Characterization of CTP: Phosphoethanolamine cytidylyltransferase alpha and beta isoforms

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Authors

Tie Ten Quee, Angela

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University of Guelph

Abstract

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 (ET[alpha] and ET[beta]) encoded by a single gene (Pcyt2). Skipping of the 7th exon in the Pcyt2[alpha] gene produces ET[beta], which is missing an internal 18 amino acid peptide that is present in ET[alpha]. 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 ET[alpha] and ET[beta], we have generated Pcyt2[alpha] and Pcyt2[beta] cDNA expression constructs bearing C-terminal 6xHistidine and Myc or V5 tags. We have used these constructs to overexpress and purify His-tagged ET[alpha] and ET[beta] proteins in mammalian cells. The results of these studies indicate that both cDNAs are expressed as functional protein isoforms, and that ET[alpha] 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 ET[alpha] for phosphoethanolamine is 318.4 [mu]M, compared to 140.3 [mu]M for ETp. The maximal velocities of [alpha] and [beta] isoforms at saturating conditions for both substrates are 138.0 and 114.4 nmol/min/[mu]mol of enzyme, respectively. When phosphoethanolamine is used at a fixed concentration of 1 mM, the Km of ET[alpha] for CTP is 102.0 [mu]M and that of ET[beta] is 84.09 [mu]M. Both isoforms purify as homodimers and are also found to heterodimerize. The relative abundance of Pcyt2[alpha] and Pcyt2[beta] mRNAs is both tissue- and species-specific. In mouse tissues, the Pcyt2[alpha] transcript is more abundant, while in human tissues, the Pcyt2[beta] form predominates. The ratio of Pcyt2[alpha]/Pcyt2[beta] 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 ET[alpha] and ET[beta] 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 ET[alpha] 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 Pcyt2[alpha] 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.

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Keywords

alternative splicing, CTP:phosphoethanolamine cytidylyltransferase, protein function, localization

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