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• W2034777583 abstract "We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1–23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR. We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1–23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR. The aryl hydrocarbon receptor (AhR) 1The abbreviations used are: AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; DRE, dioxin response element; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; NTA, nickel nitrilotriacetic acid; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin; HRP, horseradish peroxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IEF, isoelectric focusing; HA; hemagglutinin; WT, wild type; Me2SO, dimethyl sulfoxide; IPG, immobilized pH gradient. 1The abbreviations used are: AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; DRE, dioxin response element; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; NTA, nickel nitrilotriacetic acid; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin; HRP, horseradish peroxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IEF, isoelectric focusing; HA; hemagglutinin; WT, wild type; Me2SO, dimethyl sulfoxide; IPG, immobilized pH gradient. is a ligand-activated member of the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) transcription factor family, which includes Per, AhR nuclear translocator (ARNT or hypoxia-inducible factor 1-β), Sim, and HIF 1-α (1Hoffman E.C. Reyes H. Chu F.F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Crossref PubMed Scopus (836) Google Scholar, 2Jackson F.R. Bargiello T.A. Yun S.H. Young M.W. Nature. 1986; 320: 185-188Crossref PubMed Scopus (165) Google Scholar, 3Nambu J.R. Lewis J.O. Wharton Jr., K.A. Crews S.T. Cell. 1991; 67: 1157-1167Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 4Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar). This protein is believed to mediate the biological and toxic effects of a class of environmental pollutants, best exemplified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) (5Poland A. Knutson J.C. Annu. Rev. Pharmacol. Toxicol. 1982; 22: 517-554Crossref PubMed Scopus (2326) Google Scholar). TCDD binding initiates the process of transformation, which includes dissociation of one or several of the chaperone-related proteins from the unliganded AhR in the cytoplasm, translocation of the receptor-ligand complex into the nucleus, and dimerization with ARNT, to form the active TCDD-AhR-ARNT transcription factor complex (6Rowlands J.C. Gustafsson J.A. Crit Rev. Toxicol. 1997; 27: 109-134Crossref PubMed Scopus (436) Google Scholar, 7Whitlock Jr., J.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 103-125Crossref PubMed Scopus (994) Google Scholar). The endogenous ligand for the AhR remains unknown. The active AhR-ARNT complex specifically recognizes a consensus sequence, termed a dioxin response element (DRE, 5′-CACGCNA-3′), located in the upstream regulatory region of AhR-responsive genes, such as Cyp1A1, leading to transcription initiation. The basic region of the AhR contains multiple essential structural components for direct contact to DNA. It has two basic clusters separated by about 20 amino acids, both of which appear to be engaged in direct protein-DNA contact. Mutation of certain positively charged residues in either cluster (e.g. arginine 14 in the more N-terminal basic region or arginine 39 in the nominal basic region) to alanine or lysine has been found to abolish the formation of the ternary complex between the AhR-ARNT dimer and the DRE, suggesting that it is these basic residues that directly contact the DRE (8Dong L. Ma Q. Whitlock Jr., J.P. J. Biol. Chem. 1996; 271: 7942-7948Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 9Bacsi S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Fukunaga B.N. Hankinson O. J. Biol. Chem. 1996; 271: 3743-3749Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 11Swanson H.I. Yang J. J. Biol. Chem. 1996; 271: 31657-31665Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Previous investigations had determined that when N-terminal deletion analyses were performed to map the DNA binding domain of an AhR mutant lacking the C-terminal half and the minimal ligand binding domain (AhRCΔ516), the first 9 amino acids, including