Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

Angela Tie,Marica Bakovic

doi:10.1194/jlr.m600536-jlr200

Abstract

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 Pcyt2alpha and Pcyt2beta. 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 beta proteins differ significantly in their kinetic properties. The K(m) of mPcyt2alpha for phosphoethanolamine was 318.4 microM, compared with 140.3 microM for mPcyt2beta. The maximal velocities of the alpha and beta isoforms at saturating conditions for both substrates were 138.0 and 114.4 nmol/min/mumol enzyme, respectively. When phosphoethanolamine was used at a fixed concentration of 1 mM, the K(m) of mPcyt2alpha for CTP was 102.0 microM and that of mPcyt2beta was 84.09 microM. Using a combination of nondenaturing PAGE, gel filtration chromatography, and immunoprecipitation, we provide evidence that mPcyt2alpha and mPcyt2beta 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 mPcyt2alpha/mPcyt2beta mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt2 activity is regulated.

Highlights

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDPethanolamine (Kennedy) pathway
We show that alternative splicing of the murine Pcyt2 (mPcyt2) transcript is ubiquitous but could be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2a/mPcyt2b mRNAs
The importance of the CDP-ethanolamine pathway was highlighted by the observation that ethanolamine was used in the synthesis of both PE and plasmalogens, but no plasmalogens could be derived from products of phosphatidylserine decarboxylation [12]

Summary

Introduction

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDPethanolamine (Kennedy) pathway. We show that alternative splicing of the mPcyt transcript is ubiquitous but could be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2a/mPcyt2b mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt activity is regulated.—Tie, A., and M. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the formation of CDP-ethanolamine from phosphoethanolamine and CTP in the rate-controlling reaction of the CDP-ethanolamine (Kennedy) pathway [1,2,3] This pathway is known as the sole route of de novo phosphatidylethanolamine (PE) synthesis in mammalian cells. We show variations in the tissue expression pattern of the two isoforms and present the first evidence that mPcyt2a and mPcyt2b are both functional proteins that can form products of homodimeric and heterodimeric association

Objectives

Methods

Conclusion