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


CONCLUSIONS
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.
INSERM, UMR_S 938, CDR Saint-Antoine, F-75012, Paris, France; Sorbonne Universités, UPMC Université Paris 06, UMR_S 938 and Institute of Cardiometabolism and Nutrition (ICAN), F-75005, Paris, France.
Julien
J
Gautherot
Danièle
D
Delautier
Marie-Anne
MA
Maubert
Tounsia
T
Aït-Slimane
Gérard
G
Bolbach
Jean-Louis
JL
Delaunay
Anne-Marie
AM
Durand-Schneider
Delphine
D
Firrincieli
Véronique
V
Barbu
Nicolas
N
Chignard
Chantal
C
Housset
Michèle
M
Maurice
Thomas
T
Falguières
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000293:Adolescent; D000328:Adult; D000818:Animals; D016764:Cell Polarity / Q000502:physiology; D002780:Cholestasis, Intrahepatic / Q000235:genetics* / Q000378:metabolism*; D004285:Dogs; D005260:Female; D005838:Genotype; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D010713:Phosphatidylcholines / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011247:Pregnancy; D011248:Pregnancy Complications / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary
D018435:ATP Binding Cassette Transporter, Subfamily B; D010713:Phosphatidylcholines; C095122:multidrug resistance protein 3; D017346:Protein Serine-Threonine Kinases