. Myc-His-tagged and purified mPcyt2{alpha} and mPcyt2ß were subjected to gel filtration chromatography in a Bio-Gel P60 column, and the fractions were analyzed by Western blotting with anti-myc antibody.

 
Gel filtration chromatography was performed on purified mPcyt2{alpha} and mPcyt2ß to confirm the dimeric structure of both isoforms. When each protein was subjected to gel filtration on a column with an exclusion limit of 60 kDa, all of the eluted protein was present in fractions corresponding to the void volume of the column (1.8 ml), as was determined previously using BSA. All 150 µl fractions obtained by gel filtration of mPcyt2{alpha} and mPcyt2ß were analyzed for the presence of tagged protein using the anti-myc antibody, and those that contained the protein are shown (Fig. 6B, C). No protein was detected in any of the subsequent fractions (data not shown). These results indicate that both mPcyt2 isoforms were excluded from the gel and therefore must have a molecular mass of >60 kDa. This would only be possible if the proteins were in dimeric form, as the molecular mass of the {alpha} and ß monomers would be ~51 and ~49 kDa.

To further determine whether mPcyt2{alpha}/ß heterodimers could form, we coexpressed mPcyt2{alpha}-V5-His and mPcyt2ß-myc-His, then performed immunoprecipitation with anti-V5 and detection with anti-myc. As shown in Fig. 7A , the myc epitope was clearly detected in the anti-V5 precipitates, indicating an mPcyt2{alpha}/ß association. Myc was also detected in the total lysates of cotransfected cells and the immunoprecipitation supernatants but was absent from control cells. Western blotting with anti-V5 (Fig. 7B) revealed that mPcyt2{alpha}-V5 was present in the total lysates and precipitates but absent from the supernatant, indicating that the V5 antibody was completely effective in pulling down all of the expressed mPcyt2{alpha}-V5 protein. An additional Western blot of the lysates from cells overexpressing mPcyt2{alpha}-V5, mPcyt2ß-myc-His, or both constructs together was performed to show the specificity of the anti-V5 antibody for proteins with a V5 tag. Because no mPcyt2ß-myc-His was detected with this antibody, a nonspecific immunoprecipitation of mPcyt2ß-myc-His was not likely.


Figure 7
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  		Fig. 7. Immunoprecipitation of mPcyt2{alpha}/ß complexes. A, B: Lysates from cells cotransfected with mPcyt2{alpha}-V5-His and mPcyt2ß-myc-His were subjected to immunoprecipitation (IP) using anti-V5, and Western blotting was carried out using anti-myc (A) and anti-V5 (B). Lanes 1, 3, 5, vector control cells; lanes 2, 4, 6, cotransfected cells. C: Western blot showing the specificity of anti-V5 for the V5-tagged protein. Lane 1, lysates from cells overexpressing mPcyt2{alpha}-V5-His; lane 2, lysates from mPcyt2ß-myc-His overexpressors; lane 3, lysates from cells overexpressing both myc and V5 constructs.

 
Alternative splicing of mPcyt2 is tissue-specific
Several mouse tissues were analyzed for the expression of mPcyt2{alpha} and mPcyt2ß mRNAs (Fig. 8 ). Because the single primer pair we used should anneal equally well to the priming sites (on either side of the alternatively spliced exon 7) in both mPcyt2 mRNAs, it is reasonable to compare the mRNA abundance of two splice variants within a tissue using this type of assay, as was done for the SMRT gene by Malartre, Short, and Sharpe (26). Of all tissues examined, liver and adipose showed the highest level of mPcyt2{alpha}, whereas the lowest expression was found in muscle, brain, and heart (Fig. 8A). The level of mPcyt2ß mRNA was relatively low in all tissues (Fig. 8B), in keeping with the previous observation that mPcyt2{alpha} was the more prominent of the two isoforms in the murine expressed sequence database (22). Considered together with the relatively large variation observed in mPcyt2{alpha} mRNA expression (Fig. 8A) and the higher catalytic activity of {alpha} relative to ß (Figs. 2, 3), these observations suggest that in the mouse, variations in total Pcyt2 activity are largely determined by the expression of the {alpha} form. In a few tissues, namely brain, heart, and muscle, appreciable amounts of mPcyt2ß could additionally modulate mPcyt2 function. When the ratios of {alpha}/ß mRNA abundance were calculated, the liver, kidney, and fat emerged with the highest amounts (3.2, 3.1, and 2.6), whereas muscle had nearly equal amounts of each variant (Fig. 8).