tyrosine 9, were dispensable for constitutive DNA binding activity (11Swanson H.I. Yang J. J. Biol. Chem. 1996; 271: 31657-31665Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In contrast, N-terminal deletion analyses of the full-length mouse AhR indicated that the first 9 amino acids are required for TCDD-induced DNA binding of the AhR but not required for heterodimerization with ARNT (10Fukunaga B.N. Hankinson O. J. Biol. Chem. 1996; 271: 3743-3749Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Also, amino acid scanning mutations to alanine, serine, or tryptophan showed that tyrosine 9 is solely responsible for the loss of DNA binding and transcriptional activity of these N-terminal deletion mutants (9Bacsi S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Fukunaga B.N. Hankinson O. J. Biol. Chem. 1996; 271: 3743-3749Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Besides arginines 14 and 39, AhR tyrosine 9 was the only residue in the N terminus whose mutation resulted in dramatic loss of both DNA binding and transcriptional activity (9Bacsi S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Fukunaga B.N. Hankinson O. J. Biol. Chem. 1996; 271: 3743-3749Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These data clearly demonstrate that AhR tyrosine 9 is a critical residue required for full-length AhR activity. However, the mechanism by which AhR tyrosine 9 plays a role in AhR activity remains controversial. While this residue is not expected to directly contact DNA, several mechanisms including phosphorylation of this residue have been speculated (9Bacsi S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Park S. Henry E.C. Gasiewicz T.A. Arch. Biochem. Biophys. 2000; 381: 302-312Crossref PubMed Scopus (38) Google Scholar, 13Minsavage G.D. Vorojeikina D.P. Gasiewicz T.A. Arch. Biochem. Biophys. 2003; 412: 95-105Crossref PubMed Scopus (18) Google Scholar). Here, we examine three known or speculated aspects of AhR activation in which tyrosine 9 may potentially play a role. We analyze the impact of a tyrosine 9 mutation on various known steps of the AhR mechanism of action, including ligand binding, nuclear localization, and ARNT heterodimerization, as well as the ability to bind to various DRE sequences. We also test the hypothesis that tyrosine 9 is required for ligand-dependent DNA binding (i.e. for full-length AhR) (11Swanson H.I. Yang J. J. Biol. Chem. 1996; 271: 31657-31665Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Kikuchi Y. Ohsawa S. Mimura J. Ema M. Takasaki C. Sogawa K. Fujii-Kuriyama Y. J. Biochem. 2003; 134: 83-90Crossref PubMed Scopus (36) Google Scholar) and the hypothesis that tyrosine 9 is required for N-terminal cleavage of the AhR prior to DNA binding (15Bradfield C.A. Glover E. Poland A. Mol. Pharmacol. 1991; 39: 13-19PubMed Google Scholar). Finally, we address the hypothesis that phosphorylation of, or mediated by, tyrosine 9 is critical for normal AhR activation (10Fukunaga B.N. Hankinson O. J. Biol. Chem. 1996; 271: 3743-3749Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 13Minsavage G.D. Vorojeikina D.P. Gasiewicz T.A. Arch. Biochem. Biophys. 2003; 412: 95-105Crossref PubMed Scopus (18) Google Scholar). While AhR tyrosine 9 does not appear to be essential for ligand-elicited nuclear localization or ARNT dimerization, it is necessary for DNA binding of the full-length protein. Although phosphorylation of the AhR tyrosine 9 is not required for DNA binding, mutating tyrosine 9 alters the isoelectric points of the AhR charged forms suggesting that tyrosine 9 plays a role in post-translational modification of other AhR residue(s). We demonstrate that protein kinase C (PKC) phosphorylates the wild-type (WT) AhR, and that mutating AhR tyrosine 9 decreases PKC-elicited phosphorylation of the AhR. Furthermore, we demonstrate that the decreased transcriptional activity of the AhRY9F mutant can be partially overcome upon co-treatment with TCDD and phorbol 12-myristate 13-acetate (PMA). These data demonstrate for the first time that, while tyrosine 9 itself is not phosphorylated, it can play a crucial role in phosphorylation of the AhR and AhR-mediated gene transcription. Generation of AhR Constructs—Site-directed mutagenesis and construction of pcDNA3/βAHR-HIS and pcDNA3/βAHRY9F-HIS were performed as described previously (9Bacsi S.G. Hankinson O. J. Biol. Chem. 1996; 271: 8843-8850Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Fukunaga B.N. Hankinson O. J. Biol. Chem. 1996; 271: 3743-3749Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Park S. Henry E.C. Gasiewicz T.A. Arch. Biochem. Biophys. 