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

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 [P-33] orthophosphate in UGTs and protein kinase C is an element of(PKC is an element of), 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 is an element of and of UGT1A10His and PKC alpha orPKC delta. Inhibition of UGT activity by PKC is an element of-specific antagonist peptide or by PKC is an element of-targeted destruction with PKC is an element of-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 is an element of 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.