Widely distributed UDP-glucuronosyltransferase (UGT) isozymes convert innumerable structurally diverse lipophilic chemicals to inactive water-soluble glucuronides that undergo facilitated excretion. Lipophiles include toxic metabolites, dietary constituents, environmental agents/carcinogens and therapeutics. Whereas seminal findings from this laboratory include identification and characterization of the bilirubin-metabolizing UGT1A1 isozyme, the novel 215-kb UGT1A locus that encodes 13 independently regulated UGT genes that include the UGT1A1 gene, and the first genetic defects in the UGT1A1 gene that lead to Crigler-Najjar diseases in children, we also identified several UGT2B isozymes. 2B7 and 2B15 detoxify the depurinating catechol estrogens and dihydrotestosterone, respectively. To add, this laboratory demonstrated that UGTs require regulated phosphorylation, which can undergo reversible or irreversible downregulation by dietary agents or select kinase inhibitors, respectively. Detail analysis of 5 out of /19 human UGTs has shown required regulated phosphorylation at Ser/Thr and/or Tyr residues that is reversibly inhibited by treatment with dietary curcumin. Model studies with 1A7 show mutations of PKC sites in the isozyme revealed regulated phosphorylation controls substrate selection that changes in parallel with changes in pH optimum for catalysis. Coimmunoprecipitation studies of 1A7His and 1A10 revealed PKC and PKC/ phosphorylate 1A7 and 1A10, respectively. Considering 2B7 in greater detail shows it metabolizes depurinating catechol estrogens (CE)--4-OH-esradiol and 4-OH-estrone--, while both 2B15 and its 95 % identical homolog, 2B17, metabolize dihydrotestosterone (DHT) and its androstanediol (AD) metabolite but at different rates. Additionally, we demonstrated that 2B7 requires Src phosphorylation that restricts its catalysis to the depurinating catechol estrogens with the exclusion of estradiol metabolism. In Src-free cells, 2B7 is phosphorylated by an unidentified TK that enables the isozyme to increases its metabolism of CEs by 100-fold and that of estradiol by 16 fold. The results indicate regulated Src phosphorylation of 2B7 dictates endogenous substrate selections. Concerning prostate luminal-cell distributed 2B15 and prostate basal cell-distributed 2B17, both require PKC phosphorylation at position S172 and regulation of activity via Src phosphorylation at position Y237 in order to metabolize DHT and AD. Analyses also show 2B15 activity is modified by phosphorylation at 3 other sites, Y99, S124 and S422. Whereas the Y99 and S422 sites are also present in 2B17, their mutants have no effect on activity. Co-immunoprecipitation studies show PKC-alpha versus PKC-alpha and epsilon modify 2B15 and 2B17 activities respectively. Finally 2B15 expressed in Src-free cells undergo proteasomal-ubiquinationation, which is inhibited by the specific MG132 inhibitor, indicating 2B15 not only requires Src for its phosphorylation, but for its maintenance. Contrary activity for 2B17 expressed in Src-containing COS-1 cells increases 50 % following Src-siRNA treatment or following expression in Src-free cells, indicating 2B17 exists in a state of reduced activity in the presence of Src. Hence, 2B15 and 2B17 are diametrically oppositely affected by Src. In summary, results from two model UGTs indicate the active site(s) of an isozymes is not fixed but is susceptible to change via ongoing regulation of phosphorylation.
