Abcc2 transport activity is stimulated by protein kinase Cα in a baculo virus co-expression system

Life Sciences 77 (2005) 539 – 550 www.elsevier.com/locate/lifescie

Mrp2/Abcc2 transport activity is stimulated by protein kinase Ca in a baculo virus co-expression system Kousei Ito, Takeshi Wakabayashi, Toshiharu HorieT Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba, 260-8675, Japan Received 9 June 2004; accepted 20 October 2004

Abstract Cholestatic and choleretic effect are well known for protein kinase C activator and inhibitor, respectively. However, post-translational regulation, especially the effect of phosphorylation status of the biliary transporters on their intrinsic transport activity has not been fully understood. In this study, effect of phosphorylation on the transport activity of Mrp2, a biliary organic anion transporter, was examined in membrane vesicles isolated from Sf 9 cells co-expressing excess amount of protein kinase Ca (PKCa). Mrp2-mediated transport activity was enhanced to three-fold by co-expressing PKCa. At the same time, phosphorylation of Mrp2 was also detected. The Km and Vmax values for the transport of [3H]estradiol-17h-D-glucuronide exhibited a 1.5-fold decrease and a 1.9fold increase, respectively. Probenecid (100 AM) and benzylpenicillin (1 mM), both are activator of Mrp2, did not stimulated the transport activity of phosphorylated Mrp2. On the other hand, transport activity was further stimulated by Estron-3-sulfate and taurocholic acid. Similar mechanism that occurred in the presence of probenecid and benzylpenicillin, but different from that occurred in the presence of Estron-3-sulfate and taurocholic acid seems to be involved in the stimulation. Considering the discrepancy between the previous in vivo inhibitory effect of PKC activators and our in vitro stimulatory effect of PKCa on Mrp2 transport activity, direct modulation of Mrp2-transport activity may be minor if any under in vivo condition. D 2005 Elsevier Inc. All rights reserved. Keywords: Mrp2; Phosphorylation; PKC

T Corresponding author. Tel./fax: +81 43 226 2886. E-mail address: [email protected] (T. Horie). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.10.071

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Introduction Multidrug resistance-associated protein 2 (Mrp2/Abcc2 for rats and MRP2/ABCC2 for humans), a member of the ATP-binding cassette (ABC) transporter family, is expressed on the bile canalicular membrane of hepatocytes, as well as on the brush border membrane of renal and intestinal epithelial cells. MRP2/Mrp2 excretes structurally diverse organic anions including reduced glutathione, glutathione conjugates, bilirubin glucuronides, sulfated and glucuronidated bile salts and nonconjugated organic anions into bile (Suzuki and Sugiyama, 1998; Keppler and Konig, 2000), which accounts for most of the bile salt-independent bile flow (Lauterburg et al., 1984). A genetic defect in this protein causes the hyperbilirubinemia observed in Dubin-Johnson syndrome in humans (Paulusma et al., 1997; Wada et al., 1998; Tsujii et al., 1999) and also in animal models (Paulusma et al., 1996; Ito et al., 1997). Moreover, in normal subjects, a change in the transport activity of Mrp2 could result in the altered pharmacokinetics of ligand drugs (Takikawa et al., 2002), or induce cholestasis/choleresis. Mrp2 mRNA expression is decreased by bile duct ligation (Trauner et al., 1997; Paulusma et al., 2000; Tanaka et al., 2002) or endotoxin treatment (Vos et al., 1998; Nakamura et al., 1999; Tanaka et al., 2002), possibly via RARa/RXRa-mediated regulation (Denson et al., 2002) and increased by several nuclear receptor ligands including phenobarbital (ligand for CAR), rifampicin (ligand for PXR) and bile salts (ligand for FXR) (Kast et al., 2002). In addition to the transcriptional regulation, post-translational regulation has also been suggested to account for the alteration in the transport activity of Mrp2. Some protein kinase inhibitors or stimulators have been reported to alter the transport activity and/or localization of MRP2/Mrp2 (Roelofsen et al., 1991; Misra et al., 1998, 1999; Kubitz et al., 2001). Roelofsen et al. were the first to report that efflux of Mrp2 substrates from isolated rat hepatocytes was quickly stimulated, as soon as 3 min, after preincubation with phorbol 12-myristate 13-acetate (PMA), a common protein kinase C (PKC) activator, and was inhibited by staurosporine, a PK inhibitor implying the stimulation of Mrp2 transport activity by phosphorylation (Roelofsen et al., 1991). However, opposite inhibitory effects of protein kinase C activators on the bile flow have also been observed in in situ rat liver perfusion study (Corasanti et al., 1989; Raiford et al., 1991; Kubitz et al., 2001). Kubitz et al. demonstrated abnormal basolateral localization of human MRP2 and rat Mrp2 in HepG2 cells and PMA perfused rat liver, respectively (Kubitz et al., 2001). PMA treatment induces cholestasis in perfused rat liver (Kubitz et al., 2001), while H-7, a PKC inhibitor, treatment induces choleresis (Corasanti et al., 1989; Raiford et al., 1991). In spite of the above circumstantial evidence implying the relationship between phosphorylation and Mrp2 function and/or localization, direct observation of the phosphorylation of Mrp2 and its effect on transport activity has not yet been reported. It is essential to differentiate the effect on the intrinsic transport activity from the effect on the redistribution of the transporter. Using computer modeling based on the hydropathy analysis, it has been shown that rat Mrp2 consists of 17 putative transmembrane-spanning domains (Borst et al., 1999) and multiple presumable phosphorylation sites. There are 26, 5 and 1 putative PKC, PKA and tyrosine kinase sites, respectively in rat Mrp2. Of these, the 24, 1 and 1 sites are predicted to be located in the cytoplasmic region. In rat liver, five PKC isozymes including PKCa, PKChII, PKCy, PKCq and PKC~, have been reported to be expressed (Croquet et al., 1996). Based on the fact that a change in the function of Mrp2 is related to PMA (Roelofsen et al., 1991), PKC(s) sensitive to PMA (i.e. conventional PKC; cPKC) are likely to be involved. Moreover, PKCa, PKCy and PKCe have been reported to be sorted to the plasma membrane of the hepatocytes after PMA treatment (Beuers et al.,

