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J. Biol. Chem., Vol. 283, Issue 34, 23048-23061, August 22, 2008

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The Major Chemical-detoxifying System of UDP-glucuronosyltransferases Requires Regulated Phosphorylation Supported by Protein Kinase C^*

From the Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-1830

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 C (PKC), 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 PKC and of UGT1A10His and PKC or PKC. Inhibition of UGT activity by PKC-specific antagonist peptide or by PKC-targeted destruction with PKC-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 PKC 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.

Received for publication, January 2, 2008 , and in revised form, May 23, 2008.

^* This work was supported, in whole or in part, by the National Institutes of Health NICHD Intramural Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: NICHD, National Institutes of Health, 9000 Rockville Pike, Bldg. 10, Rm. 9D-42, Bethesda, MD 20892-1830. Tel.: 301-496-6091; Fax: 301-480-8042; E-mail: owensi{at}mail.nih.gov.

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