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

Authors

  • Julien Gautherot,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
    Current affiliation:
    1. Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
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  • Danièle Delautier,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Marie-Anne Maubert,

    1. INSERM and Université Pierre et Marie Curie–Université Paris 06, ERL U1057, Paris, France
    2. Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Plateforme de métabolomique, peptidomique et dosage de médicaments, Paris, France
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  • Tounsia Aït-Slimane,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Gérard Bolbach,

    1. Université Pierre et Marie Curie–Université Paris 06, Plateforme de Spectrométrie de masse et Protéomique, Paris, France
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  • Jean-Louis Delaunay,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Anne-Marie Durand-Schneider,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Delphine Firrincieli,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Véronique Barbu,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
    3. Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Antoine, Laboratoire de Biologie et Génétique Moléculaires, Paris, France
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  • Nicolas Chignard,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Chantal Housset,

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
    3. 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
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  • Michèle Maurice,

    Corresponding author
    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
    • 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: michele.maurice@inserm.fr; fax: +33 1-40-01-14-32.

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  • Thomas Falguières

    1. INSERM, UMR_S 938, CDR Saint-Antoine, Paris, France
    2. Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), Paris, France
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  • Potential conflict of interest: Nothing to report.

  • This 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)

Abbreviations
Ab

antibody

ABC

ATP-binding cassette

ATP

adenosine triphosphate

BSA

bovine serum albumin

DMEM

Dulbecco's modified Eagle's medium

HEK

human embryonic kidney

ICP

intrahepatic cholestasis of pregnancy

IF

immunofluorescence

LPAC

low-phospholipid–associated cholelithiasis

MALDI-TOF

matrix-associated laser desorption/ionization-time of flight

MDCK

Madin-Darby Canine Kidney

MDR3

multidrug resistance protein 3

MS

mass spectrometry

PC

phosphatidylcholine

PCR

polymerase chain reaction

PFIC3

progressive familial intrahepatic cholestasis type 3

PKA/C

protein kinase A/C

PMA

phorbol 12-myristate 13-acetate

WT

wild type

Transporters localized at the canalicular membrane of hepatocytes play a key role in bile secretion. They mainly belong to the adenosine triphosphate (ATP)-binding cassette (ABC) protein superfamily and their gene defects cause rare biliary diseases.[1] ABCB4, or MDR3 (multidrug resistance protein 3), functions as a phosphatidylcholine (PC) translocator.[2, 3] PC secreted into bile forms mixed micelles with bile salts and cholesterol, thereby ensuring solubilization of cholesterol and protection of the hepatobiliary epithelia against the damaging effects of free bile acids.[4, 5]

More than 250 ABCB4 gene variations have been identified, typically with a biallelic status in progressive familial intrahepatic cholestasis type 3 (PFIC3), and a monoallelic status in low-phospholipid–associated cholelithiasis (LPAC) syndrome or intrahepatic cholestasis of pregnancy (ICP).[6-14] The majority (approximately 70%) are missense single-nucleotide variants. They are distributed throughout the entire sequence and cause disease phenotypes by mechanisms that remain largely unexplored.

ABCB4 has a high sequence identity with the multidrug transporter, ABCB1/MDR1.[15] Both transporters are polytopic transmembrane glycoproteins with two transmembrane modules each spanning the membrane six times and two ATP-binding domains.[16] Based on studies of ABCB1/ABCB4 chimeras, it is assumed that the transmembrane domains are involved in substrate specificity and secretion, whereas the nucleotide-binding domains provide the energy necessary for transport.[17] The N-terminal domain of ABCB4, as opposed to other regions, is poorly conserved with ABCB1, suggesting that it may have a specific role. In order to investigate the role of ABCB4 N-terminal domain, we searched for disease-associated gene variations in this region of 54 amino acids. Two point mutations (i.e., T34M and R47G) were identified in patients with LPAC syndrome or ICP and their effects were investigated after expression in cell culture models. We found that the N-terminal domain of ABCB4 contained phosphorylation sites and that phosphorylation of this domain regulated ABCB4-mediated PC secretion. Our results also indicate that both disease-associated mutations identified in the N-terminal region of ABCB4 affected its phosphorylation-dependent secretion activity.

