The Equilibrative Nucleoside Transporter (ENT1) can be phosphorylated at multiple sites by PKC and PKA

Abstract

Equilibrative Nucleoside Transporters (SLC29) are a family of proteins that transport nucleosides, nucleobases and nucleoside analogue drugs across cellular membranes. ENT1 is expressed ubiquitously in mammalian tissues and responsible for a significant portion of nucleoside analog drug uptake in humans. Despite the important clinical role of ENT1, many aspects of the regulation of this protein remain unknown. A major outstanding question in this field is the whether ENT1 is phosphorylated directly. To answer this question, we overexpressed tagged human (h) and mouse (m) ENT1, affinity purified protein using the tag, conducted phosphoamino acid analysis and found that m/hENT1 is predominantly phosphorylated at serine residues. The large intracellular loop of ENT1, between transmembrane domains 6 and 7, has been suggested to be a site of regulation by phosphorylation, therefore we generated His/Ubiquitin tagged peptides of this region and used them for in vitro kinase assays to identify target serines. Our data support a role for PKA and PKC in the phosphorylation of ENT1 within the intracellular loop and show that PKA can phosphorylate multiple sites within this loop while PKC specifically targets serines 279 and 286 and threonine 274. These data demonstrate, for the first time, that ENT1 is a phosphoprotein that can be directly phosphorylated at several sites by more than one kinase. The presence of multiple kinase targets within the loop suggests that ENT1 phosphorylation is considerably more complex than previously thought and thus ENT1 may be subject to phosphorylation by multiple pathways.

In Vitro and In Vivo Comparison of Gemcitabine and the Gemcitabine Analog 1-(2′-deoxy-2′-fluoroarabinofuranosyl) Cytosine (FAC) in Human Orthotopic and Genetically Modified Mouse Pancreatic Cancer Models

“…However, we were unable to detect ENT2 with western blotting. ENT1 is known to have multiple phosphorylation sites, suggesting the possibility for post-translational modification [22, 23], and it may be that this accounts for the discrepancy between the inhibitor studies and protein expression. RNA interference could very usefully discriminate between receptors.…”

The adenosine transporter, ENT1, in cardiomyocytes is sensitive to inhibition by ethanol in a kinase-dependent manner: implications for ethanol-dependent cardioprotection and nucleoside analog drug cytotoxicity

“…Ethanol is thought to interact with ENT1 at a specific site in the protein as has been described for other ethanol-sensitive membrane proteins such as the N-methyl-D-aspartate receptors, gammaamino butyric acid receptors, serotonin receptors, calciumand voltage-activated potassium channels (BK Ca ), and ligand-gated ion channels [22,31,32]. Since very little is known about the structure of ENT1, we cannot identify the location of a putative ethanol binding pocket although the large intracellular loop between transmembrane domains 6 and 7 is the region of ENT1 with the least similarity to ENT2, and also the location of known phosphorylation targets [31], suggesting that this region of the protein may be the region with which ethanol interacts. Previous data [19] suggested that the adenosine transport system in neuronal cells is "primed" by phosphorylation of ENT1 before ethanol exposure, and this "priming" is critical in regulating the accessibility of the putative hydrophobic sites of ENT1 to ethanol by causing a conformational change in protein.…”

“…Ethanol-specific inhibition of ENT1 uptake (rather than ENT2) in cardiomyocytes is also consistent with previous studies showing that ENT2, which is expressed in much lower amounts than ENT1 in HL-1 cells [5], mouse, and human heart tissue [3,30], is insensitive to ethanol [8]. Ethanol is thought to interact with ENT1 at a specific site in the protein as has been described for other ethanol-sensitive membrane proteins such as the N-methyl-D-aspartate receptors, gammaamino butyric acid receptors, serotonin receptors, calciumand voltage-activated potassium channels (BK Ca ), and ligand-gated ion channels [22,31,32]. Since very little is known about the structure of ENT1, we cannot identify the location of a putative ethanol binding pocket although the large intracellular loop between transmembrane domains 6 and 7 is the region of ENT1 with the least similarity to ENT2, and also the location of known phosphorylation targets [31], suggesting that this region of the protein may be the region with which ethanol interacts.…”

Equilibrative nucleoside transporters—A review

“…Ethanol has also been observed to attenuate transport mediated by purified and reconstituted yeast ENT, FUN26 (Boswell-Casteel, unpublished data). Moreover, ENT1 sensitivity to ethanol has been shown to be regulated in a kinase-dependent manner by PKA and PKC (107, 111, 112), which have been previously shown to phosphorylate mouse ENT1 in the intercellular loop region between TMDs 6 and 7 (115). The means by which ethanol may modulate ENT function is unknown and the effect may be indirect of ENT-mediated binding.…”