Identification of protein kinase A and protein kinase C phosphorylation sites in CTP synthetase from Saccharomyces cerevisiae

CTP synthetase in Saccharomyces cerevisiae is phosphorylated on serine residues and activated by protein kinase A and protein kinase C in vitro. We examined the hypothesis that Ser424, contained in a protein kinase A sequence motif in CTP synthetase, is a target site for protein kinase A. We examined if Ser 36, Ser330, Ser354, and Ser454 , contained in a protein kinase C sequence motif in CTP synthetase, are target sites for protein kinase C. S424A, S36A, S330A, S354A, and S454A mutants were constructed by site-directed mutagenesis. CTP synthetase activity in cells bearing the S424A mutant enzyme was reduced when compared with control cells. The S424A mutated enzyme was not phosphorylated in response to protein kinase A activation in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced V_max, and elevated K_m values for ATP and UTP when compared with control enzyme. The S424A mutant enzyme was more sensitive to CTP product inhibition when compared with the control. CTP synthetase activity in cells bearing the S36A, S354A, and S454A mutant enzymes were reduced, whereas activity in cells with the S330A mutant enzyme was elevated when compared with control cells. The levels of CTP synthetase activity in the mutants generally correlated with the phosphorylation of the enzyme in vivo and with the cellular levels of CTP. Purified S36A and S354A mutant enzymes had reduced V_max values with respect to ATP and UTP. The S330A mutant enzyme had an elevated V_max value for both substrates. In vitro phosphorylation data with the CTP synthetase mutants indicated that the phosphorylation of the enzyme at one site affected the phosphorylation of the enzyme at another site. Cells with the S424A, and with the S36A, S354A, and S454A mutations exhibited a decrease in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis. Cells with the S330A mutation exhibited an increased utilization of the CDP-choline pathway. Overall, these data indicated that the protein kinase A target site in CTP synthetase was Ser424 , and that Ser36, Ser330, Ser 354, and Ser454 were target sites for protein kinase C.