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

Hepatology. 2013 Jul;58(1):284-92. doi: 10.1002/hep.26333. Epub 2013 May 14.

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.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Male
  • Membrane Proteins / metabolism*
  • Multidrug Resistance-Associated Proteins / drug effects
  • Multidrug Resistance-Associated Proteins / metabolism
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Protein Kinase C-delta / physiology*
  • Rats
  • Taurolithocholic Acid / metabolism*

Substances

  • Intracellular Signaling Peptides and Proteins
  • MARCKS protein, human
  • Marcks protein, rat
  • Membrane Proteins
  • Multidrug Resistance-Associated Proteins
  • Myristoylated Alanine-Rich C Kinase Substrate
  • multidrug resistance-associated protein 2
  • Taurolithocholic Acid
  • Protein Kinase C-delta