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1999). In particular, PKCa belonging to the cPKC family, was specifically sorted to the plasma membrane after treating the liver with cholereteic bile salts such as tauroursodeoxicholic acid (TUDCA) and ursodeoxycholic acid (UDCA) (Beuers et al., 1996; Bouscarel et al., 1999). In addition, the choleretic effect of TUDCA was associated with the sorting of Mrp2 to the canalicular membrane surface as well as PKCa to the plasma membrane fraction (Beuers et al., 2001). However, it is not yet elucidated what is happening at that time where PKCa and Mrp2 are present around the membrane. In this report, we initially and simply examined that rat Mrp2 could be a substrate of rat PKCa when co-expressed in Sf9 insect cells and also showed the effect on the transport activity and property.

Materials and methods Chemicals [3H]estradiol-17h-D-glucuronide (E217hG, 55 Ci/mmol) and [3H]leukotriene C4 (LTC4, 100 Ci/ mmol) were from NEN Life Science Products (Boston, MA). [32P]-orthophosphate (100 – 200 mCi/ml) was from American Radiolabeled Chemicals Inc. (St. Louis, MO). E217hG, probenecid, estron-3-sulfate (E1S), taurocholate (TC), ATP and AMP were from Sigma (St. Louis, MO). Benzylpenicillin (PCG) was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). MK571 was from Cayman Chemical Co. (Ann Arbor, MI). Other chemicals were of analytical grade. Cloning of rat PKCa Male Sprague-Dawley (SD) rat brain mRNA was reverse transcribed with oligo(dT)12–18 and SuperScriptk II RT (Life Technologies, Gaitthersburg, MD). PCR was performed with primers designed to encompass the entire coding region of rat PKCa (Genbank accession: X07286). The forward primer, 5V-AAAAAAgaattcACCATGGCTGACGTTTACCCGGCCAACGACTCC-3V, spanned the start codon (underlined) as well as the EcoR I restriction site (small letters). The reverse primer, 5VTTTGGGgtcgacTCATACTGCACTTTGCAAGATTGGGTGCAC-3V, covered the stop codon (underline) and the Sal I restriction site (small letters). The PCR product was inserted into pSKII(-) vector and its sequence was verified in both directions. The EcoR I and Sal I fragment was digested and inserted into the EcoR I and Sal I site of the pFASTBAC1 expression vector (Invitrogen Corporation, Carlsbad, CA). Production and infection of recombinant baculovirus Recombinant baculo viruses carrying the green fluorescent protein (GFP, as a negative control), Mrp2 cDNA and PKCa were prepared as described previously (Ito et al., 2001a). Sf 9 cells were maintained as suspension culture at 278C with serum-free Excel 420 (Nichirei Corporation, Tokyo, Japan). For infection, Sf9 cells were seeded onto 10 cm dishes then appropriate amount of the respective viruses (multiplicity of infection (MOI) ~ 10) were applied and cultured in the presence of 5 % fetal bovine serum. Cells were harvested 60 h after infection and membrane vesicles were isolated from Sf9 cells using the method described previously (Ito et al., 2001a).

