Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations.
Résumé
: The ABCB4 transporter mediates phosphatidylcholine secretion at the canalicular membrane of hepatocytes and its genetic defects cause biliary diseases. While ABCB4 shares high sequence identity with the multidrug transporter ABCB1, its N-terminal domain is poorly conserved, leading us to hypothesize a functional specificity of this domain. A database of ABCB4 genotyping in a large series of patients was screened for variations altering residues of the N-terminal domain. Identified variants were then expressed in cell models to investigate their biological consequences. Two missense variations, T34M and R47G, were identified in patients with low-phospholipid associated cholelithiasis or intrahepatic cholestasis of pregnancy. The T34M and R47G mutated proteins showed no or minor defect, respectively, in maturation and targeting to the apical membrane, in polarized Madin-Darby Canine Kidney and HepG2 cells, while their stability was similar to that of wild type ABCB4. By contrast, the phosphatidylcholine secretion activity of both mutants was markedly decreased. In silico analysis indicated that the identified variants were likely to affect ABCB4 phosphorylation. Mass spectrometry analyses confirmed that the N-terminal domain of wild type ABCB4 could undergo phosphorylation in vitro and revealed that the T34M and R47G mutations impaired such phosphorylation. ABCB4-mediated phosphatidylcholine secretion was also increased by pharmacological activation of protein kinases A or C and decreased by the inhibition of these kinases. Furthermore, the secretion activity of the T34M and R47G mutants was less responsive than that of wild type ABCB4 to protein kinase modulators. Conclusion: We identified disease-associated variants of ABCB4 involved in the phosphorylation of its N-terminal domain and leading to decreased phosphatidylcholine secretion. Our results also indicate that ABCB4 activity is regulated by phosphorylation, in particular of N-terminal residues. (Hepatology 2014;).