Patients and Methods

Patients' Data Analyses

ABCB4 gene analysis was performed in patients referred to the Reference Center for Inflammatory Biliary Diseases (Hôpital Saint-Antoine, Paris, France), as previously reported,[18] upon informed consent from the patients and approval by the local ethical committee. In brief, the 27 coding exons of the ABCB4 gene were amplified together with their exon/intron boundaries from genomic DNA and sequencing was performed on an ABI 3130 Genetic Analyzer (Applied Biosystems–Life Technologies, Saint-Aubin, France). Gene variations were assessed by sequence comparisons with SeqScape software (version 2.5; Applied Biosystems–Life Technologies). Clinical phenotypes of patients were classified according to current spectrum of liver diseases related to ABCB4 gene variations.[11]

DNA Constructs, Mutagenesis, and Polymerase Chain Reaction

The construction of the human wild type (WT) ABCB4, isoform A (NM_000443.3), in pcDNA3.1 vector has been described.[19] Site-directed mutagenesis was performed using the QuikChange II XL mutagenesis kit from Agilent Technologies (Massy, France). The I541F mutagenesis has been published.[19] DNA primers used for other ABCB4 mutagenesis were from Invitrogen-Life Technologies (Saint-Aubin, France) and Eurogentec (Angers, France) and are described in Supporting Table 1. For multiple-point mutants, mutations were done sequentially. To produce recombinant N-terminal cytoplasmic domains of ABCB4, the region encoding the first 58 amino acids was amplified by polymerase chain reaction (PCR), adding EcoRI and XhoI restriction sites in 5′ and 3′, respectively, by using the following primers: 5′-GAATTCGATCTTGAGGCGGCAAAG-3′ (sense) and 5′-CTCGAGTTACGACATAAACAATTTATCCT GCC-3′ (antisense). After amplification and digestion, the N-terminal fragment was subcloned into pGex4T-1 (GE Healthcare, Vélizy-Villacoublay, France), using the same restriction sites. The same primers as for pcDNA-ABCB4 were used for site-directed mutagenesis (Supporting Table 1). All constructs were systematically verified by automated sequencing. Quantitative real-time PCR was performed using a Lightcycler 480 (Roche, Basel, Switzerland) device, as previously described,[20] with β-actin used as a reference gene and primers described in Supporting Table 2.

Cell Models

Madin-Darby Canine Kidney (MDCK) type II cells and human hepatocellular carcinoma HepG2 cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM), as previously described.[19] WT or mutated ABCB4 were stably expressed in MDCK cells or transiently transfected in HepG2, as previously published.[19, 21] Noteworthy is that, in these conditions, HepG2 form canaliculi, but displayed virtually no endogenous ABCB4 expression, as ascertained either by immunofluorescence (IF) or immunoblotting (data not shown). Human embryonic kidney (HEK) 293 cells were cultured as previously reported.[22] Human hepatocytes were isolated from surgical liver samples, by previously established methods,[23] upon agreement (L 1232-3) from the Biomedicine Agency. Culture media were purchased from GE Healthcare.

Immunoanalyses

The monoclonal P3II-26 anti-ABCB4 antibody (Ab) was obtained from Enzo Life Sciences (Villeurbanne, France) and the monoclonal anti-β actin from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Alexa Fluor–labeled secondary Abs were from Invitrogen-Life Technologies. Peroxidase-conjugated secondary Abs were from Rockland Immunochemicals (Gilbertsville, PA). IF and confocal imaging on MDCK and HepG2 cells were performed as previously reported.[21] Western blotting and deglycosylation treatments with endo-β-N-acetylglucosaminidase H (EndoH; Roche Diagnostics, Basel, Switzerland) or peptide-N glycosidase F (PNGaseF; Roche Diagnostics) were previously described.[21] Blot exposure times were within the linear range of detection, and signal intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Protein concentrations were determined by Uptima bicinchoninic acid protein assay (Interchim, Montluçon, France).

Phosphorylation status of ABCB4 was assayed in freshly isolated human hepatocytes, which were lysed in TNE buffer (20 mmol/L of Tris-HCl [pH 7.4], 150 mmol/L of NaCl, 1 mmol/L of ethylenediaminetetraacetic acid) in the presence of protease inhibitor cocktail (Roche Diagnostics) and phosphatase inhibitor cocktail A (Santa Cruz Biotechnology, Dallas, TX). Total proteins (3 mg) were incubated with 20 μg of anti-phosphoserine or -phosphothreonine polyclonal Abs (Enzo Life Sciences) or 20 μg of immunoglobulins from normal rabbit serum (produced in our laboratory), overnight at 4°C in the presence of Protein A Sepharose (GE Healthcare), and immunoprecipitated proteins were then subjected to ABCB4 immunoblotting.