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Transport study The uptake of [3H]E217hG (50 nM) or [3H]LTC4 (5 nM) was measured as described (Ito et al., 2001a) with some modification. Briefly, 16 Al transport medium (10 mM Tris, 250 mM sucrose, 10 mM MgCl2, 6.25 mM ATP or AMP, pH 7.4), containing radiolabeled compounds, was preincubated at 378C for 3 minutes and then rapidly mixed with 4 Al membrane vesicle suspension (2–5 Ag protein). After 2 min incubation, the transport reaction was stopped by the addition of 1 ml ice-cold buffer containing 250 mM sucrose, 0.1 M NaCl, 10 mM Tris-HCl (pH 7.4). The stopped reaction mixture was filtered through a 0.45 Am HAWP filter (Millipore Corp., Bedford, MA) and then washed twice with 5 ml stop solution. The radioactivity retained on the filter was measured in a liquid scintillation counter (LSC5000, Aloka Co., Tokyo, Japan). Detection of phosphorylated Mrp2 8  106 cells plated on the 10 cm plate were infected with baculo virus carrying the respective cDNAs and cultured for 60 h in the presence of 5 % fetal bovine serum. Cells were washed with phosphate-free Grace’s medium and suspended in 2 ml phosphate-free medium containing 0.4 mCi [32P]orthophosphoric acid and poured onto a 35 mm plate. Two hours after incubation at 278C, cells were collected and solubilized with 500 Al solubilization buffer (PBS, 1% of Triton X-100, 1 mM orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 Ag/ml leupeptin, 1 Ag/ml pepstatin and 5 Ag/ml aprotinin) for 20 min on ice with occasional vortexing. The cell lysates were centrifuged at 15,000  g for 15 min at 48C and the supernatants were mixed with PANSORBIN Cells (Calbiochem, San Diego, CA) for 30 min at 48C and then centrifuged at 15,000  g for 5 min at 48C. The supernatants were mixed with an Anti-Mrp2 IgG raised against rat Mrp2 (CP-2 (Ito et al., 2001a), kindly gifted from Dr. J. Nakayama in Kumamoto University, Kumamoto, Japan). After 2 h at 48C, 20 Al protein A-Sepharose (Amersham Biosciences Corp., Piscataway, NJ) suspension was added and further incubated for 1 h at 48C. After several washing with PBS, samples were electrophoresed on a 8.5% SDS-PAGE gel then vacuum dried for 2 h at 808C. The gel was exposed to a BAS imaging plate and analyzed with a BAS2000 imaging analyzer (Fuji Photo Film Co., Ltd., Kanagawa, Japan). Preparation of canalicular membrane vesicle (CMV) Male SD rats (250–300 g) were purchased from Japan SLC Inc. (Shizuoka, Japan). CMV fraction was prepared from the liver as described previously (Kobayashi et al., 1990) and stored at 808C until use. Western blotting Membrane vesicles were fractionated in an 8.5% polyacrylamide slab gel containing 0.1% SDS and transferred to Immobilon Transfer Membrane (Millipore) by electroblotting. The membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 3% bovine serum albumin for 1 h at room temperature and probed for 1 h at room temperature with CP-2 (1:1000) or anti-PKCa antibody (Sigma). The membranes were then allowed to bind donkey anti-rabbit antibody (Santa Cruz Biotech. Inc., Santa Cruz, CA) (1:3000) and analyzed using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences Corp.).

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Data analysis Statistical analysis was performed by Student’s t-test to identify any significant differences between two groups. ANOVA followed by post hoc test (Student-Newman-Keuls) was performed for the comparison among more than three groups. Kinetic parameters were computationally obtained by fitting the data to the Michaelis-Menten equation with the MULTI program (Yamaoka et al., 1981).

