The Major Chemical-detoxifying System of UDP-glucuronosyltransferases Requires Regulated Phosphorylation Supported by Protein Kinase C
2008; Elsevier BV; Volume: 283; Issue: 34 Linguagem: Inglês
10.1074/jbc.m800032200
ISSN1083-351X
AutoresNikhil K. Basu, Labanyamoy Kole, Mousumi Basu, Kushal Chakraborty, Partha Mitra, Ida S. Owens,
Tópico(s)Pancreatic function and diabetes
ResumoFinding rapid, reversible down-regulation of human UDP-glucuronosyltransferases (UGTs) in LS180 cells following curcumin treatment led to the discovery that UGTs require phosphorylation. UGTs, distributed primarily in liver, kidney, and gastrointestinal tract, inactivate aromatic-like metabolites and a vast number of dietary and environmental chemicals, which reduces the risk of toxicities, mutagenesis, and carcinogenesis. Our aim here is to determine relevant kinases and mechanism(s) regulating phosphorylation of constitutive UGTs in LS180 cells and 10 different human UGT cDNA-transfected COS-1 systems. Time- and concentration-dependent inhibition of immunodetectable [33P]orthophosphate in UGTs and protein kinase Cϵ (PKCϵ), following treatment of LS180 cells with curcumin or the PKC inhibitor calphostin-C, suggested UGT phosphorylation is supported by active PKC(s). Immunofluorescent and co-immunoprecipitation studies with UGT-transfected cells showed co-localization of UGT1A7His and PKCϵ and of UGT1A10His and PKCα or PKCδ. Inhibition of UGT activity by PKCϵ-specific antagonist peptide or by PKCϵ-targeted destruction with PKCϵ-specific small interference RNA and activation of curcumin-down-regulated UGTs with typical PKC agonists verified a central PKC role in glucuronidation. Moreover, in vitro phosphorylation of nascent UGT1A7His by PKCϵ confirms it is a bona fide PKC substrate. Finally, catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide-activated-UGTs demonstrated that reactive oxygen species-related oxidants act as second messengers in maintaining constitutive PKC-dependent signaling evidently sustaining UGT phosphorylation and activity. Because cells use signal transduction collectively to detect and respond appropriately to environmental changes, this report, combined with our earlier demonstration that specific phospho-groups in UGT1A7 determined substrate selections, suggests regulated phosphorylation allows adaptations regarding differential phosphate utilization by UGTs to function efficiently. Finding rapid, reversible down-regulation of human UDP-glucuronosyltransferases (UGTs) in LS180 cells following curcumin treatment led to the discovery that UGTs require phosphorylation. UGTs, distributed primarily in liver, kidney, and gastrointestinal tract, inactivate aromatic-like metabolites and a vast number of dietary and environmental chemicals, which reduces the risk of toxicities, mutagenesis, and carcinogenesis. Our aim here is to determine relevant kinases and mechanism(s) regulating phosphorylation of constitutive UGTs in LS180 cells and 10 different human UGT cDNA-transfected COS-1 systems. Time- and concentration-dependent inhibition of immunodetectable [33P]orthophosphate in UGTs and protein kinase Cϵ (PKCϵ), following treatment of LS180 cells with curcumin or the PKC inhibitor calphostin-C, suggested UGT phosphorylation is supported by active PKC(s). Immunofluorescent and co-immunoprecipitation studies with UGT-transfected cells showed co-localization of UGT1A7His and PKCϵ and of UGT1A10His and PKCα or PKCδ. Inhibition of UGT activity by PKCϵ-specific antagonist peptide or by PKCϵ-targeted destruction with PKCϵ-specific small interference RNA and activation of curcumin-down-regulated UGTs with typical PKC agonists verified a central PKC role in glucuronidation. Moreover, in vitro phosphorylation of nascent UGT1A7His by PKCϵ confirms