Role of protein kinase C isoforms in bile formation and cholestasis

Abstract

Transhepatic solute transport provides the osmotic driving force for canalicular bile formation. Choleretic and cholestatic agents affect bile formation, in part, by altering plasma membrane localizations of transporters involved in bile formation. These short-term dynamic changes in transporter location are highly regulated posttranslational events requiring various cellular signaling pathways. Interestingly, both choleretic and cholestatic agents activate the same intracellular signaling kinases, such as phosphoinositide-3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK). An emerging theme is that choleretic and cholestatic effects may be mediated by different isoforms of these kinases. This is most evident for PKC-mediated regulation of plasma membrane localization of Na+-taurocholate cotransporting polypeptide (NTCP) and multidrug resistance-associated protein 2 (MRP2) by conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ). aPKCζ may mediate choleretic effects by inserting NTCP into the plasma membrane, and nPKCε may mediate cholestatic effects by retrieving MRP2 from the plasma membrane. On the other hand, cPKCα and nPKCδ may be involved in choleretic, cholestatic, and anticholestatic effects by inserting, retrieving, and inhibiting retrieval of transporters, respectively. The effects of PKC isoforms may be mediated by phosphorylation of the transporters, actin binding proteins (radixin and myristoylated alanine-rich C kinase substrate), and Rab proteins. Human NTCP plays an important role in the entry of hepatitis B and D viruses into hepatocytes and consequent infection. Thus, PKCs, by regulating NTCP trafficking, may also play an important role in hepatic viral infections.

Fig. 1

Primary structures of PKCs. There are four structurally conserved domains (C1-C4) in PKC isoforms divided into the N-terminal regulatory domain (C1-C2) and the C-terminal catalytic domain (C3-C4). The regulatory domain contains the binding sites for pseudosubstrate (PS), DAG (C1) and Ca⁺⁺ (C2 or C2-like). The catalytic domain contains the binding sites of ATP (C3) and substrate (C4). C-regions (C1-C4) represent conserved domains and V-regions represent variable domains (V1-V5). The regulatory and the catalytic domains are separated by a flexible hinge domain (V3), which is cleaved by caspase-3 in apoptotic cells. Novel isoforms contain a C2-like domain which is unable to bind Ca⁺⁺ and hence do not require Ca⁺⁺ for activation. Atypical isozymes contain a variant of the C1 domain, which lacks the ligand-binding pocket for DAG and lacks C2 domain. As a result aPKCs are not regulated by DAG and Ca⁺⁺; they are regulated by protein-protein interactions via PB1 domain. The intermolecular binding between PS and catalytic domain is highly regulated by membrane interactions, PKC conformation and phosphorylation.

Figure 2

Proposed regulation of NTCP and MRP2 by PKC isoforms. Activation of aPKCζ and nPKCδ by cAMP leads to translocations of NTCP and MRP2 to PM. Activation of cPKC (most likely cPKCα) by PMA and TCDC induces retrieval of NTCP from PM, while activation of cPKCα by TUDC facilitates MRP2 translocation to PM. Activations of cPKCα, nPKC and nPKCε have been implicated in MRP2 retrieval from PM by estradiol 17β-D-glucuronide (E17G), ethacrynic acid (EA) induced oxidative stress and taurolithocholate (TLC), respectively. Retrieval of BSEP by E17G, PMA and oxidative stress has been proposed to be mediated via cPKCs (not shown).

Figure 3

Role of radixin and MARCKS in PM localization of MRP2. Phosphorylated radixin (active form) stabilizes MRP2 in the membrane by binding to actin and NHERF-1, which binds MRP2. MARCKS also binds actin and possibly MRP2 via an unknown protein X. Dephosphorylation of radixin (inactive form) by PP-1 activated by cPKCα/cPKCε and phosphorylation of MARCKS by nPKCε result in the loss of their binding to actin leading to retrieval from PM. Removal of radixin and MARCKS from PM results in the retrieval of MRP2 (dotted line), mostly likely due to the loss of PM anchoring proteins for MRP2. Similar mechanisms may also be involved in BSEP retrieval (not shown).

Figure 4

Postulated role of PI3K/PKCδ, PI3K/PKCζ in Rab4-mediated insertion of NTCP into PM. NTCP containing vesicles co-localize with Rab4, which cycles between GTP bound (Rab4-GTP) active form and GDP bound (Rab4-GDP) inactive form. Activation of PKCδ leads to the conversion of inactive Rab4-GDP to active Rab4-GTP followed by recruitment of kinesin-1, which moves NTCP containing vesicles towards the plus end of microtubules (Mt). This movement is further facilitated by PKCζ.