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, standard dietary constituents that include endocrine disruptors, environmental wood and petroleum pyrolysates ncluding known carcinogens, consumer-products derived-chemicals, and therapeutics. Whereas seminal findings from this laboratory include identification and characterization of the bilirubin-metabolizing UGT1A1 isozyme, the novel 215-kb UGT1A locus encoding 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 cloned 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, as well as other agents. Model studies with 1A7 show mutations of PKC sites in the isozyme revealed regulated phosphorylation controls substrate selection that can change in parallel with changes in pH optimum for catalysis. Following mutation of 1/3 PKC phosphorylation sites, 1A7 flip its pH optimum from 8.6 to 6.5;mutation of the same 1/3 phosphorylation sites in 1A10 leads to pH 6.0 to 6.5. Whereas S432A-1A10 substrate-profile did not change, S432A-1A7 substrate profile changed to became identical to 1A10, phosphate signaling enabled 2 isozymes to metabolize the same set of substrate, effectively via differential signaling at the 3 phosphorylation sites in the two isozymes. Communoprecipitation studies of 1A7His and 1A10 revealed PKC-epsilon and PKC-delta phosphorylate 1A7 and 1A10, respectively. Becausa mutation of 1/3 sites in 1A7 leads to identical subsrate selection for 1A7 and 1A10. This finding indicates the two isozymes substrate-selections are not fixed, but is dictated by phosphorylation and that sting the isozymes have unlimited substrate capacity due to signaling. Findings with 2B7 show that it metabolizes depurinating depurinating catechol estrogens, but not estradiol when expressed in COS-1 cells. Following expression in Src-free cells, Catechol estrogen meabolism in creases 16 fold and estradiol metabolism increases 9-fold. To verify Src caused the difference in substrate selection, Src-inhibitor treatment of COS-1 cells caused a decrease in catehol estrogen metabolism to a constant 50 % level, as estradiol activity continued to increase with incresaing Src-inhibitor. Since Src is present in all cells, the effect indicated Src restricts 2B7 conversion of catechol estrogen levelboth cell The findings indicated Src-phosphorylaton of 2B7 to a level that prevents metabolism of estradiol to avoid conterproductive effects. Whereas the prostate luminal cell-distributed UDP-glucuronosyltransferase(UGT)-2B15 metabolizes dihydrotestosterone (DHT) and 5-alpha-androstane-3-alpha,17-beta-diol metabolite, we demonstrated for the first time that the isozyme undergoes required regulated phosphorylation similar to UGTs already analyzed. Mutation of 3 predicted PKC and 2 tyrosine kinase sites in 2B15 caused 70 to 100 % and 80 to 90 % inactivation, respectively. Anti-UGT-1168 trapped UGT2B15-His-containing co-immunoprecipitates of PKC. Complexes bound to 2B15-His-wt remained intact during electrophoresis, while 2B15-His mutants at phosphorylation sites differentially dissociated. PKC-alpha-siRNA treatment inactivated more than 50 per-cent of COS-1-expressed 2B15. Treatment with Src-specific PP2 inhibitor or Src-siRNA inhibited more than 50 per-cent activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in-vitro gamma 33-PATP-dependent phosphorylation by PKC-alpha and/or Src, affinity-purification, and SDS-gel analysis revealed 2-fold greater radiolabeling of 2B15-His by PKC-alpha than by Src. Labeling by the 2 kinases was additive. Collectively, evidence indicates 2B15 requires regulated phosphorylation by both PKC-alpha and Src kinase. Comparatively, very low-activity luminal-cell 2B15 undergoes a complex pattern of regulated phosphorylation necessarily to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions. Moreover, we have demonstrated for the first time that prostate basal-cell distributed 2B17 requires regulated phosphorylation. Regulated phosphorylation is demonstrated by reversible and irreversible inhibition following curcumin or general PKC inhibitors, respectively. PKC-epsilon-siRNA, Src kinase-specific PP2 inhibitor or Src-siRNA treatment of 2B17 expressed in COS-1 caused increases in DHT and the 5-alpha-androstane-3alpha,17beta-diol metabolite activity, whereas treatment with PKC-alpha-siRNA or PMA in COS-1 downregulated activity. 2B17 phosphorylation-sites mutants expressed in COS-1 cells, S172A and S422A were null or unaffected, respectively, whereas Y99F and Y237F mutants revealed selective downregulated DHT glucuronidation over 5alpha-androstane-5alpha,17-beta-diol by 62% versus 20% and 87 % versus 55%, respectively. Contrariwise, activities for 2B17-mutants expressed in SYF-free cells closely equalized at 50 % without evidence of substrate preference and increasing PKCepsilon-siRNA treatment uniquely switched to downregulation, confirming previous upregulation via Src. Western blot analysis of co-immunoprecipitates of Y237F-2B17His mutant compared to 2B17His-wt elicited significantly greater co-migration of PKCalpha and PKCepsilon in 80 to >250-kDa complexe(s). The fact that Y237F-2B17 showed superior PKCepsilon binding indicates Y99 is a critical residue for tyrosine kinase phosphorylation. Moreover, in-vitro studies showed PKCalpha, PKCepsilon and Src individually and additively phosphorylated 2B17. For 95% identical prostate-distributed UGT2B17 and 2B15, we show strong PKCepsilon binding at a modified Src Y99 site in 2B17 compared to that in 2B15 that downregulates 2B17 activity. Whereas our earlier findings suggest Src alone phosphorylates 2B15 upregulating its luminal-cell activity, evidence presented here indicates UGT2B17 is programmed at its Src-site to undergo Src downregulation.
