Phosphorylation of Saccharomyces cerevisiae Choline Kinase on Ser30 and Ser85 by Protein Kinase A Regulates Phosphatidylcholine Synthesis
Ying Yu, Avula Sreenivas, Darin B. Ostrander‡, and George M. Carman
02/09/2011
The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser30 and Ser85 contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (Vmax/Km = 10.8 µM-1 nmol min-1 mg-1) and GPRRASATDV (Vmax/Km 0.15 µM-1 nmol min-1 mg-1) containing the protein kinase A motif for Ser30 and Ser85, respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser30 to Ala (S30A) and Ser85 to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Δ eki1Δ double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of 3H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser30 and Ser85 by protein kinase A regulates PC synthesis by the CDP-choline pathway.