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