Analysis of ABCB4 Protein Stability

MDCK cells that were stably transfected with WT or mutant ABCB4 were treated with 10 mmol/L of sodium butyrate for 16 hours to induce ABCB4 expression and then chased for 0-3 days. ABCB4 expression was quantified from western blotting analyses. Results are expressed as a percentage of remaining ABCB4, compared to the starting point of the chase and after background subtraction, that is, ABCB4 expression without sodium butyrate treatment.

Measurement of PC Secretion

HEK cells were seeded on poly-lysine precoated dishes at 70% confluency. Six hours after seeding, cells were transiently transfected with ABCB4-encoding constructs (1 μg for a 10-cm2 well) using Turbofect (Fermentas France, Villebon-sur-Yvette, France) and following the manufacturer's instructions. Sixteen hours post-transfection, the medium was replaced by phenol red-free DMEM containing 0.5 mmol/L sodium taurocholate (omitted when indicated) and 0.02% fatty acid–free bovine serum albumin (BSA), as previously reported,[22] and then collected after 24 hours. To test the effect of protein kinase A (PKA) and protein kinase C (PKC) activation or inhibition, 10 μmol/L of forskolin (Enzo Life Sciences), 10 μmol/L of sp-adenosine-3′,5′-cyclic monophosphorothioate triethylamine salt (Sp-cAMPS; Enzo Life Sciences), 10 nmol/L of phorbol 12-myristate 13-acetate (PMA; Tocris Bioscience, Bristol, UK), 10 μmol/L of 1,2-dioctanoyl-sn-glycerol (DOG; Enzo Life Sciences), 10 μmol/L of 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö6983; Tocris Bioscience), or 10 μmol/L of N-[2-[[3-(4-Bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamidedihydrochloride (H-89; Enzo Life Sciences) were added to the medium containing sodium taurocholate and BSA, and the medium was collected after 8 hours, to minimize potential cytotoxic effects of the drugs. Collected media were centrifuged at 15,000×g for 15 minutes at 4°C to remove aggregates and cell debris. Total lipid extracts were prepared from supernatants by chloroform/methanol/water partition, as previously described.[24] After collection and evaporation of the organic phase, lipid samples were resuspended in 100 μL of phosphate-buffered saline containing 0.1% Triton X-100 (% w/v) and incubated for 2 hours at 60°C. Measurement of PC content was based on the measurement of choline release after phospholipase D treatment,[25] with some modifications.[26] Samples (50 μL) were incubated with 50 μL of reaction mix composed of 0.5 U/mL of choline oxidase, 1 U/mL of horseradish peroxidase, 1 mmol/L of 3-(4-hydroxyphenyl) propionic acid with (total choline), or without (background choline) 250 U/mL of phospholipase D (Enzo Life Sciences) as final concentrations. After a 30-minute incubation at 37°C, fluorescence was read (λexc, 320 nm; λem, 404 nm) with a multiplate cytofluorimeter SpectraFluor from Tecan (MTX Lab Systems, Vienna, VA). Signal intensities were always within the linearity of the standard PC calibration curve, from 0 to 2.5 nmol of PC (data not shown). Results were normalized to the expression levels of ABCB4, which were quantified from immunoblottings obtained from the corresponding cell lysates. Cytotoxicity was assessed by measuring lactate dehydrogenase release using the Cytotox-ONE kit (Promega, Charbonnières-les-Bains, France).

Production of Recombinant Peptides and In Vitro Phosphorylation Analysis

BL-21-DE3 competent bacteria (New England Biolabs, Evry, France) were transformed with pGex4T1 encoding WT or mutant ABCB4 N-terminal domain. After bacterial growth, expression of GST-tagged ABCB4 N-terminal peptides was induced by a 4-hour treatment with 0.4 mmol/L of isopropyl β-D-1-thiogalactopyranoside. Peptides were purified from bacteria lysates using glutathione sepharose (GE Healthcare). Recombinant peptides were cleaved from the GST tag by an overnight digestion at room temperature with 5 U/mL of thrombin (GE Healthcare), then eluted with 50 mmol/L of Na2CO3 (pH 11.0), and further dialyzed against 50 mmol/L of Tris-HCl (pH 7.4).

