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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

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


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