Figure 8
Figure 8
Figure 8
Figure 8
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  		Fig. 8. A: Tissue-specific distribution of mPcyt2{alpha} and mPcyt2ß transcripts. PCR primers flanking the alternatively spliced exon 7 were used to amplify 224 and 170 bp fragments of the mPcyt2{alpha} and mPcyt2ß cDNAs, respectively. In B and C, mPcyt2 mRNA expression was normalized to that of GAPDH. D: Ratio of mPcyt2{alpha} to mPcyt2ß expression. Results are means ± SD of three individual animals, each analyzed in duplicate. ANOVA was performed at P < 0.05 (*) or at P < 0.01 (**) relative to muscle.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We report for the first time the functional expression, purification, and kinetics of two structurally distinct isoforms of mPcyt2. The aims of our study were to characterize the function and differences between these two proteins and to gain insight into the role of alternative splicing in the regulation of enzyme function. Results obtained using three independent approaches (in vitro activity assays, metabolic labeling, and kinetic assays of purified proteins) demonstrate that the catalytic activities of Pcyt2{alpha} and Pcyt2ß isoforms differ significantly. Because the only structural difference between the mPcyt2{alpha} and mPcyt2ß proteins used in these studies is the 18 amino acid "linker peptide" (180-PPHPTPAGDTLSSEVSSQ-197) that is present only in the {alpha} variant, it is highly probable that this peptide is a major factor in the modulation of mPcyt2 function. This study provides the first direct evidence linking this particular exon-skipping mechanism to the functional consequence of decreased enzymatic activity. Several observations led to this hypothesis. First, the linker peptide is situated between two conserved catalytic domains (17), indicating that its presence or absence would affect the molecular distance between the two putative catalytic sites. Second, our examination of the amino acid composition of the linker peptide indicated a loop sequence containing four proline residues, which are known to introduce random coil structures between {alpha}-helical and/or ß-sheet domains (27). Third, a similar alternative splicing mechanism for the Pcyt2 gene is highly conserved throughout the animal kingdom, suggesting that the presence of Pcyt2{alpha} and Pcyt2ß isoforms bears evolutionary significance (22). Our kinetic analyses indicate that mPcyt2{alpha} has both a lower affinity for phosphoethanolamine (indicated by the Km values) and a higher maximal velocity than mPcyt2ß when phosphoethanolamine and CTP are at saturating conditions.

When rat Pcyt2 was initially purified by Sundler (5), the behavior of the protein on gel chromatography indicated a dimeric structure. Using a combination of native gradient PAGE, gel filtration chromatography, and Western blotting, we evaluated the individual ability of Pcyt2{alpha} and Pcyt2ß to form homodimers. Because all bands that were detected using the Pcyt2 antibody were well above the estimated monomeric sizes of 49 and 51 kDa, we conclude that monomeric Pcyt2 does not exist or is not present in appreciable quantities and that each isoform is able to associate into homodimers in the absence of the other. In addition, gel filtration chromatography indicated that both isoforms existed only as dimers. Our immunoprecipitation experiments on coexpressed proteins, in which myc-tagged mPcyt2ß was shown to coprecipitate with V5-tagged mPcyt2{alpha}, provide the first known evidence of a heteromeric Pcyt2{alpha}/ß association.

The possibility exists that homodimerization and heterodimerization itself play a role in the overall regulation of Pcyt2 activity. There are two levels at which this could occur. The first is the activity of the spliceosome complex toward the mPcyt2 primary mRNA transcript, which would directly influence the relative amounts of each isoform expressed. This would in turn determine, in whole or in part, the position of the dimerization equilibrium (the proportion of Pcyt2 dimers as {alpha}/{alpha}, {alpha}/ß, and ß/ß). Results from our multiple tissue mRNA expression study indicated that the relative levels of mPcyt2{alpha} and Pcyt2ß transcripts do indeed vary from one tissue to another, as is often the case with alternatively spliced mRNAs (28).

The second possibility is that there is posttranslational control over the formation of dimers. This process could be modulated by structural aspects of the linker peptide, such as its phosphorylation state (L. Zhu and M. Bakovic, unpublished data), that could either promote or inhibit dimer formation between like or unlike monomers. In the theoretical case in which equal amounts of {alpha} and ß monomer units were expressed, one would expect {alpha}/{alpha}, {alpha}/ß, and ß/ß dimers to occur in ratios of 1:2:1 if the process of dimerization was completely random. If, however, the affinity of the {alpha}/{alpha} or ß/ß association far exceeded that of the {alpha}/ß association, then a greater than expected proportion of the total enzyme molecules would be in homodimeric form and the net activity should be close to the average of activities we observed for homodimers. If, on the other hand, the tendency toward heterodimerization was greater than or equal to the affinity of like monomer units for each other, then the net activity would depend on both the (as yet unknown) catalytic properties of the heterodimer and the extent of heterodimerization.

The existence of multiple isoforms among enzymes of the CDP-choline and CDP-ethanolamine pathways is not unique to Pcyt2. In fact, there is a known functional redundancy at each enzymatic step, with at least two isoforms encoded by at least two different genes for choline/ethanolamine kinase (29), ethanolamine kinase (30, 31), CTP:phosphocholine cytidylyltransferase (32), and choline/ethanolamine phosphotransferase (33). In this regard, Pcyt2 is no different, except that both isoforms are encoded by the same gene and, to the best of current knowledge, only alternative splicing is responsible for enzyme diversity in Pcyt2. We have just begun to characterize two isoforms of Pcyt2, about which little has been revealed to date. This enzyme can now be included in a growing list of proteins for which alternative splicing generates functional diversity.


   ACKNOWLEDGMENTS
 
The authors thank the Canadian Institutes for Health Research (Grant 129345 to M.B.) and the Ontario Graduate Scholarship program (for financial support to A.T.).

Manuscript received December 19, 2006 and in revised form May 9, 2007 and in re-revised form July 6, 2007.


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L. Zhu, C. Johnson, and M. Bakovic
Stimulation of the human CTP:phosphoethanolamine cytidylyltransferase gene by early growth response protein 1
J. Lipid Res., October 1, 2008; 49(10): 2197 - 2211.
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