2000; 381: 302-312Crossref PubMed Scopus (38) Google Scholar, 13Minsavage G.D. Vorojeikina D.P. Gasiewicz T.A. Arch. Biochem. Biophys. 2003; 412: 95-105Crossref PubMed Scopus (18) Google Scholar). For generation of the pHM6/AhRs that code for N-terminally HA-tagged and C-terminally 6× histidine-tagged AhRs, the wild-type and mutant AhR coding sequences were amplified from pcDNA3/βAHR or pcDNA3/βAHR mutant DNA by Pfu Turbo DNA polymerase using the forward primer, 5′-CGA CCC AAG CTT GCT AGC AAT GTC TAG CGG CGC CAA C-3′ and the reverse primer, 5′-GGT TAC CCG CGG CTC GAG GGA TCC CAC TCT GCA CCT TGC TTA G-3′. HindIII and KspI were used to digest the PCR product and pHM6 to allow subcloning of the amplified AhR coding sequences into pHM6 (Roche Applied Science). For generation of the AhR346 truncation mutants, the wild-type and mutant AhR coding sequence was amplified from pcDNA3/βAHR and pcDNA3/βAHRY9F by Pfu Turbo DNA polymerase using T7 forward primer and the reverse primer, 5′-CCG CTC GAG CGG TCA GTG ATG GTG ATG GTG ATG CCG GAA AAC TGT CAT GCC-3′, containing coding sequences for 6 consecutive histidines, a stop codon, and a XhoI site. The PCR products were digested with HindIII and XhoI and subcloned into the similarly restricted pcDNA3/Neo to generate pcDNA3/βAHR346-HIS and pcDNA3/βAHR346Y9F-HIS. Cell Culture—Wild-type (Hepa1c1c7) and AhR-deficient mouse hepatoma cells (BPrc1TAO) were kind gifts from Dr. J. Whitlock, Jr. (Stanford University). These cells were incubated at 37 °C in a humidified incubator and grown in α-MEM supplemented with 10% fetal bovine serum. COS-7 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and were incubated at 37 °C in a humidified incubator. Subcellular Localization of AhR-GFP Fusion Proteins—The GFP coding sequence was amplified from pEGFP-C1 (Clontech, Palo Alto, CA) by Pfu Turbo DNA polymerase using the forward primer, 5′-ATA AGA ATG CGG CCG CTA AAA TGG TGA GCA AGG GC-3′ containing the NotI site and the reverse primer, 5′-GGT ATG GCT GAT TAT GAT CAG-3′. The PCR products were digested with XhoI and NotI and subcloned into the similarly restricted pcDNA3/βAHRs to generate pcDNA3/βAHR495-GFP. These vectors were transiently transfected into Hepa1c1c7 cells on coverslips. After treatment with either Me2SO- or TCDD-containing medium for an hour, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, mounted on slides, and examined under a fluorescence microscope. In Vitro Translation of Wild-type and Mutant AhRs and ARNT, Electrophoretic Mobility Shift Assay (EMSA), and Supershift EMSA— EMSAs were performed essentially as described previously (13Minsavage G.D. Vorojeikina D.P. Gasiewicz T.A. Arch. Biochem. Biophys. 2003; 412: 95-105Crossref PubMed Scopus (18) Google Scholar, 16Henry E.C. Kent T.A. Gasiewicz T.A. Arch. Biochem. Biophys. 1997; 339: 305-314Crossref PubMed Scopus (9) Google Scholar). Equal amounts of in vitro translated AhRs and ARNT were incubated with either 0.1% Me2SO or 10 nm TCDD (Cambridge Isotopes, Cambridge, MA) for 90 min at room temperature and then mixed with 50,000 cpm of 32P-labeled DRE at 0.085 m NaCl and 10 mm DTT and analyzed in a 4% nondenaturing gel. The DNA binding form of the AhR-ARNT complex was visualized by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For DRE synthesis, one strand of each DRE (17Lusska A. Shen E. Whitlock Jr., J.P. J. Biol. Chem. 1993; 268: 6575-6580Abstract Full Text PDF PubMed Google Scholar) was labeled at the 5′-end using T4 polynucleotide kinase prior to annealing with the unlabelled complementary strand; DRE D (AhRE3), 5′-GAT CCG GCT CTT CT CACGCAACTC CGA GCT CA-3′; DRE E (AhRE2), 5′-CCC AGT GCT GT CACGCTAGCT GGG GGA GGG GAA-3′; DRE A (AhRE5), 5′-TGC GCT TCT CACGCGAGCT TGG-3′. The duplex oligonucleotide contains a single AhR binding sequence (underlined). Supershift assays were performed as described for EMSAs with the exception of the use of the HA-tagged AhR expression vectors and the inclusion of antibody at the incubation step just prior to DRE addition. The antibodies that recognize the hemagglutinin epitope (HA.11, Covance, Berkeley, CA), the AhR (Rpt-9), a nonspecific rabbit anti-mouse IgG (H+L) (Jackson Immunoresearch Laboratories), or the anti-AhR phosphotyrosine 9 antibody were used for supershift assays. In Vitro Protein-Protein Interaction Study—Interaction of AhR with ARNT was investigated by using radiolabeled ARNT expressed in the TnT® (Promega) system as described above with the exception of the inclusion of 15 μCi of [35S]methionine in the reaction mixture. 