Dephosphorylation of Ser-226 Facilitates Plasma Membrane Retention of Ntcp

Abstract

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 [ 32 P]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.Hepatic uptake of conjugated bile acids, such as TC, 2 across the sinusoidal membrane has been proposed to be mediated via three different transporters, and Ntcp is considered to be the major transporter (1-3). Ntcp, which mediates sinusoidal Na ϩ /TC cotransport (4 -6), is a serine/threonine phosphoprotein (7) with seven transmembrane domains (4). Cyclic AMP stimulates Na ϩ /TC cotransport by translocating Ntcp to the plasma membrane (8, 9), and this is associated with dephosphorylation of Ntcp (7). Experimental manipulations that inhibit the ability of cAMP to dephosphorylate Ntcp also inhibit the ability of cAMP to stimulate TC uptake and translocate Ntcp (7,10,11). These studies alsoshow that cAMP-induced dephosphorylation of Ntcp is dependent on its ability to increase cytosolic Ca 2ϩ and activate protein phosphatase 2B, a calcium/calmodulin-dependent serine/threonine protein phosphatase (7, 11). Okadaic acid, an inhibitor of protein phosphatase 1/2A, inhibits cAMP-induced increases in cytosolic Ca 2ϩ and Ntcp translocation (10). Based on these observations, we proposed that cAMP-induced translocation of Ntcp may involve dephosphorylation of Ntcp by protein phosphatase 2B (11). However, the identity of Ntcp phosphorylation site(s) is not known and hence it is difficult to design experiments to determine whether dephosphorylation of...

Short-term feedback regulation of bile salt uptake by bile salts in rodent liver

“…Furthermore, PP2B is known to dephosphorylate Ntcp at Serine 226 , which is involved in Ntcp exocytosis. 24 Here, the PP2B inhibitor cypermethrin decreased the amount of TC* in the effluent in TCDC stimulated livers from 66 6 9% to 54 6 8% TC* from total input ( Fig. 7A; Supporting Fig.…”

“…The inhibitor okadaic acid, an inhibitor of PP1/2A, which inhibits cAMP-induced increases in cytosolic Ca þ and Ntcp translocation to the plasma membrane 24 was also tested. It was shown that okadaic acid does not significantly alter TCDC-mediated decreases in TC* uptake ( Fig.…”

Age‐dependent glycosylation of the sodium taurocholate cotransporter polypeptide: From fetal to adult human livers

“…In addition to glycosylation, other NTCP posttranslational modifications might participate in modulating NTCP subcellular localization during development. In particular, phosphorylation/dephosphorylation, a cyclic adenosine monophosphate‐dependent process, regulates NTCP localization in mature hepatocytes 1, 39. It would now be interesting to investigate how phosphorylation/dephosphorylation regulates NTCP localization in fetal hepatocytes.…”

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships

“…In these experiments, only the S226A mutant significantly reduced Ntcp phosphorylation and increased Ntcp membrane retention, indicating that S 226 in the third intracellular loop of the protein is the main site of Ntcp phosphorylation. Accordingly, cAMP may increase Ntcp translocation to the plasma membrane mainly by S 226 dephosphorylation (Anwer et al 2005).…”