Abstract
BackgroundTwo isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. However, there is no information available regarding the unique functional aspects or regulation of these isoforms.ResultsWe show that the two AdK isoforms differ only in their first exons and the promoter regions; hence they arise via differential splicing of their first exons with the other exons common to both isoforms. The expression of these isoforms also varied greatly in different rat tissues and cell lines with some tissues expressing both isoforms and others expressing only one of the isoforms. To gain insights into cellular functions of these isoforms, mutants resistant to toxic adenosine analogs formycin A and tubercidin were selected from Chinese hamster (CH) cell lines expressing either one or both isoforms. The AdK activity in most of these mutants was reduced to <5% of wild-type cells and they also showed large differences in the expression of the two isoforms. Thus, the genetic alterations in these mutants likely affected both regulatory and structural regions of AdK. We have characterized the molecular alterations in a number of these mutants. One of these mutants lacking AdK activity was affected in the conserved NxxE motif thereby providing evidence that this motif involved in the binding of Mg2+ and phosphate ions is essential for AdK function. Another mutant, FomR-4, exhibiting increased resistance to only C-adenosine analogs and whose resistance was expressed dominantly in cell-hybrids contained a single mutation leading to Ser191Phe alteration in AdK. We demonstrate that this mutation in AdK is sufficient to confer the novel genetic and biochemical characteristics of this mutant. The unusual genetic and biochemical characteristics of the FomR-4 mutant suggest that AdK in this mutant might be complexed with the enzyme AMP-kinase. Several other AdK mutants were altered in surface residues that likely affect its binding to the adenosine analogs and its interaction with other cellular proteins.ConclusionsThese AdK mutants provide important insights as well as novel tools for understanding the cellular functions of the two isoforms and their regulation in mammalian cells.
Highlights
Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells
Our results show that these mutants exhibit interesting differences in their cross-resistance pattern towards the N- and C- Ado analogs and in the expression profiles of the two AdK isoforms. (Note: In N-nucleosides the purine base is linked to ribose via a N-C bond, whereas in C-nucleosides this linkage involves a C-C bond [34,35])
We showed that a single point mutation in AdK is responsible for its novel genetic and biochemical characteristics
Summary
Two isoforms of the enzyme adenosine kinase (AdK), which differ at their N-terminal ends, are found in mammalian cells. Adenosine kinase (AdK) is a major purine salvage pathway enzyme belonging to the ribokinase family of proteins [1,2,3,4]. It plays a central role in regulating the intracellular and interstitial concentrations of the purine nucleoside adenosine (Ado), which exhibits potent serves to minimize damage to the brain [6,11]. In the S-adenosylmethionine (SAM) dependent methylation pathway, Ado and homocysteine (Hcy) are produced as a result of hydrolysis of S-adenosyl-homocysteine (SAH), which is the common end product of all methylation reactions [1,14,15,16,17]. The deficiency of AdK due to its pivotal role in the maintenance of transmethylation reaction causes developmental abnormalities and reduced salt stress in plants [20,21]
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