35S-labeled ARNT was incubated with the similarly expressed but unlabeled histidine-tagged AhRs in the presence or absence of 10 nm TCDD (in 0.1% Me2SO) for 90 min at room temperature. AhR-bound ARNT was precipitated with 70 μl of Ni-NTA-agarose for 30 min at 4 °C in the His tag lysis buffer with 0.3 m NaCl. The pellet was washed twice with His tag lysis buffer containing 40 mm imidazole and boiled with SDS-PAGE buffer. The co-precipitated ARNT was analyzed by SDS-PAGE and visualized by autoradiography. Co-transfection Assay of AhR-deficient Cells—To determine AhRWT and AhRY9F transactivation activity, 2 × 105 AhR-deficient cells (BPrc1TAO or COS-7) were co-transfected with 0.45 μg of AhR expression vectors (pcDNA3/βAHRs), 0.45 μg of DRE-reporter gene (p2DLuc), and 0.1 μg of normalization vector (pRSVLacZ) using LipofectAMINE (Invitrogen) or GenePORTER (Gene Therapy Systems, San Diego, CA) in 6-well plates as described previously (12Park S. Henry E.C. Gasiewicz T.A. Arch. Biochem. Biophys. 2000; 381: 302-312Crossref PubMed Scopus (38) Google Scholar). After the recovery from transfection, the cells were treated with either 0.1% Me2SO or 4 nm TCDD (in 0.1% Me2SO) for 22 h and harvested for the reporter gene assay. DRE-driven luciferase activity was measured with the luciferase assay kit (Promega), and β-galactosidase activity was determined with Galacto-Light Plus (Tropix, Bedford, MA) according to the manufacturer using a Turner Model TD-20e Luminometer (Turner Designs, Sunnyvale, CA). Immunoblot Analysis of Partially Purified Full-length AhRs with Anti-phosphotyrosine Antibody—The pcDNA3/βAHR-HIS and pcDNA3/βAHRY9F-HIS vectors were transiently transfected into the AhR-deficient TAO cells using GenePORTER according to the manufacturer and partially purified exactly as previously described using Ni-NTA-agarose (12Park S. Henry E.C. Gasiewicz T.A. Arch. Biochem. Biophys. 2000; 381: 302-312Crossref PubMed Scopus (38) Google Scholar). After separation by SDS-PAGE, the proteins were transferred to a PVDF membrane, and the membrane was probed with PY-20 monoclonal anti-phosphotyrosine antibodies (Transduction Laboratory, Lexington, KY) in 3% BSA-TBST. The membrane was stripped and reprobed with anti-AhR antibody (Rpt.1; ascites prepared using hybridoma cells that were a kind gift from Dr. G. Perdew (Pennsylvania State University)). The primary antibody was located with HRP-conjugated goat anti-mouse IgG and visualized with LumiGLO chemiluminescent substrate (KPL, Gaithersburg, MD). Two-dimensional Gel Electrophoresis of Wild-type AhR and AhRY9F— Two-dimensional gel electrophoresis was performed utilizing radiolabeled AhRs separately expressed in the TnT® system as described above with the exception of inclusion of 15 μCi of [35S]methionine in the reaction mixture, methods similar to those previously described (13Minsavage G.D. Vorojeikina D.P. Gasiewicz T.A. Arch. Biochem. Biophys. 2003; 412: 95-105Crossref PubMed Scopus (18) Google Scholar). 50 μl of the TnT® lysate containing 35S-labeled AhR was diluted with 150 μl of HEDG buffer (25 mm Hepes, 1.5 mm Na2EDTA, 1 mm DTT, 10% (v/v) glycerol; adjusted to pH 7.6) containing protease inhibitors. 