Identification of Ser 424 as the Protein Kinase A Phosphorylation Site in CTP Synthetase from Saccharomyces cereVisiae ² Tae-Sik Park, Darin B. Ostrander, ‡ Apostolos Pappas, and George M. Carman* Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers UniVersity, 65 Dudley Road, New Brunswick, New Jersey 08901 ReceiVed April 5, 1999; ReVised Manuscript ReceiVed May 12, 1999 ABSTRACT: The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cereVisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser 424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (K m ) 30 µM) for protein kinase A. This peptide also inhibited (IC 50 ) 45 µM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser 424 f Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity 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 the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser 424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity. In the yeast Saccharomyces cereVisiae, CTP synthetase (EC 6.3.4.2) catalyzes the final step in the pyrimidine biosynthetic pathway (1). The enzyme catalyzes the ATP- dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. GTP is an allosteric effector which accelerates the formation of a covalent glutaminyl enzyme catalytic intermediate (2-4). Two du- plicate genes in S. cereVisiae named URA7 (1) and URA8 (5) encode CTP synthetase. The deduced protein products of the URA7 and URA8 genes contain a conserved glutamine amide transfer domain common to CTP synthetases from other organisms (6-9). The URA7-encoded CTP synthetase is more abundant than the URA8-encoded enzyme (10) and is responsible for the majority of the CTP synthesized in vivo (5). Neither the URA7 nor the URA8 gene is essential as long as cells possess one functional CTP synthetase gene (1, 5). The essential nature of CTP synthetase emanates from the fact that the reaction product CTP is required for the synthesis of RNA, DNA, membrane phospholipids, and sialoglycoproteins (11). The URA7 (4)- and URA8-encoded (12) CTP synthetases have been purified to homogeneity from the cytosolic fraction of the cell and characterized with respect to their enzymo- logical and kinetic properties. These CTP synthetases exhibit positive cooperative kinetics with respect to UTP and ATP (4, 12). Studies with the URA7-encoded CTP synthetase indicate that the cooperative kinetics of the enzyme are due to the nucleotide-dependent oligomerization of an inactive dimeric form to an active tetrameric form of the enzyme (13). The URA7 (4)- and URA8-encoded (12) CTP synthetases are regulated by CTP product inhibition. The inhibition of CTP synthetase activity by CTP regulates the cellular concentration of CTP in growing cells (4, 10, 14). An E161K mutation in the URA7-encoded CTP synthetase renders the enzyme defective in the regulation of activity by CTP product inhibition (14). Cells carrying this mutant enzyme exhibit elevated levels of CTP and alterations in the regulation of phospholipid metabolism (14). The major consequence of the mutation on phospholipid metabolism is an increase in phosphatidylcholine content and an increase the level of utilization of the CDP-choline pathway for phosphatidyl- choline synthesis (14). The URA7-encoded CTP synthetase is also regulated by phosphorylation via protein kinases A (15) and C (16, 17). In vitro, phosphorylation of the URA7-encoded CTP syn- thetase by protein kinases A (15) and C (16, 17) results in ² This work was supported by U.S. Public Health Service Grant GM- 50679 from the National Institutes of Health. * To whom correspondence and reprint requests should be addressed. Telephone: (732) 932-9611 (217). Fax: (732) 932-6776. E-mail: carman@aesop.rutgers.edu. ‡ Present address: Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX 77225. 8839 Biochemistry 1999, 38, 8839-8848 10.1021/bi990784x CCC: $18.00 © 1999 American Chemical Society Published on Web 06/15/1999