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. 2016 Jun;44(6):871–876. doi: 10.1124/dmd.116.070235

p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

Takeshi Hori 1, Rick Moore 1, Masahiko Negishi 1,✉
PMCID: PMC4885487  PMID: 27074912

Abstract

Nuclear receptor constitutive androstane receptor (CAR, NR1I3), which regulates hepatic drug and energy metabolisms as well as cell growth and death, is sequestered in the cytoplasm as its inactive form phosphorylated at threonine 38. CAR activators elicit dephosphorylation, and nonphosphorylated CAR translocates into the nucleus to activate its target genes. CAR was previously found to require p38 mitogen-activated protein kinase (MAPK) to transactivate the cytochrome P450 2B (CYP2B) genes. Here we have demonstrated that p38 MAPK forms a complex with CAR, enables it to bind to the response sequence, phenobarbital-responsive enhancer module (PBREM), within the CYP2B promoter, and thus recruits RNA polymerase II to activate transcription. Subsequently, p38 MAPK elicited rephosphorylation of threonine 38 to inactivate CAR and exclude it from the nucleus. Thus, nuclear p38 MAPK exerted dual regulation by sequentially activating and inactivating CAR-mediated transcription through phosphorylation of threonine 38.

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Introduction

The nuclear receptor constitutive androstane receptor (CAR, NR1I3), a member of the thyroid and steroid hormone receptor superfamily, activates genes encoding for enzymes and transporters that metabolize and excrete therapeutic drugs and is activated by these drugs and xenobiotics (Kobayashi et al., 2015). With these functions, CAR critically regulates hepatic capability for drug disposition. CAR gains its drug responsiveness by suppressing its high constitutive activity by phosphorylation at threonine 38 within the DNA-binding domain (DBD) (Mutoh et al., 2009). Phosphorylation of CAR abolishes its DNA binding ability and is sequestered in the cytoplasm. Phenobarbital, the classic drug that indirectly activates CAR, elicits a cell signal that stimulates protein phosphatase 2A (PP2A) to dephosphorylate threonine 38 for activation (Mutoh et al., 2013). Thus, the cell signal–mediated process of CAR activation as it occurs in the cytoplasm is now well documented. Here we have investigated p38 mitogen-activated protein kinase (MAPK) that regulates CAR in the nucleus.

Phosphorylation has long been investigated in both ligand-dependent and -independent regulation of nuclear receptors. It is involved in degradation, cofactor recruitment, and dimerization (Shao and Lazar, 1999; Tremblay et al., 1999; Hong et al., 2003; Picard et al., 2008). The amino acid residues targeted for studies reside within the activation function 1 (AF-1) or ligand-binding domain (LBD) region and have been investigated in a given but not in any other nuclear receptors. Compared with these regions, much less emphasis has been placed on phosphorylation within the DBD. One phosphorylation site resides within a protein kinase C (PKC) motif in the DBD and is conserved in 41 out of 48 human nuclear receptors. Phosphorylation of this conserved site is the most well characterized within threonine 38 of CAR (Mutoh et al., 2009; Mutoh et al., 2013). CAR is, in fact, phosphorylated at threonine 38 in mouse hepatocytes to regulate its drug activation. Recently, estrogen receptor alpha (ERα) phosphorylated at the corresponding serine 216 was also found in mouse neutrophils and appears to regulate their infiltration into the uterus (Shindo et al., 2013). Although their phosphorylation in tissues in vivo have not yet been confirmed, studies with phosphomimetic mutants suggest that these residues in hepatocyte nuclear factor 4 alpha (HNF-4α), vitamin D receptor (VDR), peroxisome proliferator-activated receptor alpha, retinoid X receptor alpha (RXRα), and farnesoid X receptor may regulate various functionalities of these nuclear receptors, such as cytoplasmic retention, degradation, and transactivation (Hsieh et al., 1993; Sun et al., 2007; Gineste et al., 2008). Therefore, conserved phosphorylation has provided us with the opportunity to uniformly investigate nuclear receptors but has only been studied as the target of PKC or dephosphorylation by PP2A.

p38 MAPK is activated in response to various extracellular stimuli, such as growth factors, UV radiation, inflammatory cytokines, oxidative stress, and hyperosmosis (Freshney et al., 1994; Han et al., 1994; Rouse et al., 1994; Huot et al., 1997; Zhang and Jope, 1999). In liver cells, phosphorylated p38 MAPK is accumulated in the nucleus to activate various transcription factors and protein kinases. We have recently demonstrated that p38 MAPK is essential for CAR to activate the cytochrome P450 2B6 (CYP2B6) gene in human hepatoma HepG2-derived cells (Saito et al., 2013b). In the present study, we revealed that p38 MAPK forms a complex with CAR to promote the CAR-mediated transcription in the nucleus. Moreover, p38 MAPK linked CAR transactivation and inactivation by stimulating phosphorylation of threonine 38.

Materials and Methods

Materials.

Phenobarbital sodium salt, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), anisomycin, SB 239063, anti-FLAG M2 affinity gel, anti-FLAG M2-horseradish peroxidase (HRP) antibodies, phosphatase inhibitor cocktails 2 and 3, and l-glutathione reduced were purchased from Sigma-Aldrich (St. Louis, MO); anti-p38α (ab7952) and anti-phospho-RNA polymerase II CTD (ab5131) from Abcam (Cambridge, MA); anti-phospho-p38 MAPK (Thr180/Tyr182; #4511) and anti-p38 MAPK from Cell Signaling Technology (Danvers, MA); anti-RXRα (sc-553 X) and HRP-conjugated anti-mouse or rabbit IgG antibodies from Santa Cruz Biotechnology (Dallas, TX); and anti-V5 from Invitrogen/ThermoFisher Scientific (Carlsbad, CA). Anti-phospho-Thr38 peptide antibodies for CAR were produced in our previous work (Mutoh et al., 2009). Anti-phospho-Ser51 peptide antibodies (GFFRR-pS-MKRKALFTC) for VDR were produced by AnaSpec Inc. (San Jose, CA). A Lipofectamine 2000 reagent and Dynabeads Protein G were obtained from Life Technologies/ThermoFisher Scientific (Grand Island, NY); TaqMan Gene Expression Assays (probe and primer sets) for CYP2B6 (AssayID: Hs00167937_m1) (FAM), Cyp2b10 (AssayID: Mm00456591_m1) (FAM), Cyp2c55 (AssayID: Mm00472168_m1) (FAM), and human and mouse GAPDH (FAM) from Applied Biosystems (Foster, CA); cOmplete mini protease inhibitor cocktail tablets from Roche Diagnostics Corp. (Indianapolis, IN); recombinant active p38 MAPK (#14-587) from Millipore UK Limited (Dundee, UK).

Animals.

Both Car+/+and Car−/− mice in C3H/HeNCrlBR background were produced in house (Yamamoto et al., 2004). Phenobarbital (100 mg/kg body weight) in phosphate-buffered saline (PBS), TCPOBOP (3 mg/kg body weight) in dimethyl sulfoxide (DMSO) in corn oil, or a control solution, was intraperitoneally injected into 7- to 8-week-old male mice for a treatment of 6 hours, from which liver RNAs and nuclei were prepared for real-time polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) assays, respectively. Mice were maintained under the standard condition at the National Institute of Environmental Health Sciences, and animal experiments were conducted according to protocols approved by the animal ethics committee at NIEHS/National Institutes of Health.

Plasmid Construction.

