Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C IDENTIFICATION OF Ser AND Thr AS MAJOR SITES OF PHOSPHORYLATION*

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coliexpressed CTP synthetase 1 as a substrate, protein kinase C activity was timeand 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 PKC1R398A-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 andThr455were substrates formammalianprotein kinase C, and S462A and T455Amutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressedCTP synthetase 1mutant enzymes phosphorylatedwith 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., andCarman,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.