title: Isoform-specific and protein kinase C-mediated regulation of CTP:phosphoethanolamine cytidylyltransferase phosphorylation.
abstract: CTP:phosphoethanolamine 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 -β; 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 -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, 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, PKCβI, and PKCβII. Pcyt2α Ser-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 in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.
authors: From the Department of Human Health and Nutritional Sciences, University of Guelph, Ontario N1G 2W1 and.
authors: Zvezdan
authors: Z
authors: Pavlovic
authors: Lin
authors: L
authors: Zhu
authors: Leanne
authors: L
authors: Pereira
authors: Ratnesh Kumar
authors: RK
authors: Singh
authors: Rosemary B
authors: RB
authors: Cornell
authors: Marica
authors: M
authors: Bakovic
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D061986:MCF-7 Cells; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010703:Phorbol Esters / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D044967:Serum / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D010703:Phorbol Esters; D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors; D011493:Protein Kinase C; D012316:RNA Nucleotidyltransferases; C020126:Ethanolamine-phosphate cytidylyltransferase
keywords: CTP:Phosphoethanolamine Cytidylyltransferase; Ethanolamine Phospholipids; Lipid Synthesis; Pcyt2; Phospholipid; Phospholipid Metabolism; Phosphorylation; Protein Kinase C; Protein Kinase C (PKC)