it is a bona fide PKC substrate. Finally, catalase or herbimycin-A inhibition of constitutive or hydrogen peroxide-activated-UGTs demonstrated that reactive oxygen species-related oxidants act as second messengers in maintaining constitutive PKC-dependent signaling evidently sustaining UGT phosphorylation and activity. Because cells use signal transduction collectively to detect and respond appropriately to environmental changes, this report, combined with our earlier demonstration that specific phospho-groups in UGT1A7 determined substrate selections, suggests regulated phosphorylation allows adaptations regarding differential phosphate utilization by UGTs to function efficiently. Mammalian endoplasmic reticulum (ER) 3The abbreviations used are:ERendoplasmic reticulumUGTUDP-glucuronosyltransferasePKCprotein kinase CDAG1,2-dihexanoyl-sn-glycerolPMAphorbol 12-myristate 13-acetatePSphosphatidylserineMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideCEcommon endRACKreceptor for activated C-kinaseFITCfluorescein isothiocyanateTRITCtetramethylrhodamine isothiocyanatesiRNAsmall interference RNADAPI4′,6-diamidino-2-phenylindoleMOPS4-morpholinepropanesulfonic acidCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidROSreactive oxygen speciesMAPmitogen-activated proteinEGFepidermal growth factor. -bound UDP-glucuronosyltransferase (UGT) isozymes carry out the broad and critical function of detoxifying endogenous and exogenous lipophilic phenols that include toxic metabolites, dietary constituents, environmental carcinogens, and, inadvertently, therapeutic agents. UGT isozymes inactivate a vast number of structurally diverse chemicals by attaching glucuronic acid to generate water-soluble products with high excretability. Because lipid solubility of chemicals taken into the body largely determines membrane permeability governing their absorption, distribution, and excretion, glucuronidation is the primary cellular process that protects against such chemicals (1Dutton G.J. Dutton G.J. Glucuronidation of Drugs and Other Compounds. CRC Press, Boca Raton, FL1980: 69-78Google Scholar). Chemicals are encountered on a regular basis due to their ubiquitous presence in dietary plants, pyrolysates of wood and petroleum products, distributions related to consumer and agricultural activities, and therapeutic agents. Critical endogenous substrates include bilirubin, steroids and their metabolites, bile acids, retinoic acids, thyroid hormones, and others (1Dutton G.J. Dutton G.J. Glucuronidation of Drugs and Other Compounds. CRC Press, Boca Raton, FL1980: 69-78Google Scholar). In the case of defective UGT–/–, lipophiles accumulate in tissues leading to deleterious effects. Defective bilirubin-conjugating UGT1A1 (2Ritter J.K. Crawford J.M. Owens I.S. J. Biol. Chem. 1991; 266: 1043-1047Abstract Full Text PDF PubMed Google Scholar) in Crigler-Najjar (UGT1–/–) children causes elevated bilirubin levels that deposit in the central nervous system leading to kernicterus. Importantly, ingested dietary and environmental polyphenols are sometimes general toxins and genotoxins that can initiate cancer (3Wells P.G. Mackenzie P.I. Chowdhury J.R. Guillemette C. Gregory P.A. Ishii Y. Hansen A.J. Kessler F.K. Kim P.M. Chowdhury N.R. Ritter J.K. Drug Metab. Dispos. 2004; 32: 281-290Crossref PubMed Scopus (216) Google Scholar). To a detriment, glucuronidation converts medicinal chemicals, which lead to premature termination of therapeutic activity. Hence, it is essential to understand the mechanism(s) controlling this vital process. endoplasmic reticulum UDP-glucuronosyltransferase protein kinase C 1,2-dihexanoyl-sn-glycerol phorbol 12-myristate 13-acetate phosphatidylserine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide common end receptor for activated C-kinase fluorescein isothiocyanate tetramethylrhodamine isothiocyanate small interference RNA 4′,6-diamidino-2-phenylindole 4-morpholinepropanesulfonic acid 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid reactive oxygen species mitogen-activated protein epidermal growth factor. Because individual recombinant UGTs are shown to have broad substrate activity toward aromatic-like chemicals (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), the 17 known viable members of the human superfamily of UGTs, distributed in families A, B, and C, collectively convert an unlimited number of chemicals. Because the ER-bound UGT isozymes have proven difficult to purify for tertiary structural analyses, the critical properties and mechanism(s) controlling catalytic activity are not understood. Whereas it has been demonstrated that UGT catalysis requires ongoing regulated phosphorylation (5Basu N.K. Kole L. Owens I.S. Biochem. Biophys. Res. Commun. 2003; 303: 98-104Crossref PubMed Scopus (43) Google Scholar, 6Basu N.K. Kubota S. Meselhy M.R. Ciotti M. Chowdhury B. Hartori M. Owens I.S. J. Biol. Chem. 2004; 279: 28320-28329Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar), and that substrate selection may relate to phospho-group participation (7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar), it is critical to understand factors controlling the regulated phosphorylation. Moreover, high concentrations of certain oxidizable flavonoids and polyphenols found in the diet are or become inhibitors of critical enzymes (8Lang D.R. Racker E. Biochim. Biophys. Acta. 1974; 333: 180-186Crossref PubMed Scopus (159) Google Scholar, 9Kellis J.T. Vickery L.E. Science. 1984; 225: 1032-1034Crossref PubMed Scopus (294) Google Scholar), which we have now shown to include UGTs (5Basu N.K. Kole L. Owens I.S. Biochem. Biophys. Res. Commun. 2003; 303: 98-104Crossref PubMed Scopus (43) Google Scholar, 6Basu N.K. Kubota S. Meselhy M.R. Ciotti M. Chowdhury B. Hartori M. Owens I.S. J. Biol. Chem. 2004; 279: 28320-28329Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar). By contrast, low levels of such chemicals often serve as antioxidants and chemopreventive agents (10Gopalakrishna R. Jaken S. Free Radic. Biol. Med. 2000; 28: 1349-1361Crossref PubMed Scopus (622) Google Scholar), demonstrating the importance of chemical homeostasis. Here we demonstrate the relevant kinases and mechanism(s) involved in the regulation of ongoing phosphorylation of UGTs mediated by PKC signal transduction, which accounts for UGTs vulnerability to high concentrations of antioxidants. Materials—Human LS180 colon and COS-1 cells were from ATCC (Manassas, VA). Tissue culture medium was from Cellgro (Rockville, MD), and serum was from Intergen (Purchase, NY). UGT substrates, curcumin, calphostin-C, calyculin-A, 1,2-dihexanoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate (PMA), and phosphatidylserine (PS), kinase inhibitors, the MTT kit, catalase, and herbimycin-A were from either Sigma or Calbiochem; [14C]UDP-glucuronic acid was from PerkinElmer Life Sciences. PKCϵ translocation activator and inhibitor peptides were from Calbiochem or Dr. D. Mochly-Rosen (Stanford University). Monoclonal anti-β-COP (ϵ-RACK) was from Sigma; anti-His was from Amersham Biosciences; and antibodies toward phosphoserine and protein kinase C isozymes were from Upstate Biotechnology (Lake Placid, NY) or Calbiochem. Donkey secondary antibodies conjugated with FITC or TRITC were from Jackson ImmunoResearch Laboratories (West Grove, PA). The immunogenic phospho-group in PKCϵ is 729 (Upstate, cat. #06-821). PKCϵ-specific siRNA (ON TARGET plus SMART Pool, Human PRKCE), and its non-targeting siRNA control were from Dharmacon (Chicago, IL). TnT® T7 Coupled Wheat Germ extract system was from Promega (Madison, WI). Rabbit anti-calnexin and rabbit anti-calreticulin were from Stressgen Biotechnologies (Victoria, Canada). The rabbit UGT1A-common end (CE) (11Ciotti M. Basu N. Brangi M. Owens I.S. Biochem. Biophys. Res. Commun. 1999; 260: 199-202Crossref PubMed Scopus (132) Google Scholar) and goat UGT-1168 antibodies have been described (12Ritter J.K. Sheen Y.Y. Owens I.S. J. Biol. Chem. 1990; 265: 7900-7906Abstract Full Text PDF PubMed Google Scholar). pSVL-based constructs containing UGT1A1 (2Ritter J.K. Crawford J.M. Owens I.S. J. Biol. Chem. 1991; 266: 1043-1047Abstract Full Text PDF PubMed Google Scholar), UGT1A3 (6Basu N.K. Kubota S. Meselhy M.R. Ciotti M. Chowdhury B. Hartori M. Owens I.S. J. Biol. Chem. 2004; 279: 28320-28329Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), UGT1A4 (2Ritter J.K. Crawford J.M. Owens I.S. J. Biol. Chem. 1991; 266: 1043-1047Abstract Full Text PDF PubMed Google Scholar), UGT1A6 (13Ciotti M. Marone A. Potter C. Owens I.S. Pharmacogenetics. 1997; 7: 485-495Crossref PubMed Scopus (178) Google Scholar), UGT1A7-10 (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), UGT2B7 (12Ritter J.K. Sheen Y.Y. Owens I.S. J. Biol. Chem. 1990; 265: 7900-7906Abstract Full Text PDF PubMed Google Scholar), or UGT1B15 (14Chen F. Ritter J.K. Wang M.G. McBride O.W. Lubet R.A. Owens I.S. Biochemistry. 1993; 32: 10648-10657Crossref PubMed Scopus (111) Google Scholar) were cloned or custom synthesized. Growth and Transfection of Cells—LS180 colon and COS-1 cells were grown in Dulbecco's modified Eagle's medium with 10 and 4% fetal calf serum, respectively, and exposed to reagents solubilized in fresh Me2SO diluted to <0.5%. Cell viability was checked with a MTT kit after treatment with curcumin, calphostin-C, herbimycin-A, and other inhibitors as described in the text. Ten different pSVL-based UGT-cDNAs were transfected into COS-1 cells for specific protein expression as previously described (2Ritter J.K. Crawford J.M. Owens I.S. J. Biol. Chem. 1991; 266: 1043-1047Abstract Full Text PDF PubMed Google Scholar). Also, His tag affinity ligand from the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) was fused in-frame at the 3′-end of the cDNA in pSVL-UGT1A7 and pSVL-UGT1A10. Glucuronidation Assay—Cellular extracts were analyzed for in vitro glucuronidation with abolition of UGT latency as previously described (15Ciotti M. Owens I.S. Biochemistry. 1996; 35: 10119-10124Crossref PubMed Scopus (30) Google Scholar, 16Ritter J.K. Yeatman M.T. Kaiser C. Gridelli B. Owens I.S. J. Biol. Chem. 1993; 268: 23573-23579Abstract Full Text PDF PubMed Google Scholar). The common donor substrate, UDP-[14C]glucuronic acid (1.4 mm, 1.4 μCi/μmol), was used in all reactions with unlabeled acceptor substrate. Incubations used 150 or 300 μg of cellular protein at pH 6.4 or 7.6 for 2 h at 37 °C (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and glucuronides were separated by TLC. The product was quantified as previously described (15Ciotti M. Owens I.S. Biochemistry. 1996; 35: 10119-10124Crossref PubMed Scopus (30) Google Scholar) using appropriate controls. Protein content was estimated using the BCA kit (Pierce), and it represents picomoles of glucuronide/mg of protein/per unit time. Western Blot Analysis of UGTs and Protein Kinases in Cells—Relative amounts of UGT in transfected-COS-1 and LS180 cells were established by Western blot as previously established (5Basu N.K. Kole L. Owens I.S. Biochem. Biophys. Res. Commun. 2003; 303: 98-104Crossref PubMed Scopus (43) Google Scholar, 11Ciotti M. Basu N. Brangi M. Owens I.S. Biochem. Biophys. Res. Commun. 1999; 260: 199-202Crossref PubMed Scopus (132) Google Scholar). 25 μg of cellular homogenate were solubilized in SDS-sample buffer, applied to polyacrylamide (PAGE)-SDS gel and subjected to electrophoresis. Following protein electrotransblotting onto nitrocellulose membrane, regular blots were processed as previously described (11Ciotti M. Basu N. Brangi M. Owens I.S. Biochem. Biophys. Res. Commun. 1999; 260: 199-202Crossref PubMed Scopus (132) Google Scholar) for exposure to x-ray films. Membranes used with antibodies to target phospho-groups were blocked and processed as described (7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar). Blots were exposed to antibody specific for the protein of βII-, δ-, γ-, and ζ-PKC (not shown), of α- and ϵ-PKC, of MAP44/42 or to antibody specific for the phosphate group in PKCϵ (Ser-729), PKCα (Ser-657), or MAP44/42 according to supplier's instructions. Membranes were exposed to appropriate secondary antibody-horseradish peroxidase conjugate and visualized as described for anti-UGT. In Situ Labeling of UGT and PKCϵ with [33P]Orthophosphate—To determine whether UGTs undergo phosphorylation, LS180 cells were grown to nearly 100% confluency and processed as previously described (7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar, 17Akhand A.A. Pu M. Senga T. Kato M. Suzuki H. Miyata T. Hamaguchi M. Nakashima I. J. Biol. Chem. 1999; 274: 25821-25826Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). All conditioned cells were exposed to [33P]orthophosphate and to curcumin for 1–8 h to allow maximum inhibition and recovery or to calphostin-C for the final hour. Cells were harvested and solubilized; equal protein was allowed to immunocomplex with anti-UGT-CE, washed (12Ritter J.K. Sheen Y.Y. Owens I.S. J. Biol. Chem. 1990; 265: 7900-7906Abstract Full Text PDF PubMed Google Scholar), and subjected to electrophoresis in a SDS-7.5% PAGE system. Similarly, solubilized cell extracts were immunocomplexed with anti-PKCϵ protein backbone, washed, and resolved by SDS-PAGE. Finally, the UGT-or PKCϵ-containing gel was fixed, dried, and scanned for quantitation as described for [14C]glucuronides (15Ciotti M. Owens I.S. Biochemistry. 1996; 35: 10119-10124Crossref PubMed Scopus (30) Google Scholar). Both gels were exposed to x-ray film. Co-localization Studies—To determine whether UGT1A7His and phospho-PKCϵ co-localize, COS-1 cells, grown on Lab-Tek slides (Nalgene, Frederick, MD), were transfected (7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar) with pUGT1A7His. Cells were processed for immunofluorescence (7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar) by exposure to primary antibodies: rabbit anti-phospho(Ser-729)PKCϵ and mouse anti-His tag (Amersham Biosciences). Phospho-PKCϵ and 1A7His were visualized with donkey anti-rabbit-FITC conjugate and donkey anti-mouse-TRITC conjugate, respectively. Their images were assembled by computer using a photometric Zeiss microscope (Germany). For UGT1A10 and PKCα co-localization, rabbit anti-UGT-CE (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 5Basu N.K. Kole L. Owens I.S. Biochem. Biophys. Res. Commun. 2003; 303: 98-104Crossref PubMed Scopus (43) Google Scholar, 11Ciotti M. Basu N. Brangi M. Owens I.S. Biochem. Biophys. Res. Commun. 1999; 260: 199-202Crossref PubMed Scopus (132) Google Scholar) and mouse anti-PKCα were added to UGT1A10His-transfected cells, and subsequently visualized with donkey anti-rabbit-FITC conjugate and donkey anti-mouse TRITC conjugate, respectively. For PKCδ and UGT1A10His co-localization, rabbit anti-PKCδ and mouse anti-His tag were added to UGT1A10His-transfected cells and subsequently visualized with donkey anti-rabbit-FITC conjugate and donkey anti-mouse TRITC conjugate, respectively. All cells were exposed to DAPI fluorescein to identify nuclei. Co-localization of PKCα and ER marker proteins, calnexin or calreticulin, in control cells was analyzed as follows: mouse anti-PKCα and rabbit anti-calnexin were added to cells and visualized with donkey anti-mouse TRITC and donkey anti-rabbit FITC conjugate, respectively. Also, co-localization of PKCα and calreticulin was determined by exposing cells to mouse anti-PKCα and rabbit anti-calreticulin and visualized with donkey anti-mouse TRITC conjugate and donkey anti-rabbit FITC conjugate, respectively. Treatment of LS180 Cells with Agonists and Antagonists for PKCs—To show significant enzyme activation, we first inhibited cellular UGT activity with curcumin treatment before exposure to calyculin-A, DAG, PMA, or PS. Additionally, LS180 cells were treated with agonist or antagonist PKCϵ-specific peptide for different times as shown. For peptides lacking a cellular-permeating conjugate, cells were grown to confluency before permeabilizing with 45 μg/ml saponin (18Johnson J.A. Gray M.O. Karliner J.S. Chen C.-H. Mochly-Rosen D. Circ. Res. 1996; 79: 1086-1099Crossref PubMed Scopus (80) Google Scholar). Permeabilized cells were exposed to medium containing octapeptide (Oct) represented in the V1 region of PKCϵ (19Csukai M. Chen C.-H. De Matteis M.A. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 29200-29206Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) or to its scrambled (Scr) control and allowed to uptake test peptide. A sequence also in the VI region of PKCϵ, Antennapedia-conjugated pseudo ϵ-RACK agonist peptide, was introduced into cells following curcumin pretreatment. Extracts of cells were assayed in vitro. (ϵ-RACK receptor is commonly known as β-COP (7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar, 19Csukai M. Chen C.-H. De Matteis M.A. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 29200-29206Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar).) Treatment of UGT1A7-transfected COS-1 Cells with PKCϵ-specific siRNA—PKCϵ-specific siRNA (100 nm) or its control (Dharmacon) was transfected according to the source's protocol into COS-1 cells, which had expressed UGT1A7His for 24 h; treatment continued for 48 h before harvesting cells for eugenol and mycophenolic acid (shown) glucuronidation as described above in 2-h incubations. Co-immunoprecipitation Studies—COS-1 cells that expressed UGT1A7-, UGT1A10-, or each corresponding His construct were untreated or treated with curcumin or calphostin-C, harvested, solubilized in cold Kinexus lysis buffer (Kinexus, Vancouver, Canada) (modified Kinexus lysis buffer contained 20 mm MOPS buffer, pH 7.0, 2 mm EGTA, 5 mm EDTA, 30 mm NaF, 40 mm β-glycerophosphate, pH 3.2, 20 mm pyrophosphate, 1 mm sodium orthovanadate, 1 mm phenylmethysulfonyl fluoride, 3 mm benzamidine, 5 μm pepstatin, 10 μm leupeptin, 0.5% Nonidet P-40, and 5 mm CHAPS adjusted to pH 7.2), fractionated by centrifugation at 60,000 × g for 30 min, and subjected to co-immunoprecipitation as previously described using anti-UGT-CE or anti-His. Immunoprecipitates were resolved in SDS-PAGE gel systems and transblotted as described for Western blot analysis using antibodies given in respective figure legends. In Vitro Transcription, Translation, and PKCϵ-dependent Phosphorylation of Nascent UGT1A7—The UGT1A7cDNA was inserted at the XbaI/BamH1 cloning site of pcDNA3.1 (–)/Myc-HisB (Invitrogen) downstream of the T7 RNA polymerase promoter followed by the polyadenylation consensus sequence; the construct was linearized by digesting with SmaI. Purified 1A7-containing construct was transcribed into mRNA, which was subsequently translated into UGT1A7Myc-His using a TnTR T7 Coupled Wheat Germ extract system (Promega) that was incubated 90 min at 30 °C according to the manufacturer's protocol. To newly synthesized UGT1A7His, 10 ng of PKCϵ, 75 μm [γ-33P]ATP (2.2 μCi/pmol), and other kinasing reactants (Upstate) were combined in 60 μl and allowed to react for 20 min at 30 °C according to the manufacturer's protocol. Aliquots (10 μl) of the reaction mixture were added to P81 cellulose membrane, washed with 0.75% phosphoric acid and acetone, air dried, and counted using a liquid scintillation system according to a Promega protocol. Also, the 250-μl reaction system was solubilized with an equal volume of 2× Kinexus buffer, centrifuged at 10,000 × g for 20 min, the supernatant was added to Ni+-charged resin (Sigma), and the purification strategy followed the source's protocol. Duplicate 4–15% gradient SDS-PAGE systems were loaded with processed samples and electrophoresed; one gel was dried and exposed to x-ray film for development, and the duplicate gel was transblotted onto a nitrocellulose membrane and analyzed by Western blot as previously described using anti-His and anti-UGT-CE as already described. Treatment of LS180 Cells with Catalase and Hydrogen Peroxide—To test effects of reactive oxygen species (ROS) on constitutive UGT activity, untreated LS180 cells were exposed to different concentrations of catalase or herbimycin-A. Also, cells were treated with different concentrations of H2O2 or combinations of H2O2 and catalase or herbimycin-A (20Konishi H. Tanaka M. Takemura Y. Matsuzaki H. Ono Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11233-11237Crossref PubMed Scopus (550) Google Scholar, 21Min D.S. Kim E.-G. Exton J.H. J. Biol. Chem. 1998; 273: 29986-29994Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 22Sundaresan M. Yu Z. Farrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2342) Google Scholar, 23Suzuki Y.J. Forman H.J. Sevanian A. Free Radic. Biol. Med. 1997; 22: 269-285Crossref PubMed Scopus (1269) Google Scholar), or of H2O2 with curcumin or calphostin-C. Cellular extracts were tested for glucuronidation of eugenol and capsaicin (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Inhibition of UGTs Expressed in COS-1 and LS180 Cells by Curcumin or Calphostin-C Treatment—Our attempts to uncover special UGT properties associated with function began with a screen to discover new regulatory molecules among UGT substrates. While comparing in-cell treatment of two members, UGT1A7 and 1A10, of human UGT1A family that have similar properties, 1A7 was dramatically down-regulated by 200 μm curcumin, but 1A10 showed minor inhibition (Fig. 1A). With appropriate concentrations, there was rapid inhibition with recovery for all UGTs tested, re-exposure to curcumin caused the cycle to repeat for the three times tested (data not shown), suggesting the involvement of signaling events (24Surh Y.-J. Food Chem. 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Effects of curcumin treatment of ten independently expressed UGTs, UGT1A1, -1A3, -1A4, -1A6, -1A7 through -1A10, -2B7, and -2B15, in COS-1 cells showed that each was inhibited without affecting specific protein (Fig. 2), and the inhibitory level of curcumin was isozyme-dependent (Fig. 2). Analysis of concentration dependence of curcumin and calphostin-C for two isozymes with contrasting characteristics (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 7Basu N.K. Kovarova M. Garza A. Kubota S. Saha T. Mitra P.S. Banerjee R. Rivera J. Owens I.S. Proc. Natl. Acad. Sci., U. S. A. 2005; 102: 6285-6290Crossref PubMed Scopus (50) Google Scholar) showed UGT1A7 was among the most sensitive to the reagents, and UGT1A10 was the most resistant (supplemental Fig. S1). The concentration profiles for the eight other isozymes are not shown. Immunoprecipitates of UGT1A7 and -1A10 with anti-UGT-CE again (4Basu N.K. Ciotti M. Hwang M.S. Kole L. Mitra P.S. Cho J.W. Owens I.S. J. Biol. Chem. 2004; 279: 1429-1441Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Basu N.K. Kubota
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