Sodium Taurocholate Cotransporting Polypeptide Is A Serine, Threonine Phosphoprotein and Is Dephosphorylated by Cyclic Adenosine Monophosphate

Abstract

Na ؉ /taurocholate (Na ؉ /TC) cotransport in hepatocytes is mediated primarily by Na ؉ /TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na ؉ /TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier-free 32 PO 4 for 2 hours, followed by incubation with 10 mol/L 8-chlorophenylthio adenosin 3Ј:5Ј-cyclic monophosphate (CPT-cAMP) for 15 minutes. Subcellular fractions isolated from 32 P-labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,NЈ,NЈ-tetraacetic acid (MAPTA), a Ca 2؉ buffering agent, and the effect of CPT-cAMP on TC uptake, cytosolic [Ca 2؉ ], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPTcAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA-loaded hepatocytes, CPT-cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca 2؉ ], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP-mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na ؉ /TC cotransport.

Role of Protein Phosphatases in Cyclic AMP-mediated Stimulation of Hepatic Na+/Taurocholate Cotransport

“…We have reported previously (10) that the stimulatory effect of cAMP on Na ϩ /TC cotransport is potentiated by cAMP-induced increases in cytosolic [Ca 2ϩ ]. We also observed that cAMP failed to increase plasma membrane Ntcp when cytosolic Ca 2ϩ was buffered (15). Since okadaic acid inhibited the ability of cAMP to increase cytosolic [Ca 2ϩ ] (Fig.…”

“…Since treatment of hepatocytes with cAMP results in an increase in plasma membrane and a decrease in endosomal Ntcp mass, we proposed that cAMP increases transport maximum of Na ϩ /TC cotransport by translocating Ntcp from an endosomal pool to the plasma membrane (14). In addition, our recent study showed that Ntcp is a serine/threonine phosphoprotein, phosphorylated Ntcp is detectable in the plasma membrane, but not in microsomes and Golgi complex, and treatment of hepatocytes with cAMP results in Ntcp dephosphorylation (15). These results raise the possibility that the regulation of Na ϩ /TC cotransport by cAMP may also involve phosphorylation/dephosphorylation of Ntcp.…”

Hepatobiliary transporters and drug‐induced cholestasis†

“…On the posttranscriptional level, sodium-dependent and -independent hepatocellular uptake systems are mainly regulated by cyclic adenosine monophosphate-mediated dephosphorylation processes, which is controlled by phophoinositide-3-kinase/protein kinase B. [21][22][23] Furthermore, PDZK1 was demonstrated to be a critical determinant for the proper subcellular localization and function of rat Oatp1a1. 24 The transcriptional regulation of basolaterally expressed MRPs is not fully elucidated.…”

Cross-talk between Protein Kinases Cζ and B in Cyclic AMP-mediated Sodium Taurocholate Co-transporting Polypeptide Translocation in Hepatocytes

“…Conjugated bile acids, such as TC, are efficiently taken up by hepatocytes primarily via a Na ϩ /TC cotransport mechanism; Ntcp, a serine/threonine phosphoprotein (21), is believed to be the major protein involved in Na ϩ /TC cotransport (22)(23)(24). Cyclic AMP and cell swelling rapidly increase Na ϩ /TC cotransport and Ntcp translocation to the plasma membrane (25)(26)(27), and this effect is mediated via the PI3K/PKB signaling pathway (11).…”