The phosphorylation reaction was performed in a reaction volume of 20 μL as follows: 1.0 μg of recombinant peptides were incubated for 60 minutes at 30°C, in 50 mmol/L of Tris-HCl (pH 7.4) with 200 μmol/L of ATP, 10 mmol/L of MgCl2, with or without 10 μg/mL of PKA catalytic subunit (Promega) or 0.5 μg/mL of PKC catalytic fragment (Enzo Life Sciences). Then, samples were immediately desalted with ZipTip C18 (Millipore, Molsheim, France) and analyzed with an externally calibrated matrix-associated laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer (Voyager DE-Pro; ABSciex, Les Ulis, France). Mass spectra were obtained in the linear positive mode, using a pulsed nitrogen laser (λ = 337 nm; 3 Hz; pulse, 3 ns), an extraction delay of 200 ns, an accelerating voltage of 20 kV, and an average of 500 laser shots per condition. The matrix solution was 100 mmol/L of α-cyano-4-hydroxycinnamic acid in 70%/30% (% v/v) acetonitrile/water with 0.1% trifluoroacetic acid. Mass values displayed in linear mode correspond to the average masses. Additional mass spectrometry (MS) analyses on a truncated peptide were achieved by partial de novo sequencing, using tandem MS (MS/MS), as previously described.[27] Briefly, MS/MS spectra were acquired on a 4700 MALDI-TOF/TOF mass spectrometer (ABSciex). MALDI samples were prepared with the same matrix as described above. The collision energy was 1 keV, and the collision gas was air. Typically, 2,000 lasers shots were used for MS/MS acquisition. Background was removed from each spectra using DataExplorer software (ABSciex).

Statistical Analysis

The Student t test was used for comparisons. A P value of less than 0.05 was considered significant.

Results

Identification of Disease-Causing ABCB4 Variants in the N-Terminal Domain of ABCB4

The database of ABCB4 gene variations identified in patients referred to our center for biliary symptoms from 2001 to 2010, some of which were previously published,[8, 9, 12, 14, 18, 28] was screened for single-nucleotide variants that would change one of the amino acids of the ABCB4 N-terminal domain. Two point mutations localized in this domain, that is, T34M and R47G (Fig. 1A), were identified in 5 patients. These patients were diagnosed with LPAC syndrome or manifestations belonging to the spectrum of ABCB4-related liver diseases, including ICP.[11] All 5 patients, including 2 previously reported cases,[14, 28] were heterozygous and their main characteristics are shown in Table 1.

Figure 1.

Expression and maturation of the T34M and R47G ABCB4 mutants. (A) Schematic representation of ABCB4. Nucleotide binding domains (NBD1 and NBD2) and glycosylation in the first extracellular loop are indicated. The amino acid sequence of the intracytoplasmic N-terminal domain of the human ABCB4 isoform A (NP_000434.1) is shown. Blue arrows and letters indicate the position of T34M and R47G mutations. Potential phosphorylation sites identified using the NetPhos server (http://www.cbs.dtu.dk/services/NetPhos/) are shown in red. (B) WT and mutant (T34M or R47G) ABCB4 were expressed in MDCK (stable) or HepG2 (transient) cells and visualized by confocal microscopy (green). For MDCK cells, both xy and xz sections are shown. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, blue). Asterisks indicate bile canaliculi. Bars, 10 µm. (C) ABCB4 was detected by immunoblotting from cell lysates of MDCK cells stably expressing ABCB4 (WT or mutants). Additional EndoH or PNGaseF treatment was performed on lysates from ABCB4wt-expressing cells before immunoblotting analysis. Ctrl, control.

Table 1. Characteristics of Patients With T34M and R47G Heterozygous Variations of ABCB4
  Variant  
Patients (No./Gender)Age at First Symptoms (Years)Nucleotide ChangeEffect on Amino AcidDiagnosisPublication Status
  1. Abbreviations: F, female; M, male.

