Phosphorylation of the Yeast Choline Kinase by Protein Kinase C: Identification of Ser super(25) and Ser super(30) as major sites of phosphorylation

• Published in: The Journal of biological chemistry Vol. 280; no. 28; pp. 26105 - 26112
• Main Authors: Choi, Mal-Gi, Kurnov, Vladlen, Kersting, Michael C, Sreenivas, Avula, Carman, George M
• Format: Journal Article
• Language: English
• Published: 01.07.2005
• Subjects: Saccharomyces cerevisiae
• ISSN: 0021-9258; 1083-351X

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K sub(m) = 27 mu g/ml) and ATP (K sub(m) = 15 mu M). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5 sub(max)/K sub(m) = 17.5 mM super(-1) mu mol min super(-1) mg super(-1)) that contains the protein kinase C motif for Ser super(25) was a substrate for protein kinase C. A Ser super(25) to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser super(25) was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser super(30) was a substrate (V sub(max)/K sub(m) = 3.0 mM super(-1) mu mol min super(-1) mg super(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser super(30) was also a target site for protein kinase C.