Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA.
Am J Physiol Gastrointest Liver Physiol. 2012 Sep 1;303(5):G657-65. doi: 10.1152/ajpgi.00529.2011. Epub 2012 Jun 28.
Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.
环磷酸腺苷（cAMP）刺激钠离子/牛磺胆酸钠共转运多肽（NTCP）从细胞质向窦膜易位，以及多药耐药相关蛋白 2（MRP2）向胆小管膜易位。最近的一项研究表明，蛋白激酶 Cδ（PKCδ）可能介导 cAMP 诱导的 Ntcp 和 Mrp2 易位。此外，已经表明 cAMP 通过 Rab4 部分刺激 NTCP 易位。本研究旨在确定 cAMP 诱导的 NTCP 和 MRP2 易位是否需要 PKCδ 的激酶活性，并检验 cAMP 诱导的 Rab4 激活是通过 PKCδ 介导的假设。该研究在 HuH-NTCP 细胞（稳定转染 NTCP 的 HuH-7 细胞）中进行。细胞转染野生型 PKCδ 增加了质膜 PKCδ 和 NTCP，并增加了 Rab4 活性。矛盾的是，过表达激酶失活的显性负 PKCδ 也增加了质膜 PKCδ 和 NTCP 以及 Rab4 活性。尽管 PKCδ 总量减少，但在 PKCδ 敲低实验中也获得了类似的结果。这些结果提出了一种可能性，即 PKCδ 的质膜定位而不是激酶活性对于 NTCP 易位和 Rab4 活性是必要的。这一假设得到了以下结果的支持：先前已显示抑制 cAMP 诱导的 PKCδ 和 NTCP 质膜易位的罗特林抑制 cAMP 诱导的 Rab4 活性。此外，已显示抑制 cAMP 诱导的 NTCP 易位的 LY294002（一种磷酸肌醇 3-激酶抑制剂）也抑制 cAMP 诱导的 PKCδ 易位。与 NTCP 的结果相反，在转染 DN-PKCδ 和 PKCδ 小干扰 RNA 的细胞中，cAMP 诱导的 MRP2 易位受到抑制。综上所述，这些结果表明，PKCδ 的质膜定位而不是激酶活性在 cAMP 诱导的 NTCP 易位和 Rab4 活性中发挥重要作用，而 PKCδ 的激酶活性对于 cAMP 诱导的 MRP2 易位是必要的。