1/F15c.101 C>TT34MDrug-induced liver injury; ICP; cirrhosisThis study
2/F42c.101 C>TT34MLPACWendum et al.28: patient no. 3
3/M32c.101 C>TT34MLPACThis study
4/F42c.101 C>TT34MLPACThis study
5/F47c.139 C>GR47GLPACPoupon et al.14: patient no. 1 Wendum et al.28: patient no. 5

Targeting of the T34M and R47G ABCB4 Mutants to the Apical Plasma Membrane

To determine the molecular mechanisms whereby the T34M and R47G mutations of ABCB4 induce a pathological phenotype, we reproduced these mutations in ABCB4 complementary DNA. After expression in MDCK and HepG2 cells, WT (ABCB4wt) and mutated proteins were localized at the apical plasma membrane in both cell types (Fig. 1B). However, ABCB4R47G was also partly intracellular in both MDCK and HepG2 cells (Fig. 1B). Immunoblottings showed that ABCB4wt migrated as two bands of apparent molecular weights of ∼140 and ∼160 kDa (Fig. 1C), as previously reported.[21] These bands correspond to different states of maturation, as indicated by the effect of treatment with endoglycosidases: (1) The ∼140-kDa band being sensitive to EndoH digestion, which removes high-mannose glycosylated chains, is the immature form found in the endoplasmic reticulum; (2) the ∼160-kDa band is the mature form because it was sensitive only to PNGaseF, which cleaves both immature and complex glycosylation chains. Electrophoretic behavior of the mutants was comparable to that of the WT protein (Fig. 1C), although the immature form of ABCB4R47G was more abundant. Indeed, western blotting quantifications showed that 38.5% ± 1.7% of ABCB4R47G was immature, whereas it was only 17.2% ± 1.3% and 15.2% ± 1.3% of the WT protein and the T34M mutant, respectively (Supporting Fig. 1). This 2-fold increase of immaturity for ABCB4R47G, compared to WT and T34M, was also observed in HepG2 cells (data not shown), consistent with slightly less-efficient processing of this mutant to the apical plasma membrane. Overall, we concluded that the T34M and R47G mutants were either fully or largely matured and targeted to the apical membrane.

Stability of the T34M and R47G mutants

Next, we compared the stability of the mutant and WT proteins. We took advantage of the fact that ABCB4 constructs were cloned into the pcDNA3.1 vector under the control of the cytomegalovirus promoter, known to be strongly activated by sodium butyrate treatment,[29] which we confirmed by showing that ABCB4 messenger RNA increased by 22-fold after overnight treatment with sodium butyrate (Supporting Fig. 2). After this treatment, MDCK cells stably expressing WT or mutant ABCB4 were chased for 0-3 days without sodium butyrate and then analyzed by immunoblotting (Fig. 2A). These analyses showed that the decay of both ABCB4T34M and ABCB4R47G were not significantly modified, compared to that of the WT protein (Fig. 2B). These results indicated that the T34M and R47G mutations did not alter ABCB4 stability.

Figure 2.

Stability of T34M and R47G ABCB4 mutants. (A) Expression of ABCB4 (WT or mutant) was induced in stably transfected MDCK cells by an overnight treatment with 10 mmol/L of sodium butyrate. Then, sodium butyrate was removed and ABCB4 was detected at the indicated time points in cell lysates by immunoblotting, using equal amounts of total proteins per lane. n.i., noninduced. (B) Amounts of ABCB4 were quantified from chase experiments as in (A). After background subtraction (corresponding to the noninduced lanes), ABCB4 levels were expressed as a percentage of the baseline (time point 0). Means (± standard error of the mean) of three independent experiments are shown. P > 0.1 at all time points.

Phosphatidylcholine Secretion Activity of the T34M and R47G ABCB4 Mutants

We then examined whether the PC secretion activity of the T34M and R47G mutants was impaired. For this purpose, we used transiently transfected HEK cells. The transfection efficiency of these cells was approximately 50%. Compared with Groen et al., who previously reported on an important cytotoxicity of ABCB4 transfection,[30] we found less cytotoxicity, and only to some extent, when ABCB4wt-expressing cells were serum deprived (Supporting Fig. 3A-C). ABCB4wt-expressing cells secreted approximately 20 times more PC than control cells transfected with an empty vector or expressing ABCB4I541F, a mutant previously shown to be improperly matured and processed[19, 21] (Fig. 3A,B). The average quantity of PC secreted over 24 hours by ABCB4wt-expressing HEK cells was 12.9 ± 0.9 nmol/mg of total proteins. By comparison, PC secretion by the ABCB4T34M- and ABCB4R47G-expressing HEK cells represented only 58.3% ± 6.6% and 33.2% ± 5.6% of ABCB4wt-expressing cells after normalization to the amount of ABCB4 mature form, respectively (Fig. 3A,C). These results inferred that Thr34 and Arg47 residues affect ABCB4 transport function.

