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J. Biol. Chem., Vol. 282, Issue 24, 17613-17622, June 15, 2007

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Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C

IDENTIFICATION OF Ser⁴⁶² AND Thr⁴⁵⁵ AS MAJOR SITES OF PHOSPHORYLATION^*

Yu-Fang Chang, Shelley S. Martin, Enoch P. Baldwin, and George M. Carman¹

From the Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901 and Departments of Molecular and Cellular Biology and Chemistry, University of California, Davis, California 95616

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1^R398A-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7 ura8 mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser⁴⁶² and Thr⁴⁵⁵ were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser⁴⁶² and Thr⁴⁵⁵ were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367–5377). These data indicated that protein kinase C phosphorylation at Ser⁴⁶² stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr⁴⁵⁵ inhibits activity.

Received for publication, April 2, 2007 , and in revised form, April 25, 2007.

^* This work was supported in part by United States Public Health Service, National Institutes of Health Grants GM-50679 (to G. M. C.) and GM-63109 (to E. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); E-mail: carman{at}aesop.rutgers.edu.

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