50 μl of this mixture was added to 30 μl of HEDG containing 1.5 μg/μl carrier protein and added to 215 μl of rehydration buffer (6.4 m urea, 2.6 m thiourea, 4% CHAPS, 39 mm DTT, 0.2% Bio-Lytes 3/10 ampholytes, 0.001% bromphenol blue). The Bio-Rad Protean IEF Cell and reagents were used for isoelectric focusing (IEF) and the Criterion Precast Gel System was used for SDS-PAGE (Bio-Rad). ReadyStrip IPG 11-cm strips (pH range 3–10) were actively rehydrated (application of 50 V) at 16 °C covered with mineral oil for 26 h. For IEF, a slow ramping protocol was followed: a conditioning step of 250 V for 15 min, followed by voltage ramping up to 8000 V over 2.5 h, and final focusing at 8000 V for 80,000 VH. Maximum current limit was set at 50 μA per gel, with total VH not exceeding 100,000 VH. Following IEF, the IPG strips were equilibrated for 30 min in DTT equilibration buffer (5 m urea, 0.8 m thiourea, 4% SDS, 0.375 m Tris-HCl pH 8.8, 20% glycerol, and 130 mm DTT), followed by 30 min in iodoacetamide equilibration buffer (5 m urea, 0.8 m thiourea, 4% SDS, 0.375 m Tris-HCl pH 8.8, 20% glycerol, and 135 mm iodoacetamide). IPG strips were loaded to the well of a 7.5% Tris-HCl precast gel and run at a constant voltage of 200 V for 1 h. Proteins from the 2nd dimension gels were transferred to Sequi-Blot PVDF membranes (Bio-Rad) and 35S-labeled AhRs were visualized with a PhosphorImager (Amersham Biosciences). Anti-AhR Phosphotyrosine 9 Production and Immunodetection of Phosphorylated AhR Tyrosine 9 —Synthesis of peptides, BSA conjugation, and generation of the phosphopeptide antibody was carried out by Alpha Diagnostic International (San Antonio, TX) as previously described (18Bernard A. Kazlauskas A. Exp. Cell Res. 1999; 253: 704-712Crossref PubMed Scopus (15) Google Scholar, 19Keshvara L. Benhayon D. Magdaleno S. Curran T. J. Biol. Chem. 2001; 276: 16008-16014Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 20Wang Z. Wilson G.F. Griffith L.C. J. Biol. Chem. 2002; 277: 24022-24029Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). A BLAST search was performed to optimize selection of sequence that would be most likely antigenic and exhibit minimal cross-reactivity with nonspecific proteins. The hydrophilic region, residues 4–16 (GANIT(pY)ASRKRRK) with a cysteine on the C terminus for keyhole limpet hemocyanin conjugation, was used as the phosphotyrosine 9 peptide antigen purified to >90% for presentation to rabbits. For affinity purification, a non-phosphopeptide control of the same sequence without a phosphorylated tyrosine 9 was used as an additional affinity support to remove nonspecific antibodies prior to use of the phosphotyrosine 9 peptide affinity column for capture of the antibodies of interest. Both the control and phosphopeptide were used to test antibody cross-reactivity with non-phosphorylated peptides by ELISA. BSA was conjugated to the phosphopeptide (BSA-pY9) using the glutaraldehyde conjugation method to allow optimization for immunodetection of phosphorylated tyrosine 9. To determine the specificity of the anti-AhRpY9 antibody, the BSA-pY9 conjugate was incubated with a mixture of alkaline phosphatase and potato acid phosphatase to remove the phosphate group from tyrosine 9 to serve as a negative control. For detection of AhR from transfected cells, AhR-deficient cells were transfected with 8 μg of an AhR expression vector (pHM6/AHR) using GenePORTER in 100-mm plates 24 h after plating 2 × 106 cells in each plate. AhR-deficient cells from confluent plates were not transfected as a negative control. After 48 h of recovery, cells were treated for 1 h with either 0.1% Me2SO or 10 nm TCDD. Cells were washed with Hank's Buffer, and total protein was collected by triturating the cells in 150 μl of high salt HEG buffer (25 mm Hepes, 1.5 mm Na2EDTA, 10% (v/v) glycerol, 0.45 m NaCl, adjusted to pH 7.6) containing protease inhibitors (Complete Mini Tablets, Roche Applied Science). The AhR was immunoprecipitated from the lysate preparation with an anti-HA antibody. The pelleted protein was loaded on a 7.5% polyacrylamide gel and separated by SDS-PAGE. Protein was transferred to a PVDF membrane and analyzed by immunoblot analysis. The primary antibodies were located with HRP-conjugated goat anti-rabbit IgG and visualized with LumiGLO chemiluminescent substrate. To determine if the anti-AhRpY9 antibody was capable of immunoprecipitating the AhR, wild-type and AhRY9F HA-tagged receptors were translated in a total volume of 50 μl containing TnT®-coupled rabbit reticulocyte lysate in the presence of T7 RNA polymerase for 90 min at 30 °C as described above with the exception of inclusion of 15 μCi of [35S]methionine in the reaction mixture. Equal amounts of in vitro translated AhRs and non-labeled ARNT were incubated with either 0.1% Me2SO or 10 nm TCDD (Cambridge Isotopes, Andover, MA) for 90 min at room temperature. Aliquots of the mixture were immunoprecipitated