Figure 3.

PC secretion by T34M and R47G ABCB4 mutants. (A) ABCB4 expression was analyzed in HEK cells after transient transfection with the indicated plasmids. This immunoblotting is representative of five independent experiments. (B and C) HEK cells as shown in (A) were incubated with serum-free media containing sodium taurocholate and BSA, which, after 24 hours, were collected to measure their PC content. Results are expressed as a percentage of PC secreted by ABCB4wt-transfected cells, with (B) or without (C) normalization to the amount of the mature ABCB4. Means (± standard error of the mean) of five independent experiments are shown. *P < 0.001; wt, wild type.

In Vitro Phosphorylation of ABCB4 N-Terminal Residues

In silico predictions using the NetPhos server (http://www.cbs.dtu.dk/services/NetPhos/) indicated that Thr34 and the Arg47 neighboring residues, Thr44 and Ser49, are potential phosphorylation sites and that the R47G mutation could modulate phosphorylation of Thr44 and Ser49 (Fig. 1A). In addition, immunoprecipitation assays performed with anti-phosphoSer or anti-phosphoThr Abs in isolated human hepatocytes demonstrated that full-size ABCB4 was phosphorylated on serine and threonine residues in vivo (Supporting Fig. 4), although this could occur in different regions of the protein. We postulated that T34M and/or R47G mutations could affect PC secretion by impairing phosphorylation. To address this possibility, we produced soluble recombinant peptides of the N-terminal region of WT or mutated ABCB4, with the following changes: T34M; R47G; T34M combined with T44A or S49A; and T34M combined with T44A and S49A (Supporting Table 3). These peptides were used as substrates for in vitro phosphorylation assays using either PKA or PKC. The phosphorylation profile of these peptides was then analyzed by MALDI-TOF MS. MALDI-MS spectra of the WT peptide, with a mass of 7,028 Da, showed two additional ions with +80- and +160-Da shifts after incubation with either PKA or PKC. The latter observation indicated that the ABCB4 N-terminal region was phosphorylated (HPO3 = 80 Da) on two distinct residues (Fig. 4A, left panels). When the same experiment was performed with the T34M and R47G mutant peptides, only one phosphorylated ion (+80 Da) was detected, which clearly indicated that both mutations caused the loss of one phosphorylation site (Fig. 4A, middle and right panels). Moreover, a smaller peptide in the mass range m/z 4,600-4,800 was also detected on the MALDI-MS spectra for both WT and mutant peptides (Supporting Figs. 5A and Fig. 6). MALDI-TOF/TOF MS analysis and de novo sequencing revealed that it was a truncated form of the peptide likely resulting from proteolytical cleavage and containing the first 38 amino acids of ABCB4 (Supporting Fig. 5B,C). The absence of phosphorylation of the T34M truncated peptide in the presence of protein kinases validated the Thr34 residue as the unique phosphorylation site in the 1-38 N-terminal region of ABCB4 (Supporting Fig. 6). According to in silico predictions, the R47G mutation could possibly alter the phosphorylation of Thr44 and/or Ser49. Therefore, we separately or simultaneously mutated these two residues into nonphosphorylatable amino acids (alanines), in addition to the T34M mutation, and then analyzed the phosphorylation of these peptides by MS. The double-mutant peptides, T34M-T44A and T34M-S49A, still displayed a single phosphorylated ion with a mass shift of +80 Da in response to both PKA and PKC, whereas the triple-mutant T34M-T44A-S49A was not phosphorylated at all (Fig. 4B). These results suggested that besides Thr34, Thr44 and Ser49 residues provide secondary phosphorylation sites that can be alternatively phosphorylated. Overall, these results from in vitro phosphorylation assays (summarized in Supporting Table 4) provided evidence that residues Thr34, Thr44, and Ser49 could be phosphorylated.

Figure 4.

In vitro phosphorylation analysis of ABCB4 N-terminal recombinant peptides. (A) WT, T34M, and R47G N-terminal ABCB4 recombinant peptides were incubated in vitro in the absence or presence of PKA or PKC, as indicated, and analyzed by MALDI-TOF MS. Average masses of the peptides are indicated on the MALDI-MS spectra zoom scan from m/z 6,700 to 7,500 (also see Supporting Table 3), as well as single (+80 Da) and double (+160 Da) phosphorylated ions. (B) Same analysis as in (A) with ABCB4 double- and triple-mutant peptides, as indicated. For (A) and (B), each MS profile is representative of at least three independent experiments. Results are summarized in Supporting Table 4.

