Isoform-specific and PKC mediated regulation of CTP: phosphoethanolamine cytidylyltransferase phosphorylation [Membrane Biology]

February 10th, 2014 by Pavlovic, Z., Zhu, L., Pereira, L., Singh, R. K., Cornell, R. B., Bakovic, M.

CTP: phosphoethanolamine cytidylyl-transferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β, however very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased PE biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β, and on two PKC consensus serine residues, S-215 and S-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity and PE synthesis by 50-90%. The phosphorylation and activity of endogenous Pcyt2 was dramatically increased with phorbol esters and diminished by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI and PKCβII. Pcyt2αS-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α and -β phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences between Pcyt2 isoforms by phosphorylation and to uncover the role of PKC regulated phosphorylation.