Bone Derek B J, Robillard Kevin R, Stolk Meaghan, Hammond James R
Department of Physiology & Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.
Mol Membr Biol. 2007 Jul-Aug;24(4):294-303. doi: 10.1080/09687860701210617.
Nucleosides are accumulated by cells via a family of equilibrative transport proteins (ENTs). An alternative splice variant of the most common subtype of mouse ENT (ENT1) has been identified which is missing a protein kinase CK2 (casein kinase 2) consensus site (Ser(254)) in the central intracellular loop of the protein. We hypothesized that this variant (mENT1a) would be less susceptible to modulation by CK2-mediated phosphorylation compared to the variant containing the serine at position 254 (mENT1b). Each splice variant was transfected into nucleoside transporter deficient PK15 cells, and stable transfectants assessed for their ability to bind the ENT1-selective probe [(3)H]nitrobenzylthioinosine (NBMPR) and to mediate the cellular uptake of [(3)H]2-chloroadenosine, with or without treatment with the CK2 selective inhibitor, 4,5,6,7-tetrabromobenzotriazole (TBB). mENT1a had a higher affinity for NBMPR relative to mENT1b - measured both directly by the binding of [(3)H]NBMPR, and indirectly via inhibition of [(3)H]2-chloroadenosine influx by NBMPR. Furthermore, incubation of mENT1b-expressing cells with 10 microM TBB for 48 h decreased both the K(D) and B(max) of [(3)H]NBMPR binding, as well as the V(max) of 2-chloroadenosine uptake, whereas similar treatment of mENT1a-expressing cells with TBB had no effect. PK15 cells transfected with hENT1, which has Ser(254), was similar to mENT1b in its response to TBB. In conclusion, inhibition of CK2 activity, or deletion of Ser(254) from mENT1, enhances transporter affinity for the inhibitor, NBMPR, and reduces the number of ENT1 proteins functioning at the level of the plasma membrane.
核苷通过一类平衡型转运蛋白（ENTs）被细胞摄取。已鉴定出小鼠ENT最常见亚型（ENT1）的一种可变剪接变体，该变体在蛋白的中央细胞内环中缺少蛋白激酶CK2（酪蛋白激酶2）的共有位点（Ser(254)）。我们推测，与含有第254位丝氨酸的变体（mENT1b）相比，这种变体（mENT1a）对CK2介导的磷酸化调节的敏感性较低。将每种剪接变体转染到缺乏核苷转运体的PK15细胞中，并评估稳定转染体结合ENT1选择性探针[(3)H]硝基苄硫肌苷（NBMPR）以及介导[(3)H]2-氯腺苷细胞摄取的能力，无论是否用CK2选择性抑制剂4,5,6,7-四溴苯并三唑（TBB）处理。相对于mENT1b，mENT1a对NBMPR具有更高的亲和力——这既可通过[(3)H]NBMPR的结合直接测量，也可通过NBMPR对[(3)H]2-氯腺苷内流的抑制间接测量。此外，用10 microM TBB孵育表达mENT1b的细胞48小时，会降低[(3)H]NBMPR结合的K(D)和B(max)，以及2-氯腺苷摄取的V(max)，而用TBB对表达mENT1a的细胞进行类似处理则没有效果。用具有Ser(254)的hENT1转染的PK15细胞对TBB的反应与mENT1b相似。总之，抑制CK2活性或从mENT1中缺失Ser(254)，会增强转运体对抑制剂NBMPR的亲和力，并减少在质膜水平发挥作用的ENT1蛋白数量。