Figure 5.

Regulation of ABCB4 secretion activity by phosphorylation. (A and B) PC secretion over 24 hours was measured in HEK cells transfected with the indicated ABCB4 encoding plasmids, as in Fig. 3C. *P < 0.001; n.s., not significant. (C) PC secretion over 8 hours of exposure to the indicated treatments was measured in transfected HEK cells. Results are expressed as a percentage of PC secretion by untreated ABCB4wt-expressing cells, after normalization to ABCB4 mature protein levels. Means (± standard error of the mean) of at least three independent experiments are shown. *P < 0.05; n.s., not significant; wt, wild type.

Influence of N-Terminal Phosphorylation on ABCB4 Transport Activity

To determine the role of phosphorylatable residues in PC secretion activity of ABCB4, we mutated Thr34 into an aspartate (T34D) to mimic constitutive phosphorylation. ABCB4T34D-expressing cells showed a rescue of PC secretion activity, comparable to the ABCB4wt-expressing cells (Fig. 5A), demonstrating that phosphorylation of Thr34 is required for basal activity of ABCB4. We evaluated the role of Thr44 or Ser49 phosphorylation by mutating these residues into alanines, which cannot be phosphorylated. We observed that both ABCB4T44A- and ABCB4S49A-expressing cells had a significantly reduced PC secretion activity (Fig. 5B). These additional mutants were all expressed at the apical membrane, as with ABCB4wt (data not shown). These data argued for an essential role of the phosphorylation of ABCB4 N-terminal residues, namely, Thr34, Thr44, and Ser49, in PC secretion activity.

Regulation of ABCB4 Transport Function by Serine/Threonine Kinases

To more generally assess the role of phosphorylation on ABCB4 activity, we treated ABCB4-transfected HEK cells with several protein kinase activators and inhibitors. None of these treatments modified the background levels of PC secreted by control cells transfected with the empty vector, nor the plasma membrane localization of ABCB4 (data not shown). In ABCB4wt-transfected cells, the activators significantly increased PC secretion, whereas the inhibitors diminished this secretion (Fig. 5C and Supporting Fig. 7). Compared to the untreated condition, PC secretion activity was increased to 143% ± 13% and 235% ± 34% by forskolin (PKA activator) and PMA (PKC activator), respectively, and decreased to 51% ± 5% and 28% ± 5% by H-89 (PKA inhibitor) and Gö6983 (PKC inhibitor), respectively. The PKA/C inhibitors also decreased the activity of the T34M and R47G mutants (Fig. 5C). However, the PKA/C-activating drugs had weak or no effect on the secretory function of these mutants (Fig. 5C). We inferred, from these results, that ABCB4 function can be regulated by both PKA and PKC, and that this regulation is attenuated by the T34M and R47G mutations.

Discussion

In the present study, through the analysis of disease-associated mutations, we show that the N-terminal domain of ABCB4 is involved in its function of PC translocation, and that phosphorylation by serine/threonine kinases plays a crucial role in this process.

Heterozygous T34M and R47G mutations were identified in 5 patients, including 2 with LPAC (nos. 2 and 5; Table 1), whose cases were previously reported.[14, 28] Immunohistochemical analysis of ABCB4 in liver biopsies from these 2 patients showed staining at the bile canaliculi, suggesting that both mutants were targeted, at least to a large extent, to the bile canalicular membrane.[28] In agreement with these observations, we found that the T34M and R47G mutants were localized at the apical plasma membrane in polarized cell (including hepatocellular) culture models, although the R47G mutant proved to be less efficiently processed. For both mutants, the main observed defect was the reduced activity of PC secretion, which was measured in HEK transfected cells. Groen et al. previously reported on an important cytotoxicity of ABCB4 transfection in HEK cells unless they were cotransfected with ATP8B1 and CDC50A.[30] There was more limited evidence for ABCB4 cytotoxicity in our study, because transfection resulted in only a marginally significant increase in lactate dehydrogenase release (Supporting Fig. 3B). Furthermore, the observation that PC secretion could be enhanced in this cell system by either taurocholate or PMA (Supporting Fig. 3B,C) provided evidence for the specificity of this model.

