Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

Choline kinase beta (CK beta) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CK beta is important for normal mitochondrial function and muscle development as the lack of the ck beta gene in human and mice results in the development of muscular dystrophy. In contrast, CK alpha is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CK alpha was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CK beta phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CK beta by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CK beta phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CK beta was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CK beta residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CK beta phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CK beta to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CK beta, in concert with CK alpha, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.