title: Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Departments of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA.
authors: Christopher M
authors: CM
authors: Schonhoff
authors: Cynthia R L
authors: CR
authors: Webster
authors: M Sawkat
authors: MS
authors: Anwer
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000187:drug effects / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D051745:Protein Kinase C-delta / Q000502:physiology*; D051381:Rats; D013658:Taurolithocholic Acid / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D051745:Protein Kinase C-delta; D013658:Taurolithocholic Acid; C000715087:ABCC2 protein, human; C000619710:MARCKS protein, human; C000619712:Marcks protein, rat