Phosphorylation of ABCB4 impacts its function: Insights... : Hepatology (original) (raw)

Autoimmune, Cholestatic and Biliary Disease

Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations

Gautherot, Julien1,2,8; Delautier, Danièle1,2; Maubert, Marie-Anne3,4; Aït-Slimane, Tounsia1,2; Bolbach, Gérard5; Delaunay, Jean-Louis1,2; Durand-Schneider, Anne-Marie1,2; Firrincieli, Delphine1,2; Barbu, Véronique1,2,6; Chignard, Nicolas1,2; Housset, Chantal1,2,7; Maurice, Michèle1,2; Falguières, Thomas1,2

1_INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France_

2_Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), F-75005, Paris, France_

3_INSERM and Université Pierre et Marie Curie–Université Paris 06, ERL U1057, UMR 7203, Paris, France_

4_Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Plateforme de métabolomique, peptidomique et dosage de médicaments, Paris, France_

5_Université Pierre et Marie Curie–Université Paris 06, Plateforme de Spectrométrie de masse et Protéomique, IFR83/FRE3595, Paris, France_

6_Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Laboratoire de Biologie et Génétique Moléculaires, Paris, France_

7_Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Centre de Référence des Maladies Inflammatoires des Voies Biliaires (CMR MIVB) and Service d'Hépatologie, Paris, France_

8_Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria_

Received 10 December 2013; Revised 7 April 2014; Accepted 8 April 2014

Address reprint requests to: Michèle Maurice, Ph.D., UMR_S 938, CdR Saint Antoine, Faculté de Médecine Pierre et Marie Curie, 27 rue Chaligny, 75571 Paris cedex 12, France. E-mail: [email protected]; fax: +33 1-40-01-14-32.

1Potential conflict of interest: Nothing to report.

3This work was supported by grants from the French National Society of Gastroenterology (SNFGE), the Association Mucoviscidose ABCF2, and the French Association for the Study of the Liver (AFEF). J.G. received fellowships from Albi, Robert Debré. and VLM associations. T.F. was supported by fellowships from INSERM and Fondation pour la Recherche Médicale.

Abstract

The ABCB4 transporter mediates phosphatidylcholine (PC) secretion at the canalicular membrane of hepatocytes and its genetic defects cause biliary diseases. Whereas 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, whereas their stability was similar to that of wild-type (WT) ABCB4. By contrast, the PC 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 WT ABCB4 could undergo phosphorylation in vitro and revealed that the T34M and R47G mutations impaired such phosphorylation. ABCB4-mediated PC secretion was also increased by pharmacological activation of protein kinases A or C and decreased by inhibition of these kinases. Furthermore, secretion activity of the T34M and R47G mutants was less responsive than that of WT 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 PC secretion. Our results also indicate that ABCB4 activity is regulated by phosphorylation, in particular, of N-terminal residues. (Hepatology 2014;60:610–621)