Cyclic adenosine monophosphate (cAMP) stimulates translocation of Na ؉ -taurocholate (TC) cotransporting polypeptide (Ntcp) and multidrug resistant associated protein 2 (Mrp2) to the plasma membrane. Because cAMP activates phosphoinositide-3-kinase (PI3K) and protein kinase C (PKC) activation is PI3K-dependent, the aim of the current study was to determine whether cAMP activates conventional and novel PKCs in hepatocytes and whether such activation plays a role in cAMP-stimulated Ntcp and Mrp2 translocation. The effect of cAMP on PKCs, TC uptake, and Ntcp and Mrp2 translocation was studied in isolated rat hepatocytes using a cell-permeable cAMP analog, CPT-cAMP. The activity of PKCs was assessed from membrane translocation of individual PKCs, and phospho-specific antibodies were used to determine PKC␦ phosphorylation. TC uptake was determined from time-dependent uptake of 14 P rotein kinase Cs (PKCs) comprise a family of at least 12 isozymes. 1 These include conventional (cPKC␣, cPKC ␤I, cPKC ␤II, and cPKC␥), novel (nPKC␦, nPKC⑀, nPKC, and nPKC), atypical (aPKC and aPKC) isoforms, and PKC. These isoforms differ in their dependency on Ca 2ϩ and phospholipids, such that cPKCs are dependent on Ca 2ϩ and diacylglycerol, nPKCs are Ca 2ϩ -independent, and aPKCs are independent of both Ca 2ϩ and diacylglycerol. PKCs present in rat hepatocytes include cPKC␣, nPKC␦, nPKC⑀, aPKC, and probably cPKC␤II. 2-5 Activation of most PKCs, if not all, is phosphoinositide-3-kinase (PI3K) dependent. 1 Membrane transporter translocation and activity appear to be PKC-isoform specific. 6 Tauroursodeoxycholate-induced activation of cPKC␣ may be involved in Mrp2 translocation to the canalicular plasma membrane. 7 Taurocholate (TC) activates nPKC␦ 4 and translocates Bsep to the canalicular membrane. 8,9 cPKC␣, but not nPKC⑀ phosphorylate Bsep; this phosphorylation appears to increase transport activity without affecting translocation. 10 Studies in adipocytes and skeletal muscles suggest a role for nPKC␦ and aPKC in insulin-mediated glucose transporter 4 translocation. 11,12

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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...