UDP-glucuronosyltransferase (UGT) isozymes, distributed primarily in liver, kidney, gastrointestinal tract and steroid responsive tissues, carry out the essential function of converting innumerable and frequently encountered, but structurally diverse, lipophilic chemicals to glucuronides. Such lipophiles include toxic metabolites, dietary constituents, environmental agents/carcinogens, and therapeutics. Conversion of chemicals to glucuronides inactivates each and hastens excretion from the body to prevent tissue accumulation and toxicities. Neurotoxic bilirubin is the most important endogenous substrate, followed by genotoxic catechol estrogens. The UGT isozyme system prevents bilirubin neurotoxicities in children, inactivates common environmental mutagens and carcinogens and prematurely converts therapeutics, it is important to understand the mechanism of glucuronidation. An understanding would allow development of methods and strategies for accelerating removal of toxic chemicals, while extending therapeutic benefits of glucuronidatable medications. Our cloning and characterization of the bilirubin-specific UGT1A1 enabled us to identify the genetic defect in hyperbilirubinemic Crigler-Najjar children. Dependence of UGT2B7 activity on phosphorylation by c-Src kinase The UGT2B7 isozyme, which preferentially metabolizes genotoxic catechol estrogens associated with breast cancer initiation, undergoes required tyrosine phosphorylation. UGT2B7 incorporation of immunoprecipitable 33Porthophosphate confirmed phosphorylation. The activity of Y236F-UGT2B7 and Y438F-UGT2B7 mutants was reduced by 90100%. Affinity-purified UGT2B7His, which was initially expressed in SYF(Src,Yes,Fyn)−/−cells and phosphorylated in vitro with Src and 33PATP versus 33PATP alone confirmed Src supports UGT2B7 phosphorylation. Unexpectedly, 2B7 expressed in SYF-/-- and SYF+/--cells metabolized 4-OH-E1 at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed 17β-estradiol (E2) metabolism was 16- and 9-fold higher than in COS-1 cells. As anti-phospho-Y438-2B7 detected phospho-Y438-2B7 in each cell-line, results indicated non-Src tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF-/- cells. 2B7-Transfected COS-1 treated with increasing concentrations of Src-specific inhibitor, PP2, down-regulated 4-OH-E1 glucuronidation reaching 60 % maximum, while simultaneously increasing E2 metabolism linearly. This finding indicates increasing PP2-inhibition of Src allows increasing E2 metabolism due to 2B7 phosphorylation by non-Src TK(s). Importantly, 2B7 expressed in SYF-/- is more competent at metabolizing E2 in-cellulo than 2B7 expressed in COS-1. We also showed 2B7-transfected COS-1 efficiently protected against 4-OH-E1-mediated depurination. Finally, our results indicate Src-dependent phosphorylation of 2B7 allows metabolism of 4-OH-E1, but not E2, in COS-1, while non-Src phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined 2B7 substrate selection is not fixed, but varies depending upon the TK(s) that carry out its required phosphorylation. Dependence of UGT2B15 on PKC phosphorylation with regulation by Src kinase phosphorylation Glucuronidation of dihydrotestosterone (DHT) by UGT2B15 or UGT2B17 is the primary reaction for inactivating the potent androgen associated with benign prostate hyperplasia and prostate cancer. 3α-Hydroxysteroid dehydrogenase converts DHT to its androstane-3α,17β-diol (3α-diol) metabolite that is more efficiently glucuronidated by 2B15 and 2B17 than DHT. In prostate, 2B15 is distributed in luminal, and 2B17 is distributed in basal cells. The literature shows both basal and luminal cells are associated with the initiation of prostate cancer. Transient down-regulation of UGTs following curcumin exposure signified that UGT2B15 required regulated phosphorylation. In addition, PKC inhibitors irreversibly inhibited 2B15 activity by 80100%. In addition, the c-Srcspecific PP2 inhibitor and c-Src-siRNA inhibited activity in a concentration-dependent manner. Computer analysis of consensus phosphorylation sites in 2B15 uncovered 3 PKC and 2 tyrosine kinase sites. Site-directed mutagenesis of these residues revealed null activity for S172A and 70 to 90 % loss of activity for S124A, Y99F and Y237F mutants. S422A mutant led to minor activity changes. Whereas UGT2B15His and PKCαwere co-immunoprecipitated with an anti-His preparation, the proteins were dissociated by pretreatment with BIM, G6976, or Rtlerin. This finding indicated UGT2B15 requires PKCαphosphorylation. Further treatment of 2B15 wild-type or S422A-2B15 mutant with PKCα-siRNA led to loss of both DHT and 3α-diol activities, whereas similar treatment of S124A-UGT2B15 and S124A/S422A-UGT2B15 double-mutant showed a selective increase and decrease in DHT metabolism, respectively, without affecting 3α-diol glucuronidation. We also examined effects of PP2 on activity for mutants. PP2-treatment of 2B15 wild-type and S422A-2B15 mutant inhibited and increased both substrate-activities, respectively. Contrariwise, PP2 did not affect activity for S124A-2B15, while similar treatment of S124A/S422A-2B15 selectively increased 3α-diol turnover and inhibited DHT conversion. A consideration of the co-immunoprecipitation findings indicates S172 strictly require the modification for activity, while all other PKC sites and evidently Src-phosphorylated Y99 and/or Y237 residues act in a regulatory role to support 2B15 activity. Because DHT conversion to the 3α-diol metabolite by 3α-hydroxysteroid dehydrogenase is reversibly controlled by the cofactor ratio, NAD:NADPH, our results suggest selective inhibition of DHT glucuronidation via differential phosphorylation of 2B15 allows for 3α-diol glucuronidation only. The possibility of differential inhibition of DHT and 3α-diol glucuronidation by 2B15 allows for enhanced control of DHT levels in the luminal cells. Dependence of UGT2B17 on PKC phosphorylation UGT2B17, distributed prostate basal cells and liver, metabolizes dihydrotestosterone (DHT) and 3α-androstanediol (3α-diol) at equal rates. 2B17 converts DHT and 3α-diol at 40- and 20-fold higher rates than UGT2B15 under in vitro conditions. We found curcumin transiently down-regulated UGT2B17, which suggested that the isozyme also requires phosphorylation. Although UGT2B17 has two PKC and two tyrosine kinase sites, the enzyme is inhibited up to 80 % by 4 typical PKC inhibitors and is activated 50% by PP2 and c-SrcsiRNA. Computer searches for consensus phosphorylation sites in 2B17 detected S172, S422, Y99 and Y237. Site-directed mutagenesis revealed null activity for S172A. Low level of inhibition of Y99F and Y237F mutants was alleviated by PP2 treatment suggesting c-Src suppressed 2B17. In conclusion, our results indicate UGT2B17 requires PKC phosphorylation at S172, while c-Src appears to suppress its activity via a regulatory mechanism. Tertiary structure of UGT(s) To obtain further details for phosphorylation requirements for UGTs, we attempted to purify active UGT protein for structural analysis. In summary, our studies indicate UGT1A7 exists as a complex(es) that sustains activity via protein kinase(s) signaling. Our findings lay the foundation for further structural studies concerning this critical endogenous chemical defense enzyme system. Studies related to UGT2B7 uncovered recently are described in the following manuscript: Mitra, PS, Basu, NK, Basu, M, Chakraborty, S, Saha, T, Owens, IS. Regulated phosphorylation of a major UDP-glucuronosyltlransferase isozyme by tyrosine kinases dictates endogenous substrate-selection for detoxification. J Biol Chem Under Revision.