Result Expression of PKCa and Mrp2 in Sf 9 cells Expression of Mrp2 and PKCa were confirmed by western blot. As shown in Fig. 1A and B, single immunoreactive bands corresponding to Mrp2 (175kD) and PKCa (80kD) were observed while no signals were observed in control Sf9 cells (data not shown). Densitometry analysis revealed about 2-fold higher expression of Mrp2 in the membrane fraction of Sf9 than that in canalicular membrane fraction of SD rat liver (CMV: 190kD). Extremely higher expression of rat PKCa in crude membrane fraction of Sf9 cells compared to that of CMV (~ 150-fold). The expression level of each protein was not significantly affected by co-expression (Fig. 1C). Detection of phosphorylated Mrp2 Phosphorylated Mrp2 was detected by metabolic labeling with [32P]-orthophosphoric acid followed by immunoprecipitation with Anti-Mrp2 IgG (Fig. 2A). Phosphorylated Mrp2 was detected

Fig. 1. Expression of rat Mrp2 and PKCa gene product in Sf9 insect cells. Rat Mrp2 (A) and rat PKCa (B) were detected with anti Mrp2 and PKCa antiserum, respectively. Membrane vesicles isolated from Sf9 cells infected with virus carrying the Mrp2 (Mrp2/Sf9: 5Ag/lane) or PKCa (PKCa/Sf9: 0.5Ag/lane) alone, respectively. Canalicular membrane vesicles (CMV) isolated from SD rat liver is used as positive control of the blot (5 Ag/lane). (C) Expression of rat Mrp2 (5Ag/lane) and PKCa (0.5Ag/ lane) in membrane vesicles isolated from Mrp2/PKCa co-expressing cells (+/+).

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Fig. 2. Phosphorylation of Mrp2 by PKCa and stimulation of the transport activity. A) Phosphorylation of Mrp2. Cells expressing rat Mrp2 alone or Mrp2/PKCa were labeled with [32P]-orthophosphoric acid for 2 h. They were lysed and immunoprecipitated with an anti-Mrp2 antiserum and resolved by SDS-PAGE 8.5%. The dried gel was analyzed by BAS imaging. B) Uptake of [3H]E217hG (50 nM) and [3H]LTC4 (5 nM) into membrane vesicles from Sf9 cells expressing GFP, Mrp2 or Mrp2/PKCa are shown. Two to five Ag of membrane vesicles were used for transport experiments at 378C in the presence of AMP (open columns) or ATP (closed columns). Each column and bar represents the mean F S.D. of triplicate determinations.

Fig. 3. PKCa-dependent stimulation of Mrp2 transport activity. A) Uptake of [3H]E217hG (50 nM) into membrane vesicles from Sf9 cells infected with various amount of PKCa-containing virus (MOI = 0~10) and fixed amount of Mrp2-containing virus supernatant (MOI = 10). Uptake was carried out in the presence of AMP (open columns) or ATP (closed columns). Each column and bar represents the mean F S.D. of triplicate determinations. B) Expression of Mrp2 and PKCa in the vesicles was detected by the specific antibodies. Relative expression of Mrp2 and PKCa was indicated in the parenthesis.

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Fig. 4. Concentration-dependence of the transport of [3H]E217hG. ATP-dependent transport of [3H]E217hG into membrane vesicles expressing Mrp2 alone (open symbols) or Mrp2/PKCa (closed symbols) was plotted (Initial 2 min uptake). Each point represents the mean of duplicate determinations of a typical experiment. Solid lines represent the fitted lines obtained by the MULTI program.

in cells expressing Mrp2 alone and was further enhanced by co-expressing PKCa (2.9-fold). Background signal may be due to an endogenous insect kinase in Sf9 cells as reported previously (Geiges et al., 1997). Stimulation of the transport activity by PKCa The ATP-dependent transport of [3H]E217hG was significantly stimulated by co-expressing PKCa (3.1-fold). Similarly, transport of [3H]LTC4 was also stimulated by 2.7-fold by co-expressing PKCa (Fig. 2B). ATP-dependent transport of these substrates into the vesicles from cells expressing PKCa only was minimum and the same as those of GFP (data not shown). Transport stimulation was well correlated with the expression of PKCa as shown in Fig. 3A. The expression of Mrp2 was not altered by increasing the expression of PKCa, indicating the stimulation of intrinsic transport activity of Mrp2 molecule (Fig. 3B). Effect of phosphorylation on the kinetics of E217bG transport To investigate the kinetic parameters of [3H]E217hG transport by Mrp2, the transport experiment was performed in the presence of various concentrations of unlabeled E217hG (50 nM to 75 AM) and 5 mM ATP (Fig. 4). As shown in Table 1, the Vmax value was significantly increased while the Km value was decreased when co-expressing PKCa with Mrp2. Slight but significant increase in the non-saturable component (PSns) was also observed. Stimulation of the transport was achieved by the changes in both parameters.

Table 1 Kinetic parameters for the transport of E217hG Mrp2 Mrp2/PKCa

Vmax (pmol/min/mg)

Km (AM)

PSns (Al/min/mg)

179 F 5 340 F 6TT

3.19 F 0.73 2.18 F 0.25

11.1 F 0.3 15.1 F 0.1TT

Values are means F computer calculated S.D. TT p b 0.01, significant difference by Student’s t-test.

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Table 2 Effect of Mrp2-stimulators and inhibitor on the uptake of [3H]E217hG Mrp2 Control Probenecid (100 AM) PCG (1 mM) E1S (100 AM) TC (100 AM) MK571 (10 AM)

5.45 11.4 9.34 15.7 22.3 0.85

F F F F F F

Mrp2/PKCa 0.93 (100 %) 1.1 (210 %)TT 0.34 (171 %)T 0.3 (289 %)TTT 1.2 (409 %)TTT 0.80 (15.6 %)T

17.1 F 2.5 (100 %) 20.1 F 0.9 (118 %) 19.9 F 2.3 (117 %) 73.5 F 6.8 (431 %)TTT 46.5 F 1.6 (273 %)TTT 1.27 F 0.24 (7.5 %)TTT pmol/mg/2min

ATP-dependent transport [3H]E217hG (50 nM) into membrane vesicles expressing Mrp2 alone or Mrp2/PKCa were examined in the presence Mrp2-stimulators or inhibitor. ATP-dependent transport was shown after subtracting the uptake in the presence of AMP from that in the presence of ATP. Negligible effects were observed on the transport in the presence of AMP or control GFP-expressing vesicle (data not shown). Results are given as the mean F SD of three to five determinations. Values in parentheses indicate the relative activity (% of the respective control). Tp b 0.05, TTp b 0.01 and TTTp b 0.001 significantly different from the respective control, ANOVA followed by Student-Newman-Keuls test.

Effect of stimulators and inhibitor on the transport activity Sensitivity to known Mrp2 stimulators (probenecid, PCG, E1S and TC) and inhibitor (MK571) was examined for the transport of [3H]E217hG (Table 2). ATP-dependent transport was completely inhibited by 10 AM MK571 in both Mrp2 alone and Mrp2/PKCa co-expressing vesicles. 100 AM E1S and 100 AM TC significantly stimulated both vesicles. On the other hand, probenecid and PCG stimulated only in vesicles expressing Mrp2 alone but not further in vesicles expressing Mrp2/PKCa.

Discussion We investigated the PKCa-mediated regulation of the transport activity in a simple baculo virus coexpression system. Direct phosphorylation of Mrp2 occurred and resulted in stimulation of the intrinsic transport activity. Similar phosphorylation-dependent stimulation of transport activity has been reported in MRP1/ABCC1 (Ma et al., 1995). Ma et al. identified the phosphorylation of P190, likely MRP1, endogenously expressed in multidrug resistant HL60/ADR cells and found that reduction of phosphorylation by protein kinase inhibitors resulted in accumulation of the substrate (Ma et al., 1995). While our results seem consistent with the previous observation using isolated hepatocytes (Roelofsen et al., 1991), in vivo effects of stimulators or inhibitors of PKs are contrary to the prediction from our present results. Perfusion with vasopressor hormones, known to activate PKCs, reduced the biliary excretion of reduced glutathione, an endogenous substrate of Mrp2 (Raiford et al., 1991). Moreover, the PKC activator phorbol dibutyrate reduced the biliary excretion of reduced glutathione while an inhibitor such as H-7 and staurosporine increased it (Raiford et al., 1991). These effects are independent of tight junction permeability and rather related to the transporter-mediated efflux process. There are three possible reasons for the contradiction between in vitro ((Roelofsen et al., 1991) and our present study) and in vivo effect of PKC modulators. Firstly, heterologous PKCs may be affected in vivo situation. Notably, PMA is a potent but nonspecific activator of both conventional and novel PKCs including PKCa, PKCy, and PKCq in hepatocytes. Indeed, PMA translocates all these three isozymes (Beuers et

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al., 1996), while choleretic and cholestatic bile salts specifically translocate PKCa (Beuers et al., 1996, 2001) and PKCq (Beuers et al., 1999), respectively. Secondly, the expression ratio of PKCa to Mrp2 in our co-expression system is quite high compared to that in the hepatocyte (Fig. 1A and B). Considering the expression level of PKCa in the liver, it corresponds to Sf9 cells infected with even lower than the lowest MOI of PKCa-carrying baculovirus (MOI b 10 3) (Fig. 3). Under such condition, stimulatory effect on Mrp2 transport activity seems hard to be detected (Fig. 3). Finally, effect of redistribution of the canalicular transporters should be considered (Beuers et al., 2001). Mrp2 was abnormally redistributed to the basolateral membrane of hepatocyte after PMA treatment in rat liver perfusion system and resulted in cholestasis (Kubitz et al., 2001). More recently, thymeleatoxin (a specific activator of Ca2+-dependent PKCs including PKCa) also induced cholestasis in rat liver perfusion system via internalization of Bsep, although localization of Mrp2 was not examined there (Kubitz et al., 2004). Heterologous regulatory mechanism and redistribution of these kinases and transporters may determine the final effect on biliary excretion activity (Bouscarel et al., 1999). Phosphorylation and activation of Mrp2 transport activity by PKCa might be submerged in the redistribution of transporter molecule if any. Mechanism of the stimulation of Mrp2 transport activity by phosphorylation can not be described without speculation. Some information is available from the data obtained in the transport kinetics and inhibitor/stimulator sensitivity of E217hG uptake. Both transport maximum and affinity are similarly contributing (Table 1). This may be also favorable for the transport of other substrates including LTC4 (Fig. 2B). More interestingly, the mechanism may be similar to that occurred in the presence of PCG and probenecid (Bakos et al., 2000; Zelcer et al., 2003; Ito et al., 2004) but different from that in the presence of E1S and TC (Ito et al., 2001b) (Table 2). In line with this complex observation, multiplicity of the substrate/regulator interacting sites is proposed in human MRP2 (Zelcer et al., 2003). Among MRP/ABCC families in human, MRP2 and MRP5/ABCC5 have so many PKC consensus sites (27 and 25 sites, respectively) compared with MRP1/ABCC1 (13 sites), MRP3/ABCC3 (13 sites), MRP4/ABCC4 (18 sites) and MRP6/ABCC6 (12 sites). Rat Mrp2 has also as many as 26 putative PKC consensus sites and most of them are located within the predicted cytoplasmic region. There are some reports exploring the phosphorylation sites in other ABC transporters, especially cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7) (Rich et al., 1993) and P-glycoprotein (MDR1/ ABCB1) (Chambers et al., 1993; Ahmad et al., 1994). Both proteins have characteristic linker regions. In CFTR, phosphorylation of the linker (so-called bR-domainQ) has been shown to regulate the channel gating (Rich et al., 1993). Although its physiological significance is not elucidated (Goodfellow et al., 1996), linker region is somehow related to the regulation of MDR1 function (Chambers et al., 1993; Sachs et al., 1999). One intriguing fact is the existence of similar linker region in MRP2/Mrp2. Six potential PKC sites (S904, S912, S916, S917, S922 and S926) are found in the linker region of rat Mrp2. Notably, S912, S922 and S926 are all conserved in MRP2/Mrp2 among different species. In a preliminary experiment, mutation of all these 6 consensus serines of rat Mrp2 into neutral alanines significantly reduced the basal transport activity while mutation into acidic aspartates significantly increased it (unpublished data). Importance of the charge valence in the linker region and its possible regulation by phosphorylation is a plausible mechanism as other ABC family proteins (Chambers et al., 1993; Rich et al., 1993; Ahmad et al., 1994). In conclusion, Mrp2 is phosphorylated by PKCa and its transport activity is concomitantly enhanced in enforced co-expression system. Although a temporal change in the transport activity of Mrp2 following phosphorylation is possible, it might be minor or submerged in the redistribution of transporter

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molecules under in vivo condition. Further experiments are required to address the physiological significance of this phenomenon under various cholestatic or choleretic conditions.

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