Abstract
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 ura7Δ ura8Δ
mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser
462
and Thr
455
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 Ser
462
and Thr
455
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 exhibits 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 Ser
462
stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr
455
inhibits activity.