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

Christopher M Schonhoff1, Cynthia R L Webster, M Sawkat Anwer

  • 1Departments of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA.

Hepatology (Baltimore, Md.)
|February 21, 2013
PubMed
Abstract

Insights

Taurolithocholate (TLC) triggers retrieval of multidrug-resistance-associated protein 2 (Mrp2) from liver cell membranes. This process involves protein kinase C epsilon (PKCϵ) activation and subsequent phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS).

Area of Science:

  • Hepatobiliary transport mechanisms
  • Cellular signaling pathways
  • Protein trafficking and regulation

Background:

  • Taurolithocholate (TLC) acutely inhibits biliary excretion of multidrug-resistance-associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane.
  • Cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2 localization.
  • The effect of TLC may involve protein kinase C epsilon (PKCϵ) and Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) phosphorylation, which is implicated in endocytosis.

Purpose of the Study:

  • To investigate the hypothesis that TLC-induced Mrp2 retrieval is mediated by PKCϵ-dependent MARCKS phosphorylation.

Main Methods:

  • Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in primary rat hepatocytes.
  • Investigated the effects of TLC and cAMP on PM-PKCϵ, PM-MRP2, and MARCKS phosphorylation.
  • Utilized dominant-negative (DN) PKCϵ and phosphorylation-deficient MARCKS constructs to elucidate the signaling pathway.

Main Results:

  • TLC increased PM-PKCϵ and decreased PM-MRP2 in both cell types, while cAMP increased PM-MRP2 without affecting PM-PKCϵ.
  • Dominant-negative PKCϵ reversed TLC-induced Mrp2 retrieval but did not affect cAMP-induced Mrp2 increases.
  • TLC significantly increased MARCKS phosphorylation in a PKCϵ-dependent manner, leading to its detachment from the membrane.

Conclusions:

  • TLC-induced Mrp2 retrieval is dependent on the activation of PKCϵ.
  • PKCϵ activation leads to MARCKS phosphorylation and subsequent detachment from the membrane.
  • This signaling cascade explains the mechanism of TLC-induced Mrp2 retrieval from the canalicular membrane.

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