Subcellular localization of adenosine kinase in mammalian cells: The long isoform of AdK is localized in the nucleus
Introduction
Adenosine kinase (AdK) belongs to the ribokinase (RK) family of proteins and is the one of the most abundant nucleoside kinases in mammalian tissues [1], [2]. This enzyme is well conserved among eukaryotic species both at sequence and structural level [3], [4]. AdK knockout mice have been made and they showed a lethal phenotype indicating AdK is indispensible in eukaryotic organisms [5]. Adk is the first enzyme in the purine salvage pathway and catalyzes the phosphorylation of adenosine (Ado) to AMP, using ATP as a phosphate donor and produces ADP and AMP [1], [6]. By performing this reaction, it controls intracellular and extracellular Ado concentration in the cell, which is a potent cardioprotective agent and neuromodulator [2], [7], [8]. Adenosine is also one of the obligate end products of all methylation reactions, which exhibits end product inhibition on the upstream reactions including various methyltransferases [1], [5], [9], [10], [11]. Therefore, besides its convention role in purine salvage, AdK also ensures the continuance of methylation reaction without impedance.
As a cardioprotective agents, Ado has been implicated in tissue-protective mechanism during and after instances of ischemia by activating adenosine receptors on the cell surface [2], [8]. The AdK over-expression in neurons, which removes the inhibitory effect of Ado on neuronal excitability, was recently shown as the underlying mechanism for chronic epilepsies [12], [13]. Because of the short half life of Ado in physiological fluids, there has been much interest in developing Ado inhibitors as they provide potential means of amplifying the beneficiary effects of Ado in both cardiovascular diseases and chronic epilepsies [13], [14], [15].
Two isoforms of AdK have been identified in various mammalian organisms and the recombinant proteins from both are functional and they show no differences in their biochemical or kinetic properties [3], [4], [16], [17]. The two isoforms are identical except at the N-terminus where the long AdK isoform (AdK-long) contains extra 20–21 amino acids, which replace the first four amino acids in the short isoform (AdK-short) (see Fig. 1). Western blot analysis indicates that these two isoforms are differentially expressed in rat tissues [16]. However, there is no information available regarding possible significance of these two isoforms or differences in their functions. To date, the subcellular localization of AdK has not been experimentally determined. Most conventional programs for prediction of cellular localization (e.g. PSORT, BaCelLo) reveal no specific localization of these isoforms and thus they are both assumed to be present in the cytoplasm [18], [19]. However, in this paper we show that in contrast to the cytoplasmic localization of AdK-short, the AdK-long isoform is localized in the nucleus and the extra 20–21 amino acids present at its N-terminus are capable of directing AdK as well as other proteins to the nucleus. The significance of nuclear localization of AdK is discussed.
Section snippets
Material and methods
Cell lines, cell culture conditions and plasmid constructs. The origin of various cell lines used in this study (viz. CHO, HeLa, HT-1080 and LM (TK−)) has been described in our earlier work [20]. All of the cells were grown in α-MEM supplemented with 5% fetal bovine serum at 37 °C in a 95% humid air–5% CO2 atmosphere. The full-length sequences for AdK-long and AdK-short isoforms were PCR amplified using cDNA from HT-1080 cells with PCR primers based on known sequences (Accession No. NM_006721 and
Differential expression and subcellular localization of the two AdK isoforms
To investigate the subcellular localization of AdK in mammalian cells a polyclonal antibody against human recombinant AdK was raised. Using this antibody, the presence of AdK-antibody cross-reactive protein(s) in a number of mammalian cell lines was studied (Fig. 2A). The cell lines used in these studies included WT Chinese hamster ovary (CHO) cells, HeLa cells—a human epithelial cell line derived from cervix, HT-1080 cell line—a human epithelial cell line from connective tissue and mouse LM (TK
Discussion
This study reports for the first time subcellular localization of the enzyme AdK in mammalian cells. We have presented evidence that of the two AdK isoforms that are found in mammalian organisms, AdK-long is localized within the nucleus, whereas AdK-short is found in the cytoplasm. We have also demonstrated that the NTS in the AdK-long has the ability to transport proteins to the nucleus. Results presented here also show that mammalian cell lines differ in terms of expression of these AdK
Acknowledgement
The work from the authors’ lab in this area has been supported by a research grant (T-6177) from the Heart and Stroke Foundation of Canada.
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