Dephosphorylation of Ser-226 facilitates plasma membrane retention of Ntcp.
Ntcp is a phosphoprotein, and its translocation by cAMP to the plasma membrane is associated with dephosphorylation. However, the phosphorylation site(s) of Ntcp is not known. Thus, the aim of the present study was to determine the potential Ntcp phosphorylation sites and whether any of these phosphorylation sites is involved in Ntcp translocation. To determine the potential phosphorylation sites, metabolically labeled [32P]Ntcp isolated from hepatocytes was digested with clostripain and then subjected to SDS-PAGE followed by autoradiography. Clostripain digestion resulted in two phosphorylated peptides, and cAMP decreased phosphorylation of one of the peptides (7.8 K(d)), which contains the putative third cytoplasmic loop with three serine (Ser-213, Ser-226, and Ser-227) and two threonine (Thr-219 and Thr-225) residues. To determine whether any one of these serine/threonine residues is phosphorylated and/or is involved in Ntcp translocation, each of these serine/threonine residues were mutated to alanine. HuH-7 cells were transiently transfected with the wild-type and the mutated Ntcps followed by determination of taurocholate uptake and Ntcp expression, translocation and phosphorylation. Mutation of only Ser-226 resulted in 30% decrease in Ntcp phosphorylation and in 2.5 and 3.2-fold increases in taurocholate uptake and Ntcp retention in the plasma membrane, respectively. Cyclic AMP failed to further decrease phosphorylation and increase translocation of S226A-Ntcp. Taken together, these results suggest that the Ser-226 in the third cytoplasmic loop of Ntcp is phosphorylated and cAMP may increase Ntcp translocation to the plasma membrane by dephosphorylating Ntcp at this site.
Anwer
M Sawkat
MS
Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA. Sawkat.Anwer@Tufts.Edu
Gillin
Henry
H
Mukhopadhyay
Sunil
S
Balasubramaniyan
Natarajan
N
Suchy
Federick J
FJ
Ananthanarayanan
Meenakshisundaram
M
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D022781:Hepatocytes / Q000737:chemistry; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D017354:Point Mutation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D051381:Rats; D012694:Serine / Q000737:chemistry / Q000378:metabolism*; D027981:Symporters; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection
D026901:Membrane Transport Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D027981:Symporters; C071908:sodium-bile acid cotransporter; D012694:Serine; D013656:Taurocholic Acid; D000242:Cyclic AMP; D003546:Cysteine Endopeptidases; C019913:clostripain; D000409:Alanine