Taurolithocholate‐induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH‐NTCP Cells

Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug‐resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)–MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine‐rich C kinase substrate (MARCKS) is a membrane‐bound F‐actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine‐rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC‐induced MRP2 retrieval involves PKCϵ‐mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH‐NTCP cells) and in rat hepatocytes. TLC increased PM–PKCϵ and decreased PM‐MRP2 in both HuH‐NTCP cells and hepatocytes. cAMP did not affect PM‐PKCϵ and increased PM‐MRP2 in these cells. In HuH‐NTCP cells, dominant‐negative (DN) PKCϵ reversed TLC‐induced decreases in PM‐MRP2 without affecting cAMP‐induced increases in PM‐MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH‐NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM‐MARCKS in HuH‐NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH‐NTCP cells transfected with DN‐PKCϵ, and this suggested PKCϵ‐mediated phosphorylation of MARCKS by TLC. In HuH‐NTCP cells transfected with phosphorylation‐deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM‐MRP2. Conclusion: Taken together, these results support the hypothesis that TLC‐induced MRP2 retrieval involves TLC‐mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane. (HEPATOLOGY 2013;)