Phosphorylation of a UDP-glucuronosyltransferase regulates substrate specificity

Abstract

UDP-glucuronosyltransferase (UGT) isozymes catalyze detoxification of numerous chemical toxins present in our daily diet and environment by conjugation to glucuronic acid. The special properties and enzymatic mechanism(s) that enable endoplasmic reticulum-bound UGT isozymes to convert innumerable structurally diverse lipophiles to excretable glucuronides are unknown. Inhibition of cellular UGT1A7 and UGT1A10 activities and of [ 33 P]orthophosphate incorporation into immunoprecipitable proteins after exposure to curcumin or calphostin-C indicated that the isozymes are phosphorylated. Furthermore, inhibition of UGT phosphorylation and activity by treatment with PKC-specific inhibitor peptide supported PKC involvement. Coimmunoprecipitation, colocalization by means of immunofluorescence, and crosslinking studies of PKC and UGT1A7His revealed that the proteins reside within 11.4 Å of each other. Moreover, mutation of three PKC sites in each UGT isozyme demonstrated that T73A͞G and T202A͞G caused null activity, whereas S432G-UGT1A7 caused a major shift of its pH-8.5 optimum to 6.4 with new substrate selections, including 17␤-estradiol. S432G-UGT1A10 exhibited a minor pH shift without substrate alterations. PKC involvement was confirmed by the demonstration that PKC overexpression enhanced activity of UGT1A7 but not of its S432 mutant and the conversion of 17␤-[ 14 C]estradiol by S432G-UGT1A7 but not by UGT1A7. Consistent with these observations, treatment of UGT1A7-transfected cells with PKC-specific inhibitor peptide or general PKC inhibitors increased 17␤-estradiol catalysis between 5-and 11-fold, with parallel decreases in phosphoserine-432. Here, we report a mechanism involving PKC-mediated phosphorylation of UGT such that phosphoserine͞threonine regulates substrate specificity in response to chemical exposures, which possibly confers survival benefit.PKC ͉ PKC inhibitors ͉ detoxifying enzymes ͉ UDP-glucuronosyltransferase mutants ͉ immunofluorescence M ammalian UDP-glucuronosyltransferase (UGT) isozymes detoxify numerous lipid-soluble chemicals, including metabolites, such as neurotoxic bilirubin, as well as dietary and environmental agents͞toxins and medicines (1). UGT facilitates detoxification of chemicals by attaching glucuronic acid molecules to form water-soluble, readily excretable glucuronides. Failure to attach glucuronic acid (glucuronidation) to toxins allows high levels to accumulate in tissues, causing deleterious effects, including mutations of genes and the development of cancer (2). Mutations in UGT1 genes lead to lethal hyperbilirubinemic Crigler-Najjar disease (3) and to toxic tissue levels of the commonly used analgesic acetaminophen (Tylenol) in rats (4). The fact that a limited number of UGT isozymes facilitate excretion of a vast number of structurally diverse chemicals suggests a mechanism evolved to confer flexibility on an isozyme to metabolize multiple toxins. Because UGTs are bound to membranes of the endoplasmic reticulum (ER) that cause difficulties associated with purification and s...

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice—A model

“…These UGT isozymes, encoded at the UGT1 complex locus, were found strategically and differentially expressed in the gastrointestinal (GI) mucosa [1]. In this report, we established under both in-vitro and in-vivo conditions that mouse Ugt1a1 also requires phosphorylation, which can be transiently downregulated by nontoxic kinase inhibitor, curcumin [2,3,13], and irreversibly by calphostin-C, similar to that for human UGTs [2,3]. Moreover, effects of curcumin-pretreatment elicited a marked increase in free MPA levels in blood and a comparable improvement in immunosuppression in antigen-stimulated mice detected by the spleen CTL assay [6].…”

“…While UGT location in GI mucosa cells [1] suggests it is a strategic site for manipulating glucuronidation by either negative or positive regulators of activities, it is important that evidence for each UGT tested with curcumin [2,3] requires on-going phosphorylation. At appropriate concentrations, the literature indicates chemopreventive curcumin acts to scavenge free radicals and interrupt PKC signaling triggered by oxidants [23].…”

“…Despite identification of UGTs in GI mucosa using molecular biology techniques [1][2][3]15], it has not been possible to determine the impact of these isozymes [1] on chemical uptake due to the lack of a strategy to reversibly remove all GI activities. As liver UGTs are critical to systemically-distributed chemicals, it is likely that GI glucuronidation in mucosal cells has a significant impact on removing ingested substrates.…”

“…2, bottom). The parallel loss and recovery of both activity and phosphoserine content for Ugt1a1 following curcumin treatment indicates the mouse isozyme, like human UGTs [1][2][3], undergoes required phosphorylation without affecting Ugt1a1 protein (Fig. 2, bottom).…”

“…Unfortunately, it has not been possible to establish an effective, reversible biochemical inhibitor for all UGT activities. Because our recent studies demonstrated all UGTs tested require protein kinase C (PKC)-mediated phosphorylation [2,3] that is subject to reversible antioxidant down-regulation, we have developed a strategy to determine the impact of UGTs found in the mucosa layer of the gastrointestinal (GI) tract [1] on drug efficacy. We have downregulated glucuronidation by orally administering the kinase inhibitor and dietary condiment, curcumin, to block UGT phosphorylation on a transient basis [3], followed by administration of a test drug.…”