FUNCTIONS OF THE C-TERMINAL DOMAIN OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE - EFFECTS OF C-TERMINAL DELETIONS ON ENZYME-ACTIVITY, INTRACELLULAR-LOCALIZATION AND PHOSPHORYLATION POTENTIAL

The role of the C-terminal domain of CTP: phosphocholine cytidylyltran sferase (CT) was explored by the creation of a series of deletion muta tions 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-terminu s had no effect on the activity of the enzyme, its stimulation by lipi d vesicles or on its intracellular distribution between soluble and me mbrane-bound forms, However, deletion of the C-terminal 139 amino acid s resulted in a 90% decrease in activity, loss of response to lipid ve sicles and a significant decrease in the fraction of membrane-bound en zyme. Identification of the domain that is phosphorylated in vivo was determined by analysis of P-32-labelled CT mutants and by chymotrypsin proteolysis of purified CT that was P-32-labelled in vivo. Phosphoryl ation was restricted to the C-terminal 52 amino acids (domain P) and o ccurred on multiple sites. CT phosphorylation in vitro was catalysed b y casein kinase II, cell division control 2 kinase (cdc2 kinase), prot ein 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 phospho rylated by cdc2 kinase and GSK-3 were restricted to several serines wi thin three proline-rich motifs of domain P. Sites phosphorylated in vi tro 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 de tected. This study supports a tripartite structure for CT with an N-te rminal catalytic domain and a C-terminal regulatory domain comprised o f a membrane-binding domain (domain M) and a phosphorylation domain (d omain P). It also identifies three kinases as potential regulators in vivo of CT, casein kinase II, cyclin-dependent kinase and GSK-3.