Phosphorylation and Regulation of Choline Kinase fromSaccharomyces cerevisiae by Protein Kinase A

Kee-Hong Kim,George M Carman

doi:10.1074/jbc.274.14.9531

Kee-Hong Kim, George M Carman

Open Access

https://doi.org/10.1074/jbc.274.14.9531

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Abstract

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.

Highlights

Phosphatidylcholine (PC)1is the most abundant phospholipid in the yeast Saccharomyces cerevisiae [1, 2]
To examine whether the phosphorylation of choline kinase was mediated by protein kinase A in vivo, the extent of enzyme phosphorylation was measured in cells that were activated in the RAS-cAMP pathway
Choline kinase activity (G) was measured from cell extracts prepared from unlabeled cells that were activated in the RAS-cAMP pathway

Summary

Protein Kinase A Phosphorylation of Yeast Choline Kinase

Choline, ammonium reinecke, phenylmethanesulfonyl fluoride, benzamide, aprotinin, leupeptin, pepstatin, bovine serum albumin, phosphoamino acids, TPCK-trypsin, and nitrocellulose paper were from Sigma. The incorporation of phosphate into choline kinase was determined by scintillation counting of phosphorylated enzyme excised from SDS-polyacrylamide gel slices. Phosphoamino Acid Analysis—Choline kinase was phosphorylated with protein kinase A and [␥-32P]ATP for 10 min and subjected to SDS-polyacrylamide gel electrophoresis. Gel slices containing 32P-labeled choline kinase were treated with 50 mM ammonium bicarbonate (pH 8.0) and 0.1% SDS at 37 °C for 30 h to elute the enzyme. Tryptic Digestion and Two-dimensional Peptide Mapping—SDSpolyacrylamide gel slices containing 32P-labeled choline kinase were subjected to proteolysis using TPCK-trypsin (0.15 mg/ml) in 50 mM ammonium bicarbonate for 16 h at 37 °C [26]. Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT enzyme kinetic model fitting program [31]

RESULTS

DISCUSSION

ATPa Phosphorylated Dephosphorylated