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的机制。我们目前对胆汁淤积发病机制的理解是基于对参与胆汁形成的转运体的生理调节及其在胆汁淤积中的解除的研究。在上一个授权期内，我们定义了aPKC、Rab4和磷酸化在Ntcp易位中的作用，并开始定义nPKC、nPKC和p38 MAPK在Mrp2易位中的作用。本研究扩展了这些研究，进一步定义了nPKC和nPKC在Ntcp/ Ntcp和Mrp2/ Mrp2易位/检索中的作用。其中一个关键目标是确定这些激酶在肝细胞中的相反作用的机制。提出了以下假设：1)nPKC -pThr505介导Ntcp/ Ntcp通过cAMP易位，Mrp2/ Mrp2通过cAMP和TUDC易位至质膜；2)nPKC通过磷酸化MARCKS和/或Mrp2/ Mrp2介导tlc诱导的Mrp2/ Mrp2从质膜上恢复，而cAMP和TUDC通过抑制tlc诱导的nPKC激活来逆转这一作用。拟议的研究将在灌注肝和来自大鼠肝脏的肝细胞、原代人肝细胞和肝细胞系中进行。此外，研究将在nPKC -/-和nPKC -/-小鼠的灌注肝脏和分离的肝细胞中进行，以进一步证实这些激酶的作用。这些激酶的作用将通过使用化学抑制剂、野生型、组成型活性和显性负质粒以及Si和/或ShRNA来控制它们的活性和表达来评估。免疫荧光和共免疫沉淀研究将用于确定所需蛋白质在亚细胞细胞器和与其他蛋白质的共定位。总的来说，今后的研究应进一步明确nPKC在Ntcp/ Ntcp和Mrp2/ Mrp2易位中的作用，nPKC在Mrp2/ Mrp2检索中的作用，以及cAMP和TUDC逆转tlc诱导的胆汁淤积的机制。由于一种激酶可能以同种异构体特异性的方式产生有益或有毒的作用，更好地了解同种异构体特异性的作用应该允许更好的治疗药物，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.