Nikhil K. Basu

Gastrointestinally Distributed UDP-glucuronosyltransferase 1A10, Which Metabolizes Estrogens and Nonsteroidal Anti-inflammatory Drugs, Depends upon Phosphorylation*

Among gastrointestinal distributed isozymes encoded at the UGT1 locus, UDP-glucuronosyltransferase 1A10 (UGT1A10) metabolizes a number of important chemicals. Similar to broad conversion of phytoestrogens (Basu, N. K., Ciotti, M., Hwang, M. S., Kole, L., Mitra, P. S., Cho, J. W., and Owens, I. S. (2004) J. Biol. Chem. 279, 1429–1441), UGT1A10 metabolized estrogens and their derivatives, whereas UGT1A1, -1A3, -1A7, and -1A8 differentially exhibited reduced activity toward the same. UGT1A10 compared with UGT1A7, -1A8, and -1A3 generally exhibited high activity toward acidic nonsteroidal anti-inflammatory drugs and natural benzaldehyde derivatives, while UGT1A3 metabolized most efficiently aromatic transcinnamic acids known to be generated from flavonoid glycosides by microflora in the lower gastrointestinal tract. Finally UGT1A10, -1A7, -1A8, and -1A3 converted plant-based salicylic acids; methylsalicylic acid was transformed at high levels, and acetylsalicylic (aspirin) and salicylic acid were transformed at moderate to low levels. Atypically UGT1A10 transformed estrogens between pH 6 and 8 but acidic structures preferentially at pH 6.4. Furthermore evidence indicates UGT1A10 expressed in COS-1 cells depends upon phosphorylation; UGT1A10 versus its single, double, and triple mutants at three predicted protein kinase C phosphorylation sites incorporated [33P]-orthophosphate and showed a progressive decrease with no detectable label or activity for the triple T73A/T202A/S432G-1A10 mutant. Single and double mutants revealed either null/full activity or null/additive activity, respectively. Additionally UGT1A10-expressing cultures glucuronidated 17β-[14C]estradiol, whereas cultures containing null mutants at protein kinase C sites showed no estrogen conversion. Importantly UGT1A10 in cells supported 10-fold higher glucuronidation of 17β-estradiol than UGT1A1. In summary, our results suggest gastrointestinally distributed UGT1A10 is important for detoxifying estrogens/phytoestrogens and aromatic acids with complementary activity by UGT1A7, -1A8, -1A3, and/or -1A1 evidently dependent upon phosphorylation.

Evidence for phosphorylation requirement for human bilirubin UDP-glucuronosyltransferase (UGT1A1) activity

Our discovery of rapid down-regulation of human bilirubin UDP-glucuronosyltransferase (UGT) in colon cell lines that was transient and irreversible following curcumin- and calphostin-C-treatment, respectively, suggested phosphorylation event(s) were involved in activity. Likewise, bilirubin-UGT1A1 expressed in COS-1 cells was inhibited by curcumin and calphostin-C. Because calphostin-C is a highly specific protein kinase C (PKC) inhibitor, we examined and found 4 to 5 predicted PKC phosphorylation sites in 11 UGTs examined. UGT1A1 incorporated [33P]orthophosphate, which was inhibited by calphostin-C. Also triple mutant, T75A/T112A/S435G-UGT1A1, at predicted PKC sites failed to incorporate [33P]orthophosphate. Individual or double mutants exhibited dominant-negative, additive, or no effect, while the triple mutant retained 10-15% activity towards bilirubin and two xenobiotics. Compared to wild-type, S435G and T112A/S435G shifted pH-optimum for eugenol, but not for bilirubin or anthraflavic acid, toward alkaline and acid conditions, respectively. This represents the first evidence that a UGT isozyme requires phosphorylation for activity.

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 [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.

Src supports UDP-glucuronosyltransferase-2B7 detoxification of catechol estrogens associated with breast cancer.

