Role of Protein Kinase C in Isoforms in Bile formation and Cholestasis

描述（由申请人提供）：本提案的长期目标是了解。我们目前对胆汁淤积症发病机制的理解是基于研究，以确定参与胆汁形成的转运蛋白的生理调节及其在胆汁淤积症中的失调。在最后一个授权期间，我们已经确定了aPKC、Rab 4和磷酸化在Ntcp易位中的作用，并开始确定nPKC、nPKC和p38 MAPK在Mrp 2易位中的作用。本建议扩展这些研究，以进一步确定nPKC和nPKC在Ntcp/NTCP和Mrp 2/MRP 2易位/检索中的作用。关键目标之一是确定这些激酶在肝细胞中的对抗作用所涉及的机制。提出了以下假设：1）nPKC-pThr 505介导由cAMP引起的Ntcp/NTCP的易位以及由cAMP和TUDC引起的Mrp 2/MRP 2的易位至质膜，2）nPKC通过磷酸化MARCKS和/或Mrp 2/MRP 2介导TLC诱导的Mrp 2/MRP 2从质膜的回收，并且cAMP和TUDC通过抑制TLC诱导的nPKC活化来逆转该效应。拟定研究将在灌注肝脏和大鼠肝脏肝细胞、原代人肝细胞和肝细胞系中进行。此外，将在从nPKC -/-和nPKC -/-小鼠分离的灌注肝脏和肝细胞中进行研究，以进一步证实这些激酶的作用。将通过使用化学抑制剂、野生型、组成型活性和显性阴性质粒以及Si和/或shRNA操纵其活性和表达来评价这些激酶的作用。免疫荧光和免疫共沉淀研究将用于确定所需蛋白质在亚细胞器中的共定位以及与其他蛋白质的共定位。总的来说，拟议的研究应进一步确定nPKC在Ntcp/NTCP和Mrp 2/MRP 2易位中的作用，nPKC在Mrp 2/MRP 2恢复中的作用，以及cAMP和TUDC逆转TLC诱导的胆汁淤积的机制。由于激酶可能以同种型特异性方式产生有益或毒性作用，因此更好地理解同种型特异性作用应该允许更好的治疗剂，其可以避免潜在的肝毒性。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the mechanism of. Our present understanding of the pathogenesis of cholestasis is based on studies to define the physiological regulation of transporters involved in bile formation and their deregulation in cholestasis. During the last granting period we have defined the role of aPKC , Rab4 and phosphorylation in Ntcp translocation and started defining the role of nPKC , nPKC and p38 MAPK in Mrp2 translocation. The present proposal extends these studies to further define the role of nPKC and nPKC in Ntcp/NTCP and Mrp2/MRP2 translocation/retrieval. One of the key goals is to define the mechanism involved in opposing effects of these kinases in hepatocytes. The following hypotheses are proposed: 1) nPKC -pThr505 mediates translocation of Ntcp/NTCP by cAMP and translocation of Mrp2/MRP2 by cAMP and TUDC to the plasma membrane, 2) nPKC mediates TLC-induced retrieval of Mrp2/MRP2 from the plasma membrane by phosphorylating MARCKS and/or Mrp2/MRP2, and cAMP and TUDC reverse this effect by inhibiting TLC-induced nPKC activation. Proposed studies will be conducted in perfused livers and hepatocytes from rat livers, primary human hepatocytes and hepatic cell lines. In addition, studies will be conducted in perfused livers and hepatocytes isolated from nPKC -/- and nPKC -/- mice to further confirm the role of these kinases. Role of these kinases will be evaluated by manipulating their activity and expression using chemical inhibitors, wild type-, constitutively active- and dominant negative-plasmids and Si- and/or ShRNA. Immunofluorescence and co-immunoprecipitation studies will be used to determine colocalization of desired proteins in subcellular organelles and with other proteins. Collectively, proposed studies should further define the role of nPKC in Ntcp/NTCP and Mrp2/MRP2 translocation, the role of nPKC in Mrp2/MRP2 retrieval, and mechanism of reversal of TLC-induced cholestasis by cAMP and TUDC. Since a kinase may produce beneficial or toxic effect in an isoform specific manner, a better understanding of isoform specific effects should allow for better therapeutic agents that could avoid potential liver toxicity.