Han X, Budreau A M, Chesney R W
Department of Pediatrics, University of Tennessee, Memphis, LeBonheur Children's Medical Center 38103, USA.
J Am Soc Nephrol. 1996 Oct;7(10):2088-96. doi: 10.1681/ASN.V7102088.
Activation of protein kinase C (PKC) by the active phorbol ester 12-myristate 13-acetate (PMA, 100 nM) or phorbol-12, 13-dibutyrate (100 nM) reduced taurine uptake by 80% in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine uptake by 20% and 400%, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity K(m) (from 5.6 microM to 18.1 microM) with minimal effect on the maximal velocity (25% decrease) of the transporter. A polyclonal antibody (AbS4) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of AbS4 was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of AbS4 (12 ng/ oocyte). In contrast, downregulation of the transporter by PMA could not be restored by AbS4 when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.
活性佛波酯12 - 肉豆蔻酸13 - 乙酸酯（PMA，100 nM）或佛波醇 - 12,13 - 二丁酸酯（100 nM）激活蛋白激酶C（PKC）后，在用来自表达Madin - Darby犬肾细胞牛磺酸转运体pNCT的cRNA注射的卵母细胞中，牛磺酸摄取减少了80%。钙磷蛋白C或星形孢菌素对PKC的抑制分别使牛磺酸摄取增加了20%和400%。PKC的特异性抑制剂钙磷蛋白C阻断了PMA对牛磺酸摄取的抑制作用。PMA的调节主要影响转运体的表观亲和力K(m)（从5.6 microM变为18.1 microM），而对最大速度的影响最小（降低25%）。针对Madin - Darby犬肾细胞牛磺酸转运体保守细胞内区段（S4）的多克隆抗体（AbS4）增强了pNCT cRNA处理的卵母细胞对牛磺酸的摄取。与相应的肽抗原孵育可阻断AbS4的作用。免疫前IgG和肽抗原对卵母细胞中表达的牛磺酸转运体活性没有影响。调节似乎是通过转运体S4区段上一个共有PKC位点的磷酸化发生的，因为预先注射AbS4（12 ng/卵母细胞）可消除PMA（100 nM）对转运体的下调作用。相反，当用PMA（50 nM）预处理表达pNCT的卵母细胞时，AbS4不能恢复PMA对转运体的下调作用。总之，该抗体识别的肽段似乎直接参与了由PKC磷酸化调节的牛磺酸转运体失活过程。
