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 ura7Delta ura8Delta 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 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 Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.
Highlights
Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined
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
To examine the hypothesis that human CTP synthetase 1 is a substrate for mammalian protein kinase C, we utilized a homogeneous preparation of the enzyme that was expressed and purified from E. coli [52]
Summary
Growth medium supplies were obtained from Difco. New England Biolabs was the source of modifying enzymes, recombinant Vent DNA polymerase, and restriction endonucleases. DNA gel extraction kit, plasmid DNA purification kit, and Ni2ϩ-NTA2agarose resins were purchased from Qiagen. Oligonucleotides were synthesized by Genosys Biotechnologies, Inc. The QuikChange site-directed mutagenesis kit was purchased from Stratagene. Carrier DNA for yeast transformation was from Clontech. Peptides were synthesized and purified by Bio-Synthesis, Inc. Radiochemicals were purchased from PerkinElmer Life Sciences. Nucleotides, 5-fluoroorotic acid, aprotinin, benzamidine, bovine serum albumin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, phosphoamino acids, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin were purchased from Sigma. Bio-Rad was the source of DNA size
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