Sodium taurocholate cotransporting polypeptide is a serine, threonine phosphoprotein and is dephosphorylated by cyclic adenosine monophosphate.
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 micromol/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.
Mukhopadhyay
S
S
Department of Biomedical Sciences, Tufts University School of Veterinary Medicine, North Grafton, MA, USA.
Ananthanarayanan
M
M
Stieger
B
B
Meier
P J
PJ
Suchy
F J
FJ
Anwer
M S
MS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D012694:Serine / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D027981:Symporters; D013912:Threonine / Q000378:metabolism*; D014018:Tissue Distribution
D000596:Amino Acids; D002352:Carrier Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D010750:Phosphoproteins; D027981:Symporters; C071908:sodium-bile acid cotransporter; D013912:Threonine; D012694:Serine; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases