Phosphorylation And Regulation Of Human Choline Kinase Beta By Protein Kinase A
| dc.contributor.author | Chang, Ching Ching | |
| dc.date.accessioned | 2017-09-06T02:48:32Z | |
| dc.date.available | 2017-09-06T02:48:32Z | |
| dc.date.issued | 2015-01 | |
| dc.description.abstract | Choline kinase (CK) is the first enzyme involved in CDP-choline pathway for the biosynthesis of phosphatidylcholine, the major component of membrane phospholipid. CK exists as three isoforms, which are CKα1, CKα2 and CKβ. The regulation of these enzymes is physiologically important. Metabolic alterations of CKα are associated with tumorigenesis, while mutation or deletion of chkβ gene leads to the development of muscular dystrophy. In anticancer research, inhibition of CK activity has been explored as a potential therapeutic strategy. Post-translational modification is one of the mechanisms to regulate the function of CK. Growing evidences support that yeast and human CKα are regulated by phosphorylation but the phosphorylation of CKβ has never been reported. In this study, protein kinase A (PKA) was identified as the protein kinase responsible for the phosphorylation of CKβ by in-gel kinase assay. PKA phosphorylation was confirmed with specific PKA inhibitor and Western blotting. In vitro assay with commercial PKA further supported CKβ as the substrate for PKA phosphorylation. The phosphorylation occurred at serine 39 and 40 residues in the N-terminal region of CKβ. Phosphorylation of CKβ was observed in human embryonic kidney cells (HEK293) and liver hepatocellular carcinoma cells (HepG2). Forskolin and 3-isobutyl-1-methylxanthine treatment increased the phosphorylation level of CKβ, while the phosphorylation was inhibited by PKA inhibitor (H-89). The phosphorylation level of CKβ was also increased by epidermal growth factor. The effects of PKA xxii phosphorylation on the biochemical properties of CKβ were subsequently examined. PKA phosphorylation increased the catalytic activities of CKβ with choline, ethanolamine and ATP as substrates. The Vmax values for choline, ethanolamine and ATP were increased by 47.1%, 81.8% and 50.8%, respectively. PKA phosphorylation improved the affinity of CKβ for choline and ATP, but decreased the affinity of CKβ for ethanolamine. Consequently, the catalytic efficiencies of CKβ for choline and ATP were increased by 121.0% and 97.5%, respectively. The same effects of PKA phosphorylation on the biochemical properties of CKβ were mimicked by double mutation of the phosphorylated serines to aspartates. PKA phosphorylation also dramatically increased the sensitivity of CKβ to hemicholinium-3 (HC-3), a potent inhibitor of CK. The IC50 value for phosphorylated CKβ (50 μM) was 29 times lower than the unphosphorylated enzyme (1.45 mM). In addition, PKA phosphorylation also decreased the stability of CKβ protein against urea denaturation. On the contrary, phosphorylation did not affect the optimum pH, subcellular location and oligomeric state of CKβ. This study reports the phosphorylation and regulation of CKβ by PKA for the first time. The knowledge provides new insight into the intracellular regulation of CKβ catalytic properties by phosphorylation that might be an important mechanism to modulate lipid metabolism and cell growth. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/4474 | |
| dc.language.iso | en | en_US |
| dc.publisher | Universiti Sains Malaysia | en_US |
| dc.subject | Choline kinase | en_US |
| dc.subject | biosynthesis of phosphatidylcholine, | en_US |
| dc.title | Phosphorylation And Regulation Of Human Choline Kinase Beta By Protein Kinase A | en_US |
| dc.type | Thesis | en_US |
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