Biological consequences of ABCB4 mutations and their role in pathogenesis have been addressed in a limited number of studies. We have previously shown that the missense mutation, I541F, identified in a PFIC3 patient, led to defective traffic and early degradation of the mutant protein.[19, 21] A similar conclusion was drawn for another missense mutation associated with intrahepatic cholestasis of pregnancy.[31] Recently, a study demonstrated that two mutations found in patients with biliary diseases, Y403H and L481R, decreased ABCB4 activity without altering its membrane targeting.[32] These mutations were localized in the first nucleotide-binding domain and therefore may affect ABCB4 function by interfering with ATP binding and/or hydrolysis. In our study, the T34M and R47G mutations were located in the cytoplasmic N-terminal domain, the role of which was unknown. Our results demonstrate that the N-terminal domain takes part in ABCB4 function.

The N-terminal domain of ABCB4 contains several serines and threonines that are potential targets for protein kinases. The two mutations studied here were susceptible to affect phosphorylation, either directly (T34M) or indirectly (R47G). Our analysis of phosphorylation of the N-terminal peptides showed that only Thr34, Thr44, and Ser49 were phosphorylatable in vitro. Whereas the T34M mutation directly abrogated phosphorylation at position 34, the R47G mutation inhibited the phosphorylation of neighboring residues, either Thr44 or Ser49. The importance of phosphorylation of these residues for ABCB4 activity is strengthened by the fact that (1) T34M mutation markedly decreased PC secretion, whereas the T34D mutation, which mimics constitutive phosphorylation, restored secretion, although below the levels reached in response to protein kinase activators, suggesting that several residues need to be phosphorylated for maximal activity, and (2) the mutation of either Thr44 or Ser49 into alanines decreased PC secretion. A link between phosphorylation and activity of ABCB4 was also provided by agonists and inhibitors of protein kinases A and C. The effect of agonists and inhibitors was less pronounced for ABCB4T34M- and ABCB4R47G-expressing cells, arguing for a role of phosphorylation of N-terminal residues in ABCB4 activity. However, we cannot rule out effects of PKA/C activating and inhibiting drugs on other phosphorylatable residues of ABCB4 outside the N-terminal region. In addition, we cannot exclude that the phosphorylation of some residues also affects maturation of ABCB4, as suggested by the observation that the R47G mutant was less efficiently processed. ABCB1 can be phosphorylated on serine residues located in the linker domain, the region that separates the two halves of the transporter, and there is evidence to indicate that phosphorylation is a mechanism regulating ABCB1 function (previously reviewed[33]). It has been shown that the phosphorylation of ABCB1 modulated its interaction with transported molecules affecting substrate specificity and affinity, but was not required for ATPase activity.[34] The linker region, which is phosphorylated in ABCB1, occupies the same position in the second module as the N-terminal domain does in the first. We may thus speculate that phosphorylation of the N-terminal domain could regulate the specific interaction of ABCB4 with its substrate.

The regulation of ABCB4 by phosphorylation has major potential implication in physiology. The effect of PKC-targeting drugs (PMA and Gö6983) were more pronounced on PC secretion than PKA-targeting ones (forskolin and H-89), suggesting that PKC is the major kinase involved. Likewise, in vitro phosphorylation was more efficient with PKC than with PKA, again suggesting that the phosphorylation sites in the N-terminal domain have a greater affinity for PKC. It is of particular interest in this respect that bile acids are activators of protein kinases, and, more specifically, of PKC,[35] and that bile acids trigger secretion of biliary phospholipids.[36] Phosphorylation of ABCB4 may thus be one of the mechanisms whereby bile acids promote phospholipid secretion, which needs to be combined with their own secretion to ensure efficient, controlled bile secretion.

Acknowledgment

The authors thank Philippe Fontanges and Romain Morichon (IFR65, Université Pierre et Marie Curie, Université Paris 06) for confocal microscopy imaging, Gilles Clodic (IFR83, Université Pierre et Marie Curie, Université Paris 06) for his help with MS/MS experiments, and Raoul Poupon (Centre de Référence des Maladies Inflammatoires des Voies Biliaires [CMR MIVB] and Service d'Hépatologie, Hôpital Saint-Antoine) for his help and support with this project. The authors also acknowledge Dominique Wendum (Service d'Anatomie Pathologique, Hôpital Saint-Antoine), who provided the pathological data from patients, and Lynda Aoudjehane (Human HepCell) who provided isolated human hepatocytes.

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