Mammary gland-distributed and ER-bound UDP-glucuronosyltransferase (UGT)-2B7 metabolizes genotoxic catechol-estrogens (CE) associated with breast cancer initiation. Although UGT2B7 has 3 PKC- and 2 tyrosine kinase (TK)-sites, its inhibition by genistein, herbimycin-A and PP2 with parallel losses in phospho-tyrosine and phospho-Y438-2B7 content indicated it requires tyrosine phosphorylation, unlike required PKC phosphorylation of UGT1A isozymes. 2B7 mutants at PKC-sites had essentially normal activity, while its TK-sites mutants, Y236F- and Y438F-2B7, were essentially inactive. Overexpression of regular or active Src, but not dominant-negative Src, in 2B7-transfected COS-1 cells increased 2B7 activity and phospho-Y438-2B7 by 50%. Co-localization of 2B7 and regular SrcTK in COS-1 cells that was dissociated by pretreatment with Src-specific PP2-inhibitor provided strong evidence Src supports 2B7 activity. Consistent with these findings, evidence indicates an appropriate set of ER proteins with Src-homology binding-domains, including 2B7 and well-known multi-functional Src-engaged AKAP12 scaffold, supports Src-dependent phosphorylation of CE-metabolizing 2B7 enabling it to function as a tumor suppressor.

Regulated Phosphorylation of a Major UDP-glucuronosyltransferase Isozyme by Tyrosine Kinases Dictates Endogenous Substrate Selection for Detoxification

Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE1) with barely detectable 17β-estradiol (E2) conversion following expression in COS-1 cells. Consistent with the UDP-glucuronosyltransferase requirement for regulated phosphorylation, we discovered that 2B7 requires Src-dependent tyrosine phosphorylation. Y236F-2B7 and Y438F-2B7 mutants were null and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [33P]orthophosphate and that 2B7His, previously expressed in SYF-(Src,Yes,Fyn)−/− cells, was Src-supported or phosphorylated under in vitro conditions. Unexpectedly, 2B7 expressed in SYF−/− and SYF+/− cells metabolized 4-OHE1 at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E2 metabolism was 16- and 9-fold higher than in COS-1. Because anti-Tyr(P)-438–2B7 detected Tyr(P)-438–2B7 in each cell line, results indicated that unidentified tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF−/−. 2B7-transfected COS-1 treated with increasing concentrations of the Src-specific inhibitor PP2 down-regulated 4-OHE1 glucuronidation reaching 60% maximum while simultaneously increasing E2 metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E2 metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF−/− is more competent at metabolizing E2 in cellulo than 2B7 expressed in COS-1. To confirm Src-controlled 2B7 prevents toxicity, we showed that 2B7-transfected COS-1 efficiently protected against 4-OH-E1-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE1, but not E2, in COS-1, whereas non-Src-phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined that 2B7 substrate selection is not fixed but varies depending upon the TK(s) that carry out its required phosphorylation.

Protein Kinase Cα and Src Kinase Support Human Prostate-distributed Dihydrotestosterone-metabolizing UDP-glucuronosyltransferase 2B15 Activity

Background: UDP-glucuronosyltransferase 2B15 (UGT2B15) is the only 5α-dihydrotestosterone (DHT)-metabolizing enzyme in prostate luminal cells. Results: UGT2B15 requires regulated phosphorylation to metabolize DHT. Conclusion: PKCα and Src support phosphorylation of UGT2B15 at five sites involving two signaling pathways and cross-talk. Significance: Strategically controlled DHT synthesis and complex UGT2B15 phosphorylation impose low DHT turnover and presumably homeostatic levels of DHT-occupied androgen receptor for prostate-specific functions. Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17β-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70–100 and 80–90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130–140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D3 enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-33P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55–58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.

RESPONSE TO THE LETTER TO THE EDITOR BY DRS. N. PICARD AND P. MARQUET REGARDING A PUBLICATION: BASU ET AL., DRUG METABOLISM AND DISPOSITION (32:768–773, (2004)

What is the value of studying mycophenolic acid (MPA) glucuronidation in the esophagus? Whereas the authors would consider the esophagus not worthy of study viz. MPA bioavailability, our stated aim was to carry out in vitro characterization of the primary metabolizers of mycophenolic acid among