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

ABSTRACT: Choline kinase beta (CK?) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CK? is important for normal mitochondrial function and muscle development as the lack of the ck? gene in human and mice results in the development of muscular dystrophy. In contrast, CK? is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CK? was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CK? phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CK? 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? phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CK? was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CK? 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? phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CK? to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CK?, in concert with CK?, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.

SUBMITTER: Chang CC 

PROVIDER: S-EPMC4858151 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature