MRCK-Alpha and Its Effector Myosin II Regulatory Light Chain Bind ABCB4 and Regulate Its Membrane Expression.

Alix Bruneau,Thomas Falguières,Lynda Aoudjehane,Jérémie Gautheron,Chantal Housset,Tounsia Aït-Slimane,Romain Morichon,Haquima El Mourabit,Virginie Vauthier,Amel Ben Saad,Anne-Marie Durand-Schneider,Jean-Louis Delaunay

doi:10.3390/cells11040617

Abstract

ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.

Highlights

The ATP-binding cassette (ABC) transporter ABCB4, called MDR3, is functionally expressed in hepatocytes, where it mediates ATP-dependent translocation of the membrane phospholipid phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of hepatocytes canalicular membranes
ABCB4 deficiency causes progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare autosomal recessive disease occurring early in childhood that may be lethal in the absence of liver transplantation [4], and less-severe diseases which occur in young adults, including low-phospholipid-associated cholelithiasis (LPAC) syndrome and intrahepatic cholestasis of pregnancy (ICP) [5,6,7]
ABCB4 co-precipitated together with GFP-Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) from co-transfected HEK-293 cells. These results demonstrated that MRCKα associates with ABCB4, suggesting that this serine/threonine kinase may regulate the expression and/or function of ABCB4

Summary

Introduction

The ATP-binding cassette (ABC) transporter ABCB4, called MDR3 (multidrug resistance 3), is functionally expressed in hepatocytes, where it mediates ATP-dependent translocation of the membrane phospholipid phosphatidylcholine (PC) from the inner leaflet to the outer leaflet of hepatocytes canalicular membranes (for review, see [1]). HS1-associated protein X-1 (HAX-1) and myosin II regulatory light chain (MRLC) have been identified as direct binding partners of the linker domain of three ABC transporters located in the canalicular membrane of hepatocytes, i.e., the drug export pump ABCB1 (MDR1), the bile salt export pump ABCB11 (BSEP), and ABCB4. Regarding ABCB11, its apical trafficking was shown to involve MRLC, whereas clathrin-mediated endocytosis of the protein involves HAX-1 [13,14]. Whether these two molecules play a role in the apical trafficking and potential internalization of ABCB4 is unknown. We showed that the stability and fate of ABCB4 after reaching the canalicular membrane required a carboxyl-terminal PDZ-like motif (QNL) that binds the PDZ domain protein NHERF/EBP50 [15]

Methods

Results

Conclusion