Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A.
The CKI1-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple serine residues. Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase. This phosphorylation was accompanied by a stimulation in choline kinase activity. In vitro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent with one phosphorylation site/choline kinase subunit. The major phosphopeptide derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphorylated in vivo. Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (Km 2.1 microM) and choline kinase (Km 0.12 microM). Phosphorylation of choline kinase with protein kinase A resulted in a stimulation (1.9-fold) in choline kinase activity whereas alkaline phosphatase treatment of choline kinase resulted in a 60% decrease in choline kinase activity. The mechanism of the protein kinase A-mediated stimulation in choline kinase activity involved an increase in the apparent Vmax 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 kinase was regulated by protein kinase A phosphorylation.
Kim
K H
KH
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08901, USA.
Carman
G M
GM
D000469:Alkaline Phosphatase / Q000378:metabolism; D002797:Choline Kinase / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D010449:Peptide Mapping; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism
D000242:Cyclic AMP; D002797:Choline Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D018631:ras Proteins