Mouse CAR cDNA (GenBank accession no. NM 009803.5) was previously cloned into pGEX-4T-3 vector (GE Healthcare, Piscataway, NJ) for glutathione S-transferase (GST)-CAR fusion proteins and pCR3 vector (Invitrogen). CAR Thr48Ala (T48A) and CAR Thr48Asp (T48D) mutants were generated by a site-directed mutagenesis method with the following primers: 5′-GGCTTCTTCAGACGAgCAGTCAGCAAAACCATT-3′ and 5′-AATGGTTTTGCTGACTGcTCGTCTGAAGAAGCC-3′ for CAR T48A; 5′-GGCTTCTTCAGACGAgatGTCAGCAAAACCATT-3′ and 5′-AATGGTTTTGCTGACatcTCGTCTGAAGAAGCC-3′ for CAR T48D. Mouse p38α cDNA (NM 011951.3) was cloned into pcDNA3.1 vector (Invitrogen). The FLAG tag was inserted into the 5′-flanking region of CAR in pCR3 or p38α in pcDNA3.1.

Cell Cultures.

Primary hepatocytes were isolated using a collagenase two-step perfusion method as described previously (Honkakoski et al., 1996). Hepatocytes (6 × 105 cells/ml per well) were seeded on collagen-coated wells and cultured in Williams’ medium E containing 10% fetal bovine serum (FBS) and penicillin/streptomycin. After 3 hours of seeding, cells were treated with 2 mM phenobarbital or 250 nM TCPOBOP for 12 hours in the presence or absence of 20 μM SB 239063, and total RNA was extracted for real-time PCR analysis. Huh-7 cells or HepG2-derived Ym17 cells were cultured in minimum essential medium (Invitrogen) or Dulbecco’s modified Eagle’s medium (Invitrogen), respectively, supplemented with 10% FBS and penicillin/streptomycin at 37°C with 5% CO2. Plasmids were transfected into Huh-7 or Ym17 cells with a Lipofectamine 2000 reagent according to the manufacturer’s instructions.

Real-Time PCR.

Total RNA was extracted from mouse livers, hepatocytes, or Ym17 cell using TRIzol reagent (Life Technologies), with which cDNAs were synthesized using a High Capacity cDNA Archive kit (Life Technologies). Real-time PCR was conducted using a TaqMan Universal PCR Master mix and TaqMan probes and primers with a 7900HT Fast Real-Time PCR System (Applied Biosystems).

Immunoprecipitation.

Huh-7 cells were lysed in a lysis buffer [20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 100 mM NaCl, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktails 2 and 3]. After sonication and centrifugation, the resulting supernatant was incubated with Dynabeads with a given antibody or an anti-FLAG M2 affinity gel (FLAG gel). Dynabeads or FLAG gel were washed three to four times in a lysis buffer or Tris-buffered saline [TBS; 25 mM Tris-HCl (pH 7.4), 140 mM NaCl, and 2.7 mM KCl], respectively. Washed FLAG gel was heat-treated in 2× SDS sample buffer [157 mM Tris-HCl (pH 6.8), 4% SDS, 25% glycerol, and 0.01% bromophenol blue]. Washed Dynabeads were incubated and centrifuged in 0.1 M glycine buffer (pH 2.0) to elute proteins that were subsequently heat-treated in the above-mentioned 2× SDS sample buffer.

ChIP Assays.

