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 Km 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 (nH = 0.64) and phosphatidylcholine/diacylglycerol (nH = 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.
Yang
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W
Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Jackowski
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S
D000818:Animals; D001483:Base Sequence; D019916:Choline-Phosphate Cytidylyltransferase; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology; D010766:Phosphorylation
D004075:Diglycerides; D008055:Lipids; D009829:Oleic Acids; D010713:Phosphatidylcholines; D019301:Oleic Acid; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase