Isoform-specific and protein kinase C-mediated regulation of CTP:phosphoethanolamine cytidylyltransferase phosphorylation.

ABSTRACT

CTPphosphoethanolamine cytidylyltransferase (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 -ß; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -ß 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. Pcyt2ß was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2ß and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50-90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCßI, and PKCßII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -ß-phosphorylated sites to the solved structure of a human Pcyt2ß 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 in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.