ChIP assay was performed using ChIP-IT Express kit (Active Motif, Carlsbad, CA) as described previously (Saito et al., 2013a; Gotoh and Negishi, 2015) with some modifications. Sheared chromatin (10 μg) obtained from mouse livers or Ym17 cells was incubated with the indicated antibodies (0.3–1 μg; anti-phospho RNA polymerase II, anti-RXRα, anti-phospho-p38, anti-V5, or normal IgG) and magnetic beads. After washing beads, DNA was eluted and purified. DNA fragments were amplified by PCR with the following specific primers: 5′-TTACTGTGTGTAAAGCACTTC-3′ and 5′-GACAAACAGTCCTATTTGTAAG-3′ [for the phenobarbital-responsive enhancer module (PBREM) of the CYP2B6 gene; PBREM/CYP2B6] (the amplicon is from –1863 to –1674; –1863/–1674); 5′-GCTAATGCCTGTCTGGATCAGGA-3′ and 5′-GGAATACTGACCCAAGTTCAGTG-3′ (PBREM/Cyp2b10) (–2434/–2232); 5′-AAGGGAATGAGGAGTGAGC-3′ and 5′-CAAGAAGCCCACAAGGAGAG-3′ (TATA box/Cyp2b10) (–149/+75); 5′-GCTTCTCTTTGCCCTCGATA-3′ and 5′-ACCCAAGTCCCCTGTACCTTAC-3′ (direct repeat 4 (DR4)/Cyp2c55) (–1860/–1623); 5′-GGCCAGAGTCCATTCAGAAG-3′ and 5′-GAGCTTCCCTCTCCCAGAGT-3′ (TATA box/Cyp2c55) (–115/+114).

In Vitro Kinase Assay.

Huh-7 cells, which expressed ectopic FLAG-p38 MAPK, were treated with 1 μM anisomycin for 10 minutes. These cells were lysed in the above-mentioned lysis buffer containing 2.5 mM Na4P2O7 and 1 mM Na3VO4. Anti-FLAG M2 affinity gels, which were pretreated with dimethyl pimelimidate (1 mg/ml) and triethanolamine (100 mM), were incubated with lysates for 3 hours at 4°C and were washed four to five times with TBS. Resulting gels were used as an enzyme source for active p38 MAPK. For substrates, GST-CAR wild-type and the T48A mutant were expressed and purified as previously reported (Mutoh et al., 2009). In kinase reaction, GST-CAR (1 μg) and FLAG-p38 MAPK-bound gel were mixed in a kinase buffer consisting of 25 mM Tris-HCl (pH 7.5), 2.5 mM Na4P2O7, 1 mM Na3VO4, 10 mM MgCl2, and 1 mM dithiothreitol. Reaction was started by adding 200 μM ATP, incubated for 1 hour at 37°C, and stopped by adding 1× SDS sample buffer. Phosphorylated CAR was detected by Western blot analysis using anti-pThr38 CAR antibodies and anti-rabbit IgG (light chain–specific)–HRP antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA).

Bacterially expressed active p38 MAPK (0.36 μg) was incubated with GST-CAR (0.5 μg) and ATP (250 μM) in the above-mentioned kinase buffer for 6 hours at 37°C, and then Western blot analysis was performed to detect phosphorylated CAR.

Western Blot.

Proteins were separated in a 10% SDS-polyacrylamide gel and were transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 2% skim milk or 5% bovine serum albumin in TBS containing 0.1% Tween 20 and were incubated with given primary and secondary antibodies. Protein bands were visualized by Western Bright ECL reagents (Advansta, Menlo Park, CA).

Immunohistochemistry.

Paraffin-embedded liver sections were prepared from C3H/HeNCrlBR males and subjected to immunohistochemistry as described previously (Shindo et al., 2013).

Statistical Analysis.

Multiple groups were analyzed by one-way analysis of variance followed by Tukey’s multiple comparison test. Two groups were compared by Student’s t test. These statistical analyses were conducted using a software GraphPad Prism 6.07 (GraphPad Software, San Diego, CA).

Results

A p38 MAPK Complex with CAR on PBREM.

Anisomycin, a p38 MAPK activator, was first confirmed as a synergist in the increase of CYP2B6 mRNA by a CAR ligand TCPOBOP in HepG2-derived Ym17 cells that constitutively express V5-tagged mouse CAR (Fig. 1A). The results of a previous experiment with small-interfering RNA confirmed that this synergy was, in fact, mediated by p38 MAPK (Saito et al., 2013b). Upon activation, CAR binds the DNA sequence called PBREM within the CYP2B6 promoter to activate it. In ChIP assays, anisomycin cotreatment with TCPOBOP synergistically increased CAR binding to PBREM in Ym17 cells (Fig. 1B). Coimmunoprecipitation assays were employed to examine interactions between CAR and p38 MAPK in human hepatoma Huh-7 cells. An anti-p38 MAPK antibody coprecipitated CAR (Fig. 1C). Anisomycin treatment increased CAR coprecipitated with phosphorylated p38 MAPK (Fig. 1D). The nonphosphomimetic CAR T38A mutant spontaneously accumulated in the nucleus, whereas the phosphomimetic mutant CAR T38D was retained in the cytoplasm (Mutoh et al., 2009; Osabe and Negishi, 2011). Wild-type CAR was not effectively phosphorylated at threonine 38 in Huh-7 cells, thus resembling the CAR T38A mutant in this respect. FLAG-tagged CAR wild-type, T38A, or T38D mutant was ectopically coexpressed with p38 MAPK in Huh-7 cells and subjected to coimmunoprecipitation assays (Fig. 1E). The CAR T38D mutant coprecipitated p38 MAPK far less than either CAR wild-type or T38A mutant did. These observations indicated that CAR needs to form a complex with anisomycin-activated p38 MAPK for binding to PBREM in the nucleus.

Fig. 1.

Fig. 1.

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CAR recruitment on PBREM and interaction between CAR and p38 MAPK. (A, B) Ym17 cells were treated with 250 nM TCPOBOP or DMSO for 12 hours in the presence or absence of 200 nM anisomycin (ANI). (A) CYP2B6 mRNA expression levels were determined by real-time PCR. Each value is shown as the mean ± S.D. (n = 3). Statistically significant difference: ***P < 0.001, Tukey’s multiple comparison test. (B) ChIP assays were performed to assess recruitment of V5-tagged CAR to the PBREM of the CYP2B6 promoter. (C–E) Coimmunoprecipitation assays were performed using Huh-7 cells. (C) Cells were transfected with FLAG-CAR/pCR3. Immunoprecipitation of p38 MAPK with anti-p38 MAPK antibodies (Abcam) and Western blot analysis with anti-FLAG or anti-p38 MAPK antibodies (Cell Signaling Technology) were performed. (D) Cells were transfected with FLAG-CAR/pCR3 and treated with 1 μM ANI or DMSO for 30 minutes in the absence of FBS. Immunoprecipitation of FLAG-CAR with anti-FLAG M2 affinity gel and Western blot analysis for p38 MAPK, phosphorylated p38 (p-p38) MAPK, or FLAG-CAR were performed. (E) Cells were transfected with FLAG-CAR wild-type (WT)/pCR3, the T48A mutant, or the T48D mutant together with p38 MAPK expression vectors. Mouse CAR T48 corresponds to human CAR T38. Immunoprecipitation of FLAG-CAR with anti-FLAG M2 affinity gel and Western blot analysis for p38 MAPK or FLAG-CAR were performed.

CAR-Dependent p38 Recruitment to PBREM.

First, mouse primary hepatocytes were cotreated with a specific p38 MAPK inhibitor SB 239063 (Underwood et al., 2000) and TCPOBOP or phenobarbital to enable examination of expression levels of CYP2B10 (the mouse homolog of human CYP2B6) mRNA. Whereas SB 239063 did not affect basal levels, it effectively diminished either TCPOBOP- or phenobarbital-induced increase of this mRNA (Fig. 2A). Phosphorylated p38 MAPK was found in the nuclei of mouse livers (Supplemental Fig. 1, A and B). Having these findings, we employed ChIP assays to analyze the Cyp2b10 promoter in mouse livers. Mice were treated with either phenobarbital or TCPOBOP and their liver RNAs and nuclei were prepared, respectively, for real-time PCR to confirm that CYP2B10 mRNA had increased (Fig. 2B) and for ChIP assays to measure binding between phosphorylated p38 and PBREM (Fig. 2C). Phosphorylated p38 was recruited to PBREM after phenobarbital or TCPOBOP treatment in the livers of CAR wild-type but not knockout (KO) mice (Fig. 2C). Nonphosphorylated CAR translocates to the nucleus and heterodimerizes with RXRα (Honkakoski et al., 1998). PBREM binding of a CAR/RXRα complex resembled that of the phosphorylated p38 MAPK. These binding patterns correlated with those of phosphorylated RNA polymerase II to the TATA box (Fig. 2C). Weak bands observed with either PBS or DMSO-treated nuclei in CAR wild-type and/or KO mice appeared to be nonspecific amplifications. Thus, upon CAR activation, phosphorylated p38 MAPK was recruited to PBREM, in turn recruiting active RNA polymerase II to induce transcription.