using either excess anti-HA antibody or excess anti-AhRpY9 antibodies. Pelleted protein was solubilized in SDS-loading buffer and separated on a 7.5% polyacrylamide gel by SDS-PAGE. [35S]AhRs were visualized with a PhosphorImager. PKC Activity Assays—For the peptide substrate PKC assays, peptides corresponding to the N terminus of the mouse AhR (residues 1–23) were synthesized by Alpha Diagnostic International. These peptides included the wild-type (AhR-(1–23)WT) sequence (MSSGANITYASRKRRKPVQKTVK), the Y9F (AhR-(1–23)Y9F) mutant sequence (MSSGANITFASRKRRKPVQKTVK), and a sequence containing a phosphorylated tyrosine 9 (AhR-(1–23)pY9) (MSSGANIT(pY)ASRKRRKPVQKTVK). A control peptide was synthesized corresponding to mouse AhR residues 383–406 (EEGREHLQKRSTSLPFMFATGEAVK). The specific PKC substrate peptide control (QKRPSQRSKYL) was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). All AhR peptides were conjugated with biotin on the C terminus and >96% pure as determined by mass spectrometry and high performance liquid chromatography. The aqueous peptides were monitored by amino acid analysis at the University of Rochester Proteomics Core Sequencing Facility to ensure that the appropriate amounts of peptides were used for each experiment. The PKC assays (using an active mixture of PKC α, β, and γ) with the required reagents purchased from Upstate Cell Signaling Solutions were carried out as described by the manufacturer. For the PKC-elicited phosphorylation of full-length AhR, AhRs were expressed in the TnT system as described above, immunoprecipitated using either Rpt-9 or the anti-HA antibody and protein A/G-agarose plus (Santa Cruz Biotechnology). The pelleted immunocomplexes were washed extensively with IP wash buffer (40 mm Tris, 150 mm NaCl, and 1% Triton X-100, pH 7.4), and then either incubated for 15 min at 30 °C with alkaline phosphatase or buffer and washed extensively in IP wash buffer. The washed immunocomplexes were then incubated for the indicated times at 30 °C with an active mixture of PKC and [32P]ATP, as described by the manufacturer. Pelleted protein was solubilized in SDS-loading buffer at the time point indicated and loaded to a 7.5% denaturing gel for SDS-PAGE. Protein was then transferred to PVDF membrane and detected using a PhosphorImager. The amount of AhR was analyzed by immunoblot analysis using anti-AhR antibodies or anti-HA antibodies as indicated. The primary antibodies were located with HRP-conjugated IgG and visualized with LumiGLO chemiluminescent substrate. PMA Effect on Transcriptional Activity—AhR-deficient cells (2.0 × 104) were seeded into each well of 24-well plates and incubated at 37 °C. 60–80 percent confluent cells were washed with serum-free medium and transfected with 250 μl of serum-free medium containing 400 ng of the designated pHM6/HA-AhR-HIS construct, 100 ng of the DRE-driven firefly luciferase reporter construct, p2DLuc, and 15 ng of Renilla luciferase vector pRL-TK (Promega) that was preincubated with 2.6 μl of GenePORTER transfection reagents at room temperature for 45 min. Four hours later, another 250 μl of growth media containing 20% fetal bovine serum was added into each well to achieve the final serum concentration of 10%. Fresh medium was added 24 h later. Forty-eight hours post-transfection, the triplicate wells were treated with either vehicle (0.15% Me2SO), 10 nm TCDD, 100 nm PMA, a combination of 10 nm TCDD and 100 nm PMA, or a combination of 10 nm TCDD, 100 nm PMA, 1 μm GF 109203X, and 1 μm RO 31–8220. PMA, GF 109203X, and RO 31–8220 were purchased from BioMol. In a separate experiment, similarly transfected cells were pretreated for 15 min with 4 μm chelerythrine chloride (not shown) (Sigma) before the treatment performed above, as described previously (30Long W.P. Pray-Grant M. Tsai J.C. Perdew G.H. Mol. Pharmacol. 1998; 53: 691-700Crossref PubMed Scopus (137) Google Scholar). All treatment gro" @default.
• W2034777583 created "2016-06-24" @default.
• W2034777583 creator A5007584396 @default.
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• W2034777583 title "The Aryl Hydrocarbon Receptor (AhR) Tyrosine 9, a Residue That Is Essential for AhR DNA Binding Activity, Is Not a Phosphoresidue but Augments AhR Phosphorylation" @default.
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