Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A

The CK11-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.3 2) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple ser ine residues. Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase. This phosphor ylation was accompanied by a stimulation in choline kinase activity. In vit ro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent w ith one phosphorylation site/choline kinase subunit. The major phosphopepti de derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphor ylated in vivo. Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (K-m 2.1 mu M) and choline kinase (K -m 0.12 mu M). Phosphorylation of choline kinase with protein kinase A resu lted in a stimulation (1.9-fold) in choline kinase activity whereas alkalin e phosphatase treatment of choline kinase resulted in a 60% decrease in cho line kinase activity. The mechanism of the protein kinase A-mediated stimul ation in choline kinase activity involved an increase in the apparent V-max values with respect to ATP (2.6-fold) and choline (2.7-fold). Overall, the results reported here were consistent with the conclusion that choline kin ase was regulated by protein kinase A phosphorylation.