Fig. 2.

Fig. 2.

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Effect of p38 MAPK on Cyp2b10 mRNA expression in hepatocytes and recruitment of p-p38 MAPK on the Cyp2b10 promoter in vivo. (A) Mouse primary hepatocytes were treated with 2 mM phenobarbital or 250 nM TCPOBOP for 12 hours in the presence or absence of SB 239063. Cyp2b10 mRNA expression levels were determined by real-time PCR. Each value is shown as the mean ± S.D. (n = 3). Statistically significant differences: **P < 0.01 and ***P < 0.001, Tukey’s multiple comparison test. (B) Effect of treatment with phenobarbital (100 mg/kg) or TCPOBOP (3 mg/kg) for 6 hours on Cyp2b10 mRNA expression in livers. Each value is shown as the mean ± S.D. (n = 3). Statistically significant difference: ***P < 0.001, Student’s t test. (C) Recruitment of CAR/RXRα, p-p38 MAPK, and phosphorylated RNA polymerase II (p-RNA pol II) after 6 hours of phenobarbital or TCPOBOP injection. ChIP assays were performed for the PBREM and the TATA box of the Cyp2b10 gene using specific primers as described in Materials and Methods. PB or TC indicates phenobarbital or TCPOBOP, respectively.

The Cyp2c55 gene is also a CAR-regulated gene in mouse livers (Konno et al., 2010). As observed with CYP2B10 mRNA, treatment with SB 239063 also repressed an increase in CYP2C55 mRNA by phenobarbital in mouse primary hepatocytes (Fig. 3A). ChIP assays revealed that phenobarbital treatment induced recruitment of phosphorylated p38 MAPK and CAR to the previously identified element and of active RNA polymerase II to the TATA box within the Cyp2c55 promoter (Fig. 3B). These similarities between the Cyp2b10 and Cyp2c55 promoters support the notion that p38 MAPK may be an essential factor for CAR to activate a set of its target genes including these two.

Fig. 3.

Fig. 3.

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Effect of p38 MAPK on Cyp2b55 mRNA expression in hepatocytes and recruitment of p-p38 MAPK on the Cyp2c55 promoter in vivo. (A) Mouse primary hepatocytes were treated with 2 mM phenobarbital for 12 hours in the presence or absence of SB 239063. Cyp2c55 mRNA expression levels were determined by real-time PCR. Each value is shown as the mean ± S.D. (n = 3). Statistically significant differences: ***P < 0.001, Tukey’s multiple comparison test. (B) Recruitment of CAR/RXRα, p-p38 MAPK, and p-RNA pol II after 6 hours of phenobarbital injection. ChIP assays were performed for the direct repeat (DR)-4 and the TATA box of the Cyp2c55 gene using specific primers as described in Materials and Methods.

p38 MAPK-Stimulated Phosphorylation of Threonine 38.

Inactive CAR is phosphorylated at threonine 38 and sequestered in the cytoplasm (Mutoh et al., 2009), although the protein kinase that phosphorylates it has not been identified. Unexpectedly, CAR was found to be phosphorylated at threonine 38 when CAR was coexpressed with p38 MAPK in Huh-7 cells, as indicated by Western blot analysis with an anti-phospho-peptide antibody (Fig. 4A). Ectopic p38 MAPK was similarly phosphorylated as the endogenous counterpart was. Subsequently, in vitro kinase assays were performed to further substantiate phosphorylation of CAR by p38 MAPK. To this end, FLAG-p38 MAPK was expressed in Huh-7 cells, from which kinase was purified and incubated with a recombinant CAR (either CAR wild-type or CAR T38A mutant) as a substrate. Western blot analysis showed phosphorylation of CAR wild-type but not the mutant (Fig. 4B). In a similar kinase assay, a bacterially expressed recombinant p38 MAPK also phosphorylated CAR wild-type (Fig. 4C). Thus, these results strongly suggested that p38 MAPK directly phosphorylated CAR. In VDR (NR1I1), which is another member of the nuclear receptor 1I subfamily, serine 51 is the corresponding conserved residue. Previous studies with a phosphomimetic mutant suggested that VDR could be inactivated by phosphorylation at serine 51 (Hsieh et al., 1993) and sequestered in the cytoplasm (unpublished data). As was also seen with CAR, serine 51 was phosphorylated in Huh-7 cells when VDR was coexpressed with p38 MAPK as well as in in vitro kinase assays using FLAG-p38 MAPK as the enzyme (Supplemental Fig. 2, A and B). Since the majority of nuclear receptors conserve this phosphorylation motif, p38 MAPK can be used to phosphorylate them and to regulate their activities.

Fig. 4.

Fig. 4.

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Phosphorylation of CAR at threonine 38 by p38 MAPK. (A) Expression vectors for FLAG-tagged mouse CAR wild-type (WT) or the T48A, corresponding to the T38A in humans, were transfected into Huh-7 cells with or without p38 MAPK expression vectors. After immunoprecipitation with anti-FLAG M2 affinity gel, Western blot analysis with anti-pThr38-CAR antibodies was performed to assess phosphorylation levels of CAR. (B, C) In vitro kinase assays with active p38 MAPK purified from Huh-7 cells (B) or purified from Escherichia coli (C) and GST-CAR wild-type or the T48A mutant were performed to demonstrate phosphorylation of threonine 38 by p38 MAPK under the conditions as described in Materials and Methods.

Discussion

CAR is activated by dephosphorylation at threonine 38, which occurs in the cytoplasm where phosphorylated CAR is sequestered (Mutoh et al., 2009). Nonphosphorylated CAR translocates into the nucleus to activate its targeted genes. Here, we have demonstrated that threonine 38 is rephosphorylated to inactivate CAR in the nucleus. p38 MAPK, having formed a complex with CAR to recruit it to and activate the CYP2B promoter, subsequently phosphorylates threonine 38. This rephosphorylation should inactivate CAR and exclude it from the nucleus. Thus, by regulating this series of reactions, p38 MAPK links CAR-mediated transactivation and inactivation.

Active p38 MAPK resides in the nucleus of mouse hepatocytes and forms a complex with incoming CAR from the cytoplasm in response to phenobarbital or TCPOBOP. In Ym17 cells, anisomycin activates p38 MAPK. The complexes of CAR with active p38 MAPK promote binding of CAR to the CYP2B promoter. p38 MAPK is known to interact with RNA polymerase II (Alepuz et al., 2003). Moreover, the promoter-bound kinase activates RNA polymerase–mediated gene transcription, as indicated by the fact that the LexA fusion–p38 MAPK activated a luciferase reporter gene bearing LexA DNA binding sites upstream of the luciferase reporter (Ferreiro et al., 2010). Thus, a reasonable scenario may be that p38 MAPK, as part of the CAR complex, binds the PBREM and facilitates the TATA box to recruit active RNA polymerase II for effective promoter activation. The CYP2B6 (the human CYP2B homolog) gene looped to locate the distal PBREM to the TATA box; early growth response 1 facilitated this looping, possibly enabling the promoter to recruit RNA polymerase II for effective promoter activation (Inoue and Negishi, 2008, 2009). In addition to early growth response 1, various other transcription factors were shown to interact with and activate the CYP2B promoters together with CAR, such as peroxisome proliferator-activated receptor -γ coactivator-1 alpha (PGC-1α), steroid receptor coactivator-1 (SRC-1), HNF-4α, and CCAAT/enhancer-binding protein alpha (Shiraki et al., 2003; Li et al., 2008; Benet et al., 2010). Since p38 MAPK can activate these factors (Lim et al., 1998; Knutti et al., 2001; Guo et al., 2007; Qiao et al., 2006; Fernandez-Marcos and Auwerx, 2011; Antoon et al., 2012), it may enable CAR to bind to PBREM and to loop the promoter by phosphorylating one or more of them. In addition to the CYP2B genes, p38 MAPK similarly regulated the Cyp2c55 gene in mouse primary hepatocytes. This indicates that the regulation observed with p38 MAPK may be a general mechanism by which CAR activates hepatic genes.

