title: Functions of the C-terminal domain of CTP: phosphocholine cytidylyltransferase. Effects of C-terminal deletions on enzyme activity, intracellular localization and phosphorylation potential.
abstract: The role of the C-terminal domain of CTP: phosphocholine cytidylyltransferase (CT) was explored by the creation of a series of deletion mutations in rat liver cDNA, which were expressed in COS cells as a major protein component. Deletion of up to 55 amino acids from the C-terminus had no effect on the activity of the enzyme, its stimulation by lipid vesicles or on its intracellular distribution between soluble and membrane-bound forms. However, deletion of the C-terminal 139 amino acids resulted in a 90% decrease in activity, loss of response to lipid vesicles and a significant decrease in the fraction of membrane-bound enzyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of 32P-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was 32P-labelled in vivo. Phosphorylation was restricted to the C-terminal 52 amino acids (domain P) and occurred on multiple sites. CT phosphorylation in vitro was catalysed by casein kinase II, cell division control 2 kinase (cdc2 kinase), protein kinases C alpha and beta II, and glycogen synthase kinase-3 (GSK-3), but not by mitogen-activated kinase (MAP kinase). Casein kinase II phosphorylation was directed exclusively to Ser-362. The sites phosphorylated by cdc2 kinase and GSK-3 were restricted to several serines within three proline-rich motifs of domain P. Sites phosphorylated in vitro by protein kinase C, on the other hand, were distributed over the N-terminal catalytic as well as the C-terminal regulatory domain. The stoichiometry of phosphorylation catalysed by any of these kinases was less than 0.2 mol P/mol CT, and no effects on enzyme activity were detected. This study supports a tripartite structure for CT with an N-terminal catalytic domain and a C-terminal regulatory domain comprised of a membrane-binding domain (domain M) and a phosphorylation domain (domain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.
authors.affiliation: Institute of Molecular Biology, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: R B
authors.initials: RB
authors.lastname: Cornell
authors.forename: G B
authors.initials: GB
authors.lastname: Kalmar
authors.forename: R J
authors.initials: RJ
authors.lastname: Kay
authors.forename: M A
authors.initials: MA
authors.lastname: Johnson
authors.forename: J S
authors.initials: JS
authors.lastname: Sanghera
authors.forename: S L
authors.initials: SL
authors.lastname: Pelech
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D047390:Casein Kinase II; D002455:Cell Division; D002522:Chlorocebus aethiops; D019916:Choline-Phosphate Cytidylyltransferase; D017931:DNA Primers; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D010748:Phosphopeptides; D011994:Recombinant Proteins; D038341:Glycogen Synthase Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D038362:Glycogen Synthase Kinase 3; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase