Biochemical characterization of human and yeast choline and ethanolamine kinases

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Date
2006
Authors
See, Too Wei Cun
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Abstract
Choline kinase (CK) and ethanolamine kinase (EK) are the first enzymes in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine. Increased activity of CK has been implicated in human tumors. CK inhibition has therefore been proposed as a potential anticancer strategy. Likewise, EK is suggested to be important in carcinogenesis by promoting cell growth. This study reports the cloning of CK/EK cDNAs and biochemical characterization of the full set of human CK/EK isoforms. A total of six human CK/EKs homologs (hCKα1, hCKα2, hCKβ, hEK1, hEK2α and hEK2β) were cloned from liver cDNA library and expressed in Escherichia coli. CKs were shown to phosphorylate choline and ethanolamine, but EKs were specific for ethanolamine. The length difference of only 18 amino acids between hCKα1 and α2 resulted in more than 50% lower activity in the α1 isoform. Intriguingly, the hEK2β isoform did not show any detectable enzyme activity. Besides the human enzymes, four CK/EK genes from three yeast species (yCK and yEK from Saccharomyces cerevisiae, SpCK from Schizosaccharomyces pombe, and AgCK from Ashbya gossypii) were also cloned, overexpressed and their products were biochemically characterized. Unlike their human homologs, yEK was not specific for ethanolamine while AgCK was highly specific for choline. Inhibition studies on hCK/EKs and yCK revealed that hemicholinium-3 (HC-3) was only effective in inhibiting the activity of the hCKα2 isoform and that yCK was not a good model enzyme for testing the efficacy of hCK inhibitors. Mutagenesis studies have identified a single aspartate residue that was critical for catalysis in human and yeast CK/EKs. Important residues for substrate preference towards either choline or ethanolamine were also identified. Phosphorylation experiments showed that hCKβ and hEK2α could be phosphorylated by PKA and PKC in vitro. The phosphorylations resulted in 1.5 and 2.7 fold stimulation of hCKβ and hEK2α activities. This work also demonstrated the use of the RNA interference technique to investigate the role of hCKα and β in HeLa cells. Single knockdown of hCKα resulted in lethal phenotype while single knockdown of hCKβ did not show any apparent effect on the cells. Remarkably, the simultaneous knockdown of both isoforms resulted in normal cell growth. These observations suggested that the interaction between hCKα and β was important for cell survival.
Description
PhD
Keywords
Chemical science , Biochemical , Yeast choline , Ethanolamine kinases
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