Phosphorylation of Saccharomyces cerevisiae CTP Synthetase at Ser424 by Protein Kinases A and C Regulates Phosphatidylcholine Synthesis by the CDP-choline Pathway

Mal-Gi Choi,Tae-Sik Park,George M Carman

doi:10.1074/jbc.m303337200

Mal-Gi Choi, Tae-Sik Park + Show 1 more

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https://doi.org/10.1074/jbc.m303337200

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Abstract

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.

Highlights

Valent glutaminyl enzyme catalytic intermediate [3,4,5,6]
The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme [34] and is responsible for the majority of the CTP synthesized in vivo [11]
The phosphorylation of the S424A mutant CTP synthetase by protein kinase C was time-dependent (Fig. 4) and dose-dependent (Fig. 5), but the rate and extent of phosphorylation was reduced by about 50% when compared with the wild type enzyme

Summary

Introduction

Valent glutaminyl enzyme catalytic intermediate [3,4,5,6]. In eukaryotic cells, regulation of CTP synthetase activity plays an important role in the balance of nucleotide pools (6 –12) and in the synthesis of membrane phospholipids [12,13,14]. We showed that cells bearing the S424A mutant CTP synthetase enzyme exhibited a decrease in the synthesis of the membrane phospholipid PC1 via the CDP-choline pathway. The indicated concentrations of purified wild type and S424A mutant CTP synthetase enzymes or synthetic peptide were phosphorylated with the indicated concentrations of protein kinase C in a reaction mixture that contained 50 mM Tris-HCl buffer (pH 8.0), 10 mM MgCl2, 10 mM 2-mercaptoethanol, 0.375 mM EDTA, 0.375 mM EGTA, 1.7 mM CaCl2, 20 ␮M diacylglycerol, 50 ␮M phosphatidylserine, and 50 ␮M [␥-32P]ATP (5,000 cpm/pmol).

Results

Conclusion