Phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells, and its synthesis is controlled by the activity of CDP:phosphocholine cytidylyltransferase (CCT). Enforced CCT expression accelerated the rate of PtdCho synthesis. However, the amount of cellular PtdCho did not increase as a result of the turnover of both the choline and glycerol components of PtdCho. Metabolic labeling experiments demonstrated that cells compensated for elevated CCT activity by the degradation of PtdCho to glycerophosphocholine (GPC). Phospholipase Dmediated PtdCho hydrolysis and phosphocholine formation were unaffected. Most of the GPC produced in response to excess phospholipid production was secreted into the medium. Cells also degraded the excess membrane PtdCho to GPC when phospholipid formation was increased by exposure to exogenous lysophosphatidylcholine or lysophosphatidylethanolamine. The replacement of the acyl moiety at the 1-position of PtdCho with a non-hydrolyzable alkyl moiety prevented degradation to GPC. Accumulation of alkylacyl-PtdCho was associated with the inhibition of cell proliferation, demonstrating that alternative pathways of degradation will not substitute. GPC formation was blocked by bromoenol lactone, implicating the calcium-independent phospholipase A 2 as a key participant in the response to excess phospholipid. Owing to the fact that PtdCho is biosynthetically converted to PtdEtn, excess PtdCho resulted in overproduction and exit of GPE as well as GPC. Thus, general membrane phospholipid homeostasis is achieved by a balance between the opposing activities of CCT and phospholipase A 2 .

CTP:phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCT␣, has been studied extensively and a second isoform, CCT␤, was recently identified. We identify and characterize a third cDNA, CCT␤2, that differs from CCT␤1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCT␤ gene. Like CCT␣, CCT␤2 is heavily phosphorylated in vivo, in contrast to CCT␤1. CCT␤1 and CCT␤2 mRNAs were differentially expressed by the human tissues examined, whereas CCT␣ was more uniformly represented. Using isoformspecific antibodies, both CCT␤1 and CCT␤2 localized to the endoplasmic reticulum of cells, in contrast to CCT␣ which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCT␤2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCT␣ and CCT␤1 supporting proliferation at the nonpermissive conditions. Overexpression experiments did not reveal discrete physiological functions for the three isoforms that catalyze the same biochemical reaction; however, the differential cellular localization and tissue-specific distribution suggest that CCT␤1 and CCT␤2 may play a role that is distinct from ubiquitously expressed CCT␣. PtdCho1 is the major membrane phospholipid in higher eukaryotes and is also secreted by particular tissues for important extracellular tasks. For example, it is a significant component of lung surfactant, serum lipoproteins, and bile. CCT is a key regulator of PtdCho biosynthesis (1) and membraneprotein interaction is one important mechanism that governs cellular CCT activity (1, 2). Recently a second isoform, CCT␤, was discovered which is encoded by a second gene (3). CCT␣ and CCT␤ have nearly identical amino acid sequences in the catalytic domain which extends approximately from residues 72 to 233 in both proteins, and also near identity in the membrane-interaction domain which extends approximately from residues 256 to 288. Both isoforms are dependent on interaction with phospholipids for catalytic activity (3-9), as would be predicted from the high degree of identity in the membraneinteraction domains. These domains are characterized by three 11-residue amphipathic repeats that form ␣-helices upon association with phospholipid regulators (10 -13).The amino terminus of CCT␤ bears no resemblance to the amino terminus of CCT␣ and does not include a nuclear localization sequence as was identified in the CCT␣ protein (14, 15). CCT␣ has been localized predominantly in the nucleus but the physiological significance of the nuclear localization of CCT␣ remains unclear. CCT␤ protein was localized outside the cell nucleus by indirect immunofluorescent microscopy (3). CCT␤ consists of 330 amino acids, in contrast with the 367 residues of CCT␣, and lacks most of the carboxyl-terminal phosphorylation domain that is found in the CCT␣ protein (9, 16). Phosphorylation of CCT␣ interferes with the lipid stimulation of enzym...

Brewers' yeast pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate and Mg(II)-dependent enzyme, isolated from Saccharomyces cerevisiae possesses four cysteines/subunit at positions 69, 152, 221, and 222. Earlier studies conducted on a variant of the enzyme with a single Cys at position 221 (derived from a gene that was the product of spontaneous fusion) showed that this enzyme is still subject to substrate activation [Zeng, X., Farrenkopf, B., Hohmann, S., Jordan, F., Dyda, F., & Furey, W. (1993) Biochemistry 32, 2704-2709], indicating that if Cys was responsible for this activation, it had to be C221. To further test the hypothesis, the C221S and C222S single and the C221S-C222S double mutants were constructed. It is clearly shown that the mutation at C221, but not at C222, leads to abolished substrate activation according to a number of kinetic criteria, both steady state and pre steady state. On the basis of the three-dimensional structure of the enzyme [Dyda, F., Furey, W., Swaminathan, S., Sax, M., Farrenkopf, B., Jordan, F. (1993) Biochemistry 32, 6165-6170], it is obvious that while C221 is located on the beta domain, whereas thiamin diphosphate is wedged at the interface of the alpha and gamma domains, addition of pyruvate or pyruvamide as a hemiketal adduct to the sulfur of C221 can easily bridge the gap between the beta and alpha domains. In fact, residues in one or both domains must be dislocated by this adduct formation. It is very likely that regulation as expressed in substrate activation is transmitted via this direct contact made between the two domains in the presence of the activator.(ABSTRACT TRUNCATED AT 250 WORDS)

A HeLa cell line was constructed for the regulation of CTP:phosphocholine cytidylyltransferase (CCT) expression via a tetracycline-responsive promoter to test the role of CCT in apoptosis triggered by exposure of cells to the antineoplastic phospholipid 1-O-octadecyl-2-Omethyl-rac-glycero-3-phosphocholine (ET-18-OCH 3 ). Basal CCT expression in the engineered HeLa cell line was the same as in control HeLa cells lines, and CCT activity and protein were elevated 25-fold following 48 h of induction with doxycycline. Increased CCT expression prevented ET-18-OCH 3 -induced apoptosis. Acylation of exogenous lysophosphatidylcholine circumvented the requirement for CCT activity by providing an alternate route to phosphatidylcholine, and heightened CCT expression and lysophosphatidylcholine supplementation were equally effective in reversing the cytotoxic effect of ET-18-OCH 3 . Neither CCT overexpression nor lysophosphatidylcholine supplementation allowed the HeLa cells to proliferate in the presence of ET-18-OCH 3 , indicating that the cytostatic property of ET-18-OCH 3 was independent of its effect on membrane phospholipid synthesis. These data provide compelling genetic evidence to support the conclusion that the interruption of phosphatidylcholine synthesis at the CCT step by ET-18-OCH 3 is the primary physiological imbalance that accounts for the cytotoxic action of the drug. ET-18-OCH 31 is a nonmetabolizable analog of LPC and belongs to the first generation of ether lipids tested as growth inhibitors (1). These compounds do not directly target DNA, and numerous studies have demonstrated a selective cytotoxic action of ET-18-OCH 3 against transformed cells in whole animals and tissue culture (2-8). Recent work has established that the cytotoxic effect of ET-18-OCH 3 is due to the ability of the antineoplastic phospholipids to induce apoptosis in sensitive cells (9 -12). A plethora of biological processes have been suggested as primary targets for the antineoplastic etherlinked phospholipids (for reviews, see Refs. 13-16). The long list of physiological imbalances includes the inhibition of phosphatidylinositol phospholipase C and calcium movements (17-19), protein kinase C-regulated functions (20 -24), lysophospholipid metabolism (25,26), and PtdCho synthesis (12,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). Although some of the results are contradictory, it is clear that there are multiple targets for ET-18-OCH 3 , and it is not yet possible to distinguish the physiological imbalances that are causative from those that are either derivative or unrelated to the main event. Thus, a major contemporary focus in the field is to identify the critical cellular target(s) that are responsible for the cytotoxic and cytostatic actions of ET-18-OCH 3 .Our work has focused on the role of the inhibition of PtdCho synthesis in the mechanism of antineoplastic phospholipid action. PtdCho is essential for the survival of cultured cells because it is a major structural building block of biological membranes and the precurso...

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at two cysteines on the beta domain (221 and 222) and at H92 on the alpha domain, across the domain divide from C221. While C221 has been shown to provide the trigger for substrate activation [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635], the information must be transmitted from the substrate bound at this site [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600] to the active center thiamin diphosphate located at the interface of the alpha and gamma domains. Substitution at H92 with G, A, or C leads to great reduction of the Hill coefficient (from 2.0 in the wild-type enzyme to 1.2-1.3), while substitution for Lys affords an active enzyme with a Hill coefficient of 1.5-1.6. Iodoacetate at 10 mM reduced the Hill coefficient from 2.0 to 1.1, while also causing significant inactivation of the enzyme, presumably by carboxymethylation of C221. 1,3-Dibromoacetone, a potential cross-linker when added to the H92C/C222S variant at 0.1 mM, abolished substrate activation while reducing the activity only by 30%. Therefore, 1,3-dibromoacetone may cross-link C92 and C221. It was concluded that H92 is on the information transfer pathway during the substrate activation process and the interaction between C221 on the beta domain and H92 on the alpha domain is required for substrate activation. Extensive pH studies of the steady-state kinetic constants provide support for the interaction of C221 and H92 and the transmission of regulatory information to the active center via this pathway and pKaS for the two groups. This important interaction between the C221-bound pyruvate and His92 probably has both electrostatic and steric components.