We now know that CAR can be phosphorylated in the nucleus. Phosphorylated CAR moves back to the cytoplasm, possibly being retained for reactivation by dephosphorylation. Thus, it is possible that CAR can be recycled through phosphorylation, dephosphorylation, and rephosphorylation of threonine 38 during drug inductions. Figure 5 schematizes the hypothesis that p38 MAPK plays dual functions and links the CAR-mediated activation and inactivation of the CYP2B genes through phosphorylation/dephosphorylation of threonine 38. The phosphorylation of ERα is also known to link its activation and inactivation (Zhou and Slingerland, 2014). However, unlike CAR, which can be reused, phosphorylated ERα is degraded through ubiquitination and, therefore, cannot be recycled.

Fig. 5.

Fig. 5.

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Activation and inactivation of CAR-mediated transcription by p-p38 MAPK. CAR is phosphorylated at threonine 38 under normal conditions in livers. Phenobarbital (PB) or TCPOBOP (TC) causes dephosphorylation of CAR and translocates it to the nucleus. CAR interacts with p-p38 MAPK in the nucleus, and the CAR/p-p38 MAPK complexes activate transcription of the CYP2B gene. P-p38 MAPK phosphorylates threonine 38 of CAR and translocates it to the cytoplasm as an inactive form.

In conclusion, our present study shed light on phosphorylation within the DBD that has long been ignored. Our present findings with CAR and p38 MAPK can be used to understand biologic roles of this phosphorylation among members of the nuclear receptor superfamily.

Acknowledgments

The authors thank the NIEHS DNA sequencing and histology cores.

Abbreviations

CAR

constitutive androstane receptor

ChIP

chromatin immunoprecipitation

DBD

DNA-binding domain

DMSO

dimethyl sulfoxide

ER

estrogen receptor

FBS

fetal bovine serum

GST

glutathione S-transferase

HNF

hepatocyte nuclear factor

HRP

horseradish peroxidase

LBD

ligand-binding domain

MAPK

mitogen-activated protein kinase

PBREM

phenobarbital-responsive enhancer module

PCR

polymerase chain reaction

PGC

peroxisome proliferator-activated receptor -γ coactivator

PP2A

protein phosphatase 2A

RXR

retinoid X receptor

SRC

steroid receptor coactivator

TBS

Tris-buffered saline

TCPOBOP

1,4-bis[2-(3,5-dichloropyridyloxy)]benzene

VDR

vitamin D receptor

Authorship Contributions

Participated in research design: Hori, Negishi.

Conducted experiments: Hori, Moore.

Performed data analysis: Hori.

Wrote or contributed to the writing of the manuscript: Hori, Negishi.

Footnotes

This work was supported by the Intramural Research Program of the National Institutes of Health and National Institute of Environmental Health Sciences [Grant Z01ES71005-01].

Inline graphicThis article has supplemental material available at dmd.aspetjournals.org.

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