Han X, Budreau A M, Chesney R W
Department of Pediatrics, University of Tennessee, and the Crippled Children's Foundation Research Center at Le Bonheur Children's Medical Center, Memphis 38103, USA.
J Am Soc Nephrol. 1999 Sep;10(9):1874-9. doi: 10.1681/ASN.V1091874.
Previous studies have shown that the Madin-Darby canine kidney cell taurine transporter (pNCT) is downregulated by protein kinase C (PKC) activation. In this study, it is hypothesized that the highly conserved serine-322 (Ser-322) located in the fourth intracellular segment (S4) may play an important role in the function of taurine transporter, which is modulated by PKC phosphorylation. It is demonstrated that Ser-322 is the critical site of PKC phosphorylation, as determined by site-directed mutagenesis. When Ser-322 of pNCT was changed to alanine (S322A) and this mutant was evaluated in an oocyte expression system, taurine transport activity increased threefold compared with control (wild-type pNCT). Activation of PKC by the active phorbol ester 12-myristate 13-acetate did not influence taurine transport by mutant S322A. Kinetic analysis showed that the mutation of Ser-322 essentially changed the Vmax, rather than the Km, of the transporter. Mutation of all other PKC consensus sites did not affect transporter activity when expressed in the oocyte system. Western blot analysis showed that expression of taurine transporter protein was similar in oocytes injected with either wild-type or mutant pNCT cRNA, indicating that the enhanced taurine transport activity by mutant S322A was not caused by a greater amount of transporter expressed in the oocyte. Furthermore, this study demonstrated that the taurine transporter was phosphorylated after PKC activation, and this effect was not observed in mutant S322A. In conclusion, Ser-322 is critical in PKC regulation of taurine transporter activity. The steady-state taurine transporter activity is tightly controlled by endogenous PKC phosphorylation of Ser-322, which is located in the fourth intracellular segment of the taurine transporter.
先前的研究表明，蛋白激酶C（PKC）的激活会下调马-达比犬肾细胞牛磺酸转运体（pNCT）。在本研究中，我们假设位于第四个细胞内区段（S4）的高度保守的丝氨酸322（Ser-322）可能在牛磺酸转运体的功能中发挥重要作用，该功能受PKC磷酸化调节。通过定点诱变确定，Ser-322是PKC磷酸化的关键位点。当pNCT的Ser-322被替换为丙氨酸（S322A），并在卵母细胞表达系统中评估该突变体时，与对照（野生型pNCT）相比，牛磺酸转运活性增加了三倍。活性佛波酯12-肉豆蔻酸13-乙酸酯激活PKC对突变体S322A的牛磺酸转运没有影响。动力学分析表明，Ser-322的突变主要改变了转运体的Vmax，而非Km。在卵母细胞系统中表达时，所有其他PKC共有位点的突变均不影响转运体活性。蛋白质印迹分析表明，注射野生型或突变型pNCT cRNA的卵母细胞中牛磺酸转运体蛋白的表达相似，这表明突变体S322A增强的牛磺酸转运活性不是由卵母细胞中表达的转运体数量增加所致。此外，本研究表明，PKC激活后牛磺酸转运体发生了磷酸化，而在突变体S322A中未观察到这种效应。总之，Ser-322在PKC对牛磺酸转运体活性的调节中起关键作用。牛磺酸转运体的稳态活性受位于牛磺酸转运体第四个细胞内区段的Ser-322的内源性PKC磷酸化严格控制。
