LIPID ACTIVATION OF CTP-PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE IS REGULATED BY THE PHOSPHORYLATED CARBOXYL-TERMINAL DOMAIN

The role of the phosphorylated carboxyl-terminal domain of CTP:phosphocholine cytidylyltransferase (CT) in the regulation of enzyme activity was investigated by comparing the catalytic properties of wild-type CT to two mutant proteins with altered carboxyl-terminal phosphorylation domains. CT isolated from a baculovirus expression system was extensively phosphorylated at multiple sites in the carboxyl terminal domain, The CT[S315A] mutant lacked a major CT phosphorylation site, and the carboxyl-terminal deletion mutant, CT[Delta 312-367], was not phosphorylated, The higher activities of CT[Delta 312-367] and CT[S315A] relative to CT were attributed to differences in the sensitivities of the enzymes to lipid activators. The rank order of the apparent K-m values for activation by either phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol was CT > CT[S315A] > CT[Delta 312-367]. In addition, CT exhibited negative cooperativity in its activation by phosphatidylcholine/oleic acid (n(H) = 0.64) and phosphatidylcholine/diacylglycerol (n(H) = 0.74) vesicles, whereas CT[Delta 312-367] and CT[S315A] did not. These data support the concept that the phosphorylation of the CT carboxyl-terminal domain interferes with the activation of CT by lipid regulators.