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

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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 32PO4 for 2 hours, followed by incubation with 10 μmol/L 8–chlorophenylthio adenosin 3′:5′–cyclic monophosphate (CPT–cAMP) for 15 minutes. Subcellular fractions isolated from 32P–labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfate-polyacrylamide 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,N′,N′–tetraacetic acid (MAPTA), a Ca2+ buffering agent, and the effect of CPT–cAMP on TC uptake, cytosolic [Ca2+], 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 CPT–cAMP-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 [Ca2+], 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.