Protein kinase C‐mediated phosphorylation of human CTP synthetase

Abstract

CTP synthetase is an essential enzyme that catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP. GTP stimulates the reaction by accelerating the formation of a covalent glutaminyl enzyme catalytic intermediate. The functional expression of human CTP synthetase 1 in yeast revealed that the enzyme is phosphorylated on multiple residues. Some of this phosphorylation is mediated by protein kinase A. Analysis of the enzyme sequence indicated potential phosphorylation sites for protein kinase C. In mammalian cells, protein kinase C plays a central role in the transduction of lipid second messengers generated by receptor-mediated hydrolysis of membrane phospholipids. In this work, we showed that purified human CTP synthetase was a substrate for protein kinase C. The phosphorylation of the enzyme was time- and dose-dependent, and dependent on the concentrations of CTP synthetase and ATP. Phosphopeptide mapping analysis indicated multiple sites of phosphorylation. Identification of the protein kinase C phosphorylation sites in the CTP synthetase is the subject of current studies. Supported by NIH grant GM 50679.