Characterization of CTP:phosphoethanolamine cytidylyltransferase alpha and beta isoforms

This thesis is an investigation of alternative splicing in CTP:phosphoethanolamine cytidylyltransferase (ET), and its effect on protein function and localization. ET is expressed as two alternative splice variants (ETalpha and ETbeta) encoded by a single gene (Pcyt2). Skipping of the 7th exon in the Pcyt2alpha gene produces ETbeta, which is missing an internal 18 amino acid peptide that is present in ETalpha. An identical exon skipping mechanism is observed in a diverse array of species ranging from Drosophila melanogaster to Homo sapiens (Poloumienko et al. (2004) Gene 325:145-155). We hypothesize that exon skipping in the Pcyt2 gene modulates ET function and localization. In order to characterize the similarities and differences between ETalpha and ETbeta, we have generated Pcyt2alpha and Pcyt2beta cDNA expression constructs bearing C-terminal 6xHistidine and Myc or V5 tags. We have used these constructs to overexpress and purify His-tagged ETalpha and ETbeta proteins in mammalian cells. The results of these studies indicate that both cDNAs are expressed as functional protein isoforms, and that ETalpha is the more catalytically active of the two. We demonstrate that the tagged and purified alpha and beta proteins differ significantly in their kinetic properties. The Km of ETalpha for phosphoethanolamine is 318.4 muM, compared to 140.3 muM for ETp. The maximal velocities of alpha and beta isoforms at saturating conditions for both substrates are 138.0 and 114.4 nmol/min/mumol of enzyme, respectively. When phosphoethanolamine is used at a fixed concentration of 1 mM, the Km of ETalpha for CTP is 102.0 muM and that of ETbeta is 84.09 muM. Both isoforms purify as homodimers and are also found to heterodimerize. The relative abundance of Pcyt2alpha and Pcyt2beta mRNAs is both tissue- and species-specific. In mouse tissues, the Pcyt2alpha transcript is more abundant, while in human tissues, the Pcyt2beta form predominates. The ratio of Pcyt2alpha/Pcyt2beta transcript abundance ranges from 1 in mouse muscle to greater than 3 in liver and kidney. Localization studies using immunofluorescence and subcellular fractionation indicate that ETalpha and ETbeta are present in the cytosol, endoplasmic reticulum, and nuclei of COS-7 kidney cells and C2C12 skeletal muscle cells. When overexpressed together in equal amounts, both isoforms are present in the cytoplasm, but only ETalpha is seen in the nucleus. In C2C12 myotubes, total ET protein and activity are elevated, relative to myoblasts (Zhu et al. (2009) Gene 447:51-59, in press); furthermore, our present work shows that Pcyt2alpha is the predominant mRNA form during serum deficiency in cultured C2C12 myoblasts, suggesting that down-regulation of exon skipping in the Pcyt2 gene coincides with the early stages of muscle cell differentiation.