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Journal Article

Phosphorylation of human choline kinase beta by protein kinase A: Its impact on activity and inhibition.

MPS-Authors
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Konrad,  M.
Research Group of Enzyme Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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See Too,  W. C.
Research Group of Enzyme Biochemistry, MPI for biophysical chemistry, Max Planck Society;

Fulltext (public)

2301626.pdf
(Publisher version), 2MB

Supplementary Material (public)

2301626_Suppl_1.zip
(Supplementary material), 219KB

2301626_Suppl_2.PDF
(Supplementary material), 78KB

2301626_Suppl_3.PDF
(Supplementary material), 46KB

2301626_Suppl_4.PDF
(Supplementary material), 48KB

2301626_Suppl_5.PDF
(Supplementary material), 49KB

Citation

Chang, C. C., Few, L. L., Konrad, M., & See Too, W. C. (2016). Phosphorylation of human choline kinase beta by protein kinase A: Its impact on activity and inhibition. PLoS One, 11(5): e0154702. doi:10.1371/journal.pone.0154702.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002A-C6E9-A
Abstract
Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ 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. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ 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 CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.