Identification of a novel CTP:phosphocholine cytidylyltransferase (CCT) isoform and potential CCT binding proteins.

Abstract

CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha1) catalyzes formation of CDP-choline and is the rate limiting step in phosphatidylcholine biosynthesis. The enzyme is a modular protein characterized by an N-terminal nuclear localization signal, a central catalytic, an adjacent lipid-activation segment, and an extreme C-terminal phosphorylation segment. A survey of the EST database identified multiple human ESTs which are apparent splice variants of CCTalpha1 and encode a second isoform, CCTalpha2. CCTalpha2 contains a novel C-terminus that extends 31 amino acids beyond the last conserved residue of the CCTalpha1 catalytic domain and thus, lacks both a lipid-activation and phosphorylation segment. Additionally, the CCTalpha2 mRNA contains a canonical polyadenylation signal and a BLAST analysis of the human genome indicates the novel 3<super>'</super>-sequence derives from retention of the intron immediately adjacent to the exon encoding end of the conserved catalytic domain. RT-PCR and mRNA dot blot analysis both confirm the expression of the CCTalpha2 mRNA in a wide range of tissues. Phosphatidylcholine biosynthesis and cell viability are restored upon expression of CCTalpha2 in a cytidylyltransferase deficient temperature-sensitive Chinese Hamster Ovary cell line. Finally, characterization of CCTalpha2 activity confirms that it is a choline phosphate cytidylyltransferase and that this activity is largely lipid independent. In addition to the characterization of CCTalpha2, we have also employed the two-hybrid system in an effort to identify novel protein effectors of CCT. Using CCTalpha1 as a bait, we isolated a single binding protein, pJC1, from multiple screens of a two-hybrid HeLa cell library. A BLAST analysis of clone pJC1 indicates that it encodes the C-terminal two-thirds of a larger 920 amino acid protein. This search also revealed multiple pJC1 homologs encoded by several genes, suggesting that these proteins comprise a multigene family. The function of pJC1, as well as other putative family members, is unclear as these proteins exhibits no discernable functional domains or informative motifs. However, the two-hybrid interaction does appear to be specific for the CCTalpha isoforms as pJC1 fails to interact with either CCTbeta1 or CCTbeta2 as well as multiple nonspecific baits. Moreover, western blot analyses of CCTalpha1 HeLa cell immunoprecipitates using a pJC1 antibody indicate that pJC1 does coimmunoprecipitate with CCTalpha1, suggesting that pJC1 may indeed be a true CCTalpha binding protein.