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J. Biol. Chem., Vol. 280, Issue 28, 26105-26112, July 15, 2005

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Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

IDENTIFICATION OF Ser²⁵ AND Ser³⁰ AS MAJOR SITES OF PHOSPHORYLATION^*

Mal-Gi Choi, Vladlen Kurnov, Michael C. Kersting, Avula Sreenivas, and George M. Carman

From the Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901

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_m = 27 µg/ml) and ATP (K_m = 15 µM). 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_max/K_m = 17.5 mM^–1 µmol min^–1 mg^–1) that contains the protein kinase C motif for Ser²⁵ was a substrate for protein kinase C. A Ser²⁵ 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 Ser²⁵ 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 Ser³⁰ was a substrate (V_max/K_m = 3.0 mM^–1 µmol min^–1 mg^–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 Ser³⁰ was also a target site for protein kinase C.

Received for publication, March 31, 2005 , and in revised form, May 24, 2005.

^* This work was supported in part by United States Public Health Service, National Institutes of Health Grant GM-50679. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence and reprint requests should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); E-mail: carman{at}aesop.rutgers.edu.

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